Volume 188

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FEBRUARY/MARCH, 1995

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iii

Reference: Biol. Bull 188: 1-4. (February/March.

Responses of the Medaka Fish Egg (Oryzias latipes) to

the Photolysis of Microinjected Nitrophenyl-EGTA, a

Photolabile Calcium Chelator

RICHARD A. FLUCK

Biology Department, Franklin and Marshall College, P.O. Box 3003. Lancaster, Pennsylvania 17604

Photolabile calcium dictators (calcium cages) can be
used to elevate cytosolic [Ca 2+ ] at specific sites and times
(I. 2, 3). They have been especially valuable in flash pho-
tolysis studies of muscle contraction (2) and secretion (4,
5). In the present report. I describe several responses of
medaka eggs to the photolysis ofmicroinjectednitrophenyl-
EGTA (NP-EGTA), a new calcium cage (6). IVhen unfer-
tilized eggs injected with NP-EGTA were irradiated with
ultraviolet irradiation in a small region of the egg, the eggs
were activated and ooplasm within the irradiated region
contracted and accumulated there. Eggs into which NP-
EGTA was injected could a/so be fertilized. Subsequent
irradiation of such eggs, in addition to causing the con-
traction and accumulation of ooplasm, also caused a global
contraction of dividing blastomeres and the contraction
and blebbing of embryonic cells for up to 4 days after fer-
tilization. Injection of NP-EGTA had no apparent effect
on the maturation of fertilized eggs, which developed nor-
mally and hatched.

The methods for dissection of gonads from breeding
medaka, preparation of gametes, and in vitro fertilization
of eggs have been described previously (7). Gonads, ga-
metes, and zygotes were prepared in a balanced saline
solution (BSS: 1 1 1 mM NaCl; 5.37 mM KG; 1.0 mM
CaCV, 0.6 mM MgSO 4 ; 5 mM HEPES, pH 7.3). A nor-
malized time (T n ) scale in which the time between fertil-
ization and the beginning of cytokinesis is 1 unit was used
to indicate the relative temporal positions of events. A
total of 80 eggs from 6 females were used in these exper-
iments; 44 of the eggs were monitored in detail and an
additional 36 were monitored intermittently. The exper-
iments were conducted at room temperature (23-25C).

Received 30 August 1994; accepted 14 October 1994.

Methods described previously (8, 9, 10) were used to
microinject 1.4-5.6 nl of an aqueous solution of NP-
EGTA/Ca 2+ (50 mM NP-EGTA, tetrapotassium salt;
39.6 mM CaCl 2 ; 10 mM HEPES. pH 7.3) into the thin
peripheral layer of ooplasm. Assuming an accessible vol-
ume of 27.6 nl (9). injection of these volumes would give
a final ooplasm ic concentration of 2. 5-10.0 mM NP-
EGTA. After microinjection. the eggs were either placed
in a darkened cabinet for subsequent use or fertilized
within 5 min. During the experiments, the laboratory was
only dimly illuminated with incandescent lamps.

For microscopic observation and irradiation, the eggs
were transferred to a microscope slide on which a cover
glass was supported by four pillars of petroleum jelly (7).
An Osram 100 W mercury arc lamp was used to irradiate
the eggs with ultraviolet light. The light from the lamp
was passed through a filter cube (Omega Optical) con-
taining a 360 DF 40 exciter filter, a DC 405 dichroic mir-
ror, and a 486 DF32 barrier filter. An octagonal diaphragm
was used to control the size of the light beam (in most
experiments, it was either 200 //m or 475 /urn), and neutral
density filters (Omega Optical) were used to reduce the
light intensity by either 34-fold or 286-fold. Ultraviolet
light was projected onto the egg via one of three objective
lenses (Nikon): Plan 4X.N.A. = 0. 1 ; Fluor/Ph 2 DL 10X,
N.A. = 0.5; Fluor/Ph 3 DL 20X. N.A. = 0.75. In most
experiments, the equatorial region of the egg that is, a
region along a meridian and midway between the animal
and vegetal poles was illuminated en profit (an edge of
the egg was irradiated) via the 10x objective lens. Light
intensity was measured with a UVX radiometer with a
long wave sensor (UVP, Inc.). Given a light intensity of
523 //W cm~- (referred to as "high intensity" hereafter)
and assuming that all the ultraviolet light was of wave-
length 360 nm, I calculated an incident light intensity of

R. A. FLUCK

4.9 X 10 4 quanta second" ' ^m 2 ( 10X objective lens, no
neutral density filter). To monitor the eggs during irra-
diation, they were transilluminated with light from a
quartz-halogen lamp, using a heat filter (KG5) and a 486
DF32 filter, and the images were recorded via a SIT cam-
era (Dage/MTI) and a time-lapse videocassette recorder.

To monitor the development of fertilized eggs after the
first cell cycle, they were transferred to embryo rearing
medium (17 mM NaCl; 0.4 mM KCI; 0.3 mM CaCl : ;
0.67 mM MgSCV, 0.001 g/1 methylene blue).

When unfertilized eggs into which 1.4-5. 6 nl NP-
EGTA had been injected were irradiated with UV light,
they activated within 16 3 s (X SD, n = 5), as evi-
denced by the exocytosis of cortical vesicles. Exocytosis
began within the irradiated region and spread as a wave
over the rest of the egg. Eggs were photoactivated even
after the light intensity was reduced 34-fold with a neutral
density filter; but when a second neutral density filter was
added, reducing the light intensity an additional 8.5-fold,
the eggs were not activated even after 3 min of continuous
irradiation. Irradiation of unfertilized eggs that had not
received NP-EGTA did not cause them to activate.

Continued irradiation of photoactivated eggs caused
both ooplasm and oil droplets to accumulate in and next
to the irradiated zone (Fig. 1 A). Staining with rhodamine
phalloidin showed that these accumulations of ooplasm

contained filamentous actin (F-actin, Fig. IB). Such ac-
cumulations of ooplasm and F-actin were identified in 1 8
eggs, 13 of which had been photoactivated and 5 of which
had been fertilized. Moreover, the caps of ooplasm formed
in eggs into which either 1.4 nl or 4.2 nl of NP-EGTA
had been injected and in eggs that were irradiated either
intermittently (5 s on/1 15 s off) with a high intensity of
light or continuously with a 34-fold lower light intensity.
However, when the light intensity was lowered 289-fold,
ooplasm neither contracted nor accumulated in the ir-
radiated zone. When UV irradiation was intermittent, the
ooplasm within the irradiated zone usually contracted
each time it was irradiated. For example, the region in
the egg shown in Figure 1 was irradiated 29 times for 5 s
and contracted 16 times, and a sibling egg contracted each
of the 3 1 times it was irradiated for 5 s. Each contraction
appeared to pull ooplasm and nearby oil droplets toward
the irradiated region. Eggs that were parthenogenetically
activated by the injection itself and grown in the dark
segregated normally (as do eggs that have been parthe-
nogenetically activated by pricking; Fluck, unpub. obs.),
with a cap of ooplasm forming at the animal pole and the
oil droplets segregating toward the vegetal pole.

Eggs into which NP-EGTA had been injected could
also be fertilized. In most fertilized eggs, cortical vesicles
in one small region of the egg, presumably near the in-

Kifjure 1. Accumulation of ooplasm within a UV-irradiated region of an egg. NP-EGTA/Ca ;+ (4.2 nl)
was microimected into this egg, which was then irradiated en pro/i/ in a region (approximately defined by
the filled circles) along a meridian and midway between its animal and vegetal poles. The UV light was
projected through the 10x objective lens with no neutral density filter in the light path. Ultraviolet irradiation
was intermittent, with light pulses delivered for 5 s at 2-min intervals fora total of 79 min (until 7 n = 1.0).
The egg was then fixed overnight at room temperature with formaldehyde dissolved in an actin-stabilizing
buffer(24): 3.7% formaldehyde (Electron Microscopy Sciences. Fort Washington. PA). 100 mM KCI, 5 mM
MgCl,. 2 mM EGTA. 10 mM PIPES. pH 6.8. It was then dechorionated with fine forceps, permeabilized
for 15 min with 0.3% Triton X-KH) in BSS, and stained for 30 min in 0.25 ^M rhodamine phalloidin
(Molecular Probes, Inc., Eugene. OR) dissolved in BSS. The irradiated region of the egg was photographed
with bnghtfield optics just before fixation I A) and with epi-illumination after staining the egg with rhodamine
phalloidin (B). Note the accumulation of ooplasm, oil droplets, and F-actin in the irradiated region. Scale
bars. 100 /jm

CALCIUM CAGE IN MEDAKA EGGS

jection site, did not undergo exocytosis. Irradiation of fer-
tilized eggs caused ooplasm to accumulate within the ir-
radiated region, but no such accumulations were seen in
fertilized eggs that were grown in the dark; in such dark-
grown eggs, the ooplasm and its contents appeared to seg-
regate normally. Moreover, irradiation of unfertilized eggs
that had not received NP-EGTA caused neither contrac-
tion of the ooplasm nor accumulation of ooplasm within
the irradiated region.

All eggs that received 1.4 nl of NP-EGTA underwent
cytokinesis. Of eight such fertilized eggs whose subsequent
development was monitored, four hatched and the other
four underwent extensive morphogenesis but did not
hatch. Cleavage was abnormal in eggs that received either
4.2 or 5.6 nl of NP-EGTA, and the embryos did not de-
velop further. Irradiation of eggs (that received 1.4 nl of
NP-EGTA and were subsequently fertilized) during early
cleavage caused cells in the light beam to contract globally.
Moreover, irradiation of early gastrulae caused blebbing
and global contraction of deep blastomeres, but irradiation
of the yolk sac in stage 19, 22. and 25 embryos (that is,
up to 4 days after fertilization) caused contractions that
appeared similar to those seen during the rhythmic con-
traction waves that occur in the stellate layer of the me-
daka embryo (11). Cells of embryos that did not receive
NP-EGTA failed to contract when they were irradiated
with UV light.

Taken together, these findings show NP-EGTA to be
a useful new reagent for cell and developmental biologists.
Several properties of this compound its high affinity for
Ca :+ (6), the approximately 10,000-fold decrease in its
affinity for Ca 24 upon photolysis (6), its weak fluorescence
(6), and its persistence and low toxicity in the teleost em-
bryo (this study) appear to make it particularly suitable
for studying events that have been linked to elevations in
cytosolic [Ca 2+ ]: egg activation (12, 13), ooplasmic seg-
regation (9, 10), nuclear envelope breakdown (14), mitosis
(15, 16, 17), cytokinesis (8, 18), and neuronal growth cone
motility (19, 20).

The accumulation of ooplasm and F-actin within the
UV-irradiated region of the egg is consistent with the hy-
pothesis that cytosolic calcium gradients organize devel-
opmental localization in eggs ( 10, 2 1 , 22, 23). At present,
however, the evidence consistent with this hypothesis, in-
cluding that presented in the present report, is indirect
and must be extended by using aequorin or a fluorescent
calcium indicator to measure cytosolic [Ca : ' ] in eggs dur-
ing and after photolysis of NP-EGTA. Full exploitation
of the apparent promise of NP-EGTA will require the
development of a dextran-conjugated form of the mole-
cule and the exploration of wider ranges of intracellular
concentrations of the cage, shapes of the irradiated region
(for example, a narrow rectangle that could elevate cy-

tosolic [Ca 2+ ] in a narrow band in a cell), and light inten-
sities.

Acknowledgments

Lionel Jaffe suggested the use of calcium cages to gen-
erate zones of elevated calcium in medaka eggs. I am
grateful to Jack Kaplan and Graham Ellis-Davies for pro-
viding the NP-EGTA used in these studies; to Alan Bruns
for helpful discussions about units of light intensity; and
to Andrew Miller for helping to improve the text of the
manuscript. This work was supported by NSF DCB-
9017210 and NSF MCB-9316125.

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4

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Reference: Biol. Bull 188: 5-7. (February/March, 1995)

Hemoglobin in the Symbiont-Harboring Gill of the
Marine Gastropod Alviniconcha hessleri

JONATHAN B. WITTENBERG 1 AND JEFFREY L. STEIN 2

1 Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx,
New York 10461, and 2 The Agouron Institute, La Jolla, California 92037

Hydrogen sulfide of geochemical origin, mixing at
oceanic hydrothermal vents with oxygen from oceanic sea-
water, supports dense populations ofchemoautrophic, sul-
fur-oxidizing bacteria. Those animals, the vest i men! iferan
worm Riftia pachyptila, certain bivalve molluscs, and the
recently discovered Pacific gastropod Alviniconcha hess-
leri, that interiorize the bacteria as intracellular symbionts
dominate the vent fauna (I, 2). The immense size of these
animals, the large standing crop represented in their dense
communities, and the rapid growth of individuals all attest
to the effective use of an abundant food base. Dense con-
centrations of the mesogastropod Alviniconcha hessleri
(2, 3) were recently discovered at deep-sea hydrothermal
vents at the spreading center in the Mariana Back- Arc
Basin of the Western Pacific. These animals house che-
moantrophic. sulfide-oxidizing bacteria within specialized
cells (bacteriocytes) of their modified gills (2). They are
the only reported example of a symbiotic association be-
tween a gastropod mollusc and intracellular chemoauto-
trophic bacteria. H 'e now show that the modified gill of
Alviniconcha contains hemoglobin at a concentration of
about 65 n/nol/kg wet weight gill. This value falls within
the range. 20-250 nmol hemoglobin per kilogram, en-
countered in the modified symbiont-harboring gills of
many of the sulfide- dependent clams examined but is short
of the very high concentrations, 550 ami 1200 ^mol/kg,
found in Myrtea spinifera and Lucina pectinata respec-
tively (4). Accordingly, bacteriocyte hemoglobin is a feature
common to both gastropod and bivalve symbioses.

Symbioses with intracellular carbon-fixing bacteria,
believed to be dependent on bacteriocyte hemoglobin,
have heretofore been described only in clams of the fam-
ilies Solemyidae, Lucinidae, and Vesicomyidae and in a

Received 2 September 1994; accepted 23 November 1994.

few mussels, Mytilidae, restricted to the genus Bathy-
modiolus (4). The molluscan symbionts fix carbon from
oceanic carbon dioxide into hexoses and supply almost
all of the carbon nutrition of the host (5). Ribulose bis-
phosphate carboxylase/oxygenase (RuBisCO), the enzyme
responsible for carbon dioxide fixation, has been cloned
from the Alviniconcha symbiont and expressed in Esch-
erichia coli (6). The relatively low specificity of the purified
enzyme for carbon dioxide indicates that the intracellular
environment of the endosymbionts may be microaero-
philic for RuBisCO to maintain net carboxylation (7).

Hemoglobins, probably located in the host cytoplasm
and excluded from the peribacterial space and probably
coded by host genes, are a near-constant feature of sym-
bioses between bivalve molluscs and intracellular che-
moautotrophic bacteria (4, 8); such hemoglobins are also
a constant feature of symbioses between plants and in-
tracellular prokaryotic nitrogen-fixing symbionts (9).
Clam gill hemoglobins have been investigated intensively
(10-13), and the three-dimensional structure of one is
known from x-ray diffraction analysis ( 14). The probable
role of the hemoglobin is to bring oxygen and hydrogen
sulfide to the symbiont (8, 15). In the giant tube worm
Riftia. this function is served by blood or coelomic he-
moglobin, which bathe the symbiont-harboring cells and
transport oxygen at the heme and sulfide at a site remote
from the heme (16). In the bacteria-housing clam gill,
these functions are probably served by two separate he-
moglobins of the bacteriocyte cytoplasm ( 1 7). Cytoplasmic
hemoglobins are believed to supplement the diffusion of
free oxygen by adding to it a contribution from oxygen
combined with the protein. In those few hemoglobin-
containing tissues that have been studied intensively, the
hemoglobin is maintained partially desaturated with oxy-
gen, the larger part of the oxygen flow to the intracellular
organelle is carried in combination with the protein, and

J. B. WITTENBERG AND J. L. STEIN

the oxygen pressure is held low (18). This is in accord
with the suggestion that low oxygen pressure is probably
required to allow RuBisCO to maintain net carboxylation
in the Alviniconcha gill (7). The concentration of hydrogen
sultide likewise is probably low, perhaps in the nanomolar
range (8), and the concentration of free hydrogen sulfide
may not be sufficient to support the flux of hydrogen sul-
fide to the symbiont.

A specimen of the Western Pacific hydrothermal vent
gastropod Alviniconcha hessleri was collected by the sub-
mersible Alvin at the "Snail Pit" site (18 10.95' N,
144 43.20' E. about 3650m depth) on Dive number
1 837. 23 April 1987. The gill, 0.58 g wet weight, was stored
frozen in liquid nitrogen and a clear soluble extract pre-
pared in 1.5 ml of buffer (19). Optical direct spectra of
this extract display a prominent narrow feature at 55 1 nm.
This unchanging feature ascribed to reduced soluble sym-

o

CD

tr

8

CD

500

550 600

WAVELENGTH, nm

650

Figure 1. Optical difference spectra of the extract of Alviniconcha
gill. Features ascribable to oxyhemoglobin were inconspicuous in the
direct spectrum of the extract, suggesting that some component of the
solution had consumed the dissolved oxygen. Accordingly the solution
was equilibrated with oxygen gas. The difference spectrum of the oxy-
genated solution minus that of the initial (deoxygenuted) solution (spec-
trum not shown) exhibits conspicuous features at 541 and 579 nm. di-
agnostic of oxygenated hemoglobin. A small feature near 622 nm sug-
gested the presence of feme hemoglobin. This feature increased in
magnitude with time. The difference spectrum in the Soret region (spec-
trum not shown) of a portion of the sample that had been stored for 60
mm at ice temperature minus that of a portion of the solution to which
sodium dithiomte (a reagent that removes oxygen and reduces most
hemeproteins) had been added resembled the difference spectrum of
ferric minus deoxy Lucina Hb II. and exhibited a maximum at 409 nm
and a conspicuous minimum at 434 nm. diagnostic of hemoglobin. This
confirms the presence of a hemoglobin in the solution. The oxygenated
solution was then equilibrated with carbon monoxide. (A) Difference
spectrum: Carbon-monoxide-equilibraled gill extract minus that of the
oxygenated extract. Features at 545, 567, and 581 nm are diagnostic of
hemoglobin. Conversion of oxy- to carbon monoxy hemoglobin shows
that oxygen binding by the hemoglobin is reversible. (B) Difference spec-
trum: Carbon monoxide minus oxy Liicinu Hh II.

419

0.05 ABS

0.01 ABS

400

450

500 550

WAVELENGTH, nm

600

650

Figure 2. B> adding sodium dithionite. all of the hemoglobin present
in the extract ofAlvinianuiiu gill is converted into single chemical species,
permitting quantitative estimation of concentration. Absorbance in the
visible region is amplified sixfold relative to the Soret region. (A) Dif-
ference spectrum: Carbon-monoxide-equilibrated gill extract containing
sodium dilhionile minus the same extract containing sodium dithionite
alone. Well-defined features of 419, 435, 535. 554, 570 and 590 nm are
diagnostic of hemoglobin. (B) Difference spectrum: Carbon monoxide
Lucina lib II minus deoxv (ferrous) Lucina Hb II.

biont bacterial cytochrome 1-552 (20), vanishes in the dif-
ference spectra. Optical difference spectra (Figs. 1 and
2)unequivocally establish the presence of hemoglobin in
the extract. To confirm the identity of the spectral entities,
optical difference spectra of the extract of Alviniconcha
gill are compared to those of purified Lucina Hb II, iso-
lated from the modified symbiont-harboring gill of the
clam Lucina pcciinata ( 10). The concentration of Alvini-
concha hemoglobin in the solution, about 19 ^.U(heme),
was estimated from the spectrum presented in Fig. 2A by
using molar extinction coefficients appropriate for the dif-
ference: carbon monoxide Lucina Hb II minus ferrous
Lucina Hb II. Optical spectra in the visible and soret re-
gions are expected to differ only slightly (about 10%)
among similar hemoglobins. The concentration in the tis-
sue was estimated ( 19) to be about 65 ^mol Alviniconcha
hemoglobin per kilogram wet weight gill.

Acknowledgments

We thank Dr. Robert R. Hessler for helpful discussion.
This work was supported in part by Research grants DCB
90-17722 (to JBW) and OCE-93- 17734 (to JLS) from The
National Science Foundation. JBW was a Research Career
Awardee 1-K6-733 of The U.S. Public Health Service,
National Heart Lung and Blood Institute.

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11. Kraus, D. W'., J. B. Wittenberg, J. F. Lu. and J. Peisach.
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13. Hockenhull-Johnson. J. D., M. S. Stern, J. B. Wittenberg. S. N.
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14 Rizzi, M., J. B. Wittenberg, A. Coda. M. Fasano, P. Ascenzi, and
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15. Wittenberg, J. B. 1991. Functions of cytoplasmic hemoglobins
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16. Arp, A. J., J. J. Childress, and R. D. Vetter. 1987. The sulfide-
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158.

17. Doeller, J. E., D. \V. Kraus, J. M. Colacino, and J. B. Wittenberg.
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18. Wittenberg, J. B., and B. A. Wittenberg. 1990. Mechanisms of
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19. Schuder, S., J. B. Wittenberg, B. llaseltine, and B. A. Wittenberg.
1979. Spectrophotometnc determination of myoglobin in cardiac
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change chromatography. Anal Bioc/iein. 92: 473-481.

20. Kraus, D. W ., J. E. Doeller, and J. B. Wittenberg. 1992. Hydrogen
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Reference: Binl. Hull 188: 8-15. (February/March. 1995)

Inorganic Overgrowth of Aragonite on Molluscan
Nacre Examined by Atomic Force Microscopy

R. GILES 1 *, S. MANNE 1 , S. MANN 2 , D. E. MORSE 3 , G. D. STUCKY 4 ,

AND P. K. HANSMA 1 f

1 Department o/ Physics. University of California, Santa Barbara, California 93106,

2 School oj 'Chemistry. University of Bath. Claverton Down. Bath BA2 JAY. United Kingdom,

^Marine Biotechnology Center. Marine Science Institute. University of California,

San/a Barbara. California 93106. ami 4 Department of Chemistry.

University of California, Santa Barbara. California 93106

Abstract. The nacre (mother-of-pearl) that forms the ir-
ridescent inner layers of mollusc shells is a highly ordered
microlaminate composite of aragonite crystals and bio-
polymers with a strength and fracture resistance that far
exceed those of the mineral crystals themselves. The pro-
cesses governing the biofabrication of this material by the
secretory cells of the mantle are complex and only partially
understood. We have used the atomic force microscope
( AFM) to investigate the aqueous solution conditions un-
der which mineral growth can occur on the nacreous layer
of the shell of the bivalve mollusc Atrina sp. In situ im-
aging of the mature nacre surface exposed to a pH-con-
trolled environment of natural seawater with added car-
bonate ions reveals that inorganic overgrowth of aragonite
can occur within the ranges of pH and inorganic ion con-
centrations found in the molluscan extrapallial fluid from
which the mineral is produced during biological shell
growth. Thus, we posit that once nucleation has occurred,
nacreous tablets could grow inorganically in the extra-
pallial space; the role of proteins and other macromole-
cules may be limited to initiating growth or controlling
morphology through selective adsorption and spatial
constraint on the growing crystal.

Introduction

The mineral shells of a variety of molluscs are com-
posite biomaterials consisting of crystals of calcium car-
Received 7 March 1994; accepted 4 November 1994.
* Present address: Department of Physics. Simon Fraser University,
Burnaby. British Columbia, Canada V5A 1S6.

f Author to whom correspondence should be addressed.

bonate (CaCO,) intercalated with organic materials, pri-
marily proteins and glycoproteins (reviewed in Wilbur,
1972; Towe, 1972;Watabe, 1981;Weiner, 1986;Simkiss
and Wilbur, 1989; Lowenstam and Weiner, 1989). The
CaCOi occurs in two predominate crystal phases within
shells: calcite and aragonite. A shell may contain one phase
or the other, or both, depending on the animal species,
but commonly the stronger, denser aragonite forms an
inner structural layer, while calcite forms the outer layer.
The inner structural layer, called nacre or mother-of-pearl,
is a complex microlaminate composed of polygonal "tab-
lets" of aragonite that measure 5 to 15 ^m across, but
only 0.5 to 1 /urn in thickness, packed together with a thin
(40 nm) "mortar" of organic macromolecules. The or-
ganic component thus amounts to a small portion of the
total shell: less than 10% (Addadi and Weiner, 1992). It
nevertheless is responsible for the excellent strength and
resistance to crack propagation of the molluscan shell.
Crystallographically, the a and b axes lie in the plane of
the aragonitic tablets, with the e axis uniformly perpen-
dicular to the surface.

The nacreous layer of molluscan shell has been studied
extensively for several decades, principally with x-ray and
electron microscopic techniques. This work has been
largely successful in describing the microstructure of nacre
(see Lowenstam and Weiner, 1989; Weiner. 1986; Wa-
tabe, 1981; Towe. 1972; and Wise, 1970, for reviews).
Various calcium-binding, highly acidic, water-soluble
proteins have been isolated from the shell in various de-
velopmental stages (Cariolou and Morse, 1988). Water-
insoluble proteins from the shell have been characterized

GROWTH OF ARAGONITE ON NACRE

with x-ray diffraction, leading to the conclusion that they
resemble silk fibroin (Weiner and Traub, 1980). Both the
water-soluble and the water-insoluble proteins have been
proposed as multilaminar templates for the mineral tablets
(Nakahara t>/fl/., 1982).

The mechanism of growth of the nacreous layer is
complex and not well understood. It is known that both
organic and inorganic components are secreted by epi-
thelial cells in the mantle tissue into the extrapallial
space (the extracellular cavity between the mantle and
the shell, which is sealed from the surrounding envi-
ronment), bathing the growing shell in a mixture called
the extrapallial fluid. Although the inorganic compo-
nents of the extrapallial fluid are obviously necessary
for mineral growth, it is not known whether they are
sufficient: i.e., the role of the organic components is not
well known. Kitano and Hood (1962) showed that ara-
gonite is the most favorable phase of CaCO 3 to nucleate
in seawater supersaturated with respect to that mineral:
the presence of Mg 2+ in solution apparently acts to se-
lect aragonite over calcite. Others have measured nu-
cleation rates of aragonite crystals in seawater and ar-
tificial extrapallial fluid (Pytkowicz, 1965; Wilbur and
Bernhardt, 1984). We extended these studies by making
two experimental modifications relevant to nacreous
growth. First, we studied crystallization directly on a
nacreous surface (rather than unseeded nucleation in
solution). (Recently, Sabbides and Koutsoukos [1993]
also investigated seeded growth of aragonite on a variety
of substrates in seawater.) Second, we controlled both
pH and total carbonate concentration simultaneously,
and we compare growth conditions to those values re-
ported for extrapallial fluid.

We have used the atomic force microscope (AFM)
(Binnig el al. 1986) to examine the conditions for inor-
ganic growth of the nacre tablets. (For reviews of the AFM,
see Rugar and Hansma, 1990; Sarid, 1991; Hoh and
Hansma, 1992). The AFM (also known as the scanning
force microscope) is a member of the family of scanning
probe microscopes; these instruments form images by
raster scanning a tiny probe over the surface of the sample
while mapping some local interaction, such as electron
tunneling or near-field optical effects, as a function of
position. The AFM probe consists of a flexible cantilever,
~ 100 jum long, with a sharp tip attached at the end; the
probe measures (through the elastic response of the can-
tilever) the interaction forces between the tip and the
sample. The probe can thus map surface topography by
scanning in gentle contact with the sample; the displace-
ment of the cantilever (as the tip slides over surface fea-
tures) is detected from the motion of a laser beam reflected
from the back of the cantilever onto a position-sensing
photodiode. The AFM can operate in solution and hence

allows /// situ imaging of samples from the micrometer
to the nanometer scale. It recently has been applied to
biomineralized composites such as diatom shells (Linder
et al., 1992), bone (Tao and Lindsay, 1992). teeth (Kasas
el til., 1993), pressed powders of clam shells and sea urchin
shells (Friedbacher et al.. 1991), and molluscan nacre
(Manne et ul.. 1994). It has imaged in situ dynamic pro-
cesses on relevant systems such as calcite (Gratz et al..
1993; Hillner el al.. 1992), fluorite (Hillner et al.. 1993),
and hydroxyapatite (Kasas et al.. 1993), as well as calcite
growth modification in the presence of polyamino acids
(Wierzbicki et al.. 1993) and inorganic poisons (Gratz
and Hillner, 1993; Dove and Hochella, 1993). By ex-
amining the exposed aragonite surface of mature nacreous
tablets for signs of growth under various solutions, we
bracketed and thus defined the conditions under which
aragonite growth can occur. These conditions are biolog-
ically relevant: the solutions used approximate the inor-
ganic components of the extrapallial fluid in which new
nacre is formed.

Materials and Methods

Samples of nacre from the bivalve Atrina sp. were
kindly provided by Prof. S. Weiner at the Weizmann In-
stitute of Science in Israel. For imaging, small pieces (ap-
prox. 1 X 0.5 X 0.1 mm) of mature nacre were prepared
by mechanically cleaving a shell fragment with a razor
blade and then fracturing the resulting chip down to the
desired dimensions.

The solutions tested were based on natural seawater
collected locally from the Pacific Ocean along the Santa
Barbara coast. The water was coarsely filtered, irradiated
with ultraviolet light, passed through a 0.2-nm filter, and
stored at 2-4C in a sterilized, lightproof container until
just before use. To the seawater various amounts of
NaHCO 3 were added, and the pH was adjusted to the
desired value by addition of HC1 or NaOH.

Cation concentrations for the filtered seawater were
measured by atomic absorption spectroscopy; total car-
bonate ion concentration was determined by titration
with HC1. Table I lists concentrations for the measured
ions (at about 20C). These agree well with previously
published concentrations for seawater (Crenshaw, 1972;
Smith, 1974; Wada and Fujinuki, 1976). In addition,
the cation concentrations are all within about 10% of
the published values for the extrapallial fluid of bivalves.
In particular, the concentration of Ca : + ion we deter-
mined (Table I) is about the same as found in extra-
pallial fluid (Crenshaw, 1972; Wada and Fujinuki,
1976). The major difference between seawater and the
inorganic composition of extrapallial fluid is the higher
concentration of carbonate ion, which is approximately

10

R. GILES ET AL

Table I

Concentrations o\ f inorganic ions in the filtered natural seawater

Ion

Concentration

(mA/) standard dev.

Na +

463

-i- 2

K +

10.9

0.9

Mg 2+
Ca 2+

63.1
10.3

1.4
0.1

Sr +

0.085

0.007

HCO 3 ~ + CO 3 2 ~ or total carbonate

2.3

0.1

2-fold higher in extrapallial fluid. Therefore, only car-
bonate ion was added to natural seawater to create the
growth solutions. A comparison of the values of ion
concentrations determined for seawater and extrapallial
fluid is included in Figure 5.

The samples of nacre were glued to a stainless steel disk
with epikot resin and placed in the fluid cell of a com-
mercial AFM (Nanoscope III from Digital Instruments,
Santa Barbara, CA 93 103). Samples were always oriented
with the proximal side (the side facing the animal during
life) exposed for imaging. After an appropriate area had
been selected by imaging in air, growth solution was added
to the fluid cell. The sample was examined for signs of
growth for 15-20 min under a steady gravity flow of this
solution. Typical flow rates were 5 n\/s, which corresponds
to a replacement of the fluid cell volume every few sec-
onds. The tip was then withdrawn and flow stopped for
1.5 h to allow more time for growth to occur. Only the
exit line was blocked so that the cell remained in chemical
contact with at least 40 ml of the solution in a reservoir
above the cell. The sample was then examined again under
flow for signs of growth. The procedure was repeated with
an alternate growth solution; either the pH was raised
while maintaining the same carbonate concentration, or
vice versa. After growth had occurred with a given solu-
tion, only one or two more solutions could be tried with
a given sample before the surface became so rough that
further growth could not be analyzed. All growth exper-
iments were conducted at about 20C.

Results

Figure 1 shows an AFM image of the nacreous surface.
Most of one polygonal tablet and portions of two others
can be seen. The characteristic features of bivalve nacre
(Manne el ai, 1994), such as concavity of the proximal
tablet surfaces, a depression in the center of each of tablet,
and elongate rings surrounding the depressions, are visible.
Growth assessment was somewhat difficult at this scale;
usually the characterization was made on the basis of im-

ages only 3-j/m square, such as the area outlined in the
figure.

Figure 2 illustrates the changes in surface roughness
considered indicative of growth. It shows the same area
(the inset in Fig. 1 ) before and after incubation for 1 .5 h
in seawater with a total carbonate concentration of 4.3
mM. In the sample imaged in Figure 2a, the pH was 7.9,
at which point the surface had already changed somewhat
from its initial appearance under plain seawater. The
sample then was incubated for 1.5 h in a solution with
the same total carbonate concentration, but at pH 8.1,
and then shifted once more to pH 8.3. In Figure 2b the
surface is shown 6 min after raising the pH to 8.3; further
growth had occurred. As the images are shaded propor-
tionally to height, the new growth can be seen by com-
paring the relative brightness of corresponding features
in the two images; a few prominent pairs are indicated
by arrows.

It is important to recognize that tip convolution
(Grutter et ai , 1992; Allen et al. 1992) dominates the
growth image. This is indicated by the similarity of
shape and orientation among the bumps on the surface.

Figure 1. Atomic force microscope (AFM) image of the nacre of

Atnnu sp. The image is 1 1 /am 2 . Shading is proportional to elevation,
500 nm from dark to light, with the brightest regions being the highest.
The high ridge coincides with the boundary between nacreous tablets.
Most of a tablet is visible in the center and left of the image, along with
portions of two others. Note the general concavity, central depression
and the elongate rings characteristic of bivalve nacre. The black square
marks the area shown in Figure 2. centered on the intersection of the
three tablet boundaries.

GROWTH OF ARAGONITE ON NACRE

Figure 2. AFM images of the area indicated in Figure 1 before and
after mineral overgrowth. Both images are 2.5 ^m square; the height is
200 nm trom dark to white. Comparison of corresponding features be-
tween (a) (before overgrowth) and (b) (after overgrowth) demonstrates
the characteristic increase in the height of the surface asperities indicative
of mineral growth on the nacreous surface. Details in text. Arrows indicate
corresponding prominent asperities in the two images. These images
demonstrate the topographic changes used to characterize specific solution
conditions as productive of mineral overgrowth (Fig. 5).

Figure 3. Illustration of the convolution between the AFM tip and
a sharp asperity on the sample surface as it is scanned. The tip is shown
passing from right to left over the asperity. The measured topography
(dashed line) is a "convolution" of the tip shape and the asperity; it is
largely an image of the tip itself, except at the very top of the asperity.
Note that while the true width of the asperity is completely obscured,
the height measurement is accurate.

Tip convolution is a common AFM imaging artifact;
Figure 3 illustrates the mechanism responsible for this
effect. The AFM measures topography by scanning a
tip over the surface and measuring the vertical deflection
of that tip as it slides over surface features. However,
as the tip slides over an asperity with a higher aspect
ratio than the tip itself, the deflection of the tip traces
the tip's profile, rather than that of the asperity. In Fig-
ure 3, the dashed line indicates the path that will be
traced by the tip as it passes over an asperity. Although
as a consequence of tip convolution the lateral dimen-
sions of a sharp asperity are not resolved, the overall
height of that asperity is correctly measured, as is to-
pography on the flat top (Griitter ct ai. 1992; Allen et
a/.. 1992); thus, reliance on changes in local height de-
tected by the AFM is justified as a measure of mineral
growth. Figure 2b is consistent with the observation of
surface asperities lengthening normal to the imaging
plane, as expected for aragonite needles growing in their
normal crystallographic habit along the c 1 axis.

Although our classification of specific solution condi-
tions into growth/no-growth categories was based on
qualitative comparison of surface topography between
"before" and "after" images (of which the images in Fig.
2 are an example), this method is further supported by
quantitative measurements of surface roughness. Mea-
surements of the root-mean-square deviation of height
values from their collective mean was made for corre-
sponding areas of the images in Figure 2, as well as for

12

R. GILES ET AL

Figure -4. Unfiltered AFM image of the aragonite lattice, viewed along
the c axis to show the (001) crystallographic plane, 30 nm square. The
image was obtained on an area where overgrowth had been observed.
The inset shows a Fourier transform of the image. All peaks in the trans-
form are consistent with the expected reciprocal lattice of aragonite, and
the fundamental translation sectors (circled) that define the unit cell
agree with the aragonite a and /' axis spacing (0.495 nm and 0.796 nm,
respectively) to within 5%.

one intermediate between the two (at pH 8. 1 ). Combining
such measurements from two different areas, both away
from the high tablet boundary in the center of the image,
yielded a monotonically increasing surface roughness (of
2.9 nm for Fig. 2a, 3.9 nm for Fig. 2b, and 3.4 nm for the
intermediate image) in agreement with the qualitative as-
sessment of growth.

The observed changes in topography of the nacreous
tablets we imaged were caused by crystal growth on the
tablet surfaces, rather than by ilc nnvo nucleation and
precipitation from the growth solution. Parallel growth
experiments performed on nacreous particles embedded
in epoxy showed changes in surface roughness (growth of
crystal asperities) only on the nacre, and not on the sur-
rounding epoxy. These results are described in detail else-
where (Giles ct al.. 1 993).

Atomic lattice resolution could sometimes be obtained
atop the asperities, indicating that they are terminated by
small (<50 nm) flat areas. However, a wide variety of lat-
tices were observed, perhaps indicating the presence of
high-index planes on the sidewalls of the asperities. Oc-
casionally lattices (Fig. 4) did show periodicities corre-
sponding to the expected unit cell of the (00 1) plane of
aragonite (i.e.. viewed along the c axis).

Figure 5 presents a summary of the growth results. The
dashed lines separate the values of total carbonate con-
centration and pH that define the growth and no-growth
conditions. The rectangular bands designate the ranges of
these values previously reported for molluscan extrapallial
fluid and natural seawater. Note that growth never occurs
at the carbonate concentrations of seawater, but that the
boundary between growth and no-growth cuts across the
extrapallial fluid range, suggesting the potential for dy-
namic control of shell formation by changes in extrapallial
fluid composition. This is consistent with previous data
on seasonal variation in the acidity of the extrapallial fluid,
in which high pH was correlated with a high rate of shell
growth and low pH with slow growth or shell dissolution
(Wada, 1 96 1).

Supersaturation values of specific ions with respect to
aragonite were estimated with the ION PRODUCT com-
puter program (Shellis, 1988) for each of the solutions
tested. Saturation fractions ranged from 0.3 to 3.6, but in
general (with two exceptions) overgrowth occurred at sat-
uration values greater than 1, and no overgrowth occurred
at saturation values less than 1 .

Discussion

The nacre of molluscan shell is a highly organized mi-
crolaminate composite of proteins, glycoproteins, and
calcium carbonate crystals in the aragonite phase; the

12 -

extrapalli

al range

\ I-A-"

8,2

pH of growth solution

Figure 5. Aragonite growth/no-growth results as a function of pH
and total carbonate concentration ([HCO 3 ~] + [CO 3 : ~]). Open circles
indicate conditions al which no growth was observed; filled triangles
indicate conditions at which growth occurred; no symbol (two horizontal
lines) indicates indeterminate results. Error bars account for pH drift
over the course of the 1. 5 h incubation. Dashed curves approximately
separate the regions of growth and no growth. The labeled bands indicate
the ranges of published values for molluscan extrapallial fluid and natural
seawater. Note that the range for extrapallial fluid, in which shell growth
occurs biologically, spans the boundary between the growth and no-
growth conditions.

GROWTH OF ARAGONITE ON NACRE

13

biological mechanisms that control its formation are
complex and only partially understood. Previous research
(Wilbur, 1972; Weiner, 1986; Lowenstam and Weiner.
1989; Simkiss and Wilbur. 1989; Weiner and Addadi,
1991; Addadi and Weiner. 1992) suggests that shell min-
eralization commences with isolation of the site of min-
eralization from the external seawater environment by an
insoluble matrix of macromolecules and the mantle tissue
from which these molecules are secreted as an extension
of the growing shell edge. The shell and the mantle epi-
thelium enclose the extrapallial fluid, which is ionically
enriched and pH-controlled by enzymatic pumping across
the cell membranes of the mantle epithelium (Weiner and
Traub. 1 984; Weiner and Addadi, 1 99 1 ). It has been sug-
gested that insoluble matrix molecules play essential roles
both in the control of crystal nucleation and by establish-
ing compartments that limit the spaces in which the crys-
tals grow (e.g., Wilbur, 1972; Weiner, 1984; Nakahara,
1989). New aragonite crystals on the growth surface seem
to be nucleated in pores of the organic matrix (Nakahara
el ai. 1982), forming as small crystallites that grow to
become the next nacreous layer (Wada, 1972). Crystal
growth apparently also is controlled by several polyanionic
proteins found associated with (and in some cases, oc-
cluded within) the mineral crystals; these proteins also
have been suggested to act both in nucleation and in se-
lective inhibition of crystal growth (Addadi and Weiner,
1985; Sikes and Wheeler, 1988; Addadi et ai, 1990; Wei-
ner and Addadi, 1991; Morse et ai. 1993; Albeck et ai.
1993; Herman el ai. 1993).

The experiments reported here demonstrate that an ex-
posed surface of mature nacre can continue to grow by
purely inorganic means when the ion concentrations
present in the extrapallial fluid favor the aragonite phase
(primarily due to the presence of magnesium) and are
supersaturated with respect to that form. This indicates
that once the nucleation of the mineral phase has begun,
the crystal can continue to grow without direct biological
control. Therefore the requirements for nucleation, pos-
sibly including a template of acidic proteins (Aizenberg
et ai., 1994; Morse et ai. 1993: Weiner et ai. 1983) or
mineral bridges between the aragonite tablets (Manne et
ai, 1994), can be independent of growth. Because the
supersaturation levels required for overgrowth can be quite
low, it is plausible that cells of the mantle could regulate
growth simply by adjusting carbonate concentration and
pH in the extrapallial fluid.

Although evidence for growth was decisive in most of
the observed cases, quantification of the rates of mineral
growth proved difficult since growth was not observed to
occur uniformly over the experimental period. In some
instances, growth occurred in the first few minutes of im-
aging after introducing a solution; in others, growth was

not apparent until after the 1.5-h incubation. Unlike the
case of the cleavage plane of geological calcite (Hillner et
ai, 1993), on which growth occurs by quantifiable accre-
tion of widely spaced steps, the aragonite tablet is much
rougher and has a far greater step density. This high con-
centration of reactive sites may set up complex concen-
tration gradients that affect the reaction rate in unpre-
dictable ways.

It is interesting that the aragonitic overgrowth of na-
cre occurred in the form of needlelike extensions of the
(001 ) surface, rather than as the layered growth char-
acteristic of nacreous tablets in molluscan shells. The
former is the common growth morphology of abiogenic
aragonite and often results in extensive lateral inter-
growth, producing fanlike aggregates of misaligned
needles. Development of the highly coherent tablet
morphology found in biogenic nacre, characterized by
a high degree of orientation of the crystallographic c
axes of the aragonite tablets, thus would require
suppression of the tendency for disorder observed in
the overgrowth process seen in our experiments. One
possibility is that the highly anionic, soluble proteins
(and possibly other macromolecules) found intimately
associated with the aragonite crystals in molluscan nacre
may prevent long-range incoherent intergrowth of the
needles by specific interactions at the growing crystal
surfaces (Addadi and Weiner, 1985). This would pro-
duce the coherent (001) surface and single crystal com-
position observed in mature nacre. In addition, the in-
soluble polymers of the matrix may help determine the
final crystal form by creating a preformed microstruc-
ture that delimits the space in which the tablets grow
(Wada, 1972: Nakahara, 1989). As neither the soluble
acidic proteins nor empty sheaths of the insoluble ma-
trix proteins were present in our experiments, growth
along the e axis was persistently the fastest, as in abio-
genic aragonite.

Acknowledgments

We thank Robert Petty (of the Marine Science In-
stitute Analytical Laboratory. University of California,
Santa Barbara) for performing the atomic absorption
measurements; Monika Fritz, Angela Belcher, Charlotte
Zaremba, and Deron Walters for useful discussions; and
J. M. Didymus (School of Chemistry, University of
Bath, UK) for kindly performing the calculations of
supersaturation fractions. This work was supported by
grants from the Materials Research Laboratory program
of the National Science Foundation (DMR-9 123048);
the Molecular and Cellular Biosciences and Materials
Research Divisions of the National Science Foundation
(MCB-9202775 to G.D.S., D.E.M., and P.K.H.); the

14

R. GILES ET AL

Office of Naval Research (NOOO 14-93- 1-0584 to
D.E.M., G.D.S., and P.K.H.); and a fellowship from
AT&T (S. Mamie).

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Direct Development in the Ascidian Molgula
retortiformis (Verrill, 1871)

WILLIAM R. BATES*

Department of Biology. Carlelon University. Ottawa. Ontario, Canada K1S ?B6.
ami IlnniMiiiin Marine Science Centre. St. Andrews. New Brunswick. Canada EOG 2X0

Abstract. The cellular features of the ascidian Molgula
retortiformis (Verrill, 1871), a direct developing species,
were investigated with the aid of transmission electron
microscopy, histochemistry, and immunocytochemistry.
Developmental comparisons between direct and indirect
developing ascidians will further our understanding of how
developmental processes evolve. M. relortiformis eggs are
surrounded by a follicular envelope comprising a layer of
outer follicle cells attached to an acellular chorion. The
cytoplasm of M. retortiformis eggs contains large quan-
tities of yolk and glycogen. Immediately after hatching,
at day 2.5 of development, the cells constituting a juvenile
exhibited similar ultrastructural features, except that the
larger, deeper cells contained more yolk and glycogen than
the epidermal cells. Differentiated muscle cells were absent
in newly hatched M. retortiformis juveniles, and acetyl-
cholinesterase (AChE) activity was not detected. Immu-
nocytochemistry experiments using a vertebrate inter-
mediate filament antibody (NN18) support the idea that
the failure of newly hatched M. retortiformis juveniles to
develop muscle cells may be due to the absence of a factor
localized in the egg myoplasm. This paper concludes with
a discussion of the "substrate hypothesis" and the evo-
lution of ascidian direct development.

Introduction

Most ascidians produce eggs that develop into chordate
larvae that swim for a brief time and subsequently me-
tamorphose into adulls. During metamorphosis the
chordate features of a larva are selectively destroyed, and
the adult morphology develops (Grave, 1935; Cloney,
1978, 1982). Ascidians that produce swimming larvae

Received 18 January 1994; accepted 4 November 1994.
* Present address: Bamfield Marine Station, Bamfield, British Colum-
bia. Canada VOR 1BO

are termed indirect-developing species. In striking con-
trast to indirect-developing species, about a dozen species
produce fertilized eggs that develop directly into juve-
niles, bypassing the development of a swimming larva
(de Lacaze-Duthiers, 1874; Berrill, 1931; Jeffery and
Swalla, 1990; Bates and Mallett, 199 la). Here I report
on the cellular features of a direct-developing species,
Molgula retortiformis.

N. J. Berrill (1931) wrote that M. retortiformis has a
direct mode of development; however, he provided only
one line drawing of a juvenile. His drawing shows a M.
retortiformis juvenile without a tail, lacking a sensory ves-
icle, having partially extended epidermal ampullae, and
containing a cluster of large, opaque cells, which he terms
"tail phagocytes," in the posterior region. Aside from these
general features, no information was given on the cellular
features of eggs, embryos, and juveniles in this species.
Although Berrill was not concerned primarily about the
cellular features of direct-developing ascidians. he was
among the first to recognize that comparisons between
indirect and direct modes of ascidian development can
provide valuable insights about chordate evolution. In his
1931 paper, Berrill suggested that direct development in
ascidians evolved by the elimination of the larval sensory
vesicle and larval tail structures. He argued that the de-
velopment of a swimming tadpole larva capable of se-
lecting a habitat would be unnecessary if the adult lived
in a uniform habitat. This idea, which is termed the "sub-
strate hypothesis," is based primarily on studies of Mol-
gula occitlla. a direct-developing species that inhabits the
sand flats of Brittany. I reexamine Ben-ill's substrate hy-
pothesis in the present study of M. retortiformis.

Interest in ascidian direct development was renewed
when Whittaker (1979) reported that Molgula arrenata
embryos, embryos exhibiting direct development, can ex-
press acetylcholinesterase despite the lack of tail devel-
opment. AChE activity in a species with direct develop-

16

DIRECT DEVELOPMENT IN MOLGULA

17

ment suggested to Whittaker that AChE activity is a ves-
tigial trait that has not been eliminated from an ancestral
program responsible for larval muscle cell development.
The present study tested the possibility that newly hatched
M. retortiformis juveniles can express AChE activity. Just-
hatched tadpoles from three indirect-developing species,
Halocynthia pyriformis, Boltenia echinata, and Ciona in-
testinalis, were also tested for AChE activity.

Since the publication of Whittaker's exciting results in
1979, a number of studies on ascidian direct development
have been reported, including those by Young el al.
(1988), Jeffery and Swalla ( 1990, 1991, 1992), Bates and
Mallett (1991a,b), Bates (1991), and others. In 1988,
Young e! al. were the first to report that Molgula pacifica
is a direct developer. Many of the cellular features of M.
pacifica development have been described (Bates and
Mallett, 1 99 la,b; Bates, 1991. 1993). The postfertilization
movements of the egg cytoplasm, termed ooplasmic seg-
regation, and early cleavage patterns in M. pacifica were
similar to those in eggs and embryos having indirect de-
velopment. Although most features of early development
were similar to those in indirect developers, ampulla de-
velopment in M. pacifica juveniles was triggered before
hatching (Bates and Mallett, 199 la; Bates, 1993, 1994)
instead of after larval settlement (Cloney, 1978; Grosberg,
1981; Grosberg and Quinn, 1986).

The elimination of larval muscle cell development in
direct-developing ascidians was recently studied in Mol-
gula oculata (an indirect-developer) and Molgula occulta
(a direct-developer), the same species studied by Berrill
(1931). Results of these studies suggested that the lack of
larval muscle cell development in M. occulta may be due
to the absence of a protein that is recognized by a verte-
brate intermediate filament antibody (NN18) localized in
the myoplasm of M. oculata eggs (Swalla ct al., 1991). In
the present study, I used M. retortiformis and an indirect-
developing species, Boltenia villosa. to test the correlation
between the antigen recognized by NN18 and AChE
activity.

In summary, the threefold aim of the present study was
( 1 ) to examine the general cellular features of A/, retor-
tiformis eggs, embryos, and juveniles; (2) to determine if
there is a correlation between AChE activity and a factor
localized in the egg myoplasm that reacts with NN18 in
M. retortiformis juveniles and B. villosa tadpoles; and (3)
to test Benin's substrate hypothesis by examining the
habitats of A/, retortiformis adults.

Materials and Methods

Collection of adults, eggs and sperm, and embryo
cultures

Molgula retortiformis, Halocynthia pyriformis, Boltenia
echinata, and Ciona intestinalis adults were collected in
the Bay of Fundy near Huntsman Marine Station, St.

Andrews, New Brunswick, Canada. Collections were
made with a dredge at depths ranging from 50 to 100 feet.
Boltenia villosa adults were purchased from Westwind
Sealab Supplies, Victoria, British Columbia. Adults were
maintained in aquaria containing flowing seawater under
conditions of constant light to prevent spawning. Testes
and ovaries were removed from adults and placed in a
Syracuse dish containing seawater; eggs and sperm were
collected by using forceps to macerate the gonads. Van
Name (1945) described M. retortiformis (Verrill, 1871).
The testis on the left side of an adult was situated alongside
the inner side of the lower branch of the intestinal loop
and the left ovary was situated outside the intestinal loop
along the upper branch of the intestinal loop. On the right
side, the testis was situated ventral to the kidney and the
ovary was situated along the dorsal border of the kidney.
Fertilized eggs were obtained by mixing together eggs and
sperm from two or more individuals in a Syracuse dish
containing Millipore-filtered seawater. Eggs were insem-
inated for 10 min, washed with large volumes of seawater,
and cultured at 1 1 C. Embryos were viewed at frequent
intervals with an Olympus SZ stereomicroscope.

Transmission electron microscopy

Embryos and juveniles were prepared for light micros-
copy and transmission electron microscopy as previously
described by Bates and Mallett (1991a). Specimens were
fixed in 2% glutaraldehyde in 0.1 A/ sodium phosphate
buffer, pH 7.4, for 30 min. After a wash in the same buffer,
the specimens were immersed in 1% osmium tetroxide in
the same buffer for 1 h. Specimens were dehydrated
through a graded series of ethanol dilutions ( 10%- 100%),
then immersed in propylene oxide and gradually infil-
trated with Spurr low-viscocity resin. Thick and thin sec-
tions were cut; the thick sections were stained with
methylene blue and azure B, and the thin sections were
immersed in uranyl acetate. The thin sections were viewed
with a Phillips electron microscope at 80 kV. As a positive
control, hatched B. villosa larvae were prepared for trans-
mission electron microscopy along with hatched A/, re-
tortiformis juveniles. In every B. villosa preparation ex-
amined, sarcomeres were clearly evident within the tail
muscle cells.

Acetylcholinesterase histochemistry

Day 2 M. retortiformis juveniles, Boltenia echinata.
Halocynthia pyriformis, and Ciona intestinalis larvae were
tested for acetylcholinesterase activity as previously de-
scribed by Karnowski and Roots ( 1964), Whittaker ( 1973),
and Bates and Jeffery (1987). Wholemount preparations
were viewed with an Olympus microscope and photo-
graphed with Plus X film.

18

W. R BATtS

Figures I and 2. Transmission electron micrographs of a sectioned Mnlt;iilti ri'inriilnnni.\ follicle cell ( 1 ;
and a sectioned M rclornfrmiiix gastrula (2). The swirl patterns of follicle cell droplets (d) are evident in ( 1 !
and a test cell (tc) is seen within the perivitelline space (ps) in (2). X 3300 in ( 1 ) and in (2).

Immunocytochemistry

M. retortiformis and B. villosu eggs were prepared for
immunocytochemistry, as previously described by
Mita-Miyazawa et al. (1987). Eggs were immersed for
20 min in absolute methanol, and then for 20 min in
cold absolute ethanol. Fixed eggs were infiltrated with
50% polyester wax (BDH Limited, Poole, England): ab-
solute ethanol for 1 h at 40C and then infiltrated with
100% polyester wax for 1 h at 40C. Specimens were
embedded in BEEM capsules, and 8-/jm sections were
cut from the blocks. Sections were mounted on gelatin-
coated coverslips, de-waxed through a graded series of
ethanol dilutions (100%; 90%; 80%; 70%; 50%; 30%),
and rinsed in phosphate buffered saline (PBS). The
specimens were incubated with a monoclonal antibody
( 1:25 dilution of NN18 from Sigma Chemicals) for 1 h
at room temperature, washed with PBS, and incubated
for 50 min in a 1:60 dilution of FITC-conjugated IgG
(Sigma Chemical Company), as previously described
by Swalla el til. ( 1 99 1 ). The specimens were washed in
PBS for 30 min, mounted in 80% glycerol dissolved in
PBS, and viewed with an Olympus fluorescence micro-
scope. Sections were photographed with Tri X film,
ASA 400.

Results

A large population of M retoriiformis adults was dis-
covered living on an underwater hill near Huntsman Ma-
rine Station at a depth of 50-100 feet. The animals were

attached directly to rocks and lived close to several other
ascidian species, Bolicnia ovifera. Molgitla citrina, Ascidia
callosti. and Halocynthia pyriformis. The A/, retortiformis
adults collected from the underwater hill ranged from
about 20 to 75 mm in diameter. Only a few specimens
were collected from sand and gravel sites dredged near
the underwater hill, suggesting that M. retortiformis adults
prefer a hard substrate.

Maximum egg diameters (not including the surround-
ing follicular envelope) were 230-240 ^m. The ultra-
structural features of M. retortiformis follicle cells are
shown in Figure 1. The cytoplasm of follicle cells con-
tained droplets of various sizes, the contents of which dis-
play swirl patterns. Follicle cells are attached to an acel-
lular chorion separated from the plasmalemma of the egg
by a narrow perivitelline space. Cells within the perivi-
telline space, termed test cells, were observed in a few
sections (Fig. 2).

The cytoplasm of M. retortiformis eggs contains large
quantities of yolk and glycogen. After an egg was cross-
fertilized, a thick coat of sticky adhesive material anchored
it to the bottom of the glass culture dish. Fertilization
triggered a rapid rearrangement of the egg cytoplasm,
known as ooplasmic segregation. Opaque cytoplasm
moved into one region of the egg and subsequently, just
before first cleavage, formed a narrow belt of opaque cy-
toplasm in the equatorial region. Unlike the eggs of several
other species, including B. villosa. the egg of M. retorti-
formis does not have colored pigment granules in its cor-
tex. In some of the fertilized eggs, ooplasmic movements

DIRECT DEVELOPMENT IN MOUiL'L.l

19

Kifjures 3 and 4. Transmission electron micrographs of sectioned Molgula rclorlitorniix gastrulae showing
the outer epidermal cells (ep) containing less yolk and glycogen than the large, centrally located cells (cc).
X 3300 in Fig. 3; X 4900 in Fig. 4.

were accompanied by changes in the overall shape of
the egg.

The early cleavage patterns exhibited by M. relortifor-
mis embryos appeared similar to those exhibited by other
ascidian embryos. The first cleavage plane bissected the
narrow belt of ectoplasm into two equal regions. The two
equal-sized blastomeres of a two-celled embryo continued
cell division and formed a gastrula. Cells in the vegetal
pole region invaginated in a manner similar to that seen
in Boltenia villosa gastrulae. As a result of these vegetal
cell movements, an archenteron resembling that of B. vil-
losa formed. The ultrastructural features of the various
cells that constitute a M. retortiformis gastrula are shown
in Figures 3 and 4. The cytoplasm of the large, centrally
located cells was packed with yolk and glycogen. Ecto-
dermal cells contained less yolk and glycogen than these
central cells. Other cell types, based on distinct ultrastruc-
tural features, were not evident.

Tail development was completely absent in M. retor-
tiformis. No indication of a shape change of the posterior
region or of notochord elongation was observed. Ampulla
outgrowth was always triggered at a fixed time in devel-
opment, immediately before hatching. Each juvenile de-
veloped a maximum of eight ampullae. Rhythmic con-
traction waves were evident in each ampulla by day 4 of
development. Blood cells were evident within each am-
pullar lumen.

Figures 5 and 6 show the ultrastructural features of
various cell types constituting day 2.5 juveniles. Yolk and
glycogen stored in the egg cytoplasm persisted through
day 2.5 of development and were not partitioned into any
particular cell type, but were present in varying amounts
in all cells. Epidermal cells contained fewer yolk granules
and glycogen than the larger, central cells of a juvenile.
Given that M. retortiformis juveniles do not start feeding
until after one week of development, the energy required
for all of the morphogenetic processes is likely derived
from the large, yolky cells. These cells probably make up
part of the adult rudiment. In striking contrast to species
that produce planktonic larvae, in M. retortiformis ju-
veniles have no differentiated muscle cells (compare Figs.
5 and 6 and Fig. 7). I tested the possibility that despite
the absence of differentiated muscle cells, these juveniles
might be able to express AChE activity. AChE histochem-
istry was performed on newly hatched M. retortiformis
juveniles at day 2 of development and on day-2 larvae
produced by Hulocynthia pyriformis, Boltenia echinata,
or dona intent inalis. The results of these experiments are
shown in Figures 8 through 1 1 and Table I. Larvae from
all three species that have indirect development showed
AChE activity in tail muscle cells (Fig. 9), whereas M.
retortiformis juveniles did not express AChE activity (Fig.
1 1 ). One hundred and sixty-three M. retortiformis juve-
niles from eight egg clutches collected during four sum-

20

W. R. BATES

Figures 5 and 6. Transmission electron micrographs of sectioned day 2.5 Molgula rclorlilorniis juveniles.
Yolk and glycogen were the predominant cytoplasmic feature of juvenile cells. Centrally located cells (cc)
contain large quantities of yolk and glycogen. Differentiated muscle cells were not observed in M. ri-lortil<>rnn\
sections. - 3300 in Fig. 5: 4900 in Fig. 6.

mers were tested. .17. rctortiformis juveniles lack not only
larval muscle cells, but also the sensory structures present
in the head region of tadpole larvae. NN 1 8, a monoclonal
antibody raised to vertebrate neurofilament protein,
stained the cortical region of B. villosa eggs (Fig. 8). In
contrast, NN 1 8 did not stain the cortical cytoplasm of M.
retortiformis eggs (Fig. 10). More than 100 sectioned eggs

,.,... .

* - : - ' -

V-.*V*V

;>..

e$ .

' ct - .-J'.'jjjj.. .1

"{ V, *

Figure 7. Transmission electron micrograph of a sectioned Bullcniu
f(//iiMi larva. Differentiated muscle cells were evident in It. villnsu larvae,
in contrast to A/, retortiformis preparations that lacked differentiated
muscle cells, my striated myofihril.

from different clutches were examined together with sec-
tioned B. ri/losu eggs.

Discussion

In summary, this report ( 1 ) provides new information
on the ultrastructural features of A7. ret<>nifon)ii.<i eggs,
gastrulae, and day-2.5 juveniles; (2) presents ultrastruc-
tural and histochemical evidence suggesting that M. rc-
i/>n//i>nni\ embryos do not produce differentiated larval
muscle cells; (3) burnishes immunocytochemical evi-
dence that M. retortiformis eggs lack a cortical protein
that is recognized by NN18 antibody; and (4) suggests
that Berrill's substrate hypothesis is in need of revision,
because M. rclorti/hmiis adults live on a hard, nonuni-
form substrate.

Large quantities of yolk and glycogen were present in
the cytoplasm of eggs and most cells constituting gastrulae
and day-2.5 juveniles. Two other direct-developing ascid-
ians. Mdlgulti puci/icu (Bates and Mallett, 1991a,b) and
Molgiiln oirii/ui (Jeftery and Swalla, 1990). produce eggs
containing large quantities of yolk and glycogen. In all
three of these direct-developing molgulids, as in ascidians
having indirect development (Berrill, 1975;Cloney, 1982),
feeding does not begin until after the development of adult
organs. Large quantities of yolk present in the cytoplasm

DIRECT DEVELOPMENT IN MOUiVLA

Figures 8-11. NNI8 antibody staining of Bulletin: villow and Mol-
gula retortiformis eggs and AChE expressions of B villosa larvae and
M. retortiformis juveniles. The cortical region of/?, villoxa eggs was stained
with NN18 antibody (Fig. 8). whereas the cortical region of M. retorti-
formis eggs did not stain with NNI8 antibody (Fig. 10). M. rcturti/imni.i
follicle cells are autofluorescent. g germinal vesicle. Fig. 9: Dark-stained
AChE positive muscle cells in the tail of a B villwa larva. Fig. 1 1: M
retortiformis juvenile exhibiting no AChE activity. Scale bars equal 50 ^/m
in (X); 100 jjm in (9): 50 /jm in (10). 100 ^m in ( I I )

of meroblastic types of eggs, such as those produced by
birds and reptiles, directly affect patterns of cell division
and modify cell movements associated with gastrulation.
The presence of a few test cells within the perivitelline
space of M. retortiformis eggs was surprising because such
cells are thought to be involved in the development of a
larval tail fin (Cloney. 1 982). Despite the yolky cytoplasm
of M. retortiformis eggs, early cell divisions were holo-
blastic. and gastrulation was similar to that in indirect-
developing embryos containing less yolk. Vegetal pole cells
invaginated to form an archenteron. In contrast, gastru-
lation in M. pacified embryos is highly modified (Bates
and Mallett, 199 la) and a typical archenteron never de-
velops. Instead, the large, yolky endoderm cells within the
central region of the embryo appear to physically impede
the inward movements of vegetal pole cells.

Ooplasmic segregation movements and early cleavage
patterns in M. retortiformis are similar to those in eggs
and embryos that have indirect development (Conklin,
1905; Bates and Jeffery, 1988). Unlike the eggs produced
by several species of Stye/a and by Boltenia villosa, the
eggs of Af. retortiformis do not contain colored pigment
granules associated with the cortical region. However, the
postfertilization movements of the egg cytoplasm of M.
retortiformis could be studied in live eggs due to the pres-
ence of an opaque cytoplasm presumably derived from
the contents of the germinal vesicle, as in other ascidians
(Conklin, 1905). Opaque cytoplasm first accumulated in
one region of the egg and was subsequently moved into
the equatorial region where it spread out and formed a
narrow cytoplasmic region. These cytoplasmic move-
ments that have been described in the fertilized eggs of
indirect-developing ascidians are thought to be important

in the specification of cell fates and axial development
(Conklin, 1905; Bates and Jeffery. 1988). It appears that
in M. pacified (Bates and Mallett, 1991a) and M retor-
tiformis. these precise movements of egg cytoplasm have
been evolutionarily conserved.

The absence of myofilaments and AChE activity in M.
retortiformis juveniles suggests that the developmental
program responsible for the specification of larval muscle
cells was eliminated. Myofilaments and AChE activity
were also absent in M. pacifica juveniles (Bates and Mal-
lett, 1991b). But at least two other molgulids that have
direct development can express low levels of AChE activity
(Whittaker, 1979; Jeffery and Swalla, 1990; Bates and
Mallett, 1 99 1 b). The interpretation that AChE activity in
a direct-developing ascidian is a vestige of larval muscle
cell expression is based on Berrill's assumption that direct
development evolved from species that have indirect de-
velopment (1931 ). This assumption is being tested in sev-
eral laboratories by comparing ascidian gene sequences.
DNA sequence comparisons may suggest that Af. retor-
tiformis is most closely related to another molgulid that
has direct development or to a molgulid with indirect
development. Maybe M. retortiformis is closely related to
Molgula eitrina, an indirect-developing species that lives
on the same underwater hill as M. retortiformis.

The elimination of differentiated muscle cells in M.
retortiformis may be due to an evolutionary modification
of the egg cytoskeleton, an idea first suggested by Swalla
ct a/ ( 1 99 1 ) in their study of direct-developing Af. occult a
embryos. NN18, an antibody raised to vertebrate neu-
rofilament protein, stains the cortical myoplasmic region
of B. villosa eggs, but did not stain M. retortiformis eggs.
This result suggests that a cytoplasmic factor recognized
by NN18 antibody, absent in M. retortiformis eggs, may
be involved in larval muscle cell specification. The ques-
tion of whether the antigens recognized by NN 1 8 antibody
are attached to the myoplasmic cytoskeletal domain, an
egg cytoplasmic region thought to be involved in muscle
cell specification (Jeffery and Meier, 1983), must await
future studies.

Table I

The failure / m/r hutched, day 2 Molgula retortiformis juveniles to
express acclylcholinesl erase activity

Number

Number

Species

tested

positive

Molgula retortiformis (D)

163

Ilalocynlhia pynlormis (I)

65

56

Biillenia c'chinaui (1)

78

77

Ciona intestinalii (I)

8

8

D species with direct development; I species with indirect devel-
opment. Tested at day 2 of development.

22

W. R. BATES

Data collected from field sites in the Atlantic and Pacific
oceans, on adults of M. retortiformis (present study) and
M. padficu (Bates and Mallett, 1 99 1 a) respectively, appear
to conflict with Berrill's substrate hypothesis (1931). Berrill
based his hypothesis on field and developmental studies
of Molgula ncciiltii (a direct developer) and Molgula ocu-
lata (an indirect developer), species that live on the sand-
flats along the coast of Brittany. The occurrence of M.
occulta was attributed to habitat uniformity. Berrill sug-
gested that tadpole development was eliminated from the
life cycle because tadpoles capable of selecting a habitat
are unnecessary in a uniform environment. But the largest
populations of M. retortiformis adults live in a rocky,
nonuniform habitat. Seven summers of field collections
along the west coast of Vancouver Island near Bamfield
Marine Station indicate that M pucificu adults thrive on
rocky, nonuniform habitats (Young ci <;/., 1988; Bates
and Mallett, 199 la; Bates, 1993). The evolution of mor-
phogenetic processes in ascidians has been discussed at
length elsewhere (Bates, 1993, 1994), with the suggestion
that the evolution of a fixed timing mechanism for trig-
gering a rapid deployment of ampullae may be important
to the reproductive success of direct -developing ascidians.
The finding described in the present report that ampulla
morphogenesis occurs at a fixed time in M retortiformis
development supports this idea.

Acknowledgments

Mike Swallow is thanked for providing Figure 7. Tech-
nical help with transmission electron microscopy and
darkroom assistance were provided by J. Mallett. I am
grateful to Fred Purton at Huntsman Marine Science
Centre, St. Andrews. New Brunswick, for making my visits
productive. I am grateful for the critical reviews of a pre-
vious form of this manuscript. W.R.B. is supported by
an operating grant provided by the Natural Sciences and
Engineering Council of Canada.

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ascidians. /)nv/ (inmih /)///</ 33:401-410.

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Rc\ lech 26: 285-300.

Bales, \V. R. 199-4. Ecological consequences of altering the timing
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Bates, VV. R., and W. R. Jeffery. 1988. Polarisation of ooplasmic seg-
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ascidian Mulgulu pacilica (Huntsman), dm J //ml 69: 618-627.

Bates, VV. R., and J. E. Mailed. I991b. Ultrastructural and histo-
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Berrill, N. J. 1975. Chordata: Tunicata. Pp. 24 1 -282 in Reproduction
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Cloney. R. A. 1978. Ascidian metamorphosis: review and analysis. Pp.
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Grave, C. 1935. Metamorphosis of ascidian larvae. Papers from Tor-
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Grosberg, R. K. 1981. Competitive ability influences habitat choice in
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Grosberg, R. K., and J. K. Quinn. 1986. The genetic control and con-
sequences of kin recognition by the larvae of a colonial marine in-
vertebrate. Nature 322: 456-459.

Jeffery, VV. R., and S. Meier. 1983. A yellow crescent cytoskeletal
domain in ascidian eggs and its role in early development. Dev. Biol.
96: 125-143.

.lettery, VV. R., and B. J. Swalla. 1990. Anural development in ascidians:
evolutionary modification and elimination of the tadpole larva. Sem.
Dev Biul. I: 253-261.

Jeffery, VV . R., and B. J. Swalla. 1991. An evolutionary change in the
muscle lineage of an anural ascidian embryo is restored by interspecific
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Jeffery, VV. R., and B. J. Swalla. 1992. Evolution of alternate modes
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Swalla, B. J., M. R. Badgett, and VV. R. Jeffery. 1991. Identification
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Young, C. M., R. K. Gowan, J. Dalby, C. A. Pennachetti, and I). Gagliardi.
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Reference: Bn>l Hull 188: 23-31. (February/March, 1995)

Isolation of Biologically Functional RNA During
Programmed Death of a Colonial Ascidian

WEN-TEH CHANG 1 AND ROBERT J. LAUZON' : *

1 Department of Microbiology, Immunology and Molecular Genetics, and 2 Department of Pediatrics,
Albany Medical College, Albany. New York 12208

Abstract. The blastogenic (asexual) cycle of the colonial
ascidian Botryllus schlosseri (Tunicata. Ascidiaceae) con-
cludes in a cyclical phase of programmed cell and zooid
death called takeover, in which all asexually derived adults
die synchronously by apoptosis. The characterization of
developmental^ regulated genes whose expression pat-
terns are selectively modulated during this process could
pave the way to understand how this model organism
dies. However, isolation of biologically functional RNA
in this and other colonial ascidians with conventional
phenol/chloroform-based procedures is hampered by ex-
tensive contamination of RNA preparations by pigments.
Upon cell lysis, pigments that normally reside within spe-
cialized cells in the mantle wall of the adult are released
and tightly associate with nucleic acids. Here, we report
on the usefulness of a single-step RNA isolation method
in which acid guanidinium isothiocyanate is used as an
extraction medium, followed by preparative cesium chlo-
ride ultracentrifugation. This procedure successfully iso-
lated biologically active, high-purity total RNA (OD 26 o/
OD :s(l = 1 .9-2. 1 ) from Botryllus colonies during takeover,
as well as other species of colonial ascidians (Diplosoma
macdonaldii, Botrylloides diegense) irrespective of pig-
mentation. Northern blot analysis performed with a 32 P-
labeled tunicate actin probe detected two polyadenylated
transcripts of 1.5 and 1.7 kilobases in length from both
growth phase and takeover colonies. Two-dimensional
protein gel assays from //; vitro translated mRNA prep-
arations further revealed that specific transcripts were up-
regulated during takeover, while others were repressed or
down-regulated. Growth phase and takeover-specific
cDNA libraries were constructed from pooled poly(A) +
RNA with a complexity of 1.0 X 10 7 and 1.2 X 10 7 re-
Received 8 July 1994; accepted 22 November 1994.
* Author to whom correspondence should be addressed.

combinants respectively per 100 ng of cDNA before am-
plification. The procedure described herein renders fea-
sible the cloning of developmentally regulated genes in
this organism. In addition, our findings raise the possibility
that zooid death in Botryllus involves modulated gene
expression.

Introduction

Programmed cell death is a fundamental morphoge-
netic process within developing multicellular animals ( El-
lis et al.. 1991; Schwartz and Osborne, 1993). In adult
tissues, cell death also functions as a homeostatic mech-
anism complementary to mitosis: changes to this balance
bring about pathologic abnormalities (Ellis et al.. 1991).
Recent studies in vertebrates (Owens et al.. 1991; Miura
et al.. 1993; Woronicz et al.. 1994; Liu et al.. 1994) and
invertebrates (Ellis and Horvitz, 1986; Schwartz et al..
1990; White et al.. 1994) strongly suggest that cell death
is an active process dependent on modulated gene expres-
sion. One of the most characteristic forms of cell death is
a dynamic morphological process known as apoptosis,
characterized by nuclear chromatin condensation and
margination, cellular fragmentation into membrane-
bounded bodies followed by engulfment and digestion
within phagocytic cells (Kerr, 1972).

The colonial ascidian Botryllus schlosseri contributes
a unique perspective to the study of cell death: adult col-
onies, derived from a chordate tadpole through palleal
budding and which at peak size consist of approximately
1000 asexually derived clones (zooids), undergo weekly
phases of regression (Milkman, 1967). Every 5 days at
2 1 C, the blastogenic (asexual) cycle concludes in a phase
of programmed cell and zooid death called takeover, dur-
ing which all zooids, each containing a functional heart,
nervous and digestive systems simultaneously die by an

23

24

W.-T. CHANG AND R. L. LAUZON

apoptotic process over a 24-h period and are replaced by
a new asexual generation of zooids (Lauzon et a!., 1993).

Because the temporal and morphological events of
takeover can be predicted in detail in Botryllus, we have
undertaken a multidisciplinary investigation of the mo-
lecular mechanisms underlying programmed death in this
model organism. Thus far, molecular studies have been
impeded by the lack of appropriate methods for isolation
of biologically active mRNA. Unfortunately, Botryllus
and other colonial ascidians harbor polyphenolic and
DOPA-containing pigments that bind tightly with nucleic
acids following cellular lysis with detergents and chao-
tropic agents, thus interfering with the isolation process
as well as its subsequent analysis and cloning (Kumar et
ai, 1988). Moreover, isolation of intact RNA molecules
from regressing tissues may also prove to be difficult be-
cause, during cell death, a substantial fraction of the RNA
pool is rapidly degraded through the enhanced activity of
ribonucleases (Cidlowski, 1982; Owens et a/.. 1991).
Therefore, to ensure isolation of biologically active RNA
from these organisms, a strategy had to be developed that
would eliminate both ribonuclease activity and pigments.

Here, we report on the success of a procedure by which
biologically functional RNA suitable for cDNA cloning
and other molecular applications can be rapidly isolated
from various colonial ascidian species, including Botryllus.
In addition, we present evidence which indicates that
changes in gene expression occur during takeover.

Materials and Methods

Animals

Ascidians (Botryllus schlosseri. Botrylloides diegense,
Diplosoma macdonaldii, Molgula manhattensis) were
collected on glass microscope slides contained within
wooden enclosures submerged in the Eel Pond (Woods
Hole, MA) and Monterey Bay (CA). They were subse-
quently maintained in a refrigerated aquarium ( 1 50-gallon
capacity) containing artificial sea salts, trace and bio-ele-
ments (hw-marine mix: Hawaiian Marine Imports Inc.,
Houston, TX), and were continuously fed with an algal
scrubber irradiated for 12 h each day with two 15-watt
Aurora 50:50 bulbs (Fritz Pet Products, Dallas, TX). In-
dividual Botryllus colonies were developmentally staged
with the use of a stereomicroscope (Stemi SV 6, Carl Zeiss,
Germany). Following removal of debris and encrusting
organisms, all animals were subsequently snap-frozen in
liquid nitrogen and stored at 70C until needed.

RNA extraction

RNA was extracted using a modification of the method
by Chirgwin et ai ( 1978). Individual colonies (0.5-1.0 g)
were initially ground to a fine powder with liquid nitrogen

in a precooled mortar and pestle, and subsequently ho-
mogenized in 2.0 ml of a lysis solution containing the
following components: 4 M guanidium isothiocyanate
(Gibco/BRL, Gaithersburg, MD) predissolved in a 0.75 M
sodium citrate solution (pH = 7.0; 25 mA/ final concen-
tration), 0.2 M sodium acetate (pH = 5.0). and 0.1 M /3-
mercaptoethanol. Following transfer of the lysate to a 50-
ml polypropylene tube, the DNA was sheared with a 23-
gauge needle and syringe, and sodium lauryl sarcosinate
(10% stock) was added to a final concentration of 0.5%.
The homogenate was incubated on ice for 1 5 min and
centrifuged at 3500 X g for 5 min at 4C. The supernatant
was layered on a 1 .3-ml cesium chloride (CsCl; Boehringer
Mannheim, Indianapolis, IN) solution (5.7 AI CsCl, 0.5 M
EDTA, pH = 8.0) in a 1 3- X 5 1-mm ultracentrifuge poly-
allomer tube ( Beckman Instruments Inc., Palo Alto, CA),
and centrifuged at 40,000 rpm at 20C for 1 2 h in an
SW50. 1 rotor (Beckman Instruments Inc.). Following
centrifugation, the RNA pellet was resuspended in dieth-
ylpyrocarbonate (DEPC (-treated water (Sigma Chemical
Co., St. Louis, MO), precipitated overnight at -20C in
100% ethanol, and washed in 70% ethanol. The pellet was
subsequently dried under vacuum at room temperature,
resuspended in DEPC-treated water, and stored at 70C.
For phenol/chloroform-based extractions, the method
described by Chomcynski and Sacchi ( 1987) was used.

Spectrophotometric analysis

An aliquot from each RNA preparation (1-5 /ul) was
diluted into 250 jul of DEPC-treated water, transferred to
a quartz cuvette, and scanned between 240 and 320 nm
with a Perkin-Elmer X-2 microprocessor-controlled spec-
trophotometer (Perkin-Elmer Inc., Foster City. CA). A
GeneQuant spectrophotometer (Pharmacia, Piscataway,
NJ) was used to determine total RNA concentrations as
outlined in Sambrook et al. (1989). During the course of
our studies, we observed that OD2 6 o/OD 280 ratios were
greatly affected by pH. For instance, DEPC-treated water
samples that still contained residual levels of DEPC fol-
lowing autoclaving (pH = 5.0) gave aberrant OD 260 ab-
sorbance readings (between 1.3 and 1.5). Consequently,
we routinely autoclaved all our DEPC-treated solutions
twice for 30 min each. Poly-A + RNA was isolated with
the poly-A tract mRNA isolation system from Promega
(Promega Corp., Madison, WI), and concentrations were
determined with the Dipstick kit by Invitrogen (Invitrogen
Corp., San Diego, CA). Both were used according to the
manufacturer's specifications.

Northern blot hybridization

Ten micrograms of total RNA was denatured in for-
maldehyde, size-fractionated in 1% agarose/formaldehyde
gels (4 V/cm), and transferred onto nitrocellulose mem-

ISOLATION OF RNA FROM BOTRVLLVS

25

hranes (Schleicher and Schuell, Keene, NH) with 20 x
SSC. Blots were hybridized overnight with a cytoactin
cDNA probe from Styelu c/ava (SpCAS; Beach and Jef-
fery, 1990) under high-stringency conditions ( 1 M NaCl,
10% dextran sulfate, 1% SDS. 100A<g/ml denatured
salmon sperm DNA) at 65C. The SpCAS probe was la-
beled by random hexadeoxynudeotide priming to a spe-
cific activity of 10 9 cpm/^g DNA (Feinberg and Vogel-
stein, 1983). Blots were washed three times in IX SSC
(prepared from a 20X SSC stock solution: 3.0 M sodium
chloride, 0.3 M sodium citrate. pH = 7.0) and 0.1% SDS
at 65C 5 min each, followed by two additional washes
inO.lx SSC and 0.1% SDS (65 C) 15 min each, and au-
toradiographed with Kodak XAR film.

Two-dimensional protein gel electrophoresis

Poly-A + RNA (0.2 ^g/sample) was translated /// vitro
with a rabbit reticulocyte lysate from Promega using 35 S-
methionine. Protein products were analyzed by two-di-
mensional polyacrylamide gel electrophoresis (O'Farrell,
1975; O'Farrell ft al.. 1977) on a protein II xi slab cell
(Bio-Rad, San Diego, CA). //; v/m>-labeled samples were
focused with wide range ampholytes (pH = 3-10; Bio-
Lyte 3/10, Bio-Rad) in 4 M urea and 10% NP-40, and
size-fractionated on 10% sodium-dodecyl-sulfate poly-
acrylamide gels (SDS-PAGE) along with Brome Mosaic
Virus (BMV) molecular weight markers ( 1 10, 97. 35 and
20 Kd) provided as a control with //; vitro translation kits
(Promega). Gels were fixed in methanol/acetic acid, en-
hanced with RESOLUTION (EM Corp, Chestnut Hill,
MA), dried under vacuum, and autoradiographed with
Kodak XAR film at -70C. All samples were run at least
twice to ensure reproducibility of translational profiles
observed by autoradiography.

cDNA library construction

cDNA was synthesized from poly-A + RNA of pooled
colonies isolated at onset and early stages of takeover
(stages D-l and D-2) or from representative growth stages
(A, B-l, B-2 and C-l) with the unizap cDNA synthesis
kit from Stratagene (La Jolla, CA) using 3: P-dATP. The
cDNA products were ligated to Eco RI linkers, restricted
with Xho I, and cloned unidirectionally into lambda zap
vector, according to the manufacturer's specifications. Size
range of first and second strand cDNA products was de-
termined by alkaline agarose gel electrophoresis by the
slide technique. Briefly, 10 ml of 1% molten alkaline aga-
rose (containing 1 ml of 10X alkaline agarose buffer: 3 ml
of 5 N NaOH, 2 ml of 0.5 M EDTA and 45 ml of sterile
milli-Q water) was added near the upper center of a 5- X
7.5-cm glass slide, to which a mini-gel comb had been
attached over it with high-tension clips. The gel was run
in 1 X alkaline buffer at 75 V for 2 h at room temperature.

Following electrophoresis. the gel was blotted dry with
several changes of Kimwipes EX-L (Kimberly-Clark,
Roswell, GA), sealed in an air-tight hybridization bag,
and autoradiographed with Kodak XAR film at room
temperature. The library was packaged and titered ac-
cording to Stratagene's specifications. The level of non-
recombinants was determined by plating various phage
dilutions with XL 1 -Blue MRF cells along with IPTG
(200 mg/ml in water) and X-gal (20 mg/ml in dimethyl-
formamide). For either takeover or growth phase cDNA
library, blue background plaques were not observed on
plates containing up to 10 ? PFUs (plaque forming units),
indicating that the percentage of non-recombinants was
very low (less than 1 x 10 5 PFUs/Vg of phage arms).
Lastly, the primary library was amplified in XL 1 -Blue,
phage suspensions were stored at -70C, and an aliquot
was prepared to assess the quality of the cDNA library.
The quality of each cDNA library was assayed by probing
nitrocellulose plaque lifts for representation of actin-
complementary sequences using the SpCAS cytoactin
cDNA clone. Phage transfer was performed for 1 min at
room temperature, and filters were sequentially placed
for 3 min each onto sheets of 3MM paper saturated with
the following solutions: ( 1 ) 0.5 N sodium hydroxide and
1.5 M sodium chloride, (2) 10% SDS, (3)0.5 A/Tris-HCl
pH = 8.0 and 1.5 M NaCl, and (4) 2x SSC. Membranes
were subsequently baked at 80C under vacuum for
30 min, hybridized with the 32 P-labeled SpCAS cDNA
clone, and autoradiographed with XAR film at -70C.
Hybridization conditions and post-hybridization washes
were identical to those used in the northern blot analysis.

Results

The blastogenic cycle ofB. schlosseri

Developmental staging of B. schlosseri colonies was
adapted from the nomenclature used by Mukai and Wa-
tanabe (1976), as well as Izzard (1973), and is described
in Table I and depicted in Figure 1. Following metamor-
phosis of the free-swimming tadpole, a colony arises by
weekly cycles of palleal budding, in which the bud evag-
inates from the wall of its parent zooid. Under optimal
growth conditions, two to three primary buds are pro-
duced per zooid and can be easily observed dorsally by
stage B-2 (Fig. 1, panel B). By stage C-l. organogenesis
begins in the secondary bud with the formation of primary
atrial folds, and at this time it exhibits an elongated ap-
pearance as primary organs (gut rudiment) begin to form
(not shown). At 21C. the cycle concludes on the fifth
day with the synchronous death of all parent zooids, a
process called takeover (Lauzon et al.. 1992). The onset
of takeover is characterized by the shutdown of both oral
and excurrent siphons (Fig. 1, panel C). At this stage,
while buds begin to move dorsally, zooids are still re-

26

W.-T. CHANG AND R. L. LAUZON

Table I
Developmental stages of the blaslogenic cycle

Stage

Characteristic

A Onset of new cycle; opening of oral and excurrent

siphons.
B-l Secondary hud skewing to parent /ooid's anterior

hemisphere. Heartbeat begins in primary bud
B-2 Secondary bud is a closed double-layered vesicle.

C-l Organogenesis (atnal folds) begins in secondary bud.

Secondary bud elongates along its anteroposterior axis.
C-2 Primary subdivisions completed in secondary bud.

D-l Onset of takeover: shutdown of zooid's oral and

excurrent siphons. Primary buds move dorsally.
D-2 Early takeover; contraction of zooid along its

anteropostenor axis.
D-3 Mid-takeover (zooid involution): visceral organs are

being resorbed. Apoptotic cell death and macrophage

phagocytosis are prevalent.
D-4 End of takeover: cessation of heartbeat in zooid. Siphons

of new asexual generation not yet open.

sponsive to mechanical stimulus. In the early stages of
takeover (3-5 h post-onset), the zooids contract along their
anteroposterior axis and begin to shrink in size (Fig. 1,
panel D). Pigment cells, which normally reside in the
zooid's mantle wall, begin to accumulate in the vascular
ampullae. In the middle stages of takeover (12- 15 h post-
onset), visceral organs die principally through an apoptotic
process (Lauzon el a/., 1993), although necrotic changes
can also be observed alone or in combination with an
apoptotic morphology. Takeover concludes with the ces-
sation of heartbeat in zooids, and a new cycle begins with
the opening of siphons in the next asexual generation of
zooids (panel A).

RNA isolated by preparative ultracentrifugation is
biologically functional

As shown in Figure 2A, when RNA was extracted with
the guanidine isothiocyanate/phenol/chloroform proce-
dure (Chomcynski and Sacchi, 1987), ;he spectrophoto-
metric absorbance pattern was severely disrupted, exhib-
iting a peak absorbance at 268 nm instead of 260 nm. All
preparations extracted in this manner were significantly
contaminated with blue and red pigments that could not
be removed upon further phenol/chloroform extraction.
In addition, the yields from these preparations were very
poor (10-20 jug of total RNA/g tissue), and often displayed
OD 2 6o/OD 2 8o ratios greater than 2.5, suggesting that pig-
ments were contributing to the altered ratios. Further-
more, when the preparations were size-fractionated on
formaldehyde/agarose gels, most of the sample remained
in the loading well. We surmise that since many pigments
have been reported to be polyphenolic in nature (Kumar

et /., 1988), RNA was most likely sequestered to the or-
ganic phase along with them. In contrast, RNA isolated
by means of cesium chloride ultracentrifugation was
spectrophotometrically pure (Fig. 2B), exhibited optimal
ODibo/ODigu ratios between 1 .9 and 2.1, and consistently
produced yields ranging between 0.5 and 0.8 Aig/mg of
colony. When cesium-chloride-purified samples were
electrophoresed, prominent 28S and 18S ribosomal RNA
bands were visualized irrespective of the developmental
stage of the colony (Fig. 3, lanes 2, 3, 4, 7, 8) or species
(Fig. 3, lanes 5, 6). To test the integrity of the RNA, sam-
ples from various ascidians were size-fractionated by aga-
rose/formaldehyde gel electrophoresis, transferred to a
nitrocellulose membrane, and hybridized to a 3: P-labeled
cytoactin cDNA probe from Styela clava. The results,
which are shown in Figure 4, indicate that all colonial
ascidians (Botryllus, Botrylloides, and Diplosoma) ex-
pressed two polyadenylated transcripts of 1.5 and 1.7 kb
in length (panel A, lanes 1 -4; panel B, lane 5). In contrast,
the solitary ascidian Molgula manhattensis expressed only
a single 1.5-kb transcript. Furthermore, both transcripts
were expressed during all stages of the blastogenic cycle
in Botryllus (Fig. 4, panel B).

We next sought to determine whether Botryllus RNA
isolated by this method could also be in w/ro-translated
into protein. Samples (0.2 jug) of poly-A 4 RNA from var-
ious developmental stages were translated with a rabbit
reticulocyte lysate with 35 S-methionine and analyzed by
two-dimensional polyacrylamide gel electrophoresis to
examine patterns of gene expression between different
stages of the blastogenic cycle. The results (Fig. 5) dem-
onstrate that RNA preparations could be successfully
translated and focused into a wide spectrum of acidic-
and basic-range polypeptides during both the growth phase
of the cycle and takeover. Furthermore, at the onset of a
new blastogenic cycle, several different spots were iden-
tified from the acidic and basic range that were absent in
the early stages of takeover (panel A). Additional tran-
scripts (for instance, arrow in panels A and B, Fig. 5)
appeared to be significantly down-regulated during take-
over. Conversely, other transcripts were expressed in the
early stages of takeover, but absent at the beginning of a
new asexual cycle (panel B) or other stages (not shown).

Lastly, in order to determine whether mRNA from
takeover colonies was suitable for cDNA synthesis, poly-
A + RNA was reverse-transcribed, and cDNA products
were subsequently size-fractionated by alkaline gel elec-
trophoresis. The results (Fig. 6) indicate that both first-
and second-strand cDNA products exhibited an appro-
priate size range, with the bulk distributed between 300
bases and 2.5 kb. Pooled cDNA products were then
used to construct a unidirectional library into lambda
zap. The size of the primary nonamplified library was
1.2 X 10 7 PFUs/100 ng of cDNA, as determined from

ISOLATION OF RNA FROM BOTRYLLUS

27

Figure I. The blastogenic cycle ofBotryllus M'/i/m.stv/ Individual colonies were developmental!)" staged
by stereomicroscopy and are depicted dorsally in panels A through D. Panel A shows a colony at the onset
of a new cycle. Note that the primary buds are not visible from the dorsal plane. Panel B shows a colony
during stage B-2 (see Table I for specific details of individual stages), in which primary buds are now visible.
The onset of takeover (panel C) is characterized by the shutdown of oral and excurrent siphons in all zooids
(arrow) and star-shaped systems. Buds (arrowhead) have begun their dorsal migration. In the early stages of
takeover (panel D), each zooid undergoes a synchronous polarized contraction along its anteroposterior axis.
Zooid regression is completed in approximately 24 h at 2 1 C. and a new cycle begins with the opening of
siphons from the new asexual generation of zooids. Bar represents 1 mm.

plaque counts using serial dilutions of phage suspensions.
A cDNA probe encoding cytoplasmic actin from Styela
clava (as a prototype abundant sequence) was then used
to screen nitrocellulose plaque lifts to ensure adequate
representation of this sequence. A comparable screen was
performed with a growth phase (pooled stages A, B-l and
C-l) cDNA library (1.0 X 10 7 PFUs nonamplified). The
percentages of actin-positive clones (Fig. 7) were found
to be comparable in both libraries, namely 3.0% ( 1 50 pos-
itive/5 X 10 3 plaques) in the takeover and 3.2% in the
growth phase cDNA library (58 positive/ 1.8 : 10'
plaques).

Discussion

The findings presented in this paper indicate that RNA
isolated by cesium chloride ultracentrifugation is opti-

mally suited for a wide range of molecular applications,
including northern blot analysis, /// vitro translation, and
cDNA synthesis from dying tissues ofBotryllus schlosseri
and other colonial ascidians. Because colonial ascidians
(Kumar el at., 1988) and other marine invertebrates
(Groppe and Morse, 1993) exhibit a spectacular range of
pigmentation patterns, they have been reported to pose a
distinct problem in the isolation of nucleic acids. Poly-
phenolic compounds and DOPA-containing proteins,
which interfere with nucleic acid isolation, have been
found in the adult tunic and mantle wall of both solitary
and colonial ascidians (Kumar el ul., 1988). In Botryllus
colonies found on the eastern coast of the United States,
the problem is intensified because most colonies contain
an alcohol-insoluble red pigment that cannot be removed
from nucleic acids with conventional lysis buffers followed
by phenol/chloroform-based extractions. The addition of

28

W.-T. CHANG AND R. L. LAUZON

153-
123
093-
063-
033-
003-

Figure 2. Spectrophotometnc scanning analysis of isolated total RNA
from Bolrvllus schlmseri. (A) RNA isolated from B schlosseri with a
conventional extraction method that utilizes phenol/chloroform/gua-
nidme isothiocyanate (Chomcynski and Sacchi, 1987) demonstrates an
altered ultraviolet absorption spectrum. In contrast (B), RNA isolated
with the single-step cesium-chlonde method is free of contaminants and
exhibits an optimal OD 26 o/OD 280 ratio ( 1 .9-2. 1 ).

a cesium chloride ultracentrifugation step permitted the
recovery of high yields of spectrophotometrically and
electrophoretically pure RNA preparations, irrespective
of pigmentation or species. Groppe and Morse (1993) re-
cently described a two-step cold method of isolating RNA
from Haliotis ntfescens (red abalone); the method pro-
vided high yields of pigment-free, undegraded material
suitable for cDNA cloning. The first step, a phenol/chlo-
roform extraction performed at 0C, was crucial for the
removal of ribonuclease activity, and the second step,
employing ultracentrifugation through a cesium chloride
gradient, removed an inhibitor of reverse transcriptase.
The observations reported herein indicate that in Botryllus
and other colonial ascidians, only a single preparative ul-
tracentrifugation step through cesium chloride is required
for isolation of biologically functional RNA.

However, our findings are in contrast with those of Ku-
mar el al. (1988), who reported that they successfully iso-
lated RNA from various ascidians by using only a phenol/
chloroform-based procedure. In our hands, all prepara-
tions isolated using phenol/chloroform were significantly
contaminated with pigments and gave very poor yields.
Furthermore, much of the original sample was left in the
loading well during formaldhyde/agarose gel electropho-
resis, and the efficiencies for in vitro translation reactions
and reverse-transcription for cDNA library construction
were significantly impaired (W-T.C and R.J.L., unpub.
obs.). At present, we have no explanation for the discrep-
ancy between our results and those of Kumar et al. ( 1988).

3911
2800

1898

872
562

Figure 3. Ethidium bromide staining of total RNA isolated by pre-
parative ultracentrifugation. Lane I. RNA markers. Lanes 2, 3, 7, and
8. Bolryllus vhhnscn from Eel Pond (Woods Hole. MA): lane 2 (stage
A), lane 3 (stage B-2), lane 7 (early takeover; 3 h post-onset), and lane
8 (mid-takeover, 12 h post-onset). Lane 4, B schlosseri from Monterey
Bay, CA (stage C-2). Lane 5, Bolrylloidcx dicgense (growth phase). Lane
6, Diplosoma nuicdniinklii

One possibility is that the composition of pigments found
in Botryllus and other botryllid ascidians may differ from
those found in other solitary or colonial species reported

1.7 kb
1.5kb

1.7 kb
1.5kb

.-UK-

B

H

Figure 4. Northern blot analysis of RNA isolated from various species
of colonial and solitary ascidians. Samples were electrophoresed on a
1% agarose/formaldehyde gel, transferred to nitrocellulose, and hybridized
with a 32 P-labeled cytoactin probe SpCAS from Slre/a cluru (Beach and
Jeffery, 199(1). (A) Lane 1. Bniryllns scltlm.seri (stage A) from Eel Pond
(Woods Hole, MA); lane 2. B .ichliaxcri (stage C-2) from Monterey Bay
(CA); lane 3. Boirylloides diegense (growth phase); lane 4. /)//'/< worna
macdonaldn. lane 5, Molgula iinin/nillcnsis (B) Total RNA samples of
B schlosseri from Eel Pond isolated at various stages of the blastogenic
cycle (lanes 1-4), and pooled poly-A* RNA from stages A-C colonies
(lane 5). Lane 1. early takeover, lane 2. mid-takeover; lane 3, stage A;
lane 4, stage B- 1 .

ISOLATION OF RNA FROM BOTRYLLUS

29

IEF

SDS

110 kd -

97 kd -r

35 kd -

20 kd -

B

'r^I *<

* 4*. * * \m

Figure 5. Two-dimensional protein gel analysis of in two-translated RNA with 35 S-methionine reveal
changes in gene expression during takeover. Panel A depicts a colony at the onset of a new blastogenic cycle
(stage A), whereas panel B is from a colony in early takeover (3 h post-onset). The circled spots in panel A
represent transcripts that are repressed in the early stages of takeover. The arrows in panels A and B depict
a representative polypeptide whose mRNA is down-regulated during takeover. Conversely, the circled spots
in panel B are transcripts that appear to be induced de nova during takeover. Abbreviations: SDS. sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) dimension; IEF, isoelectric focusing di-
mension.

by Kumar et al. (1988). The presence of contaminating
pigments markedly altered OD: W)/ 2 8 o ratios, presumably
by absorbing in the range that is optimal for nucleic acids
(i.e., 260 nm). In support of this hypothesis, we have re-
cently observed that pigment cells from live colonies are
fluorescent under ultraviolet light (R.J.L., unpub. obs.).
Northern blot analysis with cesium-chloride-purified
material further revealed that the RNA was not degraded
by ribonuclease activity present during zooid regression.
Previous studies have cautioned that isolation of intact
RNA molecules from dying tissues can be significantly
impeded by ribonucleases (Cidlowski, 1982; Owens et ai,
1991). In addition, all colonial ascidian species reported
in this paper (Botryllus schlosseri from the East and West
coasts, Botryloides diegense, and Diplosoma macdonaldii)
expressed two poly-A* transcripts of 1.5 and 1.7 kb in
length that hybridized to a cytoplasmic cDNA clone from
Styela clava. This was in contrast to the single 1.5 kb-
message found in the solitary ascidian Mo/gula manhal-
tensis. The significance of two mRNAs in colonial ascid-
ians is unclear, although another solitary tunicate. Stye/a
clava, was previously reported to express a single 1.8-kb

message during both embryonic and post-metamorphic
development (Beach and Jeffery, 1990). The additional
1.7-kb band in colonial species may represent a cross-
hybridizing muscle actin transcript. Tomlinson et al.
(1987) showed that a probe made exclusively from the 3'
untranslated region of a Styela muscle actin clone detected
transcripts exclusively in muscle cells, whereas one made
from the coding region, such as the cytoplasmic cDNA
clone used in this study (e.g.. SpCAS), detected both mus-
cle and nonmuscle transcripts. However, several lines of
evidence argue against this scenario. First, although both
transcripts were expressed at all phases of the blastogenic
cycle in Botryllus including takeover, the relative intensity
of the bands varied at different stages of the cycle. Second,
if the 1.7-kb transcript represented a cross-reactive muscle
mRNA. one would expect the intensity of the hybridizing
band to be less than the 1.5-kb transcript at any given
time under high-stringency conditions. This condition was
clearly not observed. Alternatively, both transcripts could
result from alternative splicing. The expression of an ad-
ditional 1.7-kb transcript could be functionally related to
the colonial life style, but seems unlikely to be associated

30

W.-T. CHANG AND R. L. LAUZON

with zooid death since Diplosoma species do not undergo
takeover. An intriguing possibility is that it may be ex-
pressed during bud development. Therefore, determina-
tion of the complete nucleotide sequences of both cDNA
clones followed by in situ hybridization with non-cross-
hybridizing probes will be required to resolve this issue.
Studies with invertebrate (Wadewitz and Lockshin,
1988) and vertebrate (Wang and Brown, 1991) develop-
mental systems indicate that individual death programs
may involve fewer than 40 up-regulated genes. For in-
stance, thyroid-hormone-mediated changes leading to tail
resorption in Xenopus laevis involve two periods of gene
expression during which all genes belonging to a specific
group are induced with identical kinetics. Conversely,
about 10 additional genes are down-regulated with iden-
tical decay kinetics (Wang and Brown, 1993). These ob-
servations indicate that in amphibians the death program
reflects a relatively simple pattern of gene expression. The
initial findings reported here with two-dimensional protein
gels from Botryllus suggest that modulated gene expression
occurs during the takeover phase of blastogenesis. We have
previously demonstrated that takeover involves the po-
larized breakdown of the perivisceral extracellular matrix
along the zooid's anteroposterior axis, followed by apop-
totic and necrotic morphological changes within dying
visceral tissues (Lauzon et a/., 1992, 1993). Changes in
gene expression may thus be associated with these mor-
phological events. Unfortunately, the shutdown of oral
siphons during takeover precluded us from analyzing 35 S-
methionine incorporation patterns in vivo. Therefore, the
possibility cannot be ruled out that differences in 2-D

B

23,130
9,146
6,557

4,361

2,320
2,037

567

Figure 6. Alkaline agarose gel electrophoresis assay of first- and sec-
ond-strand cDNA synthesis in Botryllus schlosseri. First- (lane A) and
second-strand (lane B) cDNA products were converted using pooled poly-
A* RNA isolated from colonies during takeover (onset and early take-
over). Note that both lanes exhibit a broad size distribution of cDNA
products, with the majority of material ranging between I and 2 Kb.

B

Figure 7. Nitrocellulose plaque lifts from growth stages (panel A)
and takeover (panel B) cDNA libraries hybridized with an ascidian 32 P-
labeled cytoactin probe from Si vela clava (SpCAS). Percent positive
plaque forming units in (B) was 3.0% (1 50 positive out of 5 X 10 3 PFUs)
compared to 3.2"! for the growth phase cDNA library (58 positive out of
1. 8 x I0 3 PFUs), indicating that actin was adequately represented.

protein profiles between the onset of blastogenesis and
takeover are due to inherent limitations of the in vitro
translation kits. In addition, since clonal replicates were
not used in any of these studies, the differences observed
may represent intra-species polymorphisms. Lastly, since
takeover involves the simultaneous regression of adult
zooids along with asexual growth of the future parental
generation, the possibility cannot be excluded that tran-
scriptional changes also occur in buds or in the colonial
vasculature. Therefore, assessing the specificity of tran-
scriptional changes will require isolation of takeover-spe-
cific mRNAs and analysis of their spatial distribution pat-
tern by in situ hybridization. We are currently using dif-
ferential mRNA display (Liang and Pardee, 1 992) as a
means for ultimately characterizing full-length transcripts
from the cDNA libraries. Interestingly, the percentages of
actin-positive PFUs were similar in the growth phase and
takeover libraries (3.2 versus 3.0). Libraries with reported
actin cDNA-positive frequencies above 0. l% have yielded
clones of interest for sequences of moderate to low abun-
dance, whereas percentages below 0.05% have not (Hagen
et a/.. 1988). Collectively, our findings strongly suggest
that both libraries are likely to contain cDNAs corre-
sponding to single-copy gene transcripts. The character-
ization of genes involved in zooid regression could provide
a fundamental understanding of molecular mechanisms
of programmed cell death in Botryllus and other meta-
zoans.

Acknowledgments

The authors gratefully acknowledge Dr. Craig Tomlin-
son for his generous gift of the SpCAS cytoactin cDNA
clone, and two anonymous reviewers for their efforts in
improving the focus of this manuscript. This work was
supported by a Basil O'Connor Starter Scholar Award
from the March of Dimes Birth Defects Foundation #5-

ISOLATION OF RNA FROM BOTRYU.L'S

31

FY94-0813, and from a Frederick Bang Fellowship at the
Marine Biological Laboratory, Woods Hole, Massachu-
setts.

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Prespawning Behavior, Spawning, and Development
of the Brooding Starfish Leptasterias polaris

JEAN-FRANCOIS HAMEL AND ANNIE MERCIER

Depart ement d'Oceanographie, Universite dii Quebec a Rimouski, Centre Oceanographique
de Rimouski, 310 allee des Ursulines. Rimouski (Quebec), Canada G5L 3A1

Abstract. Our study focused on the precise reproductive
behavior of the starfish Leptasterias polaris (Miiller and
Troschel) before and during spawning a subject of much
speculation and evident ecological importance. Between
the third week of December 1992 and mid-January 1993,
we observed spawning in the laboratory that roughly cor-
responded to field observations in the Lower St. Lawrence
Estuary. In experimental tanks provided with natural en-
vironmental conditions, the spawning was preceded by 7
to 8 weeks of complex aggregative interactions among the
starfish. The individuals, which usually avoid each other,
began to make discreet arm contact, which intensified
with time and eventually led to the superposition of two
or more starfish, independently of sex. The interactions
seem to be associated with decreasing temperature, be-
cause aggregative and spawning behaviors were not ob-
served under stable temperature conditions. Male spawn-
ing is first initiated when the temperature falls to about
2C during minimum daylength (<9 h-d '). In seawater,
the spermatozoa are negatively buoyant and tend to de-
posit as a sticky film on the substrate, where they enter a
state of low activity. Stimulated by male spawning, females
spawn on the layer of sperm, which is reactivated by con-
tact with the oocytes, ensuring fertilization. In the labo-
ratory, the fertilized eggs undergo first cleavage in 45 h,
become brachiolaria in 40 days, and form fully developed
young starfish within 5.5 to 6 months, synchronously with
populations in the field. The embryos develop at the same
rate even when not brooded, suggesting that the brooding
behavior in L. polaris serves mainly to keep the eggs clean,
healthy, and protected against predation.

Introduction

Successful fertilization constitutes a critical stage in
marine invertebrate reproduction, and many organisms

Received 7 December 1993; accepted 4 November 1994.

develop strategies to maximize this important step (Him-
melman. 1981; Giese and Kanatani, 1987). Starfish show
diversified reproductive behaviors. In many species, ga-
metes are broadcasted by both sexes, with fertilization in
the water being enhanced by synchronization of spawning
(Hyman, 1955; Strathmann, 1987; Chia and Walker.
199 1 ). In other starfish, males broadcast spawn in the usual
fashion, and females emit fewer gametes but brood their
embryos to fully developed young starfish (McClary and
Mladenov, 1990; Chia and Walker, 1991). Leptasterias
polaris, which protects its embryos for 5 to 6 months, is
among the few species that brood by overlaying the eggs
deposited on the substrate (Emerson, 1977; Himmelman
et ai, 1982; Boivin el a/.. 1986). Although brooding star-
fish are generally small-sized, with lecithotrophic devel-
opment (Chia and Walker, 1991). L. polaris can reach
diameters up to 50 cm (Boivin et al., 1986) and are prob-
ably among the largest brooders.

Prespawning and spawning behaviors are very impor-
tant to reproductive success in marine invertebrates.
Breeding aggregations have been observed in a number
of asteroids (Chia, 1968; Komatsu, 1983; Minchin, 1987;
Young et a/., 1992; Slattery and Bosch, 1993). Many au-
thors suggest that such aggregations could minimize sperm
dilution and increase fertilization success (Ormond et al.,
1973; Levitan, 1991; Levitan et al., 1992), as exemplified
by the pairing strategies in Archaster typicu.i (Run et al..
1988) and Neosmilaster georgianus (Slattery and Bosch,
1993). In those species, the male, after finding a female,
mounts her before spawning (Ohshima and Ikeda, 1934;
Komatsu, 1983; Run et al.. 1988; Slattery and Bosch,
1993). There is also evidence that the spatial distribution
of broadcast spawners has a major influence on the prob-
ability of fertilization due to gamete viability (Pennington,
1985; Yund, 1990; Levitan et al.. 1992; Young et al..
1992). Young et al. (1992) suggested that aggregations

32

REPRODUCTIVE BEHAVIOR OF LEPTASTER1AS POLARIS

33

could be useful in overcoming the absence of the usual
spawning cues (e.g., light, temperature) in bathyal echi-
noid populations. The possible role of pheromones and
other possible attractants on clustering and related
spawning inducement in starfish has been examined
(Lewis, 1958; Miller, 1989). Komatsu (1983) suggested
that initial heterosexual recognition and pairing in A. typ-
icus allow male spawning to be induced by release of ma-
ture oocytes from females. However, most studies on ag-
gregation have associated it with cooperative feeding or
predation avoidance (Ormond el a/., 1973; Blankley and
Branch, 1984; Sloan, 1984; Pearse and Cameron, 1991).

Although different kinds of aggregations during spawn-
ing have been observed, little is known about the involve-
ment of grouping prior to spawning other than recent
work on the bathyal sea urchin Stylocidaris lineata (Young
el a/., 1992). Moreover, prespawning interactions have
never been discussed in respect to environmental factors
such as photoperiod and temperature, which are known
to influence gametogenesis and spawning, respectively
(Giese and Pearse, 1974; Himmelman, 1981; Pearse and
Walker, 1986; Pearse el al. 1986; Pearse and Cameron,
1991). As for fertilization strategies, observations on ga-
mete interactions, other than mutual recognition and at-
traction, remain scarce. Sperm motility and respiration
activation by egg extracts have been studied in sea urchins
(Suzuki el ai, 1982) and in horseshoe crabs (Clapper and
Epel, 1981). but only sperm chemotaxis has been de-
scribed in detail for starfish (Miller, 1985).

The starfish Leptasterias polaris can be kept in labo-
ratory facilities that reproduce natural conditions, and
this has provided a chance to record and describe its ag-
gregative behavior both before and during spawning. Fur-
ther evidence from experiments on gamete behavior and
embryonic development allowed us to better understand
the evolutionary strategy that seems to link spawning, ga-
mete fertilization, and brooding activities in this species.

Materials and Methods

Using scuba, we collected 60 specimens of Leptasterias
polaris from a depth of about 1 m on the south shore of
the Lower St. Lawrence Estuary (48 21' N: 68 47' W),
eastern Canada. The animals, ranging from 150 to
200 mm in diameter, were collected in May 1992, to en-
sure that they were acclimatized well before the December
spawning that we expected on the basis of previous ob-
servations by Boivin el at. (1986). The starfish were kept
in tanks to which seawater from the collect site was sup-
plied by a flow-through system and light on natural pho-
toperiod was provided through large windows. Physical
and chemical conditions were therefore similar to the
natural environment. Preliminary determination of sex
in L. polaris demonstrated a natural sex ratio close to 1:

1 , and this was carefully reproduced in the tanks. To es-
tablish the importance of environmental factors on pre-
spawning aggregative behavior, spawning, and develop-
ment, the temperature and the salinity of the circulating
water were continuously recorded. The data for the day-
length were provided by the Canadian government (En-
vironment Canada; Atmospheric Environmental Service,
Quebec airport). The starfish were given an unlimited
quantity of mussels (Mytilus edulis, =20 mm in shell
length), their favorite prey (Himmelman and Dutil, 1991 ).
Frequently very abundant in subtidal environments
(Himmelman and Dutil, 1991), L. polaris adapts ex-
tremely well to experimental conditions.

Prespawning behavior

Starfish behavior was recorded on a regular basis be-
tween 4 and 14 times a week depending on activities ob-
served, from November 1992 to February 1993, with
complementary observations before and after this period.
The number of individuals preying on the mussels, resting
on the bottom, and climbing on the sides of the tank were
noted. The number of starfish in contact was recorded
and categorized as light, when arms touched from the
middle part to the tip; intimate, when the arms intertwined
for more then half their length; or superposition, when
the individuals overlaid one another (Fig. 1). Particular
attention was given to reactions of the starfish after re-
newal of food supply, about twice a month. Data were
subsequently combined for weekly comparison of contact
intensities. Two control groups were also observed during
all prespawning and spawning experiments. Group 1 was
maintained under constant conditions in the Quebec
Aquarium at a temperature of 6C, a salinity of 28% and
a daylength of 10 h; group 2 was kept in continuous dark-
ness with natural conditions of salinity and temperature.

Spawning behavior

As the spawning period approached (Boivin el al.,
1986), observations focused on detecting gamete release
in relation to the postures adopted by the starfish and to
the prevailing environmental conditions. When observed,
spawning events were carefully described.

To test the hypothesis that sperm induces spawning in
females, a solution (1.2 X 10 1 spermatozoa ml ' deter-
mined with a hemacytometer under a light microscope)
was prepared with freshly collected sperm from a single
male. This sperm solution was poured into a tank (4 m 3 )
among mature females, and their subsequent behavior
was recorded. This procedure was carried out four times
at different periods under the conditions previously de-
scribed for the starfish in control group 1. Male starfish
were exposed to the same sperm concentrations to see if
they would be induced to spawn.

34

J.-F. HAMEL AND A. MERCIER

Figure 1. Photographs illustrating the prespavvning aggregations of
Leptasterias polaris in the laboratory. (A) Superposition of two individ-
uals. (B) Massive aggregation of starfish.

Sperm behavior

The term dry sperm refers to undiluted, freshly removed
sperm from the mature gonads of at least two males. Ac-
tivated oocytes were obtained by spreading freshly re-
moved female gonads (including gonoducts) in a petri
dish filled with 5 ml of 1-methyladenine 1(T 5 M, yielding
naturally spawned oocytes after about 45 min of exposure.
The buoyancy, capacity for adherence to the substrate,
and motility of the sperm were used to describe its be-
havior in seawater. All experiments on sperm behavior
were conducted at a temperature close to the one recorded
during the spawning period (2-4C). All live observations
of sperm samples were made under a light microscope
( 100-400X) and the sperm was kept cool by surrounding
the slide with ice cubes. The speed of the spermatozoa
was evaluated with a graduated lens, by calculating the
number of bars traveled per second. Verifications were
undertaken to discard any behavior induced by the light
of the microscope or false sperm velocity induced by the
sperm-glass interaction (thigmotaxis).

For more than 7 h we continuously recorded the speed,
orientation, and flagellar activity of sperm before and after
the samples were diluted in seawater ( 1 50 /jl of dry sperm
in 1 I). To examine the behavior of sperm in the water

column and on the bottom, the protocol was carried out
in both still and agitated (current of 7cm-s~') water.
Samples were taken from the bottom of the beaker and
at a middle depth every minute for 15 min, then every
1 5 min for 7 h, and finally at 24-h intervals until the sper-
matozoa were dead. This protocol, which was repeated
twice using repetitive independent measurements, also
allowed the estimation of the sperm concentration, with
the proportion of resting and agglomerated sperm in the
water and on the bottom over time. To estimate the
maintenance of sperm potency, samples of sperm depos-
ited on the bottom of the beaker were collected just after
the sperm was released in the seawater and regularly over
72 h. The samples were tested on two replicates of 5-10
freshly activated oocytes. Fertilization success was deter-
mined by staining the eggs with the DNA-specific flu-
orescent dye Hoechst 33258. Using a Leitz Diaplan flu-
orescence microscope, we determined the proportion of
eggs showing a male pronucleus.

After spermatozoa reached a state of low activity (>80%
barely moved) in the beaker of the above experiment, we
tried to stimulate the sperm by exposing it to oocytes. In
separate trials, a sample of sperm collected on the bottom
of the beaker was exposed to oocytes of different levels of
maturity and of different origins. The conspecific previ-
tellogenic and mature oocytes, classified according to the
studies of Boivin ft til. (1986) and Mercier ct ul. (1994),
were first assayed. In the case of mature oocytes we tried
both activated oocytes (showing germinal vesicle break-
down) and surgically removed unactivated ones. The ac-
tivated oocytes of Asterias vulgaris. another species of
starfish, were also tested for stimulation of Leptasterias
polaris sperm. The oocytes of both species were routinely
washed in filtered seawater and immediately used for the
reactivation experiments. The speed and the flagellar ac-
tivity of sperm were noted every 10 min for the first hour,
then periodically until no movement was detectable, using
replicates and going through the whole protocol twice. A
control sample with no oocytes was tested in the same
manner.

To investigate sperm sinking, dry sperm was diluted in
seawater ( 1:60) to a concentration of 120 X 10 3 sperma-
tozoa-mi '. Five homogeneous replicates (10/^1) of this
solution were prepared from eight males. The samples
were deposited at a middle depth in a 1000-ml beaker
filled with seawater. and the time needed for about 50%
of the sperm (in visible filaments) to reach the bottom
was recorded. These times were compared to those of
sperm collected from three species in which fertilization
occurs in the water column, the starfish Asterias vulgaris.
the sea urchin Strongylocentrotus droebachiensis, and the
sea cucumber Cucumaria frondosa.

To examine sperm dispersion over time, we deposited
dry sperm on the bottom of a large dish filled with 1000 ml

REPRODUCTIVE BEHAVIOR OF LEPTASTER1AS POLARIS

35

18"

16-

14

12

ID-
S'
6
4
2"

Prespawning

Spawning

Developmental biology

Temperature (C) Metamorphosis

O

N
1992

D

M A

1993

M

Figure 2. Variation of environmental factors during the reproductive season of Leptasterias polaris in
the experimental tanks. The arrow points to the beginning of embryo metamorphosis.

of seawater and periodically collected water samples at
the surface and on the sides of the dish. The spermatozoan
concentrations of the samples were evaluated with a he-
macytometer under a light microscope. This experiment
was simultaneously performed on live and dead dry sperm
of Leptasterias polaris and on dry sperm from Asterias
vulgaris. Strongylocentrotus droebachiensis, and Cucu-
nuiria fmndosa. Sperm longevity could thus be compared
for those species under the same conditions.

Development

Whenever we discovered a naturally spawned egg mass,
whether brooded or not, it was left undisturbed in the
tank so that we could examine development under natural
variations of environmental factors. Nonbrooded egg
masses were kept clean by periodically agitating the water.
Samples were regularly collected with pipettes, from fer-
tilization to young starfish stage, and transferred to 4%
formaldehyde/seawater for later examination with light
microscopy. These embryos also served to determine de-
velopmental kinetics and growth. During the first hour,
samples were collected every 2-5 min, then about every
day until the brachiolaria stage, and finally once a week.
A new stage was considered attained when 50-60% of the
embryos reached it. Maximum embryo diameters were
measured under a light microscope equipped with a grad-
uated lens.

Results

Prespawning behavior

During summer and early fall, the well-fed starfish
clearly avoided each other. Contacts among starfish began
in mid-October, coinciding with the first significant de-
crease of temperature (Fig. 2). The proportion of contact-
free starfish decreased, reaching a minimum plateau be-
tween 1 November and 15 December (Fig. 3) when tem-
peratures fell to about 4C. In that same interval, the
proportion of starfish involved in intimate contacts in-
creased progressively, with the maximum recorded in the
last week of November. Superposition became more fre-
quent in early December and was observed most often
just before the main spawning period of late December
(Figs. 1,3) when the water temperature fluctuated between
3 and 4C. When the temperature fell below 3C (end
of December), the number of superpositions and intimate
contacts decreased while spawning was recorded (Fig. 3).
Superposition behavior ceased after January and most in-
dividuals resumed avoiding one another (Fig. 3), with
contact-free starfish accounting for more than 85% of the
observed individuals. Only some light contacts and a few
intimate ones were observed. No particular sex-specific
patterns were noticed for the aggregations, which involved
both males and females. Addition of prey to the experi-
mental tanks always provoked a migration of the starfish
toward the food source, except 1 week before spawning

36

J.-F. HAMEL AND A. MERCIER

100

u

o

40"

20

Spawning

Types of contact

B Superposition

Q Intimate

B Light

D No contact

N

D

1992

1993

Figure 3. Leptasterias polaris. Temporal evolution of the prespawning aggregative behavior recorded
several times a week for a 3-month period. For each date, the percentage of individuals involved in a
particular type of contact was recorded.

when feeding did not disturb the contact behavior between
the individuals.

Environmental factors seem to be involved in the ini-
tiation and development of the prespawning aggregative
behaviors. No aggregation occurred among individuals
maintained at constant temperature and photoperiod, but
grouping did take place among individuals kept in total
darkness with natural temperature.

Spawning

Our experimental information includes actual obser-
vations of spawning events in 4 females and 3 males and
additional indications from sperm agglutinates on many
males and on the substrate. We also observed more than
20 brooding females, which were always discovered within
24 h of spawning. We examined the correlations of all
our observations with environmental factors (Fig. 2),
which were similar in the laboratory and in the field.
Spawning occurred in our tanks from 1 9 December to 1 2
January, which roughly corresponded to the period when
we observed spawning in the field. During spawning
events, the starfish stayed close together, although there
was a net decrease in frequency of contact (Fig. 3). The
spawning individuals were not paired or superposed.

Spawning events. Figure 4 schematically illustrates the
spawning behavior we observed in the experimental tanks.
When a male spawned, it elevated the central disk, stand-

ing on the curved tip of its arms, and emitted sperm as a
whitish stream from the six interradial aboral gonopores.
Emission continued for more than an hour. Qualitative
observations showed the sperm to be negatively buoyant,
with a tendency to deposit on the substrate. The first fe-
male spawning was observed after male spawning. The
female remained flat on the substrate to release eggs while
its arms were extended, then progressively adopted the
characteristic brooding posture in "pinwheel" shape (Figs.
4, 5a, b). The average 300-500 spawned oocytes emerged
individually at a rate of about 1 oocyte every 2-5 s from
each of the six aboral gonopores located between the arms.
Almost all the spawning starfish observed were on a ver-
tical substrate (aquarium wall or rock face). The eggs had
a tendency to fall and were retained by the ambulacral
podia and the curved arms of the female (Fig. 5c). A num-
ber of eggs (possibly 50%) were, however, lost before the
end of a given spawning event. The final posture adopted
by the female was not always a perfect pinwheel shape
but seemed designed to cover the egg mass (around 20-
25 eggs -cm : ) in the best way possible. Consequently,
some brooding individuals were seen with two or three
arms extended somewhat to cover isolated groups of oo-
cytes.

Induction <>/ 'spawning. Although the first individuals
to spawn were males, spawning events were subsequently
recorded in both sexes alternately throughout the spawn-
ing period (Fig. 2). Nevertheless, some correlations con-

REPRODUCTIVE BEHAVIOR OF LEPTASTERIAS POLARIS

37

2 C

0Male spawning induced by
decreasing temperature

Active sperm in the
-^ water column (moving at

Female spawning stimulated
by sperm in the water column

Aggregation and settling
of sperm (resting state)

Spterm reactivated by
fresrrty-soawned oocytes
" rtilizationT?

Brooding and
embryonic
development

Figure 4. Schematic illustration of spawning behavior for male and female Lcplastvrias polans, showing
the relation between them.

cerning the induction of spawning could be made in the
course of our experiments. A few isolated male spawnings
occurred during minimum daylength (<9 h) when water
temperature was around 2-3C (first sight of sperm fil-
aments) in late December (Fig. 2), but most spawnings
were observed in January as the temperature fell further.
The temperature fluctuated between 2 and 4C (Fig. 2)
throughout the following weeks of spawning, and gamete
release seemed to be closely related to these variations.
The same spawning pattern was observed in control group
2, maintained in natural temperature and total darkness,
whereas no spawning occurred in control group 1, kept
at steady temperature and photoperiod. As a result of the
seasonally low primary production, the tanks provided
with seawater from the estuary, where spawnings were
recorded, contained virtually no phytoplankton. Salinity
continuously fluctuated between 26 and 32%o without any
consistent increasing or decreasing trends (data not pre-
sented). During qualitative observations, the presence of
sperm filaments seemed to be correlated with subsequent
female spawning within a few hours. Complementary ex-
periments conducted in replicates showed that the intro-
duction of sperm in the water induced spawning of several
females in the controlled environment (control group 1 ),
therefore minimizing the importance of temperature in
female spawning. No male spawning was induced by the
presence of sperm.

Sperm behavior

A microscopic examination of the sperm showed that
the head (more or less spherical) measured 3.25 0.25 ^m

and the flagellum 62 3 ^m. The negative buoyancy of
the male spawn caused it to sink (mainly as white fila-
ments), at a rate of about 2.1 mm-s" 1 . Only a small por-
tion was resuspended after reaching the bottom. We ex-
amined the motility of sperm artificially maintained in
the water column compared to the motility of sperm set-
tled on the bottom (Fig. 6). Freshly extracted dry sperm
contained nonmotile spermatozoa. Upon introduction to
the seawater, the spermatozoa immediately displayed a
major increase of activity, both in the water and on the
bottom (Fig. 6). Within the first 30 min of water contact,
100% of the spermatozoa had reached a velocity of 250-
350 p.m s~' and showed intense flagellar activity, resem-
bling a helical movement with 6-7 revolutions -s~'. The
spermatozoa maintained in suspension continued to show
the same high velocity and activity with no marked net
decrease for the whole 425 min of observation. In contrast,
after reaching a maximum velocity (after 40-50 min) in
synchrony with the sperm in suspension, the sperm on
the bottom showed an abrupt decrease of activity (reduced
velocity and flagellar movement). The settled spermatozoa
attained a low velocity (=s50Mm-s~') after 120 min of
contact with seawater (Fig. 6a). This corresponded to an
increase in the inactive population of spermatozoa, which
rose from 7% to 56% in the same period (Fig. 6a). The
spermatozoa seemed to gradually reach a state of almost
null velocity (about 0-15^m-s ') in which they only
quivered and moved by a wave along the flagellum (prox-
imal to distal) with a 10 angle. The percentage of settled
spermatozoa that attained a state of low activity was 47%

38

J.-F. HAMEL AND A. MERCIER

Figure 5. Underwater photographs showing (A) Li'ptasterias polari* brooding in its natural habitat
surrounded by sea urchins; (B) close-up of a brooding female on the rocky bottom at 30m depth: (C)
fertilized eggs under a female: (D) young starfish after about 5.5 months of growth in their natural habitat
(the female was previously removed). The scale bar represents 15mm and applies to photographs (C)
and (D).

after 120 min of contact with seawater, 62% after 250 min,
and a maximum of 80% after 380 min (Fig. 6a). On the
bottom and most probably on the glass walls, the increased
number of nearly inactive spermatozoa was responsible
for the decrease in overall velocity of the population. This
correlation could also be made for the sperm in the water
column, although the small apparent decrease in velocity
could only be visually associated with the slight increase
in inactive spermatozoa (Fig. 6b). Another progressive
phenomenon was the formation of sperm conglomerates
(dense aggregations), which began about 180 min after
the sperm came in contact with seawater and reached a
maximum (100 and more spermatozoa together) after
380 min. About 8 h after entry into still seawater, the
sperm covered the bottom and glass walls of the dish.
This also occurred in agitated conditions, although after
a longer period, showing that the sperm of Leptasterias
polaris is very adhesive.

The sperm behavior of Leptasterias polaris differed
from that observed in the other species of echinoderms

tested: the sperm of Asterias vulgaris was diluted within
2 min, before reaching bottom, and those of Cucumaria
Jrondosa and Strongylocentrotus droebachiensis sank at
1 mm-s~' and 1.5 mm-s~' respectively. The sperm of
these species did not adhere to the bottom, but was im-
mediately and almost totally redispersed, becoming well
dispersed in seawater within 80 min. In contrast, the ma-
jority of L. polaris sperm stayed on the bottom until death.
After reaching a state of almost null activity (conglom-
erated or not), the sperm of Leptasterias polaris could be
reactivated by contact with conspecific activated mature
oocytes (Fig. 7). Within 10 min of exposure to these oo-
cytes, sperm motility was reinitiated. Spermatozoa veloc-
ity increased significantly, by 485% (P < 0.01, Student's
/ test), after 20 min of contact, and attained a peak of
230 nm s~' after 50 min. This was almost 12 times the
original speed (Fig. 7). Subsequently, the sperm velocity
decreased progressively and reached a minimum after
1020 min (Fig. 7). Reactivation was unsuccessful with
unactivated conspecific mature and previtellogenic oo-

REPRODUCTIVE BEHAVIOR OF LEPTASTERIAS POLARIS

39

100 150 200 250 300 350 400 450

Time (min)

Figure 6. Leptasterias polaris Temporal changes in velocity and
activity of sperm settled on the bottom and kept in suspension. The
sperm velocity ( O ) upon contact with seawater is given with the
corresponding percentage of inactive spermatozoa ($) The error
bars represent the confidence intervals (95%).

cytes as well as with mature activated oocytes otAsterias
vulgaris. The spermatozoa exposed to those oocytes
showed a constant low velocity, comparable to that ob-
served in the unexposed sperm serving as control (Fig. 7).
The sperm of Leptasterias polaris was more resistant
than that of other species. Most spermatozoa collected
from other echinoderms were dead after 8-20 h, whereas
those of L. polaris remained capable of high fertilization
success (57%) for as long as 34 h in seawater (Fig. 8) and
showed a high percentage of mortality only after 6-7 days.

Development

Early development. The complete chronology of em-
bryonic development of Leptasterias polaris is presented
in Table I and Figure 9. The large unfertilized mature
oocytes (^0.85 mm in diameter) were mainly spherical
and yellowish, or occasionally light orange. They were
covered with a rather thick outer membrane (average of
7.04 ^m). After their fertilization, the eggs were attached
to one another by the fertilization membrane, showing
that the membrane was sticky, especially after reaching
the 2-cell stage (Fig. 9a). All the cleavages were of the
radial holoblastic type. Later in its development, from the
blastula to young gastrula, the embryo decreased in size

300 n

^ 250-

u

<u

s 20

Jl

>.

n iso

<u
Q.
CD

100

50

Control

Previiellogemc oocyks
UnacDvaled oocytes
Activated oocytes
Astenas oocytes

10

100

1000

Time (min)

Figure 7. Leptasterias polaris Influence ofoocyte maturity on sperm
reactivation. The horizontal axis has a log lo scale and the error bars
represent the confidence intervals (95%).

(zx6%) due to blastomere compaction (Table I). The fur-
rows became shallower on the pole, but tended to deepen
on the other pole, eventually forming the blastopore. After
7 1 1 h, the embryo's surface became uniform and ciliated.
The embryo then began to spin inside the fertilization
membrane and hatched 96 h later (Fig. 9e).

Many females moved away from their brood within a
week, leaving their embryos uncared for in the experi-
mental tank. No significant difference was observed be-

20

40 60

Time (h)

80

100

Figure 8. Leptaslerias polaris. Sperm effectiveness to fertilize acti-
vated mature oocytes over time. The error bars represent the confidence
intervals (95%).

40

J.-F. HAMEL AND A. MERC1ER

Table I

Leptasterias polans: A'/mY;o / cnihryaiuc i/c\-c/<>piiiciii in
experimental lank', supplied hy running Mwatcr

Developmental Stages

Time

Size (jim)

Spawning

Early stage of the fertilization

membrane elevation
Fertilization membrane completely

elevated

Emission of the first polar body
Second polar body
2-cell
4-cell
8-cell
16-cell
32-cell
64-cell
128-cell
256-cell

Blastula (compaction)
Wrinkled-blastula
Young gastrula
Late gastrula
Spinning
Hatching
Brachiolaria
Metamorphosis
Young starfish (2 pairs of ambulacra!

podia/arm)
Young starfish (preoral lobe

disappears and ocelli are present)
Free-moving starfish (visible pyloric

caeca and opening of buccal

cavity)
Small starfish (uprighting

movements)

852 36
840 38

27 min

940 48

45 min

439 26

45 h

1 079 [ 5

86 h

1032 15

92 h

1046 12

106 h (4d)

1031 18

121 h(5d)

1062 9

133 h (5-6d)

1103 62

146 h (6.1 d)

1080 23

156 h (6.5 d)

1 101 40

209 h(8-9d)

1 1 20 5 1

260 h (10-13 d)

1 144 45

493 h (20-21 d)

1056 31

666 h (27-28 d)

1288 52

711 h ( 29-30 d)

1375 27

807 h (33-34 d)

1121 42

38-84 d

1 1 90 3 1

75-40 d

1207 70

120-132 d
150-170 d

180-195 d
more than 200 d

1348 53
1534 77

2102 102
over 2500

A new stage was considered attained when 50'" -60^ of the embryos
reached it. The standard deviations about the mean size are given.

tween the developmental rate of brooded and nonbrooded
embryos during early development up to hatching (P
= 0.392, Student's / test), which is the latest brooded stage
we observed in the laboratory (Table II).

Lute development. After the loss of the fertilization
membrane upon hatching, the unciliated portion of the
late gastrula enabled it to attach to the substrate, well
before the appearance of the brachiolar arms (Fig. 9f).
The hatched larvae immediately settled on the bottom;
however, many embryos were lost by the female at this
time of development (especially for those brooding on
vertical surfaces). With the growth of the embryo, the arms
elongated, becoming very distinct from the dorsal ciliated
bulb (larval body), and served the purpose of adhesion to
the substrate. Cilia were still present, so the fixation was
mainly with the sticky ramified tips that had developed
at the end of each arm (Fig. 9g). A depression began to

grow in the central portion delimited by the arms (fixing
disk), and was used for later fixation on the substrate with
the brachiolar arms. The brachiolar stage was prolonged
as long as the water temperature remained around 1C
(February and March; Fig. 2), until a sudden warming
coincided with metamorphosis (day 75-90). Although
there was a concurrent elevated photoperiod, this factor
had been increasing for months and cannot be the decid-
ing inducer (Fig. 2). About 50% of the embryos died during
the gradual metamorphosis, which was completed around
mid-May following the complete disappearance of the
brachiolar arms (Fig. 9h-j). At this stage the characteristic
yellow color had been lost and the embryo was whitish
or translucent, indicating that a large amount of vitelline
reserves had been consumed. One month later (mid-June),
the young starfish possessed a well-developed buccal cav-
ity, stomach, and pyloric caeca, which were easily observed
across the transparent body wall on the oral surface (Fig.
91) with the madreporite and anus on the aboral surface.
The ambulacra! podia, baring suckers, became effective
in helping the uprighting movement and displacement of
the growing starfish, which were capable of coordinated
locomotion. Having the capacity to feed and move on
their own, the young were self-sufficient about 6 months
after fertilization (Figs. 5d, 9k, 1).

Discussion

Temporary aggregative behavior is common among
marine invertebrates. It has been observed in echinoderms
such as echinoids ( Pearse and Cameron, 1 99 1 ; Levitan et
til.. 1992: Young </<//.. 1992), ophiuroids( Warner. 1979)
and asteroids (Ormond et al.. 1973; Sloan, 1980, 1984;
Blankley and Branch, 1984; Run et al.. 1988) as well as
in crustaceans (Gherardi and Vannini, 1993). Those
studies have proposed many hypotheses about the mean-
ing and usefulness of aggregation, but only a few have
discussed a relationship with reproduction and spawning.

Most of the few reports of aggregation related to repro-
duction in echinoderms refer to aggregative spawning
events in the field (Hendler and Meyer. 1982; McEuen,
1988; Pearse et al.. 1988). Pseudocopulation or pairing
has been observed in Arc/Ulster typieiis (Ohshima and
Ikeda. 1934; Komatsu. 1983; Run et al.. 1988) and in
Neosmilaster georgianus (Slattery and Bosch, 1993), but
no such behavior could be detected during our study. Like
Chia (1968) in observations of Leptasteriii* lie.\actis. we
noticed that the groupings occurred only as the breeding
season approached. Grouping behavior initiated well be-
fore spawning, such as observed in L. polaris, has not
been often reported. Young et al. (1992) observed this
behavior in the bathyal sea urchin Stylocidaris lineata. in
which the individuals aggregate during autumn before
spawning. Orton (1914) and Lewis (1958) also mentioned

REPRODUCTIVE BEHAVIOR OF LEPTASTERIAS POLARIS

Figure 9. Lcplastcrias polaris. Photographic sequence showing the principal steps of development from
fertilization to young starfish (4X); see Table I for corresponding age and size of the embryos. (A) Fertilized
eggs with fertilization membrane completely elevated (arrows). (B) 4-cell stage. (C) 8-cell stage. (D) Wrinkled-
blastula stage on which it is possible to observe the furrows (arrows). (E) Early brachiolaria stage (newly
hatched) showing early shaping of brachiolar arms (ba) and the persistent blastopore (arrow). (F) Growth
of the three brachiolar arms (ba). (G) Fully developed brachiolarian embryo with the larval body (Ib) and
the slightly ramified brachiolar arms (ba). (H) Metamorphosing embryo with brachiolar arms (ba), showing
the development of the first five hydrocoelic lobes (hi). (I) Further metamorphosed embryo with two pairs
of ambulacral podia (p) and a terminal tentacle (t) on each of the five arms. The six hydrocoelic lobes are
transforming into the radial canals (re). A distinct oral disk (od) and the residual brachiolar arms (ba) with
central fixing disk (f) are visible. (J) Aboral view of a free-moving, six-rayed young starfish showing clearly
visible dorsal spines (ds), madreporite (m), and regressing preoral lobe (pi). (K) Aboral view of a small starfish
showing the well-developed terminal spines (ts). (L) Oral view of a small starfish ready to leave the brood.
The buccal cavity (be) has opened and the stomach (s) is visible surrounded by the ring canal (ri). We can
also see the amhulacral podia (p). the well-developed radial canals (re), and the ocelli (o).

42

J.-F. HAMEL AND A. MERCIER

the occurrence of this prespawning aggregative behavior
in echinoids.

What cues make the starfish come together and why
do they display this behavior? Pheromones have often
been proposed as the proximate cause (Kanatani and Shi-
rai, 1968), principally acting to synchronize the liberation
of gametes (Ormonde/ al., 1973; Young el al, 1992; Slat-
tery and Bosch, 1993). For broadcast spawners, aggrega-
tion and synchronous gamete release appear to be im-
portant in minimizing gamete dispersion, ensuring good
fertilization success (Levitan, 1988; Levitan el al, 1992).
For a protective brooder like Leptasterias polaris, syn-
chronous spawning would not be very advantageous as
the eggs laid by the female are maintained under the body
at all times. Therefore, aggregation is more likely to be
related to preparatory recognition. Contact chemorecep-
tion is suggested to be a strong sensory stimulus in aster-
oids (Sloan and Campbell, 1982), maybe to ensure ade-
quate recognition of males and females before spawning.
In Arcliaster typicus and Neosmilaster georgiamis, this
recognition is of prime importance because fertilization
is ensured by the close superposition of a male and a fe-
male (Run et al.. 1988; Slattery and Bosch, 1993). In L.
polaris, our results demonstrate a totally different pattern,
in which the prolonged intimate contacts could be related
to the chemical induction of synchronized gamete devel-
opment as demonstrated in sea cucumbers Citcumaria
frondosa (Hamel et al., unpub. manuscript).

The initiation of aggregative behaviors in Leptasterias
polaris appears to be correlated with decreasing temper-
ature. Aggregations were observed among all starfish that
were supplied with natural seawater and exposed to sea-
sonal changes of water temperature, both when main-
tained in darkness and when exposed to natural photo-
period. Lower temperatures possibly trigger or enable the
liberation of hormones through a pathway otherwise sup-
pressed and may favor the formation of clumps of starfish.
This would assure that a fairly good proportion of male
and female individuals would be close together and ready
to release their gametes during the winter spawning events.
We saw no evidence of recognition between sexes but can
assume that the male/female ratio close to equality ensures
a 50% chance of random heterosexual encounters. Young
ct al. ( 1992) found that individuals of Stylocidaris lineata
also aggregated without regard to sex. The spawning events
were not synchronized as the male began liberating
sperm before any female spawning could be detected
which differs from spawning sequences mentioned for
Hymenaster membranaceus (Pain et al., 1982) and for
Archasler typicus (Run et al., 1988). Male spawning
seemed to be triggered when the falling temperature
reached about 2C, an inducer previously suggested by
O'Bnen (1976) for L. linoralis.

The sperm behavior of Leptasterias polaris possibly
further explains the need for aggregation. Upon its release,
a portion of sperm can be dispersed by currents and re-
main active as long as it is maintained in the water column
(Fig. 6), potentially limiting the genetic isolation in a pop-
ulation. From the spawnings successfully induced by
sperm in our experimental tanks, we infer that this active
fraction is the stimulus for females to spawn. Sperm as a
stimulus of spawning has also been discussed by Starr et
al. (1990) for the sea urchin Strongylocentrotus droeba-
cliiensis. In fact, sperm suspension in seawater is suggested
to be the spawning inducer in many species of ophiuroids
and echinoids (Thorson, 1950; Lewis, 1958). The lack of
strong epidemic spawnings during natural breeding activ-
ities in our tanks could be explained by fluctuations of
water temperature around the 2C threshold at that time
(Fig. 2). A few male spawnings could have been triggered
in late December, then delayed by the rising temperature
(to almost 4C) before being induced again in the second
week of January. Females followed this scattered pattern
because they probably need to be close to a sperm source
for spawning to be induced. The negative buoyancy and
stickiness of sperm causes most spermatozoa to settle on
the substrate where they gradually enter an inactive state.
The settling ensures a minimal dispersion of sperm but
makes fertilization dependant on the proximity of indi-
viduals, which is achieved by aggregation. Further, sperm
inactivation seems an effective energy-saving behavior,
extending the viability of settled sperm up to 6 or 7 days,
which is much longer than the 2- to 3-day longevity of
sperm maintained in the water column. The extreme en-
durance of L. polaris sperm is further emphasised upon
comparison with that of the other asteroids, holothuroids,
and echinoids tested, for which the spermatozoa did not
survive longer than a day at the temperature normally
recorded during their spawning periods. These short-lived
spermatozoa display a dispersion behavior that we could
probably associate with organisms having synchronously
spawning males and females. The longevity of sperm from
L. polaris is probably an advantage given the asynchro-
nous spawning of the sexes in this species.

We could not determine whether females were attracted
by deposited sperm, but the delay between male and fe-
male spawnings seems advantageous. Because the sper-
matozoa are present on the medium before eggs are emit-
ted, they do not have to overcome the protective barrier
maintained by the brooder. Thanks to its adhesiveness,
sperm also covered the vertical substrates favored by many
spawning females in our experiments. It is probable that
as soon as a female spawns on the sperm-covered sub-
strate, the oocytes can reactivate the inactive sperm (Fig.
7), and fertilization takes place. Experimentally, the best
success (>75%) was achieved when the delay between the
male and female spawnings was no more than 1 1 h; how-

REPRODUCTIVE BEHAVIOR OF LEPTASTERIAS POLARIS

43

ever, success was still good (>50%) after as long as 30 h
(Fig. 8). A contact of 20-50 min with the oocyte seemed
to be necessary for the spermatozoan to attain an optimum
speed that probably maximizes its ability to fertilize.
Sperm inactivity and reactivation appears to be very rare
in marine habitats. Although sperm chemotaxis has been
shown in echinoderms (Miller, 1985), no significant ve-
locity increase or activation of the attracted sperm has
ever been mentioned, except in cnidarians (Miller, 1979a,
b) and larvaceans (Miller and King, 1983). The closest
example with a similarity to Leptasterias polaris is the
sperm of the horseshoe crab (Limulus polyphemus), which
undergoes a brief flurry of motility and remains nonmotile
until it encounters a sperm motility initiating molecule
(SMI) emanating from eggs (Clapper and Epel, 198 1 ). In
contrast, both our observations and previous studies (Chia,
1968) show that the sperm of most echinoderms becomes
active upon release in the water and disperses quickly.
This is probably the best strategy when both male and
female gametes are released in great numbers in the sea-
water at close intervals.

In Leptasterias polaris, the gamete behavior seems well
adapted to the brooding mode, which in turn has a pro-
tective function. Brooded and nonbrooded embryos
showed almost perfect synchrony in development through
the gastrula stage (Table II). This suggests the absence of
the obligatory exchange of nutrients between parent and
young that is seen in Pteraster militaris (McClary and
Mladenov, 1990), where a brood chamber is present. In
L. hexactis, another brooding asteroid overlaying its eggs,
the maternal presence is proposed to be essential to help
the hatching embryo tear the fertilization membrane
(Chia, 1966). Although this was thought to be the case
for L. polaris (Himmelman el ai, 1982), we observed no
evidence of that phenomenon. The hatching was not de-
layed and no loss of embryos was evident in the unbrooded
group. Brooding in L. polaris probably serves mainly to
aerate the embryos and prevent them from being covered
with sediment as they lie on the substrate. Observations
in the field (Himmelman el ai, 1982) support this hy-
pothesis, as the substrate under a brooding starfish was
always found clear of debris. Brood protection also seems
to be in direct relation to adverse environmental condi-
tions and predatory pressures. Extremely active grazers
such as sea urchins, which are abundant wherever L. po-
laris is found, would rapidly decimate any unprotected
starfish embryos exposed on a rock (Fig. 5a).

The embryonic development observed in Leptasterias
polaris is similar to that described for L. hexactis (Chia,
1968) and L. acceptances similispinis (Kubo, 1951). The
developmental kinetics of L. polaris is characteristically
slower (Table I) than in all other reports for this genus,
perhaps because of the lower temperatures (0- 1 C) found
during the breeding and the subsequent development of

Table II

Leptasterias polaris: Development of brooded and nonbrooded embryos

Nonbrooded

Brooded Embryos

Embryos

Developmental Stages

Time

Size i

:Mm)

Time

Sizei

,pm)

Fertilization

870

43

852

36

2-cell

43 h

1111

+ 22

46 h

1079

15

4-cell

81 h

1092

18

86 h

1032

15

8-cell

93 h

1032

9

92 h

1046

12

16-cell

104 h

1044

24

106 h

1032

18

32-cell

125 h

1050

33

121 h

1062

9

64-cell

140 h

1082

39

133 h

1103

62

128-cell

152 h

1086

41

146 h

1080

3

256-cell

164 h

1092

52

156 h

1101

40

Blastula

8-9 d

1090

62

8-9 d

1120

51

Gastrula

20-21 d

1288

52

20-21 d

mi

33

Hatching

33-34 d

1121

42

32-35 d

1199

63

A new stage was considered attained when 50%-60% of the embryos
reached it. The standard deviations about the mean size are given.

this species. The major differences between our results
and those of Kubo (1951) and Chia (1968) are the oc-
currence of a spinning stage and the much earlier hatching
of L. polaris. Because L. polaris embryos hatch in late
gastrula, before they develop brachiolar arms, such arms
cannot contribute to the tearing of the fertilization mem-
brane, as they are said to do in the two other species.

The freshly spawned eggs are negatively buoyant and
do not adhere to one another until a few moments later,
after fertilization. This stickiness was also observed by
Chia ( 1968) for Leptasterias hexactis, but was correlated
with a reaction to seawater rather than with fertilization.
Through its growth, the embryo undergoes many changes
in attachment capacity, which is first provided by the
sticky fertilization membrane, then by small unciliated
body areas after hatching, and later by the fixing disk and
ramified tips of the brachiolar arms. The parental protec-
tion is probably useful in preventing dispersion of embryos
during these changes in fixation ability, for instance during
hatching, when attachment to the substrate may be inef-
fective for a short time. After the metamorphosis of the
embryos (4-5 months), brooding individuals are still ob-
served in the field for at least one month. The free-moving
young starfish seem to remain under protection through
the development of the pyloric caeca and the opening of
the mouth. Environmental factors apparently play a role
in the development of the embryos, especially in initiating
metamorphosis by a considerable increase in temperature
(Fig. 2). As previously mentioned by Boivin el at. (1986),
this correlation seems to ensure that the young starfish
are ready for release at the proper time, namely spring,
when conditions are most favorable for their survival as

44

J.-F. HAMEL AND A. MERCIER

self-sufficient individuals. This timing control, together
with the possibly lower energetic cost required from the
parent under cold temperatures, is probably an advantage
of winter brooding.

Acknowledgments

We thank Dr. C. Bouland for her help during the ex-
periments, A. Caron for helpful criticism and assistance
with statistical analysis, and J.-L. Theberges for photo-
processing. We greatly appreciated the helpful comments
of Drs. S. Demers, J. H. Himmelman, C. Bouland, M. I.
El-Sabh, and two anonymous reviewers on the manu-
script. We are also grateful to J. Noel for her collaboration
on the sketches and to N. Piche for photographs of Lep-
tasterias and field observations. This work was carried
out thanks to the space and material lent to us by Drs. E.
Pelletier and F. Dube at the Station Aquicole de Pointe-
au-Pere and was supported by personal funds of J.-F. Ha-
mel and A. Mercier.

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Reference: Biol. Bull 188: 46-56. (February/March, 1995)

The Effects of Hydrodynamic Shear Stress on

Fertilization and Early Development of the Purple

Sea Urchin Strongylocentrotus purpumtus

KRISTINA S. MEAD AND MARK W. DENNY

Department of Biological Sciences, Stanford University, Hopkins Marine Station,
Pacific Grove, California 93950

Abstract. Life in the highly turbulent surf zone poses a
severe challenge to reproduction in free-spawning animals.
Not only can breaking waves quickly dilute the gametes
shed by spawning organisms, but turbulence-induced
shear stresses may limit fertilization and interfere with
normal development. A Couette cell was used to re-create
some of the effects of turbulent water motion to study
effects of environmentally relevant shear stresses on fer-
tilization in the purple sea urchin (Strongylocentrotus
purpuratiis). Although low shear stresses improved fertil-
ization success (presumably by increasing mixing), ex-
posure to high shear stresses (of the magnitude found in
the surf zone) substantially decreased fertilization success,
probably by interfering with contact between egg and
sperm. Furthermore, eggs fertilized at high shear stresses
often showed abnormal development and low survival of
eggs through the blastula stage.

Introduction

Like most intertidal invertebrates, Strongylocentrotus
purpuratiis. the purple sea urchin, reproduces by means
of external fertilization. S. purpuratiis is commonly found
on exposed intertida! rocky shores and in nearshore
benthic environments. In both of these habitats, its ga-
metes are subject to the energetic turbulent mixing char-
acteristic of these environments. Although a small amount
of turbulence can provide beneficial mixing, bringing eggs
and sperm into contact, the intense turbulence of the surf
zone or the benthic boundary layer may interfere with
fertilization through a variety of mechanisms. These in-
clude gamete dilution, gamete damage, decreased en-
Received 28 July 1994; accepted 28 November 1994.

counter rate between the egg and the sperm, and removal
of the sperm from the egg's surface before fertilization is
complete.

To date, the best studied of these mechanisms concerns
the effects of gamete dilution. The percentage of eggs fer-
tilized during spawning depends on the local concentra-
tion of viable sperm (Vogel et al., 1982, Levitan el a/.,
1 99 1 ). When subjected to the type of turbulent flows found
commonly in nearshore waters, sperm concentrations can
decrease by several orders of magnitude within a meter
of a spawning male (Denny, 1988; Denny and Shibata,
1 989; Grosberg, 1 99 1 ), resulting in low rates of fertilization
(Pennington, 1985; Levitan et al., 1992). The dilution of
sperm increases with an increase in distance downstream
from a spawning male and with an increase in current
velocity, resulting in a decrease in fertilization (Penning-
ton, 1985; Grosberg, 1987. 1991; Yund, 1990; Petersen,
1991; Petersen et al.. 1992; Levitan et at.. 1992; Oliver
and Babcock, 1992). The detrimental effects of sperm di-
lution can be partially offset through an increase in the
overall number, size, spatial density, synchronicity, and
fecundity of spawning animals (Denny and Shibata, 1989;
Levitan, 1991; Levitan et al.. 1992; Babcock and Mundy,
1992; Sewell and Levitan. 1992; Babcock et al., 1994).
Nonetheless, field studies have shown that the fraction of
eggs fertilized in the water column is typically low, often
less than 15% (Pennington, 1985; Levitan, 1991; Levitan
el al., 1992).

Under certain circumstances, however, gamete dilution
may be greatly reduced. Denny and coworkers (1992)
showed that the presence of surge channels can drastically
reduce the rate of dilution in trre surf zone of rocky shores,
to the extent that gamete concentrations could locally be
high enough to result in efficient fertilization. They tem-

46

TURBULENCE AND FERTILIZATION

47

pered this prediction, however, by noting that gamete di-
lution is only one of several factors affecting external fer-
tilization and suggested that the effects of small-scale hy-
drodynamics could impose additional limits to the efficacy
of this common form of reproduction.

Here we explore this possibility by recreating in the
laboratory one important aspect of turbulent flow (hy-
drodynamic shear stress) and measuring its effect on fer-
tilization and early development in the common purple
sea urchin, 5. purpwatus. In the next section, we describe
the eddy sizes, shear stresses, and energy dissipation rates
found in the surf zone. We then discuss how the average
values of these parameters make it possible to mimic some
aspects of turbulent flow in a rotating device called a
Couette cell. Finally, by examining fertilization under a
variety of conditions in the Couette cell, we show that
surf zone turbulence may have important consequences
for both the fertilization and the early development of sea
urchin embryos.

Theoretical Background

Surf-zone turbulence

When a wave breaks on a rocky shore, its orderly mo-
tion is disrupted as the waveform degenerates into a col-
lection of turbulent eddies (Denny, 1988). The interaction
of eddies causes the water to be sheared, and the friction
between layers of fluid (a result of viscosity) causes a shear
stress, T, to be exerted on the fluid and any gametes in it
(Okubo, 1978; Fischer, 1979). As an additional result of
friction, the wave's energy is lost to heat at a rate , called
the energy dissipation rate. Shear stress has the units
N/nr, the energy dissipation rate has the units W/m 3 ,
and for a given fluid, the two are related by viscosity:

* = T 2 /M, (1)

where n is the dynamic viscosity of seawater at 1 2C, here
taken to be 1.24 X 10~ 3 Pa -s (Denny et al., 1992). Shear
stress and energy dissipation rate are different descriptors
of the same phenomenon: the small-scale deformation of
fluid by turbulence.

Rates of energy dissipation, averaged over both time
and space, have been predicted for the surf zone (Thornton
and Guza, 1983; Svendsen, 1987; Denny et al., 1992;
George et al., 1994); increases with both wave height
and beach slope, with expected values ranging from 10-
3000 W/m 3 on wave-swept rocky shores (Fig. 1 ). The only
direct data for energy dissipation rates in the surf zone
come from hotfilm anemometer measurements of the
turbulence generated by small waves breaking on the
gently sloping beach at Scripps Pier (George et al., 1994).
For 1-m-high waves, a time-averaged value of t of about
10 W/m 3 was measured, with instantaneous values rang-
ing up to 100 W/m 3 .

3000

0.50 1.00 1.50 2.00

Wave Height (m)

Figure 1. Energy dissipation rates in the surf zone. Predicted values
given typical beach slopes and wave heights (Denny el al.. 1992).

The bent hie boundary layer

While the intense turbulence characteristic of the surf
zone is apparent to an observer, habitats occupied by 5.
purpuralits in the benthic boundary layer near shore also
experience high energy dissipation rates. In this case en-
ergy is dissipated as water is sheared against the solid sub-
stratum. The shear stress exerted on the substratum by
turbulent flow is pu\, where p is the density of the water
(about 1025 kg/m 3 for seawater) and /<*, an index of tur-
bulence intensity, is the friction velocity (Schlichting,
1979). From Equation 1, we see that this shear stress re-
sults in an energy dissipation rate of p 2 */M- The friction
velocity for flow over the rough substrata typical of rocky
shores is typically 5% of the mainstream velocity (Mid-
dleton and Southard, 1984), which in turn depends on
the height and period of the waves and the depth of the
water column. Using linear wave theory to estimate
mainstream water velocities near the substratum (Denny,
1988), it is thus possible to estimate energy dissipation
rates in near-shore benthic boundary layers for a typical
wave period of 10 s (Fig. 2). For the depths and wave
heights shown, peak wave-induced velocities vary from
0.02 to 1.35m/s, friction velocities from 0.001 to
0.067 m/s, and peak energy dissipation rates from 0.001
to 1 7,600 W/m 3 (resulting in average energy dissipation
rates of 0.0005 to 8,800 W/m 3 ).

Although energy dissipation rates predicted for the
benthic boundary layer can be quite high, their effect is
very localized. The thickness of the benthic boundary layer
under waves is approximately 0.41 u* 7/2 TT, where T is
the wave period (Grant and Madsen. 1986). Thus, for T

48

K. S. MEAD AND M W. DENNY

2000

10 m

0.50

1.00

1.50

2.00

Wave Height (m)

Figure 2. Energy dissipation rates in the benthic boundary layer.
Predicted values given typical wave heights and water depths of habitats
favored by Strongylocentrotus purpuratits. The wave period is assumed
to be 10 s.

= 10 s and the values of u* assumed here, the boundary
layer is expected to be only 0.7 mm to 4.2 cm thick. This
narrow localization of high energy dissipation rates in the
benthic boundary layer is in contrast to the situation in
the surf zone, where the entire volume encompassed by
the breaking wave is subject to high energy dissipation
rates.

In either case, once gametes or embryos leave the surf
zone or the benthic boundary layer, they are no longer
subject to high energy dissipation rates. Most other marine
habitats have turbulent energy dissipation rates that are
several orders of magnitude smaller than those found in
the surf zone and in the benthic boundary layer. For ex-
ample, energy dissipation rates in oceanic waters typically
vary from 10^ 7 to 10"' W/m 3 (Belyaev rta/., 1975; Dillon
etal.. 1981;Kitaigorodskii etal.. 1983;Osborn andLueck,
1985; Baker and Gibson, 1987), and energy dissipation
rates in tidally mixed areas vary from 10~ 2 to 10"' W/m 3
(Rothschild and Osborn, 1988).

Eddy size

To visualize how eggs and sperm respond to turbulent
flow and understand how it is possible to re-create some
of the effects of oceanic turbulence in the laboratory, it is
useful to consider the size of the smallest vortices, or ed-
dies, in a flow. This length, called the Kolmogorov length,
is

(2)

(Tennekes and Lumley, 1972) where i> is the fluid's kine-
matic viscosity (v = n/p, approximately 1.2 X 10~ 6 m 2 /s
for seawater at 12C). For example, using values for e
from 10 to 3000 W/m 3 , the Kolmogorov length in the
surf zone is predicted to be 22 to 5 /urn. Note that eddy
size decreases as the energy dissipation rate increases.

Few eddies have a diameter of less than 5-10 times the
Kolmogorov length, and the maximum shear energy den-
sity occurs in eddies about 40 times the size of the Kol-
mogorov length (Lazier and Mann, 1989; Osborn el a/.,
1 990). Therefore, the most energetically significant eddies
in the surf zone and benthic boundary layer are 200 to
880 ^m in diameter, and are larger still in less turbulent
flows. Most eddies are thus considerably bigger than a sea
urchin egg (80-1 10 nm) and sperm (head. 3 jum; flagel-
lum. 40-45 ,um). Within an eddy, the rotational nature
of the flow results in a velocity gradient (a shear) extending
from the center of the eddy to its periphery (Vogel, 1981).
It is reasonable to suppose, therefore, that the gametes
experience turbulent eddies as a temporally variable ve-
locity gradient with an associated shear stress and energy
dissipation rate (Denny ft ai. 1992).

Effects of shear

As yet, it has not been possible to directly observe eggs
and sperm in a velocity gradient of the sort found in the
surf zone. Nevertheless, general predictions about their
behavior can be made. Like all spherical objects in velocity
gradients, eggs are likely to tumble at many cycles per
second (Happel and Brenner. 1983; Kessler. 1986; Denny
et u/.. 1992), and the axis of rotation is likely to change
rapidly as different eddies shear. The rotation of the eggs
induces a secondary velocity gradient (a boundary layer)
around the egg. Although sperm are motile, their swim-
ming velocity (about 150-200 /um/s; Levitan etal., 1991)
is substantially lower than the small-scale velocities in-
duced by turbulence (approximately u*\ Denny, 1988).
Therefore, sperm are also expected to move according to
the local water motion. Due to the elongated shape and
flexibility of the sperm, however, their motion is expected
to differ from that of eggs, and it is possible that the effect
of a velocity gradient will be to align sperm with the di-
rection of flow. In the boundary layer surrounding the
egg, this alignment would cause sperm to move tangen-
tially to the egg's surface. It is therefore easy to imagine
how the induced motion of the egg and sperm might con-
spire to hinder contact between the gametes.

Shear stress in the laboratory: Taylor-Couette flow

A simple way to expose eggs and sperm to shear stress
(and thereby to mimic one aspect of turbulent flow) is to
place them in the well-defined velocity gradient of a
Couette cell (Coles, 1965; Donnelly, 1991). Couette cells

TURBULENCE AND FERTILIZATION

49

and similar instruments were recently used in several bio-
logical investigations. For example, Thomas and Gibson,
(1990a.b. 1992) used Couette cells to examine how tur-
bulent motion inhibits dinoflagellate growth, cell count,
and chlorophyll content, and Edwards el a/. (1989) used
a combination of Couette and cone and plate flows to
observe changes in cell length, septal length, hyphal di-
ameter, and branching frequency in two species of bac-
teria.

Materials and Methods

The Couette cell

The Couette cell consists of an inner stationary cylinder
and a rotating coaxial outer cylinder (Fig. 3). The inner
cylinder (50.5-mm outer radius) is constructed of stainless
steel with an acrylic plastic base. Cold water circulates
through the lumen of the inner cylinder to control the
temperature of the test solution, and an air line runs to a
small hole at the bottom of the inner cylinder, allowing
air to be bubbled into the test solution at a controlled
rate. The air bubbles keep the eggs from settling and get-
ting caught between the bases of the two cylinders. The
outer cylinder (54-mm inner radius) is made of clear
acrylic plastic and has a base that fits onto a motor shaft.
The distance between the two cylinders (3.5 mm) is more
than 30 times larger than the diameter of the 5. piirjniratiis
egg. The cylinders are 20 cm long, allowing an experi-
mental volume of more than 200 ml.

When the motor turns, the outer cylinder rotates,
shearing the liquid between the two cylinders. By treating
the cylinders as two wide plates a small distance apart,
the shear stress T in the test solution is calculated to be

(3)

where u> is the angular velocity (radians/s) of the outer
cylinder and r is its inner radius (here 54 mm), n is the
dynamic viscosity of the test solution, and /; is the radial
distance between cylinders (here 3.5 mm). Equivalently,
the flow inside the Couette cell can be described by the
energy dissipation rate t:

Air Inlet

Coolant Outlet

Coolant Inlet

ur\

~h

(4)

Note that Equations 3 and 4 hold whether flow in the cell
is laminar or turbulent (Schlichting, 1979). Both T and t
will be used to describe the flow inside the Couette cell.
By varying w (or ^) it is possible to recreate the shear
stresses (and energy dissipation rates) found in the surf
zone. In the experiments described here, filtered seawater
was used at 12C, and the dynamic viscosity n was taken
to be 1 .24 X 10~ 3 Pa s. The velocity of the outer cylinder

UL

22cm

=3

t Inner Cylinder

Outer Cylinder
( 108 mm m djameter)

! Working Space
(3 5 mm between cylinders,
100 ml working volume)

Mptor

.-L.l...^ Beanng

1

V ..III

1 , ^ ,

, b- =3 U

Figure 3. The Couette cell.

was measured by means of a magnetic pickup system.
Each time a tooth of a gear attached to the motor shaft
passed by the pickup, a current pulse was induced. The
pulses were amplified, counted, and converted into an-
gular velocities. The Couette cell was run at shear stresses
of 0.06 to 1 .45 Pa, corresponding to energy dissipation
rates of 2.8 to 1591 W/m 3 . These rates cover a large por-
tion of the range of turbulent energy dissipation rates ex-
pected on exposed rocky shores.

The Reynolds number describing the flow inside the
Couette cell is Re = pu>r/;//u- At the maximum angular
velocity used in this study (75.4 rad/s). Re = 11.600.
Dye studies indicated that flow became turbulent at
Re =a 4400.

Experimental design

S. piirpuratus males and females were induced to spawn
by injection with 0.5 A/KC1. Sperm were collected and
stored undiluted on ice. Eggs (jelly intact) were collected
in a beaker filled with filtered seawater, washed three times,
and diluted to a 0.5%- 1% suspension (by volume), which
was stirred gently and kept at 12C. Sperm concentrations
were determined by hemacytometer counts. All experi-
ments were performed within 8 h of gamete collection.
To determine the relative concentrations of eggs and
sperm to be used in each experiment, a standard fertil-
ization curve was created for each pair of urchins (Fig.
4). Small volumes of egg suspension were exposed to so-

50

K. S, MEAD AND M W DENNY

T3

S

U)
O)
O)
LU

O

c

Q)
0.

100

80

60

40

20

10 5

10 s

Sperm/mL

10'

Figure 4. Representative standard fertilization curve. Concentrations
and volumes of egg and sperm solutions giving 80%-90% fertilization
in a test tube were used in all experiments.

lutions of sperm for 2 min in gently stirred test tubes.
Fertilization was stopped by the addition of an equal vol-
ume of 0.5 Af KCI, which renders the sperm immobile
without harming the eggs (Schuel, 1 984). Concentrations
and relative volumes of eggs and sperm giving rise to 80%-
90% fertilization in a test tube were used in the Couette
cell experiments, to ensure that the decrease in fertilization
success expected as a result of shear stress would not be
concealed by an overabundance of sperm.

The rates of energy dissipation used in the experiments
ranged from 2.8 to 1 591 W/m\ In each experiment, 45 ml
of the egg suspension was put in the Couette cell. The
outer cylinder was brought up to speed over about 10 s.
after which 5 ml of newly diluted sperm was added to the
egg suspension. Fertilization was allowed to take place at
the specified energy dissipation rate for 2 min before the
reaction was stopped with 50 ml 0.5 M KCI. Eggs were
subsequently washed with filtered seawater and examined
under a microscope 4 h after fertilization. Because shear
stress can induce artificial activation and concomitant
formation of both the fertilization envelope and the hya-
line membrane (normally indicators of fertilization) in
the absence of sperm, only eggs that had divided were
counted as fertilized, possibly resulting in a slight under-
estimate of fertilization. Two hundred eggs were counted
per sample.

Each experiment was repeated between 3 and 16 times
at the same energy dissipation rates, using gametes from
different pairs of urchins. Because the relative concentra-

tion and the "fertilizability" of the gametes varied slightly
between pairs, all data were normalized to the percent
fertilization obtained when the experimental concentra-
tions of eggs and sperm were combined in a test tube in
the absence of appreciable shear.

Some urchins, such as those living on exposed rocky
shores, experience turbulence almost constantly. Other
animals, for instance those in tide pools, may experience
significant turbulence only as the waves break, or just
when the largest waves break on the shore. To approxi-
mate the time-dependent nature of this kind of environ-
mental turbulence more accurately, eggs were fertilized
under intermittent shear stress. As above, 45 ml of the
egg suspension was put in the Couette cell. Once the outer
cylinder had been brought up to speed, 5 ml of newly
diluted sperm was added to the egg suspension. Fertiliza-
tion was allowed to occur for 2 min, during which time
the Couette cell was alternately spun for 10 s, then allowed
to stop for 10s throughout the 2-min trial. Experiments
were repeated and data were normalized as above.

To determine if exposure to shear stress decreases fer-
tilization success by damaging gametes (as opposed to
some other mechanism, such as interfering with egg-sperm
binding), S pitrpnnitits eggs were sheared prior to fertil-
ization and then combined with unsheared, newly diluted
sperm in a test tube. Similarly, S. purpitralus sperm were
sheared prior to fertilization and immediately combined
with unsheared eggs in a test tube. Fertilization was
stopped after 2 min by the addition of an equal volume
of 0.5 M KCI. For comparison, eggs and sperm from the
same animals were fertilized under shear in the Couette
cell. All eggs were washed with filtered seawater and in-
cubated at 12C for 4 h before being examined. Experi-
ments were repeated three times and data were normalized
as above.

Eggs were reexamined after 24 h to determine whether
exposure to shear stress (either before or during fertiliza-
tion) had any effects extending past fertilization. Samples
were counted, and the percentage of fertilized eggs that
had developed into normal blastulae was recorded. Nor-
mal blastulae were characterized as clear, hollow balls of
cells spinning rapidly about their animal-vegetal pole axes.
Two hundred embryos were counted per sample.

Results

Fertilisation under constant shear stress

Water motion associated with low energy dissipation
rates (<70 W/m 3 ) enhanced fertilization success, pre-
sumably as a result of mixing. As the energy dissipation
rate increased from 2.8 to 69.2 W/m 3 , the mean fertiliza-
tion success increased from 78%- to 96%. Fertilization
success decreased when fertilization occurred during ex-
posure to moderate and high energy dissipation rates. As

TURBULENCE AND FERTILIZATION

51

100

T3

H
N

V)

O)
O)
UJ

C.

O

O

D.

50 75 100

500 1000 1500

Energy Dissipation Rate (W/m 3 )

Figure 5. The effect of shear stress on fertilization. Low energy dis-
sipation rates enhance fertilization success, whereas moderate and high
energy dissipation rates decrease fertilization success. Data from 16 pairs
of sea urchins. Error bars indicate the standard error.

the energy dissipation rate increased from 69.2 to
1 59 1 W/m\ the percentage of eggs that were fertilized
decreased from 96% to 19% (Fig. 5).

Intermittent shear stress

When eggs were fertilized under conditions of inter-
mittent shear stress, fertilization success decreased with
increasing energy dissipation rate, although not as dra-
matically as when the shear stress was constant. As the
maximum energy dissipation rate experienced during
the 10-s pulses of turbulence increased from 44.1 to
1 59 1 W/m\ the mean fertilization success decreased from
90% to 39% (Fig. 6). In comparison, when eggs and sperm
from the same pair of urchins were fertilized under con-
stant shear, mean fertilization success decreased from 92%
to 19%. At moderate energy dissipation rates (up to
397.7 W/m 3 ). there was no significant difference between
the effects of constant and intermittent shear stress. At
high energy dissipation rates (707 W/m 3 and above), eggs
fertilized under intermittent shear stress had greater fer-
tilization success than eggs fertilized under constant shear.

In this set of experiments, few measurements were made
at low energy dissipation rates. Therefore, the pattern seen
in constant shear stress of an increase in fertilization suc-
cess at low energy dissipation rates was not observed.

Shearing gametes before fertilization

When S. purpuratus eggs and sperm were sheared sep-
arately before fertilization and then combined with un-

"D

N

O)
O>
111

C

O

Q_

100

80

60

40

20

- Intermittent
A Constant

400 800

1200

1600

Energy Dissipation Rate (W/m 3 )

Figure 6. The effect of intermittent shear stress on fertilization. Data
are from three pairs of sea urchins. Error bars indicate standard error.

sheared gametes in a test tube in the absence of appre-
ciable shear stress, fertilization success was very high.
As the energy dissipation rate increased from 44. 1 to
1 59 1 W/m\ the fertilizability of the sheared eggs decreased
from 98% to 86%., and the fertilizability of the sheared
sperm decreased from 100%. to 92% (Fig. 7). In compar-

-o

in

O)

C

O

Q-

100

80

60

40

20

- - Sperm Sheared
-- Eggs Sheared
A Fertilized in Shear

400

800

1200

1600

Energy Dissipation Rate (W/m 3 )

Figure 7. The fertilizability of presheared gametes. Sperm and eggs
lose little fertilizability when sheared before fertilization. Data are from
three pairs of sea urchins. Error bars indicate standard error.

52

K. S. MEAD AND M. W. DENNY

ison, the fertilization success of eggs from the same female
fertilized under shear decreased from 94% to 8%.

Effect oj shear stress on early development

Although almost 100% of the 5". purpuratits eggs fertil-
ized in the absence of shear developed into normal blas-
tulae, many of the eggs fertilized while exposed to shear
stress showed the effects of shear-stress-induced damage;
typically their development was arrested at about the 64-
cell stage. As the energy dissipation rate during fertilization
increased from 44. 1 to 1591 W/m 3 , the percentage of fer-
tilized eggs that developed into normal blastulae decreased
from 92% to 22% (Fig. 8). Given that many eggs were not
fertilized, the overall fraction of eggs that developed nor-
mally was lower still. For example, as the energy dissi-
pation rate during fertilization increased from 44. 1 to
1591 W/m 1 , the percentage of eggs that developed into
normal blastulae decreased from 88% to 2%.

In comparison, almost all fertilized eggs developed
normally when the gametes were sheared separately and
then combined in a test tube (Fig. 9A). As the energy
dissipation rate increased from 44.1 to 1591 W/m 3 , the
percentage of sheared eggs that (once fertilized) developed
into normal blastulae decreased only from 99% to 89%>,
and the percentage of eggs fertilized by sheared sperm
that developed into normal blastulae decreased only from
96% to 94%;. These data can be graphed to reflect the total

100

CD

o.
o

<D
Q

15

100

80

60

40

20

- % Of Fertilized Eggs
A- % Of All Eggs

400

800

1200

1600

Energy Dissipation Rate (W/m 3 )

Figure 8. The effect of shear stress on development. Many eggs fer-
tilized in shear do not develop normally. Combining fertilization and
developmental data shows that the percentage of all eggs developing
normally at 24 h is very low. Data are from 12 pairs of urchins. Error
bars indicate standard error.

o

X
CO

<

O)

c

3

W

E

LLJ

O

X

o
<

W

<n

o>

D)
111

80 -

60

40

20

- - Sperm Sheared

-- Eggs Sheared

* Fertilization In Shear

400

800

1200
Energy Dissipation Rate (W/m 3 )

1600

100

80

60

40

20

- - Sperm Sheared
-- Eggs Sheared
A Fertilized In Shear

400

800

1200

1600

Energy Dissipation Rate (W/m 3 )

Figure 9. The effect of shearing gametes on early development. Al-
most all eggs fertilized in the absence of shear develop into normal blas-
tulae, but eggs fertilized in shear do not. (A) Development of fertilized
eggs. (B) Survival of all eggs to blastula, including eggs that were not
successfully fertilized. Data are from three pairs of urchins. Error bars
indicate standard error.

number of eggs that developed into normal blastulae, in-
cluding the effect of reduced fertilization (Fig. 9B). As
the energy dissipation rate increased from 44.1 to
1591 W/m 3 , the percentage of sheared eggs that developed
into normal blastulae decreased from 97% to 77%, and
the percentage of eggs fertilized by sheared sperm that
developed into normal blastulae decreased only from 96%
to 87%.

TURBULENCE AND FERTILIZATION

53

Discussion

How can low levels of turbulence increase fertilization
success?

Although intense turbulence limits fertilization success,
low energy dissipation rates enhance fertilization success.
This is presumably because turbulent mixing increases
contact rates between the egg and sperm. It is reasonable
to suppose that at low turbulent intensities the tumbling
of the egg in response to shifting velocity gradients may
not be rapid or abrupt enough to inhibit binding of the
sperm to the egg. Similarly, investigators have shown that
low energy dissipation rates increase rates of contact be-
tween predator and prey in the plankton (Rothschild and
Osborn, 1988; Marrase et al., 1990; Costello et at., 1990;
Saiz et al., 1992). At high energy dissipation rates, the
beneficial aspects of turbulence are outweighed by other
factors.

Why does excessive turbulence decrease fertilization
success?

Despite observations that turbulence decreases fertil-
ization success by diluting gamete concentration, the data
presented above indicate that environmentally relevant
turbulence can dramatically decrease fertilization success
even when eggs and sperm are in high concentrations.
There are several possible mechanisms for this shear-in-
duced decrease in fertilization success, including gamete
damage, a decrease in the encounter rate between egg and
sperm, and a removal of sperm from the egg's surface
during the latent period, the period after the sperm has
made initial contact with the egg, but before fertilization
is complete. The fact that shearing gametes before fertil-
ization does not dramatically reduce their fertilizability
indicates that exposure to shear does not substantially
damage gametes (Fig. 7). This suggests that high levels of
turbulence instead may limit fertilization by reducing the
effective encounter rate between gametes. If, as expected,
turbulence causes the eggs to tumble rapidly and the sperm
to align themselves in the direction of flow tangential to
the egg surface, small-scale turbulence could hinder con-
tact between the gametes. Alternatively (or additionally),
exposure to turbulence might limit the ability of the sperm
to attach to an egg once contact has been made, or it
might cause attached sperm to be removed before fertil-
ization is complete.

Our experiments provide no direct evidence that allows
us to choose among these possibilities, but indirect evi-
dence allows for reasonable speculation. We begin by ask-
ing whether it is likely that hydrodynamic shear stresses
are sufficient to tear an attached sperm from an egg. Al-
though no data are yet available for sea urchins, bond
strengths between a sperm and the zona pellucida of an

egg range in mammalian systems from 6 to 250 dyn, with
40 dyn being the most likely average value (Baltz el ui,
1988), and for the sake of argument we assume a similar
value for urchin sperm. This force can be compared to
the hydrodynamic force exerted on an urchin sperm as
follows: The shear force pulling at a sperm attached to an
egg is approximately equal to the hydrodynamic shear
stress to which the sperm is exposed multiplied by the
sperm's surface area. If a sea urchin sperm is modeled as
a 41-^m-long cylinder with a radius of 0.1 j/m (the fla-
gellum) attached to a conical head with a height of 3 /urn
and a radius of 0.5 ^m (Gray, 1955; Brokaw, 1965), its
surface area is 3 1 .4 /urn 2 . At a shear stress of 1 .45 Pa (the
maximum shear stress to which the gametes were exposed
in the Couette cell, equivalent to an energy dissipation
rate of 1591 W/m 3 ) the resulting shear force is 4.6 dyn,
much less than the expected bond strength. A still smaller
force would be exerted at lower shear stresses. It thus seems
unlikely that sperm, once well attached, are sheared off
before fertilization is complete. This suggests that the pri-
mary effect of hydrodynamic shear on fertilization is to
reduce the encounter rate between gametes, to disrupt
contact between sperm and egg before final sperm at-
tachment, or both.

This proposition is supported by our experiment re-
garding the "dose" of turbulence to which gametes are
subjected. A comparison of the effects of constant and
intermittent shear stress reveals a time-dependent re-
sponse. At high shear stresses, a smaller percentage of eggs
is fertilized if shear is constant than if shear is applied in
10-s periods alternating with 10-s periods of quiescence
(Fig. 6). In both cases gametes are exposed to the same
peak shear stress. If shear stress acted primarily by tearing
well-attached sperm from the egg before they could fer-
tilize (a process that takes about 20-30 s [Epel, 1989, 1990;
Ruiz-Bravo and Lennarz, 1989]), one would expect that
the effects of intermittent shear would be similar to those
of constant shear. If, on the other hand, the primary effect
of shear stress is to prevent sperm from encountering or
becoming strongly attached to eggs (processes that can
occur effectively in periods less than 10s [Epel, 1989,
1990; Ruiz-Bravo and Lennarz, 1989]), periods of qui-
escence would allow some sperm to encounter eggs and
attach well enough to be immune from later disruption
by shear stress. If this is indeed the case, fertilization rates
will be higher under intermittent shear, and this is the
effect that we observed. The time-dependence of the effect
of shear stress is consistent with the hypothesis that shear
stress limits fertilization by interfering with contact be-
tween the egg and sperm.

Further experiments will be necessary to test this spec-
ulation and to differentiate between the effects of turbu-
lence on encounter rates and the effects on subsequent
adhesion.

54

R. S. MEAD AND M. W. DENNY

Why does turbulence affect development'.'

Only a fraction of the eggs that are fertilized under tur-
bulent conditions develop into normal blastulae (Fig. 8),
indicating that shear stress, whether constant or inter-
mittent, affects reproductive success beyond fertilization.
Somehow, exposure to shear stress curtails or irrevocably
alters some process essential to normal development. Al-
though the mechanism or mechanisms behind this effect
are uncertain, reasonable speculation is again possible.
Hiramoto (1974) noted a brief fivefold stiffening of the
egg membrane 90 s after fertilization in the urchin Tem-
nopleurus toreumaticus. If S. purpiiratus eggs experience
a similar increase in stiffness, and if the hardened mem-
branes result in increased damage, eggs exposed to shear
stress during fertilization could sustain injuries not seen
when eggs were exposed to shear stress prior to fertiliza-
tion. These injuries could result in leakage of some essen-
tial cytoplasmic compound or in infection, either of which
could prevent the fertilized egg from completing more
than a few rounds of cell division.

Not only might different types or degrees of injury arise,
depending on whether the egg is exposed to turbulence
before or during fertilization, but the ability of the egg to
heal itself after injury might vary. Mechanically injured
cells heal themselves by releasing exocytotic vesicles,
which fuse to the plasma membrane (Steinhardt el a/.,
1994). Shortly after fertilization, these vesicles are depleted
(Vacquier, 198 1 ), presumably decreasing the cell's ability
to heal itself. This could help explain why a large fraction
of the eggs fertilized under turbulent conditions developed
abnormally.

Two further possibilities can be discounted. First, in-
juries resulting in parthenogenic stimulation or poly-
spermy cannot explain the effect of shear stress on de-
velopment, because the fertilized eggs usually divide to
about the 64-cell stage before degrading. In contrast, ar-
tificially activated sea urchin eggs tend not to divide, and
polyspermic eggs have very characteristic disruptions in
their cleavage patterns (e.g.. irregular or incomplete di-
vision, multiple asters, lobe formation), none of which
were observed here. Second, if exposure to shear stress
altered egg membranes in some way, eggs sheared prior
to fertilization would show the same defects in their de-
velopment, and this was not seen.

Ecological implications of the effect of turbulence
on fertilization

These experiments indicate that although low levels of
shear stress can aid mixing and fertilization, the intense
shear stress found in the surf zone and in the benthic
boundary layer can have severe mechanical effects on fer-
tilization and early development. These effects interact
with those of gamete dilution to narrow any window of

opportunity open to urchins attempting to reproduce by
external fertilization. For instance, although surge chan-
nels may restrict gamete dilution (Denny el al. 1992),
thereby promoting efficient fertilization, the deleterious
effects of shear stress may still limit the fraction of eggs
fertilized. Furthermore, any eggs that are fertilized are
very likely to develop abnormally. Gametes shed outside
of surge channels may be quickly swept out of the surf
zone, thereby avoiding prolonged exposure to high shear
stresses, but dilution will be rapid; consequently, the frac-
tion of eggs fertilized will again be low. Similarly, while
gametes released above the benthic boundary layer (by
large or aggregated individuals) experience lower energy
dissipation rates, they too are quickly diluted, and fertil-
ization rates will still be low.

The picture is not quite as bleak as it appears. Turbu-
lence is patchy, and even areas with high temporally av-
eraged rates of energy dissipation may experience (short)
periods of relative calm. Because of their great fecundity,
urchins able to time it right may end up with abundant
offspring.

There are plenty of sea urchins along our rocky shores,
but the chanciness of external fertilization in a turbulent
environment suggests that only a small number of them
contribute to the next generation. If this is true, then the
effective population is much smaller than the actual pop-
ulation a situation that could have populational, con-
servational, and evolutionary consequences (Quinn el al.
1993; Hedgecock, 1994).

Acknowledgments

We thank D. Epel for his constant moral and technical
support and for his excellent advice. Both he and Friday
Harbor Laboratories (University of Washington) provided
generous access to laboratory facilities. H. Crenshaw made
helpful suggestions, and B. Rees, P. Sund, J. Geller, and
B. Podolsky made insightful comments on earlier versions
ol this manuscript. Two anonymous reviewers had
thoughtful observations and helped clarify parts of the
paper. This study was funded by grants to K. Mead from
the Myers Oceanographic and Marine Biological Trust,
and by NSF grants to M. Denny (OCE-91 15688) and D.
Epel (IBN-9205393).

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Elemental Distributions in Marine Bivalve Shells as
Measured by Synchrotron X-Ray Fluorescence

KURT THORN 1 *. ROBERT M. CERRATO 2 , AND MARK L. RIVERS'

1 Department of Geophysical Sciences. University of Chicago, Chicago, Illinois 60637, and 2 Marine
Sciences Research Center, State University of New York, Stony Brook, New York 11794-5000

Abstract. The concentrations of elements from Mn to
Pb in the shells of Mercenaria mercenaria. Mya arenaria,
and Argopecten irradiam were measured using synchro-
tron x-ray fluorescence. This technique provides sensitiv-
ity as low as 1 ppm and resolution of 8 ^m. Elements
were heterogeneously distributed, both on a large scale
(several millimeters) and on a small scale (tens of mi-
crometers). Large-scale variations were observed in the
compositions of shell layers and in seasonal variations in
strontium concentration. Small-scale changes in com-
position included elevated iron levels at the boundary be-
tween the prismatic and inner homogeneous shell of the
hard clam, Mercenaria mercenaria. Variations in stron-
tium concentrations were seen over time spans of several
months, suggesting that this technique can be used to de-
termine historical water temperatures. Elemental maps
with a resolution of less than 10 ^m were produced.

Introduction

The use of marine bivalves as environmental indicators
has received considerable attention. Bivalves are believed
to incorporate trace elements into their shells in propor-
tion to the concentration of those elements in the water
(Rhoads and Lutz, 1980). This incorporation is also in-
fluenced by other circumstances, including water tem-
perature and salinity (Rosenberg. 1980). The incorpora-
tion of trace elements into marine bivalves could be used
to monitor temporal changes in aspects of the marine
environment, including the elemental composition and

Received 29 October 1993; accepted 22 November 1994.

* Current Address: Whitespruce Dr., Wading River, NY 1 1792.

Abbreviations: SXRF, synchrotron x-ray fluorescence; PIXE, proton
induced x-ray emission; EPMA, electron probe microanalysis; MDL;
minimum detectable level.

temperature of the water. Moreover, recent pollution
could be studied, as well as paleoceanographic conditions.
Strontium/calcium ratios have already been used as a
method in paleothermometry that overcomes the limi-
tations of the more common oxygen isotope paleother-
mometry (Beck el al, 1992). Additionally, many bivalves
produce visible growth increments in their shells, often
as frequently as once per day. These growth increments
provide a method of dating any observed changes in el-
emental composition of the shell.

When techniques like instrumental neutron activation
analysis (INAA) and atomic absorption analysis are ap-
plied to whole shells of marine bivalves, heavy metal con-
centrations of less than 5 ppm by weight are reported (Al-
Dabbas el al, 1984; Koide et al, 1982; Milliman, 1974).
Carriker el al (1982), using proton induced x-ray emission
analysis (PIXE), reported concentrations of Cu and Zn
in oysters (Crassostrea virginica) of 20 to 30 ppm weight.
When such concentrations are measured by bulk analysis,
fairly large amounts of shell are required for analysis, so
resolution over short time scales is impossible.

All previous attempts to measure the distribution of
elements within bivalve shells have been made with either
PIXE microprobes or electron probe microanalysis
(EPMA). Because the minimum elemental concentration
measurable with EPMA is high, only those elements oc-
curring in fairly high concentrations in the shell (Ca, Sr,
Mg, S) can be observed (Wada and Suga. 1976; Rosenberg
and Hughes, 1991). Three PIXE microprobe studies
(Carriker et a!., 1982; Carell et al., 1987; Swann et al..
1991) report variations in heavy metal concentrations,
but over fairly long (several month) time scales, although
the excellent spatial resolution of the proton microprobe
(3 ^m) used by Carell et al. (1987) would have enabled

57

58

K. THORN ET AL

them to study periods corresponding to 6-8 hours in
young mussels.

In his analysis of bivalve shell chemistry, Rosenberg
( 1980) concludes that whole-shell analysis represents the
elemental concentration incorrectly because elements are
not necessarily distributed uniformly throughout the shell.
Carriker el al (1982) list several factors that influence the
distribution of elements in shells, including structural and
chemical changes that result from environmental fluc-
tuations, fouling, adsorption, and weathering of the shell
surface, elements adsorbed at shell surfaces and integrated
into the shell matrix, and the heterogeneous distribution
of elements within the shell.

The metal concentrations typically found in bivalve
shells are considerably less than those measurable by
EPMA and are near the 20 ppm detection limit of PIXE
microprobes. Synchrotron x-ray fluorescence has mini-
mum detection limits near 1 ppm, which should permit
detection of these elements and resolution of variations
in their concentration over short time scales.

This study attempts to evaluate the potential of syn-
chrotron x-ray fluorescence as a technique for micro-
analysis of trace element distributions in bivalve shells of
three species. Some samples were also analyzed with a
PIXE microprobe in a pilot attempt to measure elemental
distributions on scales down to 1 //m.

Materials and Methods

Preparation of shell sections

Three species were examined in this study: hard clams
(Mercenaria mercenaria), soft-shell clams (Mya arenaria),
and bay scallops (Argopecten irradians). Hard clams were
obtained from a population in Moriches Bay, soft-shell
clams from a population in Stony Brook Harbor, and
scallops from a population in Peconic Bay. All collection
locations were on Long Island, New York. Two types of
sections were used in this study: thin sections (less than
250 nm thick) and thick sections (about 1 mm thick). For
studies of Mya arenaria, sections were made of the chon-
drophore, an internal structure projecting from the hinge
region. Sections of the chondrophore were used for anal-
ysis because the chondrophore contains better preserved
growth lines than the main shell (Cerrato et al.. 1991 ). In
addition, it is isolated from the environment, preventing
damage and elemental contamination.

Shells were cleaned to remove adherent debris by
scrubbing with a nylon brush in tap water. Thick sections
were prepared by cutting a section from the shells along
the axis of maximum growth. The cut surfaces were
ground with successively finer silicon carbide grits and
polished with aluminum oxide. The sections were then
attached to cardboard mounts for synchrotron x-ray flu-

orescence analysis. Thin sections were prepared in the
same manner as thick sections except that the shell sec-
tions, after grinding, were glued to pure SiO 2 glass slides
with 5-minute epoxy. X-ray fluorescence analyses of the
epoxy and slide indicated that they contained only trace
amounts of zirconium and titanium. The mounted sec-
tions were resectioned to several hundred micrometers
and ground to the desired thickness with successively finer
silicon carbide grits. The sections were then hand polished
with aluminum oxide. Previous x-ray fluorescence studies
have shown that this process neither introduces significant
amounts of elemental contamination nor smears the el-
emental distribution of the sample.

Shell microstmctiire

The shells of Mercenaria mercenaria are composed of
three distinct shell layers: an outer prismatic layer and a
middle and an inner homogeneous layer. The middle and
inner homogeneous layers are separated by the pallial
myostracum (Panella and MacClintock, 1968). All layers
of the shell are aragonitic (Taylor et al.. 1973). During
periods of stress, the organism ceases its growth and re-
tracts its mantle, producing a translucent band, known
as a growth break, in the shell. Daily growth lines are also
visible in the shells of Mercenaria mercenaria. These con-
sist of an aragonite-rich increment followed by a protein-
rich line(Kennish, 1980).

Both the shell and the chondrophore of Mya arenaria
contain daily growth patterns; however, the growth pat-
terns in the chondrophore are much better preserved than
those in the main shell. These growth increments are sim-
ilar in form to those in Mercenaria mercenaria: they are
composed of a thin line followed by a broad increment.
It is not known whether they also exhibit the same pattern
of protein-rich lines and calcium-carbonate-rich incre-
ments. The chondrophores also show strong seasonal pat-
terns: they are opaque in spring, translucent in summer,
and opaque in fall-winter. Winter and spring are separated
by a prominent translucent spawning band (Cerrato et
al. 1991).

The shell of Argopecten irradians consists of an inner
and an outer calcitic foliated layer separated by a middle
aragonitic crossed-lamellar layer (Kennedy et al.. 1969).
To our knowledge, these shells do not contain growth
lines.

Synchrotron x-ray fluorescence analyses

Synchrotron x-ray fluorescence analyses were per-
formed at beamline X26-A of the National Synchrotron
Light Source at Brookhaven National Laboratory. Sam-
ples were mounted in a sample holder in air. X rays pro-
duced by the synchrotron travel 9 m through an evacuated

ELEMENTAL DISTRIBUTIONS IN BIVALVES

59

pipe to the sample. The x rays produced by the synchro-
tron have a critical energy of 5 keV and travel through
several beryllium windows (total Be thickness = 1 mm)
and 35 mm of air before striking the sample. The air path
of the fluorescent x rays sets the effective lower detection
limit at argon (Z = 18). The x-ray beam is collimated by
an 8 nm tantalum pinhole immediately before striking
the sample. This produces a microbeam, allowing reso-
lution of fine structures. Immediately upstream of the
collimator, the intensity of the x-ray beam is measured
by an ion chamber.

The samples were mounted on an X, Y, Z, 6 stage.
They could thus be rastered under the x-ray beam, al-
lowing the measurement of elemental concentrations at
different points as well as along one- and two-dimen-
sional scans over the specimen. An optical microscope
imaging the specimen permitted precise determination
of the location being analyzed. The secondary x rays
emitted by the sample are collected in a Si(Li) detector.
The detector was filtered with 500 ^m of Kapton film,
to reduce the count rate from the Ca x rays. This prevents
saturation of the detector. The spectra are then stored
in a Micro Vax computer for further analysis. Minimum
detectable levels (MDLs) were 5 ppm for Mn, 2.5 ppm
for Fe and Pb, 2 ppm for Sr, and 1 .5 ppm for Ni through
Br, for a spectrum collected for 4 min. MDLs were cal-
culated by measuring the background under these peaks
in a typical x-ray spectrum and computing the elemental
concentration of a peak with an area equal to three stan-
dard deviations of the background. The calculated con-
centrations from the spectra were used to standardize
these measurements.

The sample is mounted with the surface oriented at 45
degrees to the incoming x-ray beam and the detector. This
exploits the angular distribution of the scattered radiation
to minimize background. However, it also results in the
beam traveling relative to the face of the sample as it
penetrates the sample. This prevents the detection of
changes in elemental concentration over small areas per-
pendicular to the beam. This effect is more pronounced
at higher x-ray energies. At Ca (3.69 keV) the horizontal
travel is only 1 1 ^m; at Sr (14.14 keV) it is 55 j/m. To
reduce this effect, one can make thinner samples; however,
it becomes difficult to optically resolve growth lines in
samples thinner than 100 ^m. Thus, the samples were
oriented so that the interfaces of interest were parallel to
the incoming beam whenever possible.

Spectra can be recorded in one of two ways: either the
entire spectrum collected by the detector is recorded, or
the net areas of the peaks of interest are recorded. Gen-
erally, when analyzing individual points, the entire spec-
trum was saved, as this allowed a more sophisticated
background and peak-fitting routine to be used. However,

when scanning a region, generally only the net areas of
the peaks were saved, because this greatly reduces data
analysis time. The Micro Vax implements automated
scanning routines that allow the collection of data over
long scans with no user intervention. The setup of the x-
ray microprobe is discussed more comprehensively by
Gordon et al. (1990).

Absolute concentrations were determined from the
peak areas fit to the spectra by use of a "standardless"
analysis program (NRLXRF). Using fundamental pa-
rameters, this program predicts the relative intensities
for the different x-ray lines in the spectrum. These pre-
dictions are based on the thickness of the sample, the
filtering conditions, and the assumed concentrations.
The concentration of Ca was assumed to be 40% by
weight, and was used as an internal standard to calculate
the composition by comparing the predicted intensities
and the actual count intensities. It was checked by pre-
dicting known standards that were measured at the be-
ginning of each measurement session. The error was
typically less than 5%.

PIXE microprobe analyses

Several samples were also analyzed by proton induced
x-ray emission (PIXE) at the University of Melbourne.
Analyses were performed with a 3 MeV proton beam,
magnetically focused to 1 and 4 jum. Samples were thick
shell sections, mounted in vacuum. The secondary x rays
were collected with a Si(Li) detector, which was generally
filtered with 50 nm Al to reduce the Ca count rate. How-
ever, some analyses were performed without filters in an
attempt to measure sulfur concentrations. Rutherford
backscattering spectroscopy (RBS) analyses were also
made to allow carbon and oxygen concentrations to be
measured.

PIXE analyses were made by sweeping the beam over
a predetermined region of interest in the shape of an un-
closed lissajous figure. Every time an x-ray event is de-
tected by the Si(Li) detector, the energy of the event and
the A and y coordinates of the beam are recorded. The x-
ray spectrum is generated by disregarding the .v and y
values and simply taking the energy spectrum. A map of
the concentration of a given element can then be generated
by placing a window over a given peak and plotting the
intensity of events in that energy range as a function of
A and y. Analyses were performed on scales from 30 by
30 ^m to 100 by 100 /urn and were generally collected for
30 min. The MDLs in calcium carbonate matrix were
about 1 8 ppm for Zn and Fe. This microprobe is described
in more detail in Legge et al. ( 1986).

Although the PIXE microprobe does not have elemen-
tal sensitivity as good as the synchrotron x-ray fluorescence

60

K. THORN ET AL

microprobe, it does have substantially better position res-
olution (a beam spot of 1 /urn rather than 8 /urn). The mean
x-ray production depth for proton excitation in calcium
carbonate is also less than that of x-ray excitation. Both
of these factors enabled higher resolution of trace element
distributions in the shell, but increased the MDLs for
many elements.

Results

Surface contamination

Both shell surfaces of all species studied were found to
be extremely contaminated with many trace elements.
Table I lists the concentrations of these elements on both
the exterior and interior surfaces of several organisms.
Transverse scans across sections of scallop and hard clam
shells showed that metal concentrations were much higher
on both the interior and exterior surfaces of the shell than
they were within the main shell. A representative transect
is shown in Figure 1. Most elements were more concen-
trated on the exterior surface than the interior surface.
However, the ratio of surface to main shell concentrations
varied widely from element to element. The Ni concen-
tration was only 1.5 times greater on the exterior surface,
but the Cu concentration was 4.5 times greater. This is
not unexpected, as the processes by which the elements
are deposited on these surfaces are very complex. Scans
in hard clams showed similar results. Trace element con-
tamination on the interior shell surface is present in only

a very thin layer. Within 100 ^m the trace element con-
tamination drops by a factor of 20-50, typically to within
a factor of 2 of its average concentration. Most elements
on the exterior surface of the hard clam behave in the
same way as those on the interior surface, except iron
(Fig. 2). Iron concentrations peaked both near the surface
(30 ^m) and farther in ( 180 ^m). This is probably due to
the periostracum and adherent debris on the shell surface;
but why only Fe is affected is not known. The other trace
elements are present in a surface layer that contains little
calcium. This effect was most pronounced on the external
surface; the maximum trace metal concentrations were
at 30 /urn from the edge of the shell and about 240 ^m
from where calcium concentrations became constant.

Shell layers

Most trace and minor elements (including Sr) are pref-
erentially incorporated into the prismatic shell of the hard
clam. The ratio of concentration in the prismatic shell to
concentration in the homogeneous shell varied consid-
erably from element to element, from about 20 for Fe to
1.5 for Sr. The reason for this increased concentration is
unknown. Similar differences between shell layers were
observed in scallops. Sr concentration in scallops was in-
versely related to both Mn and Fe concentrations; and Sr
concentrations were highest in the outer 500 ^m of shell,
while Mn concentrations were highest in the inner 400 ^m
of shell. Some feature of the biological precipitation pro-
cess is influencing the trace element concentration, as the

Table I

Elemental concentrations (ppm) in selected marine bivalves

Location

Mn

Fe

Ni

Cu

Zn

Br

Sr

Ph

Scallops (Argopecten imuiians)

Exterior surface

262

1080

152

902

308

17

814

21

Interior surface

208

636

102

201

106

21

1300

21

Outer shell layer

34 1 .4

8.5 3.5

2 +

2.5 2.1

3 1.4

1

1495 106

2

Inner shell layer

48.5 5

8.5 3.5

20

20

5 1.4

2 1.4

911.5 18

5 2.1

Hard-shell clams (Mercenana mercenana)

Prismatic shell

12.4 3

219 59

<1.5

3.6 2.2

<1.5

5.4 0.8

1247 448

<2.5

Prismatic (DEC)

8.3 3.9

38.8 35

2.8 1.5

1.5 0.6

1.5 0.6

30

1139 + 211

1

Prismatic (PIXE)

9.8 15

349 518

<18

<18

13 6.8

12.8 + 8.3

3832 961

Prism. /homog. boundary

3. 5 2.1

1085 302

<l.5

3.3 0.4

<1.5

5.5 1.1

1 1 1 5 345

<2.5

Middle homog. shell

7.2 + 2.2

11.6 + 7.3

<1.5

3 0.9

<l.5

5.4 0.5

984 353

<2.5

Middle homog. (DEC)

6.8 8.2

4 4.1

1 0.8

1.8 0.5

1.3 0.5

20

1007 336

2.3 2.5

Middle homog. (Thick)

1.25 0.5

<2.5

1

1

1.5 0.6

2.5 1.3

725 + 132

1

Inner homog. (DEC)

1.8 1.3

2. 8 2. 5

1

3 2.6

1.4 0.6

1.4 0.6

1673 + 180

1.7 + 0.6

Soft-shell clams (Atyn arenana)

Chondrophore

10 4.4

<2.5

<1.5

<1.5

2 0.6

<1.5

1383 133

<2.5

All concentrations are in parts per million. Errors are given as the standard deviation of multiple measurements within the same shell layer; they
do not reflect measurement error. Numbers without listed errors are based on single measurements. DEC: Shells came from collections in waters
closed by the Department of Environmental Conservation. Thick: Data were taken on shell thick sections.

ELEMENTAL DISTRIBUTIONS IN BIV-MVES

61

Figure 1 . Representative transects along which scans were taken in Mcrccnaria nwirciuiria (A) Transect
across daily growth increments in prismatic shell. (B) Transect across growth break. (C) Transect across
prismatic-homogeneous boundary. (D) Transect across daily growth increments in homogeneous shell. (E)
Transect across exterior shell surface.

chemistry of the scallop shell does not behave like geo-
logical carbonates. However, differences in chemical
composition between biologically precipitated carbonates
and geologically precipitated carbonates are not unknown:
Buchardt and Fritz ( 1978) described aragonitic shell pre-
cipitated by a freshwater gastropod in which the strontium
concentration of the shell is 5 times less than that of geo-
logically precipitated aragonite under the same conditions.
In addition. White el al. ( 1977) showed that Mn can re-
place Ca in aragonite formed by Mya arenaria, whereas
this is not possible in geologically precipitated aragonite.
At the boundary between the prismatic shell and the
middle homogeneous shell in the hard clam, very pro-
nounced changes in elemental composition were observed
(Figs. 3, 1 ). At this boundary, iron concentrations are typ-
ically 4.5 times greater than in the prismatic shell and
100 times greater than in the homogeneous shell. This
iron peak is very narrow, about 40 /jm wide, and was
present in every measurement made across this boundary.
The peak was always within 50 yum of where the boundary

was located optically. Strontium concentrations declined
between the prismatic and the homogeneous boundary,
but this occurred more gradually, over about 100 /urn, and
appears to be associated only with the difference in Sr
concentration in the two shell layers. Sr concentration
reaches its nominal concentration in homogeneous shell
at the same location as the peak in iron concentration.
Manganese concentrations are lower at this boundary than
in either the prismatic shell or the homogeneous shell,
perhaps because the much greater concentration of iron
is occupying the sites in the crystal lattice that are normally
occupied by manganese. No feature similar to this has
been observed in other organisms to our knowledge.

Sr concent nil ion maps

Both the x-ray microprobe and the proton microprobe
can be used to make high-resolution elemental maps, so
changes in elemental concentration can be explored over
very small areas. And with bivalve shells, fine variations

62

K. THORN ET AL.

<n

o
O

30 60 90 120 150 180 210 240 270 300

Depth (urn)

Figure 2. Scan across the outer surface of a Mercenaria mercenana
thick section. Data were recorded as individual spectra at each point.
The spectra were then fitted to a sum of gaussian peaks and a background.
The counts reported are the net areas of the peaks, less the background.
Data was recorded for 40 s per point (detector live time).

with ontogeny can also be studied. We have used this
scanning ability to elucidate changes within the shell
structure, as with the prismatic-homogeneous boundary
described above; but we have also measured the variations
of trace element concentrations with time. The measure-
ments of strontium were most successful. Because of its
high concentration in the shell, strontium can be measured
very accurately with counting times of only 5 s at each
point in the shell. One-dimensional scans can therefore
be made very quickly: a millimeter can be scanned at 10-
/xm resolution in less than 10 min. We could easily map
a 2-year area of shell deposition at a resolution of a few
days (Fig. 4) and produce very high-resolution two-di-
mensional elemental maps. A map of the hinge region of
M. mercenaria is shown in Figure 5. Note the very good
correlation between the strontium concentration and the
banding in the thin section. Such scans are also possible
for other elements, though reduced concentrations require
proportionally longer counting times. Scans to measure
Fe concentration were typically recorded at 30 s per point.
For elements at levels of a few parts per million, counting
times of several minutes are necessary.

High-resolution maps of Sr concentration made with
the PIXE microprobe have shown variations in Sr con-
centration over scales as small as 1 nm. These variations
appear to be correlated with the presence of growth breaks.
However, because only a few PIXE measurements were
made, this is not conclusive.

Growth lines

A comprehensive attempt was made to find trace ele-
ment correlations with the daily microgrowth lines in the
prismatic shell of M. mercenaria. The results strongly
suggest that, although there is variation in trace element
concentration in the prismatic shell of M. mercenaria, it
is not necessarily correlated with the presence of micro-
growth lines. All scans showed that iron concentration in
the prismatic shell was nonuniform, but in most cases,
no correlation with the microgrowth lines could be made.
However, one scan (nsls!06.10) showed very high cor-
relation between the microgrowth lines and fluctuations
in the iron content. In that scan, every microgrowth line
was accompanied by a drop in iron concentration. Other
scans showed some correlation between the presence of
microgrowth lines and iron concentration, but none was
as pronounced as this one. In an effort to improve the
detectability of any iron fluctuations, we oriented samples
so that the microgrowth lines were parallel to the incident
beam. Thus, as the beam penetrated, it would stay within
the microgrowth lines rather than transversing them. In
addition, images were captured digitally after the beam
position had been determined, enabling precise compar-
ison between the image and the elemental concentration.
Neither of these improvements showed further correla-
tions between iron concentration and the presence of mi-
crogrowth lines. PIXE microprobe analyses were not sen-
sitive enough to detect variations in the iron concentration
of less than 40 ppm. and no structures were seen in the
PIXE measurements of iron concentration. A correlation
between microgrowth lines and iron concentration in the
shell seems unlikely, but this is an area deserving further
studv.

Discussion

Synchrotron x-ray fluorescence (SXRF) microprobes
provide lower minimum detectable levels than PIXE and
EPMA microprobes. The experimentally determined
MDL for Fe in thick clamshell sections was 3 ppm for
SXRF and 1 8 ppm for PIXE. Our experience is that PIXE
microprobes can make much larger and higher resolution
maps than SXRF microprobes, although at lower sensi-
tivity. PIXE measurements are generally made for long
times (30 min) over large areas ( 1000 j/nr ), whereas SXRF
measurements are made for short times ( 1 min) over small
areas (64 /jm 2 ). Electron microprobes have MDLs of sev-
eral hundred parts per million, worse than either PIXE
or SXRF. But electron microprobes are typically more
sensitive to low-Z elements than SXRF and PIXE micro-
probes (Janssens ct ai. 1992). The minimum spot size of
the NSLS microprobe is 8 ^m; for EPMA and PIXE mi-

ELEMENTAL DISTRIBUTIONS IN BIVALVES

63

.15000

30000

25000

w

D

o
o

20000

15000 -

10000

5000

100 200 300 400

Position (urn)

500

600

Figure 3. Scan across the prismatic/homogeneous boundary in a Mercenaria mcrcenaria thin section.
Data were recorded as the area under user-defined regions of interest, with the background subtracted. Data
was recorded for 1 5 s per point (detector live time). Initial Fe concentration is 2 1 1 ppm, initial Sr concentration
is 1550 ppm. The Fe peak concentration is 1420 ppm.

croprobes it is 1 ^m. New PIXE microprobes claim spot
sizes of 300 nm.

For low-Z elements (Z < 18) EPMA has the greatest
sensitivity as well as small spot size. Because of the ready
availability of electron microprobes, EPMA is also suited
to measurements of higher-Z elements present in quan-
tities of several hundred parts per million or more. For
measurements of elements present in concentrations of a
few parts per million, SXRF is the method of choice. This
is the only technique that combines MDLs of a few parts
per million with spot sizes of a few micrometers. However,
for some measurements, the spot size of the NSLS mi-
croprobe (8 ^m) is too large. In these cases, PIXE micro-
probes can be used, if the elements are present in large
enough concentrations. The greater accessibility of PIXE
microprobes also makes them attractive for studies of ele-
ments present in concentrations of several tens of parts
per million.

Bulk analysis techniques cannot provide the same spa-
tial resolution as microprobes. The neutron activation
studies of Carell ct ai (1987) were performed on shell
samples of 10-50 mg. This corresponds to a shell section

0.5 mm thick by 6 mm long by 3 mm deep. If this section
is cut with the thin direction normal to the ventral margin,
it represents approximately 1 month of growth. Atomic
absorption analysis can detect much smaller amounts of
trace elements, hence the limiting factor is the size of the
shell section that can be obtained. This probably limits
bulk analysis techniques to time resolutions of a few
weeks. In addition, many clamshell sections must be pre-
pared to make a study at this resolution over a significant
period. In contrast, microprobe techniques require only
one section per shell. Consequently, bulk analysis tech-
niques are suitable for situations where resolution of small
time scales is not necessary and integration of elemental
concentrations over all dimensions of the shell is not a
problem. In practice, microprobe techniques are probably
more suitable for time-resolved studies of trace elements
in bivalve shells. Bulk analysis methods can, however,
detect elemental concentrations a factor of a thousand
smaller than microprobes can.

Wavelength-dispersive detectors should improve the
usefulness of SXRF microprobes for analyzing trace ele-
ments in bivalve shells. Because wavelength-dispersive

64

K. THORN ET AL.

Distance (mm)
0.0 0.5 1.0 1.5 2.6 2.5
15

10

50 100 150

Days Before Collection

15

E 10

W

< 5

15

10

I 5
w

Distance (mm)
2 3

100

200
Days Before Collection

300

400
Distance (mm)

200

400
Days Before Collection

600

800

Figure 4. Variation in Sr concentration across growth increments in the chondrophore ofAIya arenaria.
Each of the three scans was taken on a different shell; all shells were harvested at the same time. Distance
is measured in millimeters from the ventral margin of the chondrophore. Time before harvesting was calculated
hy dividing the distance from the margin by the average growth rate per day, which was determined by
measuring the distance from the margin to the spawning break which occurred approximately 90 days before
harvest. Each plot consists of three separate scans separated by up to 1 50 /jm parallel to the growth increments.
Notice the strong correlation of variations in each of the separate scans. Note also the correlations between
the shells. We believe that these variations represent changes in the water temperature over time.

spectrometers detect only a single x-ray wavelength, they
eliminate the filtering necessary to prevent saturation of
an energy-dispersive detector by Ca x rays. This filtering
also attenuates other low-energy x rays, preventing the
detection of low-Z elements and resulting in high
(200 ppm) MDLs for Ti. Use of a wavelength-dispersive
detector would allow the detection of much smaller quan-
tities of low-Z elements, and possibly improve MDLs for
higher-Z elements as well. Such a device is now in use at
the National Synchrotron Light Source at Brookhaven
National Laboratory (Rivers et at., 1992). Future SXRF
microprobes will become even more useful as they are

developed at third-generation synchrotrons, such as the
Advanced Photon Source (APS) at Argonne National
Laboratory. A planned SXRF microprobe at the APS will
have 10 4 times more photons in a given energy range.
This will allow the use of extremely small spot sizes
(0.1 nm) at current detection limits, or lower detection
limits at small spot sizes ( 1 nm).

SXRF microprobes are not limited merely to the ap-
plications discussed here. It is also possible to use SXRF
to study other calcined samples, such as corals or fish
otoliths. Studies offish otoliths with EPMA (Gunn et a/.,
1992) and PIXE (Gauldie et at.. 1992) suggest that the

ELEMENTAL DISTRIBUTIONS IN BIVALVES

65

Figure 5. Two-dimensional scan (map) of Sr and Ca concentration in the hinge region of Merccnaria
incrci'iiaria. Each pixel measures 20 ^m on a side, and was collected for 20 s. The data were recorded as
background-subtracted regions of interest and have been normalized to beam intensity and detector live
time. The box on the photomicrograph approximately corresponds to the region scanned. Note the correlation
of the variations in strontium concentration with the seasonal banding in the chondrophore.

SXRF microprobe would he a valuable tool for such stud-
ies. Spatial resolution and elemental sensitivity similar to
that obtained here should be achievable in studies of other
calcined samples. Furthermore, it is not necessary to sac-
rifice the molluscs and section their shells to use the SXRF
microprobe. Because the beam deposits relatively little
energy in the shell and stops within the first few hundred
micrometers of the shell, live molluscs can be studied,
though this raises the problems of removing surface con-
tamination and dating the sampled positions accurately.
SXRF can also be used to detect organometallic com-
pounds, provided the metals are of sufficiently high Z.
Techniques such as extended x-ray absorption fine struc-
ture (EXAFS) also promise the ability to determine the
oxidation state of metals detected bv SXRF.

The measurements made on bivalve shells have re-
vealed several interesting features. The contamination of
the exterior surface is most likely due to the adsorption
of elements onto the calcium carbonate from seawater.
However, the contamination could also be due to adherent
debris on the surface of the shell. The contamination of
the interior surface is probably not due to elements ad-
sorbed onto the calcium carbonate during active growth,
because they would then have to be removed before ad-
ditional shell was deposited. Although there is some evi-
dence that the shell undergoes structural changes after
deposition (Gordon and Carriker. 1978). it seems more
likely that the elemental contamination arose after the
shells were collected, either from adsorption of metals
from the pallial fluid or from contamination after collection.

66

K. THORN ET AL

Seasonal variations in strontium concentration were
observed in the shells of both hard- and soft-shell clams
(Fig. 4). Differences in strontium concentration of 30%
to 40% were typical between winter and summer. Each
of the high and low Sr regions was about 180 days long.
We believe these variations are a result of the seasonal
temperature change of the seawater. In addition, shorter-
term variations (100 nm long) were observed; these prob-
ably correspond to shorter-term temperature variations.
As with paleothermometry done with Sr/Ca ratios in cor-
als (Beck el al., 1992), our results show that increases in
Sr concentration correspond to decreases in temperature.
Assuming that the change in Sr concentration is linearly
related to the change in water temperature, the change in
the observed Sr count rate is 18 counts/s/C. In a 10-s
per point scan, changes of 6 counts/s are observable. This
corresponds to a temperature change of 0.33C. The use
of x-ray fluorescence to measure small (0.3C) tempera-
ture variations over short (3-day) time scales seems quite
simple. In practice, the resolution is probably limited by
the rate at which shell strontium concentrations equili-
brate to the ambient water temperature. Our scans show
good correlation between different transects on an indi-
vidual shell and between different shells harvested from
the same location at the same time. This strongly suggests
that we are observing environmental effects upon the shell.
Figure 5 also shows very good correlation between Sr con-
centration and seasonal banding in the shell.

One problem complicating the use of x-ray fluorescence
as a means of paleothermometry using Sr/Ca ratios is that
for samples thinner than 200 /urn. sample thickness influ-
ences the Sr count rate. This is especially a problem near
the edges of shells, which were often inadvertently ground
thinner during preparation than other parts of the shell.
Such "thickness effects" show up as a gradual increase in
the Sr (and in extreme cases Ca) count rate as the sample
is scanned. In practice, the best way to avoid such effects
is to use samples thicker than the mean fluorescence depth
for Sr K x rays, because preparing samples uniform in
thickness to within a few percent is difficult to do. In that
case, the Sr count rate is unaffected by small variations
in sample thickness.

The synchrotron x-ray fluorescence microprobe has
significant advantages for trace element analyses in cal-
cined structures. It combines very low detection limits
with very small spot sizes. This allows the measurement
of elemental concentrations as low as 1 ppm over scales
as small as 10 ^m. Synchrotron x-ray fluorescence mi-
croprobes have significantly better detection limits than
EPMA and PIXE microprobes, and are probably the best
instrument for most studies of trace element distributions
in calcified structures. When structures smaller than
10 nm are to be resolved, the extremely small spot size

of PIXE microprobes makes them the analytical technique
of choice.

Acknowledgments

We are grateful to David Jamieson for performing the
PIXE and RBS analyses at the University of Melbourne.

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Kennedy, \V. J., J. D. Taylor, and A. Hall. 1969. Environmental and
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kennish, M. J. 1980. Shell nucrogrowth analysis: Mercenaria mercen-
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ELEMENTAL DISTRIBUTIONS IN BIVALVES

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Milliman, J. D. 1974. Marine Carbonates, Part I of Recent Sedimentary
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Panella, G., and C. MacClintock. 1968. Biological and environmental
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Rhoads, D. C., and R. A. Lutz. 1980. Skeletal records of environmental
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Rosenberg, G. D. 1980. An ontogenetic approach to the environmental
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Reference: Biol. Bull 188: 68-77. (February/March, 1995)

Life History Patterns of Discorsopagurus schmitti,
a Hermit Crab Inhabiting Polychaete Tubes

FRANCESCA GHERARDI 1 AND PAUL M. CASSIDY 2

[ Dipartimento ill Biologia Animate e Genetica "Leo Pardi, " Universita di Firenie, Via Romana 17,

50125 Firenic, Italy: and 2 Shannon Point Marine Center, Western Washington University.

1900 Shannon Point Rd.. Anacortcs, Washington 98221

Abstract. Discorsopagurus .schmitti is a hermit crab that
inhabits empty polychaete tubes in the North Pacific. Here
we describe some aspects of its lite history (relative growth,
population structure, reproductive biology, and incidence
of parasitism) and discuss the relationships among them.
Unlike most hermits, the two sexes of this species have
similar size distributions. In both sexes, larger body size
is accompanied by a higher reproductive output (larger
clutch size in females and more intrasex competitive po-
tential in males). The energy the females expend in egg
production might be equaled in this species by the energy
the males expend in supporting parasites. In fact, the ex-
tent of infestation by two rhizocephalans [Peltogaster
boschmae and Thilacoplethus ( = Thompsonia) reinhardi]
is more pronounced in males, especially those in the larger
size classes. However, rhizocephalans have little effect on
their hosts; growth and secondary sexual characters are
not influenced. The only morphological modification is
the more frequent loss of the second pleopod. Infected
hermits also showed a mock parental behavior, fanning
the externae with the pleopods as ovigerous females fan
their eggs. Larvae are released in sequential bursts, and
hatching occurs exclusively at night, possibly to minimize
predation by diurnal fishes. Hatching is also synchronized
with neap tides, which might keep the larvae from being
flushed out into open waters. In a species whose habitat
(sabellarian bioherms) is rare and quite unpredictable, it
is beneficial to retain larvae near the parental population.

Introduction

Discorsopagurus schmitti (Stevens, 1925) is an ano-
muran crab that occurs widely along the North Pacific

Received 10 December 1993; accepted IX November 1994.

coasts from Japan to Puget Sound (McLaughlin, 1974).
In both its geographical distribution and its ecological role,
this species is strictly dependent on the polychaete Sa-
hellaria ceinentariitin Moore, 1906. The hermit uses at-
tached worm tubes as housing and occupies a niche within
the community associated with sabellarian bioherms
(Gherardi and Cassidy, 1994a). A bioherm is a rock
formed by accretions from sedentary organisms and sur-
rounded by other kinds of rocks. Within the habitat, D
schmitti has a contagious distribution, the crabs occurring
with a density averaging 6 specimens per dm 2 (Gherardi
and Cassidy. 1994b).

Despite its peculiar habits and widespread distribution,
the main life history traits of the species are still unknown.
Previous papers were concerned only with its adaptations
to the sessile worm tubes (Caine, 1980) and its ecology
(Gherardi and Cassidy, 1994b). Our study investigates the
relative growth, population structure, reproductive biol-
ogy, and incidence of parasitism by rhizocephalans in D.
schmitti.

Materials and Methods

D. schmitti was collected from a wide sabellarian bio-
herm in Burrows Channel. Fidalgo Island (northern Puget
Sound, Washington). A total of 440 specimens were col-
lected: 329 from June to August 1992, and 1 1 1 from Jan-
uary to April 1993. See Gherardi and Cassidy ( 1994a. b)
for details on habitat and sampling procedure.

Sixty-four animals were individually weighed to the
nearest 0.01 g. Chelipeds were excluded from the weight
because they are variable and sometimes absent. For each
specimen, we recorded sex, size (shield length, SL, to the
nearest 0.1 mm), missing chelipeds (i.e.. the number of
"injured" specimens), and the number and maximum

68

LIFE HISTORY OF A TUBE-DWELLING HERMIT CRAB

69

diameter of any egg present. When possible, the maximum
axis of the occupied polychaete tube was measured with
a caliper. The number and position of externae of the
parasitic rhizocephalans Peltogaster boschmae Reinhard
and Thilacopk'thus ( = Thompsonia) reinhardi Lutzen were
also noted. Because we did not assess the presence of root-
lets penetrating major organs (stage of interna), which
precedes the parasite's sexual development, we may have
underestimated the extent of infestation within the
sample.

To describe the format of relative growth (i.e.. the
change in shape with growth; Hartnoll, 1982), we mea-
sured the length of the dactyl (DL) and palm (PA) of both
chelae, and their depth (DE) in 1 30 hermits. To represent
the patterns of the relative growth of these measures (y)
with respect to the SL as an independent variable (.\). the
natural logarithmic transformation (In y = In a + h In x)
of the exponential function y = a x h was used. This re-
lationship fits nearly all the instances of allometric growth
in crustaceans (Hartnoll, 1982). The values of h define
the type of allometry (b = 1: isometry; b < 1: negative
allometry; b > 1: positive allometry). This and the other
parameters of In y on In A, calculated using the Least-
Squares Method, allowed us to use standard tests for sig-
nificance and to compare slopes and intercepts between
groups.

Within winter samples, pleopods were examined and
their configuration related to the occurrence of parasites.
The configuration of pleopods in D. schmitti was first de-
scribed by McLaughlin (1974); the species shows pleopods
2-5, with the exception of some males, in which the sec-
ond pleopod is absent.

The behavior of animals occupying pieces of transpar-
ent glass tubing was recorded with a Panasonic color
camera and played back on a Mitsubishi recorder.

1.5

-i r

25 3 3.5

SHIELD LENGTH (mm)

Figure 1. Relationship between size (shield length) and body (without
chelipeds) weight, compared between sexes. A positive correlation was
found in both males (r = 0.692. df = 41, /> < 0.01 ), and females
(r = 0.809, df = 40, /><0.01).

1.2

2 24 28 32

SIZE CLASSES (SL, mm)
n = 104

i = 100

Figure 2. Size class distributions compared between sexes from
summer collections.

Data on egg incubation and hatching were obtained
from 19 ovigerous females, collected on January 18 (5),
February 8 (5), and April 27 (9) 1993. In the laboratory,
the females were placed in individual glass bowls 20 cm
in diameter, in filtered seawater with a salinity of 28-
3 1 %o. The bowls were kept in a constant temperature unit
at 10C under a light:dark regime of 14:10. Until they
released larvae, ovigerous females were checked twice a
day for hatching, placed in bowls of clean seawater, and
fed Anemia. The number of larvae released daily was re-
corded for ten of the females.

For statistical analysis, we followed the methods and
recommendations of Siegel (1956) and Zar (1984). The
level of significance under which the null hypothesis was
rejected is a = 0.05.

Results

Population struct we

Figure 1 shows the relationship between SL and body
weight (excluding chelipeds) compared between sexes. No
between-sex difference was found in either the slope (32.12
vs. 43.96, / = 1.549. df = 81. ns) or the intercept (-49.90
vs. -78.98. t = 1.781, df = 82, ns) of the regression line.

The sizes of crabs (SL) during summer were analyzed
(Fig. 2). No significant difference in size distributions was
found between the two sexes (G = 4.442, df = 7, ns). The
smallest specimens measured 1.1 (prepubertal, showing
no gonopores), 1.4 (male), and 1.9 mm SL (female), and
the maximum size attained 3.9 mm SL in the two sexes.

The sex ratio was 50.98% (104 males to 100 females),
which did not differ from 1:1 (X 2 = 0.044, df = 1, ns).
Similarly, the sexes remained balanced when three size
classes were distinguished (<2. 6 mm SL: X : = 0.5, df= 1,
ns; 2.6-3.4 mm SL: X : = 0. df = 1, ns; >3.4 mm SL:
X 2 = 0, df = 1, ns).

70

F. GHERARDI AND P. M. CASSIDY
Table I

Isometric or allomeiric growth with sue of Discorsopagurus schmitti (shield length) of three measures of both the major (right) and the minor (left)
chela, compared between sexes

In shield length vs.:

Si

99

b* 1

In DC (major chela)

0.721

0.79

2.173

0.13

0.337

0.40

4.367*

0.65

In PA (major chela)

0.878

0.66

7.450*

0.12

0.624

0.47

7.435*

0.36

In DE (major chela)

0.900

0.84

3.010*

-0.04

0.740

0.57

6.727*

0.33

In DC (minor chela)

0.831

0.70

4.928*

-0.07

0.484

0.41

6.590*

0.31

In PA (minor chela)

0.811

0.60

7.238*

-0.04

0.682

0.51

7.371*

0.07

In DE (minor chela)

0.634

0.48

6.831*

0.19

0.752

0.56

7.445*

0.11

DC = dactyl length; PA = palm length; DE = chela depth
a = intercept of the regression line.
* /><0.01.

All the correlation coefficients (r) are significant (P < 0.01). Isometry is satisfied when the regression coefficient (b) equals 1 after Student's (-test
((), otherwise an allometric growth (here only negative) occurs. The numbers of males and females are 63 and 69. respectively.

The size and sex of specimens in three individually
collected clumps were separately analyzed; the compari-
son did not show any difference in either size distribution
(G = 6.08, df = 4, ns) or sex ratio (X 2 = 1.458, df = 2, ns).

Chelipeds

The growth of both chelae relative to hermit size (SL)
was always negatively allometric for DC, PA, and DE,
with the exception of the DC of the major chela in the
males, where it was isometric (Table I). Table II gives the
between-sex differences in the parameters of the regression
lines; the males had a more voluminous major chela than
similarly sized females, as well as a longer dactyl in the
minor chela. However, the male and female regression

lines crossed at a large crab size (major chela: 3.8, 3.4,
4. 1 mm SL for DC, PA, and DE, respectively; minor chela
DC: 3.7 mm SL).

In all the examined specimens, the right chela was the
major one. This had constantly higher values than the
left one with SL increment (equal slope, but a higher in-
tercept), with the exception of the major chela DE, where
the growth increased with size (Table III).

Injured specimens represented 24.11% of the sample,
without any significant difference between sexes (25% in
males, 24.26% in females: X 2 = 1.762, df = 1, ns). Right

Table III
Between-side comparison of chelar growth in Discorsopagurus schmitti

Table II
Between-sex comparison of chelar growth in Discorsopagurus schmitti

r vs. I

rvs.l

6

a

( t vs. 9

I i vs. 9

DC (major chela)

2.298*

PA (major chela)

2.265* >

DE (major chela)

3.190"

DC (minor chela)

2.734"

PA (minor chela)

1.003

0.301

DE (minor chela)

0.713

1.428

test not applicable.

DC = dactyl length; PA = palm length; DE = chela depth.

* /><0.05, " /><0.01.

Comparisons after Student's (-test (t) between sexes in both the slope
(b) and the intercept (a) of the regression lines (after a In-ln transfor-
mation) describing the relationships between hermit size (shield length)
and three chelar measures. The degrees of freedom are 127 and 128.

DC

0.755

16.342"

>

PA

0.847

20.505**

>

DE

3.935**

99:

b

a

t r vs. 1

l

r vs. I

DC

0.037

17.964"

>

PA

0.418

21.289"

>

DE

1.636

24.428**

>

test not applicable.

DC = dactyl length; PA = palm length; DE = chela depth.

" P<0.0\.

Comparison after Student's (-test (() between the right (r) and the left
(/) chela in both the slope (b) and the intercept (a) of the regression lines
(alter a In-ln transformation) describing the relationships between hermit
size (shield length) and three chelar measures. The degrees of freedom
are 121, 122 in males, and 134, 135 in females.

LIFE HISTORY OF A TUBE-DWELLING HERMIT CRAB

71

N

co

n = 30

i 1 1 1

OS 1 12

^SHIELD LENGTH (mm)

B

1000-1 n = 30

1
1 5

25

35

SHIELD LENGTH (mm)

Figure 3. Relationships between the size of ovigerous females (shield
length) and both the number (after a In-ln transformation. A) and the
diameter (maximum axis, B) of the spawned eggs.

chelipeds were missing more often than left ones (65.96%
vs. 34.04%, X 2 = 4.17, df = 1, P<0.05).

Eggs

Egg-bearing females occurred in winter samples only.
They were first found in January and were still present at
the end of April, though their percentage was low (20%,
9 out of 45). Their numbers did not differ from those of
nonovigerous females (January 18: 19 vs. 10, X 2 = 2.207,
df = 1 , ns; February 1:15 vs. 2 1 , X 2 = 0.694, df = 1 . ns),
and specimens in the two reproductive states shared the
same size distribution (G = 2.906, df = 5, ns) and fre-
quency per size class (<2. 6 mm SL 45.10%, vs. a uniform
distribution: G = 0.486, df = 1, ns; >2.6 mm SL 70%,
G = 1.567, df = 1, ns). The smallest and largest females
found bearing eggs measured, respectively, 1.1 and
3.2mm SL.

Egg number per clutch ranged from 1 4 to 496, averaging
287. Female size (SL) was positively correlated with the
number of eggs (after a In-ln transformation: r = 0.478,
df = 28, P < 0.01, b = 2.56, a = 2.48) (Fig. 3A). The

value of the correlation coefficient did not significantly
differ from 3 (/ = 0.495. df = 28, ns): that is, clutch size
is proportional to the cube of the SL (roughly equaling
the body mass).

The mean egg diameter was 722 ^m (SE = 19, n = 30),
ranging from 455 to 990 ^m. A positive correlation was
also found between the SL of the female and the average
diameter of her eggs (r = 0.586, df == 28, P <0.01.
b = 0. 14, a = 0.40) (Fig. 3B), showing that bigger females
produce larger (and more numerous) eggs.

Eggs are attached to the second through the fourth
pleopods, about 100 per pleopod, in bunches of 7 to 15.
They are slightly ovate and attached by a funiculus, mea-
suring around 1.2 mm. Ovigerous females kept inside
transparent tubing were seen fanning the eggs with a re-
versing current created by the second and third pleopods.

Hatching

Hatching occurred between 1 and 75 days (n = 1 7) after
collection. Because all the analyzed females bore eggs
when collected, this is only a minimum estimate of the
actual length of egg incubation.

The number of larvae per individual ranged from 80
to 541 (average = 226, SE = 46) in the 10 females ana-
lyzed, and did not differ significantly from the number of
eggs per batch (/ = 1.31. df = 38. ns). being on average
98.74%- of the eggs spawned. Larvae were released in 3-
6 days (average = 5.1 day. SE = 0.3). with a maximum
of 209 larvae in the fourth day. No correlation was found
between the length of the hatching period and the female
size (r = 0.074, df = 8, ns), but the former was positively
related to the overall number of larvae (Spearman rank
correlation test: i\ = 0.742, / = 3.134, df = 8, P < 0.02).
Larvae were not released at a constant rate; the percentage
released (Fig. 4) differed significantly throughout the
hatching period (Kruskal-Wallis one-way analysis of vari-

40 -i

TIME (days)

Figure 4. Percentage (average SE) of the larvae released by K)
females plotted against the length of hatching (in days).

72

F. GHERARDI AND P. M. CASSIDY

ance: H = 29.975, df = 5. P < 0.00 1 ). peaking in the third
day and then falling off abruptly after the fifth day.

Hatching occurred exclusively at night, and mostly
during the neap phase of the tide (Mann-Whitney test: U
= 0, H = 7 and 7. P < 0.01 ). when the mean tidal current
is consistently slower (Fig. 5). For our comparison, we
defined neap (or spring) phase as the day of the minimum
(or maximum) tidal excursion for a lunar tidal cycle plus
the 3 days preceding and following that date.

Parasite distribution

Peltogaster boschmae was the most common rhizoce-
phalan parasite in our samples, affecting 18.5% of the
specimens; Tliilacopletlnis ( = Thoipsonici) reinhardi in-
fected 6.8%; and the two rhizocephalans co-occurred in
2.5%. These figures are similar to the percentages reported
by Lutzen (1992) for a previous study in the same area.

A sexual difference in the degree of infestation was seen
in both the number of externae per individual (males:
average = 2.9, SE = 0.6; females: average = 2.3, SE = 0.9;
X 2 = 9.312, df = 2, P < 0.01) (Fig. 6) and the prevalence
of parasitism (i.e.. the percentage of the parasitized her-
mits; Margolis el al.. 1982) (males v.v. females: 28.17% vs.
14.39%, X 2 = 7.144, df = 1, P < 0.01). Similar results
were obtained from the winter samples, where parasitized
males and females scored 28.26% and 9.37%. respectively
(X 2 = 5.424, df = l,P< 0.02). The number of parasitized
specimens did not differ between sampling periods (sum-
mer: 60 out of 281. winter: 19 out of 1 10; X 2 = 0.01,
df = 1, ns).

The maximum number of externae from the summer
samples was 15 in males and 18 in females. We counted
23 externae in one female collected in winter. None of
the ovigerous females in our sample had parasites (0 vs.
20% in nonovigerous ones: X 2 = 5.334, df = \.P< 0.05).
Only one female has been collected bearing both eggs and

hatching days = 93
tide cycles = 4

LU
u_

O

SPRING

1

NEAP

^
Q

<r

D
O

z
<

01

SPRING

TIDE CYCLE (days)

Figure?. Occurrence of hatching within a lunar tidal cycle, compared
ith the mean tidal current speed per day (average SE).

180 -

160 -

140 -

S. 120 -

>

O

100 -

LU

8(H

o: 60 -

LL

40 -

20 -

NUMBER OF EXTERNAE PER HERMIT

Figure 6. Number of the externae of rhizocephalan parasites per
hermit host, compared between sexes.

three externae (P. M. Cassidy. pers. obs.), but it is plausible
that infestation occurred after spawning. The number of
externae was significantly correlated with the host size if
the host was male (Spearman rank correlation test: r s
= 0.413, / = 2.759, df = 37, P < 0.01). but not if it was
female (r, = 0.299, / = 1.331, df = 18. ns).

The frequency distribution per size class of the infested
specimens compared with respect to the healthy ones did
not show any difference in the males (G = 4.816, df = 3,
ns) (Fig. 7 A); a difference (though slight) was found in the
females, where parasites occurred more often within
smaller size classes (G = 6.913. df = 3. P cu. 0.05) (Fig.
7B). When three size classes were distinguished, no be-
tween-sex difference was found in small specimens (G
= 0.004, df = 1, ns). but the difference was significant in
larger classes) intermediate: G = 10.643, df= 1,.P<0.01;
biggest: G = 2.744. df = 1. P ca. 0.05). The minimum
size of parasitized specimens was 1.8 in females and
1.4 mm SL in males.

Peltogaster hoshnuie externae never exceeded two per
individual. They were more frequently found on the left
side of the hermit abdomen (left, center, and right vs. a
uniform distribution: X 2 = 76.513, df = 2, P < 0.001),
and at the proximal end, close to the carapace (proximal,
middle, distal v.v. a uniform distribution: X 2 = 34.241, df
= 1 . P < 0.00 1 ), without any difference between sexes
(side: G = 1.1 18, df = 2, ns; extremity: X 2 = 0.029, df
= 1, ns). Their first point of eruption corresponded to the
position of the second pleopod. In contrast, the externae
of Thihicopletluis ( = Tlioinpsoniu) rcinluinli. ranging from
1 to 23, were more clumped on the host, equally distrib-
uted on the right and left halves of the hermit body, and
more diffused, involving the dorsal side of the abdomen,
the cephalothorax, and even the pereiopods and chelipcds.

LIFE HISTORY OF A TUBE-DWELLING HERMIT CRAB

73

70 -

24 3.2

SIZE CLASSES (SL, mm)

UNINFECTEDn = 102

INFECTED n = 40

B

24 32

SIZE CLASSES (SL, mm)

I I UNINFECTEDn=119

INFECTED n = 20

Figure 7. Size frequency distributions compared between hermits
that were either uninfected or infected by rhizocephalan parasites in
males (A) and females (B).

Parasites ' effects on the host external morphology and
behavior

To evaluate the effect of parasites on their hosts, we
examined various aspects of the hermit external mor-
phology. The pleopod number did not show any signifi-
cant difference between infested and noninfested speci-
mens in either sex (distinguishing animals with 4, 3, and
less than 3 pleopods, males: G = 1 .645, df = 2, ns; females:
G = 0.31, df = 2, ns). However, the second pleopod was
absent more often in parasitized specimens (7 out of 16
vs. 5 out of 54: G= 8.333, df = l,P< 0.01). The difference
was more pronounced in the males (6 out of 12 vs. 5 out
of 3 1 : G = 4.576, df = 1 , P < 0.05) than in females ( 1
out of 5 v.v. out of 23: G = 1 .839, df = 1 , ns).

The relative growth of the DE of the major chela, one
"maleness" character, was analyzed. No difference was
found between parasitized and unparasitized specimens.

either in males (after a In-ln transformation: b, 0.63 vs.
0.93,? = 1.772, df = 56, ns; a, 0.21 vs. -0.15,? = 1.847,
df = 57. ns) or in females (/>, 0.52 vs. 0.57, / = 0.237, df
= 63, ns: a, 0.40 vs. 0.33. / = 0.01 1, df = 64, ns). Anal-
ogously, parasites seemed not to affect hermit relative body
weight in either sex (after In-ln transformation. SL vs.
weight without chelae, males: b, 3.45 vs. 5.08, / = 1.539,
df = 39, ns; a, -1.13 vs. -3.31, / = 0.043, df = 40, ns;
females: b, 1.70 vs. 3.59, / = 0.778, df = 38, ns; a, 1.32
vs. - 1 .04, / = 1 .2 1 6, df = 39, ns). The same was also true
when cheliped weight was examined (males: b, 8.80 vs.
7.26, / = 0.537. df = 24, ns; a, -15.41 vs. -11.97,
t = 0.858, df = 25, ns: females: b, -2.19 vs. 6.41, / = 1.664,
df = 22, ns, a, 13.89 v.v. -9.34, / = 1.043, df = 23, ns).

Several parasitized hermits were seen molting and pre-
served their externae after the ecdysis, even when externae
belonged to the latest stages, already containing oocytes
and embryos (Liitzen. 1992).

One possible behavioral effect of parasites is lethargy,
which might reduce the ability of naked hermits to find
empty tubes. However, in pilot experiments in the labo-
ratory, parasitized and unparasitized D. schmitti individ-
uals were about equally matched when competing for a
single empty tube. In the field, no difference was seen in
the relative opening diameter of the occupied tubes be-
tween hermits belonging to the two conditions (males: b,
t = 0.025, df = 64, ns, a, t = 0. 169, df = 65, ns; females:
b. t = 0.86, df = 68, ns, a,t= 1 .9 1 3, df = 69, ns).

Parasitized specimens of both sexes placed inside
transparent tubing were seen fanning the externae of Pel-
togaster boschiuae. The behavior was the same as that
already described for ovigerous females fanning their eggs.

Discussion

The structure oj the D. schmitti population

In the species of hermit crabs studied to date (Table
IV), sex ratio in relation to size mostly follows the "anom-
alous" pattern described by Wenner (1972). This implies
that sexes in small size classes are approximately balanced,
a large excess of females is found in intermediate size
classes, and an excess of males is found in the largest ones.
Exceptions are reported by Wenner ( 1972) in Clibanarius
zebra and Calcinus latens, by Abrams (1988) in Pagurus
ochoiensis and P. aleitticus. and by Gherardi and Mc-
Laughlin (1994) in Calcinus laevimanus from the Mas-
carenes. A further exception is D. schmitti. in which an
equal number of males and females are represented in
each size class.

One obvious bias in the size distribution analysis is the
large- or small-scale habitat segregation between sexes.
Species have been reported to show between-sex differ-
ences in habitat utilization (e.g., males of Pagurus hir-
siitiuscnlits occupy high tidepools, but females are dom-

74

F. GHERARDI AND P. M. CASSIDY

Table IV

Species ot hermit crabs reported from the literature following the
"anomalous" pattern (Wetiner. 1972) in the se.\-ratio-lo-si:e relation

Genus

Species

Reference

Coenobila coinpressus Wenner. 1972

Calcinux laevimanus Wenner, 1972

lalens Gherardi and McLaughlin, 1994

Clibananus digueti Harvey, 1988

erythropus Gherardi, 1991

laevimanus Gherardi el at., 1994

Intmilt.s Gherardi and McLaughlin, 1994

Diogenes breviroslris Walters and Griffiths, 1987

Elassochims tenuimanita Abrams, 1988

Pagurisles turgidus Abrams, 1988

Pagurux grano.si mii/uis Abrams, 1988

hirsutiusculus Abrams, 1988

samuelis Abrams, 1988

kenncrly Abrams. 1988

beringanus Abrams, 1988

dalli Abrams, 1988

inant in microhabitats without standing water at low tide;
Abrams, 1988). The size of clustering species is also seg-
regated within clumps (in Clibanarius laevimanus, Ghe-
rardi et ai, 1994). However, both sexes of/), schmitti are
restricted within sabellarian bioherms (Gherardi and Cas-
sidy, 1994b), starting from the late megalopa stage, and
although the species has a contagious distribution (Ghe-
rardi and Cassidy, 1994b), clumps do not significantly
differ in either sex ratio or size.

The sexual selection hypothesis

Under the rationale of the sexual selection hypothesis
( Bertness, 1 98 1 a), the between-sex balance in the size dis-
tribution of D. schmitti implies that the two sexes get the
same benefits (or handicaps) from larger dimension.

Reproductive potential might be enhanced with size.
From the perspective of D. schmitti females, clutch size
significantly increases with the body mass, and larger fe-
males also bear more voluminous eggs.

Larger size might also provide a higher reproductive
potential to males. A sexual dimorphism was evident in
the major chela dimensions (dactyl length, and palm
length and width): the chela (especially the biggest) was
more massive in the male than in the female. The func-
tional significance of this sexual difference has been widely
discussed for Brachyura (Hartnoll, 1974), where it was
related to the use of chelipeds in territorial defense, com-
bat, display, and courtship. In several hermit crabs, males
showed complex precopulatory behaviors, involving the
chelae, for example, either rotating and shaking the female
(Diogenidae) or jerking her toward himself (Paguridae)
(Hazlett, 1966, 1968). Sexual behavior has not yet been

observed in D. schmitti. but the importance for males of
having larger chelipeds might be associated with the in-
trasexual competition to mate. Chelipeds are widely used
in aggressive interactions, both in displays (cheliped ex-
tension, waving, and wig-wag display; F. Gherardi, in
prep.), and in fights (hits and grasps), where the bigger
and stronger the chelipeds are, the more likely the hermit
is to win.

In hermit crabs, factors that could reduce the tendency
to grow are the interspecific competition for shells and
the scarcity of large housings within the habitat. By its
ability to occupy empty polychaete tubes as a new housing,
D. schmitti has freed itself from the harsh war for shells
that occurs within the subtidal hermit crab assemblage in
northern Puget Sound (Abrams et ai, 1986). Its small
relative size must have preadapted this species to this nar-
row microhabitat, but its body mass is certainly con-
strained by the size distribution of the available empty
tubes. In his ecological notes on the endemic Bermuda
hermit Ca/cinus verrilli. Markham (1977) observed that
the mean size of the crabs occupying attached vermetid
shells was far smaller than that of crabs in mobile Ceri-
thium shells. Members of the D. schmitti population an-
alyzed here occupy the largest tubes at their disposal in
the bioherm, and size in both sexes was positively cor-
related with tube opening, suggesting that crabs must
change their housing with growth (Gherardi and Cassidy,
1994b).

The growth hypothesis

The growth hypothesis (Abrams, 1988) refers to the
between-sex difference in the available energy for growth;
the male-biased sex ratio in larger size classes in most
hermit species is attributed to the additional energy that
males can allocate to growth because they do not have to
produce eggs (Bertness, 1981b). Data are still missing for
the extent of growth through molts in D. schmitti and its
energy-time budget is unknown, but a number of clues
suggest that the distribution of the rhizocephalan parasites
might affect growth in this species.

In the population we examined, the extent of infestation
and parasite prevalence varied significantly between sexes,
reaching in the males an average of 2.9 externae per in-
dividual and a percentage of 28 infested specimens. Prev-
alence is unaffected by the male host size, but the fre-
quency of infested females decreases in the intermediate
and larger size classes, where the infestation is significantly
less diffused than in similar sized males.

Within the framework of the growth hypothesis, one
likely conclusion drawn from these data is that if (1) the
males are more frequently infected than the females, and
if (2) parasites cause a reduction in the growth rate of the
host, then the two sexes grow to the same extent because

LIFE HISTORY OF A TUBE-DWELLING HERMIT CRAB

75

the energy the females expend in producing eggs equals
that which the males consume to support parasites. How-
ever, the two assumptions require further clarification and
open new questions.

First, we do not know why the parasites are unequally
distributed between the sexes. The observed pattern could
not be explained by either an increased mortality rate of
infested females or the occurrence of sex reversal, because
the sex ratio was 50% in all the size classes. The attachment
of the parasite larvae may be impeded by the efficiency
of cleaning and grooming (Bauer, 1981), but the two sexes
did not differ in either the extent or the modes of cleaning
behavior (Gherardi, 1994). As a third explanation, im-
munological responses by the hosts might vary between
sexes. Parasitized, but not normal, Carcimis mediterra-
neus have a substance in their blood that fixes complement
in the presence of extracts ofSacculina (reviewed in Bang.
1983). However, in that parasitic relationship, electro-
phoregrams did not show any marked difference between
the parasitized males and females (Herberts. 1978). D.
schmitti females differ in their susceptibility to infection
according to their reproductive states. No parasitized fe-
males have been found in ovigerous condition (other ex-
amples in Hoggarth, 1990, and Liitzen and Jespersen,
1992; exceptions in Hoeg and Liitzen, 1985); one expla-
nation is that parasitized females lose their eggs after a
few days (Liitzen and Jespersen, 1992), but the reasons
remain unknown.

The second assumption, that growth rate of the host is
affected by the parasite, is supported by the previous lit-
erature on rhizocephalan infestation (O'Brien and Van
Wyk, 1984; Hawkes et ai. 1986; Hoggarth, 1990; Abello
and Macpherson, 1992; Bang, 1983; Overstreet, 1983).
Nevertheless, a direct investigation of molt frequency is
lacking and figures on the relative increase at ecdysis
compared between infected and uninfected individuals
are provided only by Liitzen and Jespersen ( 1992). Our
findings that parasites do not inhibit molting in D. schmitti
or influence either body or cheliped weight in either sex
make the growth hypothesis questionable, at least in this
species.

Other effects of parasites

A variety of morphological and behavioral alterations
exhibited by rhizocephalan-infected decapods and the
hormonal involvement in those phenomena are exten-
sively described by Hartnoll (1967), Nielsen (1970), and
Phillips and Cannon (1978) among others (see, e.g.. bib-
liography by Overstreet, 1983). D. schmitti males do not
undergo the process of feminization observed in other
species (Hartnoll, 1982; O'Brien and Van Wyk. 1984). as
evidenced by the preservation of some "maleness" char-
acters (e.g., the high relative depth of the major chela).

The only alteration is the frequent absence of the second
pleopod, which cannot result from an attempt by the par-
asite to provide a safe accommodation for the externae,
but seems instead to be a consequence of the eruption of
Peltogaster boschmae externae within the soft tissue lining
the host abdomen, which corresponds to the attachment
point of the second pleopod.

Neither do infected hermits exhibit behavioral altera-
tions, such as lethargy, that could decrease their ability
in direct or exploitative competition: in the laboratory,
parasitized and healthy D. schmitti had the same proba-
bility of getting an empty polychaete tube, and in the
field, they occupied equally sized housings. Besides, rel-
ative weight, and thus possibly feeding efficiency, was un-
affected by the presence of rhizocephalans. The only be-
havioral result of parasite manipulation is the initiation
of mock parental care, in which infected hermits of both
sexes ventilate Peltogaster boschmae externae in the same
way that gravid females ventilate their eggs.

Reproductive patterns

D. schmitti females attain maturity at a relatively small
size: the smallest egg-bearing specimen measured 1 . 1 mm
SL. On the other hand, the allometry of chela growth
should indicate that maturity (at least, functional matu-
rity; Hartnoll, 1969) occurs in males at larger size (over
3.4 mm SL).

A precocious onset of sexual behavior in females has
been reported in the decapod literature and associated
with a reduced possibility of encountering males; in the
parasitic females of the Pinnotheridae (Christensen and
McDermott, 1958) and in the freshwater crab Potamon
Jhtviatile(Miche\i et al.. 1990) copulation can occur even
in prepubertal females, and sperm are kept in the seminal
receptacles until ovulation.

A second remarkable feature of reproduction is the low
frequency (50%) of gravid females in all the size classes.
This is particularly evident when we consider that D.
schmitti breeds only once per year (Nyblade, 1974), and
that the breeding period (January-April) is short, but the
time necessary for eggs to mature is relatively long (ex-
ceeding, on average, 1 month).

One explanation is that due to the shortage of food,
females may have limited energy for producing clutches,
causing them to skip the reproductive season. If this were
the case here, we should expect a gradient in the clutch
size depending on the available energy. Nonetheless, egg
number is a function of female size, and the latter is not
related to feeding efficiency (Gherardi, 1 994). In addition,
the annual egg production (52.8 mg of eggs per 100 mg
female weight per year; Nyblade 1974) is high compared
with that of the other hermit crabs in northern Puget
Sound.

76

F. GHERARDI AND P. M. CASSIDV

Another hypothesis refers again to the difficulty that
this sedentary species encounters in finding a mate. D
schmitti is gonochoristic and mating in hermit crabs
requires copulation (Hazlett, 1966). hut it is still unclear
how this is effected in this species. Males, females, or
both are assumed to leave the attached tube and roam
about with their abdomens naked (or at best in broken
pieces of tubes; Nyblade as reported by Caine, 1980) to
seek receptive mates. Despite the clumped distribution
of the population, this is a risky behavior; in the labo-
ratory, wandering hermits inhabiting loose tubes are
easy prey for the crabs and fishes (F. Gherardi, in prep.),
that frequent sabellarian bioherms (Gherardi and
Cassidy. 1994a).

Hatching lasts from 1 to 6 nights, the length of time
being related to the overall number of larvae. This suggests
either that development of embryos belonging to the same
batch is out of phase or that hatching is controlled by the
embryos themselves, by the females, or by both (Saigusa,
1992). Such an extension of hatching in sequential bursts
might be a mechanism to allow survival of at least a num-
ber of larvae in a difficult, predator-filled, and unpredict-
able environment, such as the current-swept channels of
Puget Sound.

In D. schmitti. hatching occurs exclusively at night,
possibly to minimize predation on the newly released lar-
vae by diurnal fishes. In contrast to the other decapods
inhabiting enclosed habitats (estuaries and mangrove
swamps; Forward, 1987; Hartnoll. 1988). in this species
larval release is synchronized with neap tides, when the
tidal current is consistently lower than in the spring phase.
This timing seems to be controlled by an endogenous
clock (De Vries and Forward, 1989). persisting under lab-
oratory conditions in which the tidal cycle corresponding
to the rhythm is absent. This pattern of larval release seems
unrelated to salinity tolerance (Forward el ai. 1982), be-
cause salinity is nearly constant in the examined area
(SPMC, 1992). Its adaptive meaning is suggested by D.
schmiiii '.v behavioral ecology. For this species so depen-
dent upon a habitat (sabellarian bioherms) that is rare
and quite unpredictable (Gherardi and Cassidy. 1994a)
it is more beneficial if larvae are retained within the basin
near the parental population than if they are flushed out
to open waters for planktonic development (see Mc-
Conaugha. 1992. for a discussion of larval retention vs.
dispersal in decapods).

Acknowledgments

We thank Dr. Jorgen Liitzen (University of Copen-
hagen) who kindly identified parasites of D. schmitli. The
study was encouraged by Dr. Patsy A. McLaughlin
(Shannon Point Marine Center, WWLJ), to whom we are
greatly indebted. Part of the work was conducted at the

Shannon Point Marine Center, Anacortes, Washington.
Partial funding was provided by M.U.R.S.T. to the first
author.

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Point Marine Center, Anacortes. WA.
Walters, W. L., and C. L. Griffiths. 1987. Patterns of distribution,

abundance and shell utilization amongst hermit crabs, Diogenes bre-

virostris S Afr. J Zool. 22: 269-277.
\Venner, A. M. 1972. Sex ratio as a function of size in marine Crustacea.

Am. Nalitr. 106: 321-350.
Zar, J. H. 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs,

NJ.

Reference: Biol Bull 188: 78-82. (February/March, 1995)

Taurine-like Immunoreactivity in the Motor Nerve Net
of the Jellyfish Cyanea capillata

MATS CARLBERG 1 , KARIN ALFREDSSON 1 , SVEN-OLLE NIELSEN 1 ,
AND PETER A. V. ANDERSON 2

^Department of Zoology, University of Lund, Helgonavdgen 3, S-223 62 Lund, Sweden, and

2 Whitney Laboratory and Departments of Physiology and Neuroscience.

University of Florida. Si. Augustine, Florida 32086

Abstract. Two antisera against the sulfonated amino
acid taurine were applied to subumbrella tissue of the
jellyfish Cyanea capillata. Taurine-immunoreactive nerve
nets were found in both the ectoderm and endoderm. The
ectoderm had two morphologically and immunocyto-
chemically distinct populations of neurons, the motor
nerve net (MNN), which was immunoreactive to the tau-
rine-like molecule, and the diffuse nerve net (DNN), which
was immunoreactive to the neuropeptide Phe-Met-Arg-
Phe-NH : (FMRFamide). In the endoderm, immunoreac-
tivity was found in the endodermal DNN. This localiza-
tion was confirmed by double-labeling experiments, which
also revealed that the endodermal DNN neurons may
contain both taurine and FMRFamide-related peptide.
The presence of a taurine immunoreactivity in the MNN
supports the hypothesis that taurine or some chemically
related compound is the neurotransmitter at synapses
within the MNN of Cyanea.

Introduction

Cnidarians are the earliest extant animals to have a
nervous system and, as such, they may provide useful
information about the evolution of the nervous system
and its components. Furthermore, their structural sim-
plicity affords opportunities for studying functional as-
pects of these nervous systems, including the cellular
mechanisms underlying chemical synaptic transmission
(Anderson, 1985; Spencer et ai, 1989; Anderson and
Spencer, 1989). A focus of considerable interest in recent
years has been the identity of neurotransmitters in the
Cnidaria. Neuropeptides are known to be common within

Received 24 May 1994; accepted 27 October 1994.

the phylum (Grimmelikhuijzen et ai. 1989a, b: 1992),
and evidence for a role of small molecules and amino
acids as neurotransmitters is growing (Anctil, 1989;
Scemes, 1989; Chung el ai. 1989; Chung and Spencer,
1990; Umbriaco et ai, 1990), but remains limited.

The sulfonated amino acid taurine, which is ubiquitous
in animals and prokaryotes and has been implicated as
an inhibitory neurotransmitter in both vertebrates (Huxt-
able, 1989) and invertebrates (Nistri and Constant!, 1976;
Hue?/ a/.. 1979; Giles and Usherwood, 1985), has recently
been shown to depolarize neurons in the motor nerve net
(MNN) of the scyphozoan jellyfish Cyanea capillata (An-
derson and Trapido-Rosenthal, 1990). The mode of action
of taurine on these neurons is very similar to that of the
endogenous neurotransmitter, raising the possibility that
taurine may serve as an excitatory neurotransmitter at
these synapses. To determine whether taurine is present
in the tissues of Cyanea and if so, to delineate its distri-
bution, we used antisera raised against a taurine-bovine
serum albumin complex (Campistron et ai. 1986: Madsen
cl ai. 1985). The results indicate that taurine, or a taurine-
like molecule, is indeed present in the MNN and that its
distribution is consistent with a role as a neurotransmitter
in the Cyanea MNN.

Materials and Methods

Specimens of Cyanea were collected at the Tja'rno Ma-
rine Biological Laboratory on the west coast of Sweden.
Pieces of perirhopalial tissue (Anderson and Schwab,
198 1 ) were removed from the animal and pinned out to
prevent curling. In some preparations the myoepithelium
that envelops the MNN neurons was removed to expose
the nerve net (Anderson and Schwab, 1 984). Tissues were

78

TAURINE-LIKE MATERIAL IN NERVES OF A JELLYFISH

79

fixed for 3 h in freshly prepared 5% glutaraldehyde in
0.05 M Na-cacodylate buffer containing 1% sodium
metabisulphite and 2.4% sodium chloride (pH 7.5). After
fixation, the tissues were given three 15-min washes in
Tris-buffered saline (TBS; 0.05 A/TRIS-HCI buffer, pH
7.5, 1% sodium metabisulphite and 2.4% sodium chlo-
ride), followed by 30 min in 0.1 M sodium borohydride
in TBS, then a further three 1 5-min rinses in TBS. Twelve
specimens from 5 to 20 cm in diameter were used for
immunocytochemical investigations.

Samples were incubated for 4-6 days in rabbit anti-
taurine antisera diluted 1:200 (Chemicon) or l:1000(Im-
munotech S.A.) in TBS with 0.2% Triton X-100 (TBS/
TX) and 1% bovine serum albumin (BSA). After three
15-min rinses in TBS/TX, the samples were incubated
for 3 h with fluorescine isothiocyanate- (FlTC)-conjugated
swine anti-rabbit IgG (Dakopatts, Denmark) diluted 1:10
in TBS. The samples were then given three 15-min rinses
in TBS, stained in a 1% solution of Evans blue (Merck)
in phosphate-buffered saline (PBS), pH 7.4, rinsed for 2 h
in PBS, and mounted in phosphate-buffered glycerol.
Specificity was tested by preabsorbtion of antiserum with
1 [iM taurine-glutaraldehyde-BSA conjugate.

Double labeling with rabbit antisera raised against tau-
rine and the neuropeptide FMRFamide was carried out
in the manner developed by Wiirden and Homberg
(1993). Specifically, tissues were first stained with anti-
bodies to taurine. and the location of the primary antibody
was visualized by a 3-h incubation with Texas-red-con-
jugated donkey anti-rabbit IgG (Jackson Immuno Re-
search) diluted 1:40. After a 3-h incubation in rabbit IgG
(Dakopatts) diluted at 1:25, the tissues were then incu-
bated for 24 h with biotinylated (Bayer and Wilcheck.
1980) anti-FMRFamide antibodies (Incstar), diluted 1:
800. The FMRFamide immunostaining was then visu-
alized by treatment with streptavidin-FITC (Dakopatts)
at 1 :20 for 3 h. All light microscopical observations were
made with a Leitz Aristoplan microscope.

The specificity of taurine antiserum from Chemicon
has been characterized by the company. Cross-reactivity
with glutaraldehyde-conjugated hypotaurine was 0.067
(1:15) and was less than 0.002 for other glutaraldehyde-
conjugated amino acids including GABA, beta-alanine,
aspartate, glycine, cysteine. and glutamate.

Results

Light microscopy

Taurine-like immunoreactivity (Tau-IR) was found in
both the ectoderm and endoderm of the perirhopalial tis-
sue of Cyanea.

Ectodermal-specific Tau-IR was found in neurons and,
to a lesser extent, in myoepithelial cells. Ectodermal myo-
epithelial cells in this species contain a large central vac-

uole (Anderson and Schwab, 1981). Immunoreactivity
was restricted to the narrow layer of cytoplasm that sur-
rounds each vacuole; vacuolar contents were not im-
munoreactive. The Tau-IR neurons were large, bipolar
cells with lengths up to 2 mm. cell-body diameters of 15
to 20 nm, and axonal diameters from 1 to 5 /urn (Fig. 1 A).
These were clearly motor nerve net (MNN) neurons (An-
derson and Schwab, 1981) and were easily distinguished
from the FMRFamide-immunoreactive (FMRF-IR) cells
that form the diffuse nerve net (DNN) (Fig. 1C), the other
nerve net present in the perirhopalial tissue ectoderm. In
the MNN, synapses occur wherever two neurons are in
physical contact with one another (Anderson, 1985), and,
as can be seen in these micrographs (Fig. IB), such con-
tacts are abundant. The Tau-IR within the MNN was
restricted to the perirhopalial tissue; although MNN neu-
rons are known to extend into the radial and circular
muscle bands that surround the perirhopalial tissue (An-
derson and Schwab, 1981), no Tau-IR neurons were found
in the radial or circular muscle bands.

In the endoderm. at least two cells types were immu-
noreactive. One was a population of bipolar neurons.
These cells, which had cell-body diameters of 1 to 15 ^m
and axon diameters of 0.5 to 2 ^m, were at least 0.6 mm
long and formed a loose nerve net (Fig. ID). Their overall
appearance is consistent with that of the diffuse nerve net
(DNN) known to be present in this tissue. The other ob-
viously immunoreactive endodermal cell type was more
difficult to characterize. The cells in question occurred
relatively densely, and their immunoreactivity appeared
as a rather amorphous, frequently circular mass. Whether
this mass represents an intracellular compartment of the
cell or the true dimensions of the cell was not clear. Both
of these cell types were surrounded by a low level of back-
ground immunoreactivity interspersed with occasional
nonfluorescent areas that are presumably spaces in the
endodermal epithelium.

Preabsorbtion of antiserum with taurine-GA-BSA con-
jugate completely abolished all immunostaining.

Double labeling

Double labeling revealed two distinct nerve nets in the
ectoderm. Again. Tau-IR was restricted to MNN neurons,
whereas FMRF-IR was localized to a separate population
of smaller, multipolar cells (Fig. 2A). FMRF-IR was also
evident in the marginal rhopalia and in regions covered
by the circular and radial muscle bands. At no time were
the two signals co-localized in the ectoderm.

In the endoderm, both antibodies stained what ap-
peared to be the DNN. In smaller animals, the neurons
were FMRF-IR, but in larger animals they were apparently
Tau-IR. In one specimen, both FMRF-IR and Tau-IR
were evident in the same cells, indicating co-localization
(Fig. 2B).

80

M. CARLBERG ET AL

Figure 1. Whole-mount immunostaming of penrhopalial tissue of Cyanea capillata. (A) Low power
micrograph of Tau-IR in the ectoderm. Neurons in the MNN stain readily. Scale bar = 0. 1 mm. (B) Micrograph
of the Tau-IR MNN in the ectoderm. Several apparent contact sites between the axons (arrows) and thinner
elements twined together (arrowheads) can be seen. Scale bar = 50 ^m. (C) Low-power micrograph of
FMRFamide-immunoreactive diffuse nerve net (DNN) in the ectoderm. Scale bar = 0.1 mm. (D) Tau-IR
in the endoderm of the penrhopalial tissue. Immunoreactivity was present in bipolar neurons and in un-
differentiated endodermal cells. Scale bar = 50 nm.

Discussion

In cnidarians, nerve nets are located under an overlying
epithelium that forms a permeability barrier for phar-
macological agents and microelectrodes. The ectodermal
MNN in the penrhopalial tissue of Cyanea is one of the
very few instances in which a coelenterate nerve net can
be exposed, permitting access for electrophysiological and
pharmacological studies (Anderson and Schwab. 1984:
Anderson, 1985). The size of the neurons makes them
suitable for electrophysiological recordings. The accessi-

bility of this nerve net and, in particular, its synapses pro-
vides a useful preparation for studying the pharmacology
of chemical neurotransmission in a cnidarian.

The MNN is a plexus of large bipolar neurons that
innervates the swimming muscle bands and serves as the
pathway to coordinate swimming motor activity. Synapses
between the MNN neurons are fast, chemical synapses
that are bidirectional (Anderson, 1985). Previous work
(Anderson and Trapido-Rosenthal, 1990) has implicated
taurine or a closely related molecule as a potential neuro-

TAURINE-LIK.E MATERIAL IN NERVES OF A JELLYFISH

81

Figure 2. Double labeling. (A) Whole mount of perirhopalial tissue
ectoderm, stained with antibodies against taurine (red) and FMRFamide
(green ). The clear anatomical separation between the Tau-IR MNN neu-
rons and FMRFamide DNN neurons is evident. (B) Endodermal tissues
stained in the same manner. The majority of cells in this preparation
are Tau-IR. Two neurons (arrows) were more (yellow) or less (green)
intensely immunoreactive to FMRFamide, but at least one (arrowhead)
was reactive to both antibodies. Scale bars = 50 ^m.

transmitter at these synapses. The current investigation
provides immunocytochemical evidence that a taurine-
like molecule is indeed present in MNN neurons and in
an endodermal nerve net, but is not present, at least in
detectable quantities, in neurons of another ectodermal
nerve net, the diffuse nerve net (DNN). This was partic-
ularly obvious in the double-labeling experiments, in
which the ectodermal FMRFamide-IR was clearly re-
stricted to the DNN (Anderson et ai. 1992). Although
Tau-IR was also present in the ectodermal myoepithelial
cells, its absence in the DNN indicates that taurine is not
a constituent component of all nerve nets in this animal.
This distinction is important because taurine acts as an
osmoregulator in many marine organisms (Thurston et

ii/., 1980). If it were serving the same function in Cyanea,
one might expect it to be widespread in different cell types
and present in all nerve nets.

The presence of Tau-IR in the endodermal DNN in
Cyiineu was unexpected considering that immunoreac-
tivity to antibodies raised against the sea anemone neuro-
peptide AnthoRFamide was found in these neurons
(Anderson ct at.. 1992). In the present study, endodermal
Tau-IR neurons also were found to be immunoreactive
to antibodies to FMRFamide. In addition, however, mor-
phologically similar neurons in larger animals were found
to have Tau-IR, and one specimen showed apparent co-
localization of the two transmitter candidates. It may be
worth further investigations to find out if there is a pro-
gression from an FMRFamide or AnthoRFamide-like
peptide to taurine (or a related compound) as the animal
grows. To meet the requirement for faster transmission
in a large medusa, a switch from peptidergic metabo-
trophic receptors to fast excitatory ionotrophic receptors
would be functional. In either case, it is clear from this
work that cnidarian synapses may have a hitherto un-
appreciated complexity in the number of neurotransmit-
ters present in single neurons.

Tau-IR was also found in a very abundant, non-neu-
ronal cell type in the endoderm (Fig. ID). The identity
of this cell is unclear. The presence of Tau-IR in these
cells, and perhaps all endodermal cells if the light back-
ground fluorescence is indeed indicative of low levels of
taurine, may imply that it has an alternative function such
as osmoregulation. It is also possible, however, that some
of the small circular profiles represent interstitial cells dif-
ferentiating into DNN neurons.

The antibody used in the light microscopical compo-
nent of this study is known to have low cross reactivity
to the most abundant metabolites of taurine including
hypotaurine and cysteine. However, one cannot as yet
exclude the possibility that the antigen is a closely related
compound or a small taurine-containing oligopeptide
(Marnela et a/., 1985). Free taurine is, however, an abun-
dant constituent of MNN neurons (Anderson and Trap-
ido-Rosenthal, unpub.).

The MNN extends over the entire subumbrella surface,
forming a network that connects all eight marginal ganglia,
or rhopalia, with the circular and radial swimming muscle
bands. To do this, the nerve net must transit the muscle
bands, and individual, Lucifer-yellow-filled neurons have
been seen to extend from the perirhopalial tissue into ra-
dial muscle bands (Anderson and Schwab, 1981). How-
ever, Tau-IR neurons were never observed in either the
radial or circular muscle bands. Although the MNN neu-
rons located within the confines of these muscle bands
may employ a different neurotransmitter in this region,
it is also possible that failure to observe Tau-IR in these
areas is due to a technical problem. To get adequate stain-

82

M. CARLBERG F.T AL

ing of neurons in these preparations, they had to be in-
cubated with the antibodies for as long as 6 days. In con-
trast, anti-FMRFamide antibodies usually penetrate the
tissues easily, typically requiring 24 h (Anderson et a/.,
1992). The staining difficulty may reflect either a low an-
tibody liter or poor penetration by anti-Tau antibody. In
either case, the thick layer of muscle that overlies the
MNN in the radial and circular muscle bands may have
compromised the ability of the anti-Tau antibodies to
reach their targets in this region.

The major conclusion of this study is that MNN neu-
rons are Tau-IR. This, together with electrophysiological
evidence that taurine depolarizes the MNN neurons in a
manner consistent with that of the endogenous neuro-
transmitter, provides compelling evidence that taurine,
or a taurine-like molecule, is the neurotransmitter in
Cyanea. This possibility has evolutionary implications.
Taurine is one of the most abundant amino acids in the
animal cell, and it is conceivable that carnivores and
scavengers developed olfactory receptors for taurine very
early in evolution. Receptors for taurine are, indeed,
known to be present on the olfactory antennae of lobsters
(Derby and Atema, 1982), and one could envisage how
neurotransmitter receptors might have developed from
external chemoreceptors (Carr, 1989).

Acknowledgments

We are very grateful to the Director, Dr. Larz Afzelius,
and to Drs. Lars Hagstrom and Benno Magnusson for
providing us with facilities at the Tjarno Marine Station.
This work was supported by grant B-BU 1781-303 from
the Swedish Natural Research Council to Mats Carlberg
and by NSF grant BNS 91091 55 to Peter Anderson.

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Anderson, P. A. V., A. Moosler, and C. J. P. Grimmelikhuijzen. 1992.
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Carr, \V. E. S. 1989. Chemical signalling systems in lower organisms:
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Chung, J. M., and A. N. Spencer. 1990. On the potassium conductance
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Chung, J. M., A. N. Spencer, and K. H. Gahm. 1989. Dopamine in
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Giles, D. P., and P. N. R. Usherwood. 1985. The effects of putative
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Grimmelikhuijzen, C. J. P., D. Graff, O. kiozumi, J. A. Weslfall, and
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Reference: Biol Bull 188: 83-88. (February/March, 1995)

The Influence of Opponent-Related and Outcome-
Related Memory on Repeated Aggressive Encounters
in the Paradise Fish (Macropodus opercularis)

ADAM MIKLOSI*, JOZSEF HALLER**, AND VILMOS CSANYI*

*Depanment of Ethology, Eotvos Lorand University and **Imtitute of Experimental Medicine,

Hungarian Academy of Sciences

Abstract. The aggressive behavior of male paradise fish
(Macropodus opercularis) was studied. Fish were subjected
to three aggressive encounters on consecutive days. If
submissive males encountered the same opponent three
times, the last aggressive encounter was very different than
the first one. When the animals faced a new opponent
each day, the changes were much less pronounced. We
conclude that ( 1 ) fish are able to recognize their opponents
at least one day after the encounter ("social recognition"),
and (2) social recognition modifies the effect of prior defeat
("status- related memory") in subsequent encounters.

Introduction

An overwhelming amount of evidence indicates that
prior agonistic experience influences the outcome of future
aggressive encounters (Beacham and Newman. 1987;
Frank and Ribowsi, 1987). One can hypothesize that prior
aggressive experience may influence subsequent aggressive
encounters by two kinds of processes: one related to the
outcome of the encounter ("winner" or "loser" effect)
and the other specifically related to the opponent. The
significance of the former process was recently examined
in detail (Bevan et a/., 1960; Poll el ai, 1982; Francis,
1983; Beaugrand and Zayan, 1985; Beacham and New-
man, 1987; Bakker et ai. 1989). Most studies demonstrate
an asymmetrical effect of prior winning or losing on sub-
sequent winning probability. For example, in paradise fish
(Macropodus opercularis; Francis, 1983) and in stickle-
backs (Gasterosteus acideatits; Bakker and Sevenster,
1983) losing greatly enhances the probability of also losing

Received 15 April 1993; accepted 18 November 1994.
Address for correspondence: Adam Miklosi, Eotvos Lorand University,
Department of Ethology, God, Javorka S. u 14, H-2131. Hungary.

the subsequent contest. Winning usually has no strong
effect, but under some experimental conditions it might
increase the probability of winning again (Bakker and
Sevenster, 1983; Bakker et ai, 1989).

The possibility of the involvement of the second pro-
cess individual recognition in agonistic encounters has
been also demonstrated. Fricke (1973) showed that Am-
phiprion bicinctus males more frequently attacked un-
known individuals than known ones in a two-choice ex-
periment. The importance of individual recognition in
the stickleback was demonstrated by Peeke and Veno
( 1973), who observed that a resident male that had been
habituated to an intruder presented in a glass cylinder
would resume aggressive behavior if the individual in the
cylinder was changed. Thresher (1979) used a similar
method to study rival recognition in the threespot dam-
selfish (Eupomaceus planifrons), and those field obser-
vations further confirm that some fish species might be
able to recognize individuals. Myrberg and Riggio (1985)
showed that coral reef fish (Pomacentrus partitus) rec-
ognize territorial neighbors acoustically. Recently Waas
and Colgan (1994) provided experimental evidence that
male sticklebacks can distinguish between familiar rivals
on the basis of visual cues alone.

Assuming that the effects of previous encounters are
mediated by memory and that the behavioral differences
are not due to energetic consequences of aggression (Haller
and Wittenberger, 1988; Haller, 1991) the problem of
interference between social recognition and status-related
memory arises.

As a continuation of a recent study (Miklosi et ai, 1992)
on aggressive behavior in the paradise fish, we experi-
mentally examined these processes in the aggressive be-
havior of the paradise fish. To clarify the relationship be-

83

84

A. MIKLOSI ET AL

tween social recognition and status-related memory, two
questions have been posed: ( 1 ) Does the behavior of the
fish change if it encounters the same or different opponents
for two subsequent encounters? (2) Are there differences
between these experimental manipulations?

Materials and Methods

Experiments were conducted with 6-month-old, 8-cm
to 10-cm-long Macropodus operadaris males, which were
raised and kept in our laboratory. Three days before the
start of the experiment, pairs of size-matched fish were
placed in 40 X 20 X 20 cm glass tanks provided with
filtration and aeration. Each tank was separated into two
equal parts by a green opaque screen, and a single fish
was kept in each part of the tank. The animals were vi-
sually isolated from each other: all sides but the front side
of the tanks were covered with green plastic sheets. This
isolation lasted 3 days prior to the experiment. Water
temperature was kept at 28C, and a 14:10 h light:dark
cycle was maintained. The fish were fed daily on Tubifex
worms.

Animals were exposed to three aggressive encounters
on successive days. Encounters were begun by removing
the plastic partition. All encounters were videotaped until
dominance relationships were established. We defined this
as the point at which one of the fighting males no longer
participated in simultaneous or reciprocal threatening and
fighting but instead became the "subordinate," showed
fleeing and escaping behavior when approached by the
"dominant," which in turn chased and bit its opponent.
A submissive fish remains motionless for a long time in
"oblique position" near to the water surface and does not
retaliate against the winner (Miklosi el a/., unpub. obs.;
Forselius, 1957). Both fish were observed for an additional
hour to monitor the stability of the dominant-subordinate
relationship. One hour after the end of fighting, the contact
between the fish was interrupted by lowering the plastic
door, and the animals were kept in isolation for the next
24 h.

Two groups were tested: the fish in group A (n = 10)
faced the same opponent throughout the experiment; in
group B (n = 10) the dominant animals were randomly
changed between the tanks after each fight. Thus, in group
B, the submissive fish remained in the tank and faced a
new. previously dominant opponent each day. The dom-
inant fish was changed immediately after the end of the
encounter and remained isolated in its new tank until the
following day when the partition was removed again. The
time between two consecutive encounters was long enough
for the dominant to acclimate to the new place (Csanyi
ct ai, 1985), thus the advantage of prior residency of the
submissive fish was minimized.

Videotapes were later analyzed by recording behavioral
units with an event recorder (Nagy et til., 1985). On the

basis of earlier findings, each aggressive encounter was
divided into three main phases: latency, threatening, and
fighting. After the latency for initiation of the first display,
a second phase was defined, which lasted until the first
appearance of contact behavior (biting or mouthlocking).
This was called threatening, which in turn was followed
by the escalation of the fighting fighting phase until
one of the males showed submissive behavior. The be-
havior units we identified are as follows:

Display at distance (DIS): The fish stay in head-tail
position with erected tailfin, but the distance between
them is larger then one body length.

Head-head display (HHD): The fish are oriented par-
allel to one other and face in the same direction, with one
fish slightly behind the other.

Parallel swimming (PAS): The fish swim very close to
each other in the same direction.

Head-tail display (HTD): The fish in parallel orienta-
tion are facing opposite directions. Sometimes this be-
havior is associated with circling.

Shaking (SHA): This behavior is similar to the head-
tail display, but it is associated with fast circling, vigorous
body-shaking, and a downward movement of the pair;
the pattern stops when the animals reach the bottom.

Bite (BIT): One fish makes a swift dart and slashes at
the other fish.

Mouthlock (MOU): The fish reciprocally bite and hold
one another's mouths for up to 2 min.

Air gulping (AG): A fish takes an air bubble in its mouth
by breaking the surface of the water.

Each of these behavioral units was recorded in all of
the pairs investigated. Two samples of behavior were reg-
istered. The first sample, which characterized behavior
during the threatening phase, lasted for 10 min from the
raising of the door or until the first instance of contact
behavior (biting or mouthlocking). The second sample,
which characterized the fighting phase, was a 20-min ob-
servation following the first observed bite.

To permit comparison between pairs, the values of ob-
served behavior units were divided by the sampling time.
This adjustment was necessary because in many contests
fish finished the threatening or fighting phase before our
predetermined interval (10 or 20 min) of observations
ended, resulting in shorter time samples. Thus the relative
duration (minutes per hour) or frequency (number per
minute) of behavior units was used for statistical analysis.

Because the measured variables were not normally dis-
tributed (according to the Kolgomorov-Smirnov test),
nonparametric statistical methods were used. Kruskal-
Wallis's one-way ANOVA was used separately for groups
A and B to examine the change in the general pattern of
aggressive behavior.

OPPONENT-RELATED AND OUTCOME-RELATED MEMORY

85

Results

The difference between the two groups that is, the
different effects of the "treatments" can be seen in Ta-
ble I.

Repeated encounters with the same opponent, group
A, caused marked change in aggressive behavior. Although
the duration of the threatening phase did not change sig-
nificantly in the course of the three encounters, shaking
and air-gulping were significantly reduced. All measured
variables (with the exception of head-head display) of
fighting, including its duration, decreased significantly
when submissive fish faced the same opponent three times.

Interestingly, the changes in the other group (B) were
much less pronounced. When the submissive fish repeat-
edly faced new opponents, only minor changes could be
observed in their aggressive behavior. The threatening
phase did not change significantly; only the relative du-
ration of shaking and the frequency of biting showed a
marked decrease.

A comparison with current literature showed that some
behavioral elements and parameters are of special im-
portance. Thus head-tail display (e.g.. Baerends and Baer-
ends-Van Roon, 1950; Barlow, 1962; Enquist and Ja-
kobsson, 1986), biting (e.g.. Peeke and Veno, 1973; Frank
et a/.. 1985; Enquist and Jakobsson, 1986; Halperin and
Dunham, 1994), mouthlocking (e.g.. Baerends and Baer-
ends-Van Roon, 1950; Enquist and Jakobsson, 1986),
duration of threatening (e.g., Frank et al.. 1985), and du-
ration of fighting (e.g.. Enquist et al.. 1990; Haller, 1992)

were examined further when we used the nonparametric
Mann-Whitney test to compare the behavior of the two
groups in the first, second, and third encounters (Fig. 1).

The two groups did not differ in the first and second
encounter; however, with the exception of head-tail dis-
play, they differed markedly in the third encounter. The
time spent with mouthlocking (z = -2.4, P < 0.02), the
number of bites (z = -2.3, P < 0.02), and the duration
of threatening (z = -2.6, P < 0.01) and fighting (z =
-l.9,P< 0.05) were lower in the group (A) with the same
opponent than in the group (B) with different opponents.

The same variables were compared by the nonpara-
metric Wilcoxon test to show within-group differences
during the three encounters. In group A same opponent
in each encounter we found a significant change from
the first to the second encounter only in fighting duration
(z = -2.8, P < 0.01). However, significant changes oc-
curred between the second and the third encounters in
all of the selected variables (head-tail display: z = -2.8,
P < 0.01; mouthlocking: z = -2.6, P < 0.01; biting: z =
-2.8, P < 0.0 1 ; threatening: z = -2.5, P < 0.02; fighting:
z = -2. 1, P < 0.04). In contrast, no significant differences
could be found in the group (B) with unknown opponents.

Discussion

The results clearly show that the type of opponent (fa-
miliar versus nonfamiliar) has a major effect on the ag-
gressive behavior of male paradise fish. In the case of fa-
miliar opponents (group A), three consecutive encounters

Table I

Analysis of elements of aggressive behavior shown by fighting paradise fish pairs during three consecutive contests in both groups

Group A: familiar opponent

Group B: unfamiliar opponent

encounter 1
Mean (SE)

encounter 2
Mean (SE)

encounter 3
Mean (SE)

Chi

Signif.

encounter 1
Mean (SE)

encounter 2
Mean (SE)

encounter 3
Mean (SE)

Chi

Signif

Dur. of threatening

12.6(2.1)

13.4(2.9)

5.5(1.8)

4.7

ns

12.3(1.9)

12.1 (2.2)

11 (2.2)

1.7

ns

Head-head display

2.3 (0.9)

2.5 (0.7)

3.7(1.4)

0.02

ns

4.4(0.8)

6.2(1.6)

3.6(0.8)

0.7

ns

Shaking

2.3(0.6)

1.7(0.4)

0.4(0.2)

7.3

P < 0.03

2.1 (0.7)

1.4(0.3)

1.1 (0.3)

0.8

ns

Air-gulping

1.7(0.4)

1.9(0.7)

0.3 (0.1)

10.5

P<0.01

3.2(1.4)

1.9(0.8)

2.5(0.6)

2.5

ns

Parallel swimming

1.4(0.7)

3.5 (1.3)

0.7 (0.3)

2.5

ns

2.1 (0.6)

4.4 (1.6)

1.6(0.6)

2.1

ns

Head-tail display

32.2(4.5)

36.1 (6.6)

19 (4.7)

4.7

ns

28.7(4.4)

25.5(5.1)

26.5 (4.3)

0.4

ns

Display at distance

4.1 (1.3)

3.1 (0.7)

5.1 (1.9)

0.2

ns

5.5 (1.8)

6.9(3.5)

6.3(2.1)

0.2

ns

Dur. of fighting

142.6(40.1)

59.8(22.8)

7.3(3.5)

18.1

/><0.01

115 (42.4)

50.1 (16.5)

54.8(32.7)

4.9

ns

Head-head display

1.9(0.8)

1.8(0.8)

2.1 (1.4)

4.7

ns

4.4 (0.8)

6.2(1.7)

3.6(0.8)

0.1

ns

Shaking

1.3(0.5)

1.2(0.5)

0.5 (0.4)

5.9

P<Q.Q\

1.3(0.2)

0.5 (0.2)

0.5 (0.2)

9.2

P < 0.01

Air-gulping

3.4 (0.9)

1.7(0.5)

0.4(0.3)

15.1

F<0.01

3.1 (0.6)

2.6(0.9)

1.6 (0.6)

4.0

ns

Parallel swimming

22.4(6.2)

16.2 (5.7)

12.3(2.8)

8.8

P < 0.01

20.4 (7.9)

3.1 (1.4)

2.5 (0.4)

0.9

ns

Head-tail display

39.6 (2.6)

31.4(3.2)

10 (5.2)

8.8

P < 0.02

32.8 (2.2)

22 (5.5)

17 (5.4)

0.2

ns

Biting

1 (0.2)

1.1 (0.2)

0.1 (O.I)

15.3

P < 0.01

1.6(0.3)

0.9 (0.3)

0.9(0.3)

16.9

P < 0.01

Mouthlocking

13.8(2.5)

9.2(3.4)

0.8 (0.8)

17.3

P<0.01

9.1 (1.8)

6.1 (2.4)

5.9(2.2)

3.3

ns

Note: As a summary of the results, the mean (standard error) and the result of the one-way Kruskal-Wallis ANOVAs are given. The level of
significance was greater than 0.05.

86

A. MIK.LOSI ET AL

20

18

16

14

12

10

8

6

4

2

Duration of threatening (a)

1.

encounter

2.
encounter

3.
encounter

200

180

160

140

120

100

80

60

40

20

Duration of fighting (b)

1.
encounter

2.
encounter

3.

encounter

Head-tail display (c)

Mouthlocking (d)

60

, N

to .-A
50

| 40
re

I 30

$ 20
+:

m
15 1 U

1.

encounter

3.

encounter

o

a

n>

20

18

16

14

12

10

8

6

4

2

1.

encounter

3.
encounter

1.6
'P 1.2

10

I 0.8

| 0.6

0.4

0.2

Biting (e)

i.

encounter

2.

encounter

3.
encounter

Group A: familiar opponents
Group B: unfamiliar opponents

Figure 1. The mean duration of threatening (a) and fighting (h), and the mean relative duration of head-
tail display (c) and mouthlocking (d) and biting (e) in the three consecutive encounters of the experimental
groups. In group A the opponents were the same for each contest: in group B the former dominant was
replaced hy a new dominant male for each fight.

were needed to induce significant changes in aggression.
On the other hand, repeated encounters with unfamiliar
individuals (group B) caused significant changes in only
some parameters of fighting. However, the changes that

occurred in group B are much less dramatic than those
in group A.

The behavior of the contestants was markedly similar
during the first two encounters in both groups. This means

OPPONENT-RELATED AND OUTCOME-RELATED MEMORY

87

that (1) there was no significant change within a group
from the first to the second encounter, and (2) behavior
did not seem to depend on the familiarity of the opponent
during the second encounter. The significant decrease in
the duration of fighting can be explained by noting that
the first encounter occurred after 3 days of isolation, dur-
ing which fish could build up energy reserves depleted
during the rather long fight (about 2 h on average) at the
first encounter (Haller and Wittenberger, 1988; Haller,
1 99 1 ). Without these energy reserves the second encounter
became shorter. However, it is also possible that isolation
increased aggression levels.

The defeated fish in group A (same opponents) fought
very similarly during the first and second encounters. Al-
though all defeated fish lost the fight again, they gave up
fighting only after a considerable time and engaged in
both signaling behavior (e.g.. head-tail display) and
strength-testing behavior (e.g.. mouthlocking). The same
happened in the group with unfamiliar opponents (B):
although defeated fish lost against the formerly dominant
opponents, the previous defeat did not seem to change
their behavior significantly.

Thus comparing the first two encounters in both groups
we find the effect of previous experience on behavior
"status- related memory" but no direct evidence of social
recognition. Since the initial work of Ginsburg and Allee
(1942), many studies have documented the effects of prior
experience (e.g., Bakker and Sevenster, 1983, Beacham
and Newman, 1987). In the case of the paradise fish, defeat
decreases the probability of subsequent winning in an ag-
gressive encounter, but prior winning has no influence
(Francis, 1983). However, three other factors might de-
crease the difference between a first and second encounter.
(1) Following longer isolation before the first contest
(3 days) and between contests (about 22 h), fish fight
longer in both the first and second encounters (Miklosi
et a/., unpub. obs.). (2) The encounter was terminated
1 h after fighting had finished, and fish were fed only fol-
lowing separation, thus opportunities for expressing
dominance or submission were limited. (3) The weight
symmetry between contestants rendered mutual assess-
ment more difficult, according to the resource holding
power (RHP) hypothesis (Parker, 1974). Usually larger
animals initiate aggressive encounters and are more likely
to win in a shorter fight. Thus similarity in size will in-
crease both the latency of initiation and the duration of
a contest.

The third encounter separates the two groups clearly.
For fish facing familiar opponents (group A ), the duration
of the threatening phase decreased by half, and previously
submissive fish gave up fighting soon after they began. In
contrast, no significant change was observed in the be-
havior of fish facing unfamiliar opponents (group B).
There was about a sixfold difference in biting, mouth-

locking, and duration of fighting between the two groups,
which rules out the role of exhaustion. In both groups,
submissive fish lost two fights before engaging in the third
contest: thus experience in submission or dominance
cannot explain the observed difference. As a result, the
involvement of some form of social recognition should
also be taken into account for the third encounter.

Nevertheless, this experiment does not directly prove
that individual recognition takes place. As Waas and Col-
gan (1994) recently noted, "Individual recognition implies
that subjects can distinguish between animals that belong
to the same social and physical class." But it is very difficult
to tell the exact basis of this form of recognition because
individuals can be categorized into several subcategories,
and the same animal can use different arrays of variables
to categorize its opponents. Because opponents were al-
ways of the same social class in both groups (submissive
or dominant), the recognition might have occurred on a
different level, which suggests that paradise fish are capable
of categorization within dominants or submissives.
Whether this can be described as individual recognition
remains to be seen, and Waas and Colgan (1994) show a
good way to examine this subject.

On the other hand, we already have some evidence that
individual recognition exists in fish (Gandolfi et a/.. 1973;
Zayan, 1975; Myrberg and Riggio. 1985). For example,
as shown by Zayan (1975), individual recognition of
formerly dominant fish can reverse the effect of prior res-
idence. Thus, the process of individual recognition inter-
acts with the effects of both prior experience and prior
residence in a way similar to that in our present experi-
ment.

It is usually assumed that the end of the fight depends
on the decision of the future submissive fish. This idea
stems from the classical conditioning view of aggression,
in which contact behaviors (biting, mouthlocking) are seen
as punishment for the opponent, which learns during the
aggressive encounter to avoid these aversive effects
(McDonald eta/.. 1968; Bakker el <//.. 1989). In this con-
text an individual opponent becomes a conditioned signal
for future punishment, but a new opponent acts as a dis-
criminative signal, which does not predict aversive stim-
ulation.

Another theory interprets aggressive encounters in
terms of an associative habituation process (Peeke, 1969;
Lorenz, 1981). New opponents (or territorial neighbors)
disrupt habituation and release an aggressive response.
For example, Peeke and Veno ( 1973) found that resident
male sticklebacks attacked unknown territorial neighbor-
ing sticklebacks more often than they attacked familiar
ones.

The problem of small changes in a complex stimulus,
like an opponent, raises a major problem to the incor-
poration of individual recognition into the learning mod-

A. MIKLOSI ET AL.

els presented above. Either dishabituation or discrimi-
nation suppose some form of recognition of the opponent,
but we do not know whether this categorization process
is similar in the two models or not. As is the case with
other processes described mainly on a behavioral level
(e.g.. imprinting), it is very difficult to explain them in
the framework of classical learning models.

At least in paradise fish, it seems that submissive fish
try to use every occasion that offers the possibility of win-
ning. Winning a contest presumably has many advantages.

Paradise fish males defend territories and build bubble-
nests in shallow waters of rice-fields, where several males
breed at the same time near each other (Forselius. 1957).
Recognizing neighbors could be advantageous because
males spend less time in aggression, leaving time for
courtship and later caring for the fry.

Our results support the hypothesis that aggressive ex-
perience in the paradise fish influences subsequent ag-
gressive encounters by means of two kinds of memory:
one related to the outcome of the encounter ("status-re-
lated memory") and the other related to the opponent
("social recognition").

Acknowledgments

This study was supported by the Hungarian Academy
of Sciences by an OTKA grant (No. 368-08 13\ 1991).
Three anonymous reviewers gave helpful comments on
the earlier version of this paper.

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Rapid Arm Movements in Stalked Crinoids

CRAIG M. YOUNG 1 AND ROLAND H. EMSON :

'Department of Larval Ecology. Harbor Branch Oceanographic Institution, 5600 U.S. //ivy / N..

Ft. Pierce, Florida 34946, and 2 Division of Life Sciences, King's College. London,

Campden Hill Road, London. W8 7 AH, United Kingdom

Abstract. Stalked crinoids in the family Isocrinidae have
been observed to wave individual arms actively. Using
video cameras mounted on a manned submersible, we
studied these movements and investigated the factors that
elicit them. Crinoids wave their arms in response to sand
or detritus dropped on their crowns, to entanglement in
tentacles of adjacent sea anemones, and to contact by
small crustaceans that might steal from the food grooves.
There was no evidence that arm waving functions in food
collection. In most cases, the movements could be attrib-
uted directly to mechanical stimulation by some natural
stimulus. The rapid effective stroke of an arm flexure is
caused by contraction of dorsal longitudinal arm muscles.
The slower return stroke results from the elastic recoil of
large ligaments near the aboral sides of the arms.

Introduction

Stalked crinoids are passive suspension feeders with
limited mobility but are nevertheless capable of several
kinds of movements. The most characteristic behaviors
are slow movements used to orient with respect to currents
and to hold the arms and pinnules in a parabolic feeding-
fan posture (Macurda and Meyer, 1974, 1976; Conan et
al.. 1981). The mechanisms by which these postures are
maintained and controlled are poorly understood. Ori-
entation of the stalk, which contains no muscles, is de-
pendent on mutable collagenous tissues (Wilkie et al.,
1993). The tonic posture of the parabolic feeding fan is
probably maintained by a similar mechanism, but there
is as yet no morphological or physiological evidence for
mutable arm ligaments (I. Wilkie, pers. comm.).

Stalked crinoids occasionally demonstrate fast muscular
movements. Several species are thought to be capable of
moving between attachment sites (Carpenter, 1884:

Received 16 March 1993; accepted 2 December 1994.

Conan et al., 1981: Roux. 1976), and stalked crinoids
have recently been observed crawling across the bottom
(Messing, 1985; Messing et al.. 1988). When stimulated
by the manipulator arm of a submersible or by very bright
lights, this same species, Endoxocrinits parrae, rapidly
flexes some or all of its arms in an adoral direction (Mess-
ing et al.. 1988; Young and Emson, unpub.). Except for
an unpublished anecdotal observation suggesting that cri-
noids may respond to suspended sediment (W. I. Ausich.
pers. comm.), all reports of rapid active arm movements
have involved strong artificial stimuli. The natural roles
of rapid arm movements remain undocumented. Here,
we describe in detail rapid arm flexures of some bathyal
isocrinids and present evidence that this behavior defends
crinoids against various biotic and abiotic threats.

Materials and Methods

Several species of stalked crinoid were observed from
Johnson-Sea-Link (JSL) submersibles at depths ranging
from 400 to 900 m in the northern Bahamas (see map in
Young, 1992). Still photographs were taken with a Benthos
35-mm camera equipped with an 80-mm lens, mounted
on the front of the submersible and focused with twin
laser beams that converged on a fixed focal plane. Video
footage was obtained with a Photosea Camera on a pan-
and-tilt mechanism and was recorded on W or hi-8 vid-
eotape. Video still sequences were taken from the tape
with a Seikosha VP-1500 video printer.

We obtained numerical data on arm-waving frequency
and crustacean abundance directly from the videotape.
We stopped the tape every 30 s and counted the number
of arm movements, the number of crinoids involved in
arm-waving behavior, and the total number of crinoids
visible in the frame. We ran the tape forwards and back-
wards a few frames at each census point to be certain that
arms counted as waving were really in motion and not

89

90

C. M. YOUNG AND R. H. EMSON

Figure 1. (A I Ijii/ii\ncniuit pumic with arms drooping in slack current. Note the single arm waving in
the water column (arrow). (B) Cenocriniis aslerius in current, showing parabolic feeding tan characteristic
of all Bahamian isocrinids. (C) E ptimic engaged in arm-waving behavior (arrow indicates moving arm).
(D) A dense population of E purrac with numerous individuals waving arms (indicated by arrow).

being held in a static posture. The number of small crus-
taceans in a frame was estimated by repeatedly passing
the video forward and back, frame by frame, while scan-
ning each part of the frame in succession for moving or-
ganisms.

The velocity of arm movement during effective and
recovery strokes was documented by laying down a time
code on the videotape with a hi-8 video editing machine
(Sony EVO-9700), then, during frame-by-frame analysis,
recording the time that movements were initiated and
completed (resolution: 0.067 s).

To investigate the possibility that sediment particles
might elicit arm waving, we used a suction tube on the
manipulator arm of the submersible to pick up a small
amount of sediment and release it about 1 m above an
aggregation of crinoids. This experiment was repeated on

six different occasions, while recording the responses of
crinoids on videotape. On some occasions, the sediment
consisted of fine silt; at other times, it was dominated
either by coarse sand or coarse, flocculent organic parti-
cles.

Crinoid arm pieces were fixed in 4% neutral buffered
formalin, decalcified in 70% acid alcohol, then embedded
in paraffin by standard histological procedures. Sections
were cut at a thickness of 8 ^m and stained with Milligan's
trichrome (Humason, 1972).

Results

Description and mechanics oj arm waving

At times of slack current, three Bahamian isocrinids,
f'~nc/o.\ocnnu,\ parrac, Cenocriniis asterius, and Diplocri-

ARM WAVING IN SEA LILIES

91

Figure 2. Video sequence of characteristic arm waving behavior in Endo.\ocnnii.\ parrac (A-C) Sequential
steps of the effective stroke. (D) Maximum arm extension. (E-F) Recovery stroke.

mis maclearanus, stand erect with arms drooping down
near the stalk (Fig. 1 A). In a current, these same species
form their arms into a parabolic fan for feeding (Fig. IB;
see also Macurda and Meyer, 1974, 1976), though the
uppermost few arms of the fan may sometimes be ex-
tended straight up into the water column. All three species
have been observed with individual arms waving up and
down rapidly (Fig. 1A, 1C). In dense populations, large
numbers of individuals have been observed to engage in
arm-waving behavior simultaneously (Fig. ID), particu-
larly after several minutes of illumination by the sub-
mersible.

Although we have occasionally observed arm flicking
or waving in animals with their arms extended in the
feeding posture, arm-waving behavior has been observed
more commonly in animals with drooping arms. The arm
is moved rapidly away from the stalk, sweeping outward
and upward until it is fully extended above or to the side
of the calyx (Fig. 2). The arm pauses only briefly at the
end of the stroke before reflexing downward more slowly
to its initial position. This entire movement may take as
little as 2 s or as much as 21 s. Frequency histograms of

the durations of effective and recovery strokes (Fig. 3)
show that the recovery strokes were more variable and
often longer than the effective strokes, but the two distri-
butions overlapped substantially. For individual strokes,
the ratio of the effective component to the recovery was
nearly always greater than 1 (Fig. 4). and the difference
between the durations of paired effective and recovery
strokes was highly significant (paired Student's / test,
54d.f., i = 5.75, P < 0.0000). The arms were flexed
through arcs ranging from a few degrees to more than
1 80 degrees. Most arms were flexed only once before an-
other arm was brought into play. Often, one arm was
flexed while another on the same animal was in its re-
covery stroke.

Examination of histological sections of the arm of E.
parrae revealed the presence of large dorsal (oral) longi-
tudinal muscles linking the arm segments (Fig. 5). These
muscles, which are described elsewhere (Hyman, 1955)
as flexor muscles, are clearly responsible for the flexure
of the arms. There are no opposing longitudinal muscles,
but large ligaments are found ventral (aboral) to the flexor
muscles (Fig. 5). The recovery phase of arm waving must

92

C. M. YOUNG

25
20 -

p

0)

D Effect

ve Stroke

2 15-

Recoi

ery Stroke

"o

1 10-

:

5 -

,

r

III In

1 P

5 10

15

Duration of Stroke (s)

Figure 3. Frequency histogram comparing the durations of the ef-
fective strokes (hatchedl and recovery strokes (solid black) during arm
flexure of EndoMicrinus parrae.

therefore be achieved by elastic recoil. Small dark-staining
cell bodies at the insertions of the ligaments (Fig. 5B)
appear to be juxtaligamental cells (Wilkie, 1984), which
are known to regulate collagen viscosity in other echi-
noderms. The largest axons in arm cross sections mea-
sured 3.75 ^m in diameter, and most were between 2.5
and 3.5 //m in diameter.

Functions of arm waving

With the use of a close-up video camera, we determined
that flexures often occurred in response to mechanical
stimuli caused by various organisms and particles. For
example, when the arms of stalked crinoids become en-
trapped in the tentacles of adjacent sea anemones, arm
flexures allow them to escape. Arms are also flexed in
response to contacts by small crustaceans. Such crusta-
ceans are always attracted to the lights of the submersible
in large numbers, affording us increased opportunities for
observing encounters between crinoids and crustaceans.
On 17 February 1980, we came upon a rocky ndge sup-
porting more than 200 E. parrae and C. asterius at a
depth between 409 and 500 m off Booby Rocks, New
Providence Channel, Bahamas. As we passed up the ridge
without stopping, we filmed 49 crinoids in two aggrega-
tions, observing all the while only two instances of arm-
waving behavior. We then rested the submersible near a
third large aggregation and filmed it from a distance of
3 m for 7 min. The percentage of animals participating
in arm waving and the number of arm waves per indi-
vidual increased linearly with the number of crustaceans
visible (Fig. 6). Although these regressions are consistent

with the idea that crustaceans stimulate arm waving, we
could not dismiss the possibility that density of crustaceans
covaried with some other factor (e.g., illumination time)
until video cameras with higher resolution were installed
in 1991.

On 24 October 1991 at a depth of 642 m off Egg Island,
we located a large aggregation of E. parrae. By focusing
on inactive individuals, we recorded 10 instances of arm
waving that were clearly stimulated by a single crustacean.
A representative encounter is shown in Figure 7. The time
required for initiation of a visible response to the impact
of this crustacean was 0.47 s. In every case, the crustacean
contacted the crinoid on the oral side of the arm between
the pinnules and in the region of the food groove. In one
observed encounter, the crustacean remained attached
during three sequential flexures before becoming dis-
lodged; in all other instances, the crustacean was dislodged
by the initial arm movement and swam away. On sub-
sequent dives, crustacean-induced arm movements were
also recorded for C. asterius, one of which is shown in
Figure 8. Here, the crustacean was swimming upstream
in the turbulent downstream wake of a crinoid feeding
passively in the current. When the crustacean contacted
the oral side of the arm, a small flexure was elicited im-
mediately (Fig. 8), and the crustacean moved downstream.

We dropped sediment from the manipulator on six
separate occasions with two to four attempts on each ex-
periment. Sediment containing a mixture of particles
ranging in size from 1 to several millimeters elicited dis-
crete flexures of individual arms when individual particles
struck (Fig. 9). Small amounts of very fine silt did not

20

-!-

0)

n.

15 "
CO

10 -

LLJ

C

o
ro
D

5 -

10

I
15

20

Duration of Recovery Stroke (s)

Figure 4. Durations of individual effective strokes plotted against
durations of corresponding individual recovery strokes for individual
arm flexures of Endoxocrinus parrae. If flexure and recovery were of the
same duration, all points would fall on the dashed line. Most points lie
below the line, indicating that recovery strokes are generally, but not
always, longer than effective strokes.

ARM WAVING IN SEA LILIES

93

200pm

Figure 5. Cross section (A) and longitudinal section (B) of an arm
of Endoxocrimtx parrae showing longitudinal flexor muscles (M), and
extensor ligaments (L) connecting portions of ossicles (O). Individual
bundles of collagen (CB) are visible in the ligaments. Cell bodies of jux-
taligamental cells ( JC) are visible at the points where collagen fiber bundles
insert into the brachial ossicles. PM: longitudinal muscle of a pinnule
cut in cross section.

stimulate waving, but fine sediment in large quantities
sometimes elicited a dramatic arm-waving response in-
volving numerous arms. Figure 10 shows the response of
one E. parrae individual to a large piece of flocculent
organic matter that lodged firmly on an arm. The crinoid
moved the affected arm as well as adjacent arms several
times until the material was dislodged.

Various kinds of crabs and ophiuroids (e.g.. euryalids)
commonly perch on sessile organisms, including large
sponges, gorgonians, and antipatharians, on the Bahamian
slope. These same organisms live on the stalks of crinoids.
but we have never seen a single individual occupying the
crown region. We suppose that arm waving might deter
occupation of the crown by ophiuroids and crabs, but
cannot prove this with observations.

Discussion

Virtually all sessile animals have neuromuscular
mechanisms for ridding themselves of impinging organ-
isms or objects that threaten them or that interfere with
the feeding process. It is not surprising, therefore, that
stalked crinoids would have an active mechanism of pro-
tection appropriate to their form and life style. In echi-
noderms, some protective mechanisms involve the use of
giant nerve fibers and very rapid (0.25 s) reaction times
(Cobb, 1985; Cobb and Ghyoot, 1993). The nerve fibers
of E. parrae measured between 2.5 and 3.75 /urn in di-
ameter, only about 30% as large as the giant fibers in
Ophiwa ophiura (Cobb, 1985). However, these are larger
than the 1 jum diameter neurons found in most echino-
derms (Cobb, 1985). Reaction times of stalked crinoids
(about 0.5 s) were about twice as long as those reported
for ophiuroids (Moore and Cobb. 1985).

On the basis of behavioral and histological observations,
it appears that arm flexure results from the contraction
of large flexor muscles, and that recovery results from the
elastic recoil of ligaments. This interpretation is consistent

2.5

'

. 2,0 -

g. 15H
en

I 1<H

0.5 -

00

r 2 =0725

10

15

1 I '
20

25

30

30

5 10 15 20 25

Number of Crustaceans Visible

Figure 6. The relationships between crustacean density and the
number of arms waving per crinoid (top panel) and proportion of crinoids
waving arms (bottom panel) during a single 7-min taping session in a
dense bed of Endoxwrinm pumic at 409 m depth at Booby Rocks. Ba-
hamas.

94

C. M. YOUNG AND R. H. EMSON

Figure 7. Response of Endoxocrinus parrae to an individual crustacean contacting the arm. (A-C)
Crustacean (position and direction of swimming indicated by arrows) enters rapidly from the right side of
the field and moves into crown region of crinoid. (D) Arm begins to flex immediately after crustacean
contacts it, and crustacean responds by swimming rapidly away (arrow). (E-F) Arm contacted by crustacean
continues to Ilex until it is maximally extended.

with the views of most previous workers (e.g., Muller.
1843; Breimer, 1978; Grimmer and Holland, 1987). An
alternative hypothesis, invoking hydraulic pressure in the
coelomic canals of the arms as a mechanism of arm ex-

tension, has been put forward by Candia Carnevali and
Saita (1985). Grimmer and Holland (1987), however,
showed experimentally that destruction of these coelomic
canals did not affect recovery from flexure in the coma-

Figure 8. (A) Small crustacean swimming upstream (in direction of arrow) within the turbulent wake
of a feeding Ccnucrinnx aslerius. (B) Small arm flexure following contact by crustacean (arrow), which is
thrown downstream.

ARM WAVING IN SEA LILIES

95

Figure 9. Arm flexures of Cenocrimis axicrinx in response to sand particles dropped on the crown. Sand
particles (one is indicated by arrow in A) were falling throughout this sequence, which involved movements
by arms on all sides of the crown.

tulid Florometra serratissima. In fact, their experimental
results demonstrate that elastic forces in the aboral soft
tissues of the arm of a comatulid were alone sufficient to
drive a power stroke in active swimming. We suggest that
a similar mechanism is involved in the recovery stroke of
stalked crinoids such as E. parrae.

Existing evidence does not support the idea that arm
waving is involved in food collection. Unlike some other
sedentary filter-feeding echinoderms at bathyal depths
(e.g., brisingid asteroids: Emson and Young. 1994),
stalked crinoids are not known to capture large particles
(Meyer, 1982; Lawrence, 1987). Indeed, our observa-
tions indicate that large particles contacting the crown
are thrown away from the crown, not toward the mouth.
Arm waving could enhance encounters with small par-
ticles, particularly in still water. However, such a feeding
strategy would seem inefficient in the oligotrophic hab-
itats where these animals live, since arm flexure requires
considerable muscular involvement and might result in
a net energy loss. By contrast, the maintenance of pos-
ture for passive feeding may involve catch connective

tissues (Wilkie and Emson, 1988) which require little
energy expenditure. Our observation of apparent jux-
taligamental cells (Fig. 5B) is equivocal evidence for
mutable collagenous tissues in stalked crinoid arms. The
use of arm flexing for filter feeding seems unlikely, but
cannot be discounted completely.

Arm-waving behavior appears on present evidence
to be principally a mechanism to eliminate inorganic
particles from the crown and to deter small organisms
from settling on the arms of the crinoid. Specifically,
we have demonstrated that the behavior has a deterrent
effect on small crustaceans, preventing them from acting
as predators, food thiefs, or opportunistic commensals.
Arm movements may prevent colonization of the crown
by crabs, ophiuroids, and other epifauna, and they per-
mit escape from the feeding structures of adjacent sessile
organisms such as sea anemones. Inorganic and organic
particles that fall on the arms also elicit arm waving,
so the behavior may have evolved as a general mech-
anism for ridding the crown of unwanted particles. As
all stalked crinoids that have been examined histolog-

96

C. M. YOUNG AND R. H EMSON

Figure 10. Response of Cenocrinus aslerius to fine sediment and a large flocculent mass of detritus
dropped on the crown. (A) Sediment contacts the crown, and a large particle of detritus (arrow) falls toward
one arm. (B) Detrital mass lands on the arm. Note that the fine silt elicits no dramatic responses from the
arms. (C) Arm (asterisk) with large detntal particle flexes, dislodging a portion of the mass. (D) Remainder
of detrital mass (arrow) remains on the arm. (E. F) Various arms in the region of the detrital mass flex
repeatedly, apparently attempting to dislodge the attached detritus.

ically have similar flexor muscles, we suspect that arm
waving may be a behavior of great antiquity and com-
mon to many living and extinct crinoids.

Acknowledgments

We thank the skilled pilots of the JSL submersibles for
assistance with close-up video. This work was funded by
the National Science Foundation (OCE-89 16264 and
OCE-91 16560) and by a NATO Collaborative Research
Grant (CRG-900628). Harbor Branch Contribution No.
1064.

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Brcimer, A. 1978. Recent cnnoids. Pp. 9-58 in Treatise on Invertebrate
Paleontology. Part T. Eclunodermalu. I . R. C. Moore and C. Teichert,
eds. University of Kansas Press, Lawrence, and the Geological Society
of America, Inc., Boulder, CO.

Candia Carnevali, M. D., and A. Saita. 1985. Muscle system organi-
zation in the echinoderms: II. Microscopic anatomy and functional

significance of the muscle-hgament-skeleton system in the arm of

the comatulids (Anledon medilerranea). J Morph 185: 59-74.
Carpenter, P. II. 188-4. Report on the Crinoidea collected during the

voyage of HMS 'Challenger' during the years 1874-1876. The stalked

crinoids. Challenger Rep Zoo/. 11, 442 pp.
Cobb, J. I.. S. 1985. The neurobiology of the ectoneural/hyponeural

synaptic connection in an echinoderm. Bio/. Bull 168: 432-446.
Cobb, J. {.. S., and M. Ghyoot. 1993. The giant through conducting

neuron of the hnttlestar Opliinru ophiura a key neuron? Coitifi.

Biiielieni Phyii'l 105A: 697-703.
Conan, G., M. Rouv and M. Sibuel. 1981. A photographic survey of

a population of the stalked crinoid Diphcrimis (Annacrinu.i) wyvil-

lcllinin\iini (Echinodermata) from the bathyal slope of the Bay of

Biscay. Deep-Sea Res 28A(5): 441-453.
I ins. .11. R. II., and C. M. Young. 1994. The feeding mechanism of a

deep-water hrisingid asteroid, Novodinea antillensis. Mar Bio/ 1 18:

433-442.
Grimmer, J. C., and N. I). Holland. 1987. The role of ligaments in

arm extension in feather stars (Echinodermata: Cnnoidea). Ada Zool.

68: 79-82.
Ilumason, G. I.. 1972. Animal Tissue Techniques. W. H. Freeman,

San Francisco. 641 pp.

Hyman, I,. II. 1955. The Invertebrates. II'. Echinodermata. McGraw-
Hill, New York. 763 pp.

ARM WAVING IN SEA LILIES

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Lawrence,.). 1987. A Functional Biology of Echinodemu Johns Hop-
kins University Press. Baltimore. 340 pp.

Macurda, D. B., and I). 1.. Meyer. 197-1. Feeding posture of modern
stalked conoids, .\\nurc 247: 394-396.

Macurda. I). B.. and I). I.. Meyer. 1976. The identification and inter-
pretation of stalked conoids from deep-water photographs. Bull Mar,
Sci 26:205-215.

Meyer, D. L. 1982. Food and feeding mechanisms: Crinozoa. Pp. 25-
42 in Echinoderm Nutrition. M. Jangoux and J. M. Lawrence, eds.
A. A. Balkema, Rotterdam.

Messing, C. 1985. Submersible observation of deep-water crinoid as-
semblages in the tropical western Atlantic Ocean. Pp. 185-193 in
Echinodcrmata, B. F. Keegan and B. D. S. O'Connor, eds. A. A.
Balkema. Rotterdam.

Messing, C. G., M. C. RoseSmyth, S. R. Mailer, and J. E. Miller.
1988. Relocation movement in a stalked conoid (Echinodermata).
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Moore, A., and J. L. S. Cobb. 1985. Neurophysiological studies on

photic responses in Ophnira ophiura Comp. Biochem. Physiot 80A:
11-16.

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\\ ilkie, 1. C. 1984. Vaoahle tensility in echinoderm collagenous tissues:
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\\ilkie, I.C., and R. H. Kmson. 1988. Mutable collagenous tissues and
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C. R. C Paul and A. B. Smith, eds. Clarendon Press, Oxford.

\\ilkie, I. C., R. H. Emson, and C. M. Young. 1993. Smart collagen
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Reference: Bin/ Bull 188: 98-1 10. (February/March, 1995)

The Structure and Mode of Function of the

Water Vascular System of a Brittlestar,

Ophioderma appressum

JOHN C. FERGUSON

Galbraith Marine Science Laboratories. Eckerd College, P.O. Box 12560'
St. Petersburg. Florida 33733

Abstract. Unlike the asteroids, which have large mad-
reporite structures, the ophiuroid Ophioderma appressum
possesses only two small hidden madreporite pores. Ex-
periments with labeled amino acids, fluorescent micro-
beads, and surgical obstruction show that small amounts
of seawater do routinely enter these pores and become
distributed throughout the water vascular system; but this
uptake does not seem essential. The flagellated stone canal
draws its fluid from the axial sinus, to which the pores
connect through a tortuous ampulla. Thus, the stone canal
mainly recirculates fluid from hyponeural (perihemal)
passages. That perihemal fluid is augmented by seawater
from the pores. As perihemal fluid moves towards the
stone canal, it passes by or through the axial organ, where
nutritive materials may be removed and passed into the
hemal channels. Pressure generated by the stone canal
forces flow out to the oral tube feet, polian vesicles, and,
through valves, eventually to the arm tube feet. Inflation
of the tube feet also might occur through osmotic mech-
anisms, but their activity was not impeded by raising the
external osmotic level with dextran. Observations indicate
that negative coelomic pressures must be generated during
respiratory movements, and these could lead to sufficient
body fluid production (by nitration) that the need for sub-
stantive madreporitic inflows would be alleviated.

Introduction

One of the most distinctive characteristics of echino-
derms is their water vascular system a unique arrange-
ment of fluid-filled coelomic passages and associated parts.
The general form of these structures in the different echi-

Received 25 July 1994; accepted 4 November 1994.

noderm classes has been summarized by Hyman ( 1955)
and Nichols (1966) from the works of pioneer micros-
copists. In most types, the water vascular passages open
to the exterior through a hydropore or compound mad-
reporite. and fluid within the system is used hydraulically
to extend agile appendages, the tube feet. With their va-
riety of functional adaptations, the tube feet are probably
responsible for much of the evolutionary success of this
major animal group. The water vascular system, however,
is much more complex in both structure and function
than is generally realized, and it presents many attributes
that are puzzling and. as yet, little studied.

It is generally assumed, for example, that the madre-
porite admits seawater into the system, and that ciliary
pumping by the stone canal connected to it forces the
seawater taken in to flow out to the tube feet where it
replenishes fluid losses due to their activity. That view,
however, was challenged by Binyon ( 1964, 1966, 1976a,
b, 1980, 1984), who observed that starfish tube feet remain
active for long periods without access to the madreporite.
He noted that ionic elevation of the water vascular fluid,
particularly of potassium ion, could produce an osmotic
entry of water into the structures, making madreporitic
inflow unnecessary. Binyon opined that the madreporite
might be a relief valve, a supplemental mechanism, or
have some other unknown functions.

More recently, my own work with fluorescent molec-
ular tracers and microbeads, and other methods, has
shown that seawater does routinely enter the madreporite
of all starfishes tested; but this uptake is not specifically
directed at tube foot inflation. Rather, the uptake plays a
more important role in keeping the spacious asteroid body
filled with fluid (Ferguson, 1988, 1989, 1990a, 1992,
1 994). Further, the stone canal, through connections with

98

BRITTLESTAR WATER VASCULAR SYSTEM

99

Figure 1. Oral disk of Op/iioderma appressnm. The arrows mark a pair of central (C) and peripheral
(P) slits that open into one of the 10 genital bursae. Seawater is drawn from the side of the disk through the
peripheral genital bursal slits and is vented by the central ones.

Figure 2. Magnified view of the oral shield overlaying the axial complex, visible as a slightly darkish
area (arrow). The madreporite pores are out of sight in the crevice near the asterisk. Extended tube feet (TF)
can be seen in the mouth and on the arms.

HC

UAO

CRC

P V

V^-^v J

I. V

OHR

FIG.
-15

-14

-13
-12

-11

Figure 3. Organization of the water vascular system. The oral tube
feet have been omitted and the arm tube feet and axial complex are only
partially shown. A, ampulla: CRC. circumoral nng canal; OTC, oral
tube feet canal; PP. primary madreporite pore; PV, polian vesicle; RAS.
right axial sinus; RC, radial canal; SC, stone canal; SP, secondary mad-
reponte pore; TC, transverse canal.

-10

-9

-8
-7
-6
-5

Figure -4. Detailed diagram of the madreporite-axial complex, with
a scale showing the approximate level of serial sections (Figs. 5-15). A.
ampulla; CRC, circumoral ring canal; GHS, genital hemal strand; GS.
genital bursal slit; HC, hyponeural coelom; LAO, lower axial organ;
LAS, left axial sinus; OHR, oral hemal ring; PP. primary madreponte
pore; UAO, upper axial organ; RAS, right axial sinus; S, slit in axial
organ connecting left and right axial sinus; SC, stone canal; SP, secondary
madreporite pore.

100

J. C. FERGUSON

.*'/>* i 1 '- ' '

: i**-'<fe-' rii
4 1^3^ ;i\ '

Figure 5. First of a set of serial frontal sections from the madreporite oral shield upwards. It shows part
of the large, angular ampulla (A) that lies within the oral shield. The edge of the secondary madreporite
pore (SP) can be seen on left, facing the cleft formed by the edge of the genital bursal slit. (All scale bars
= 50 ^m).

Figure 6. Section located 2()(im higher than Figure 5, showing the secondary pore opening (SP) and
the lowest portion of the left axial sinus (LAS). The pore canal lends towards the site of the primary pore,
away from the direction of inflow through the tortuous ampulla (A).

BRITTLESTAR WATER VASCULAR SYSTEM

101

other parts, must maintain an internal system of circu-
lation and filtration of the body fluids. Some starfish, such
as the intertidal Pi\ii\ter ncliruccnx, cannot adequately
maintain their body fluid volume without access to the
madreporite, while others, such as Pycnopodia helian-
thoides, rely more heavily on osmotic uptake (Ferguson,
PN100,,,L,I01992, 1994). Physical differences between
species largely reflect their individual adaptations to fluid
volume maintenance. Pinaster has a heavy, stiff, and im-
permeable integument, while that of Pycnopodia is light,
flexible, and much more permeable.

Because the function of madreporite mechanism in dif-
ferent asteroids varies considerably, similar diversity
should be expected in the other classes; indeed, differences
in body form between the classes might relate to their
individual approaches to solving the problems of body
fluid maintenance. Although ophiuroids share many
characteristics with the asteroids, they are also quite dif-
ferent. Here I begin a detailed examination of the structure
and function of an ophiuroid water vascular system-
thai of Ophioderma appressnm. Comparison of its prop-
erties with those of other echinoderms should broaden
our understanding of the adaptations of these diverse or-
ganisms.

Materials and Methods

Specimens of Ophioderma appressnm were collected
from tidal grass flats in Tampa Bay and maintained in
running, fresh seawater until used for experiments. This
species is very similar to Ophioderma hrevispinum (re-
ferred to in earlier work), but grows larger and possesses
more equal-dimensioned oral shields (five large plates
surrounding the mouth). As in other ophiodermids. the
openings into its genital bursae (pairs of pouches invagi-
nated into the disk on either side of the arm bases) are
each separated into two slits a "central" one near the
mouth and a "peripheral" one on the edge of the disk. Its
madreporite cannot be seen externally, and the structure
of its water vascular system must be inferred from old
descriptions of other ophiuroid species (cf. Hyman, 1955;
Nichols, 1966).

Because the internal parts of the water vascular system
are all small and embedded in ossicle and fibrous con-
nective tissue, they are best studied with histological
methods. To locate the site of the madreporite and ex-
amine the organization of the entire system, two small
specimens (6-7 mm disk diameter) were fixed in Helly's
fixative and decalcified for one week in changes of cold
(4C) 5% disodium EDTA. After paraffin embedding, se-
rial frontal sections (from the mouth through the top of
the disk) were cut at 10 /^m thickness and stained in
Delafield's hetnatoxylin, with eosin and orange G as
counter stains.

The extent of seawater entry through the madreporite
was evaluated in two sets of experiments. The first of
these was actually an unpublished part of a former study
dealing with ingestion and hemal transport by Ophio-
derma( Ferguson, 1985). A pair of brittlestars was placed
for 8 h in seawater containing 100 ^Ci' 4 C synthetic algal
protein hydrolysate, 375 mg glycine, 990 mg glucose,
and 10 mg chloramphenicol per liter. They were then
fixed and decalcified in Bouin's solution, embedded in
paraffin, sectioned (8 ^m). and exposed for up to
6 months to Kodak AR-10 autoradiographic film. In
the second set of experiments, fluorescent microbeads
were employed. These had been used to study madre-
porite function of the starfish Leptasterias hexactis (see
Ferguson. 1990a). Two brittlestars were placed in a dish
containing about 100 ml of seawater along with about
0.5 ml of 0.2-/jm YG "Fluoresbrite" carboxylate beads
(Polysciences, Inc.). After 48 h they were removed,
rinsed in seawater, fixed for 24 h in 10% formalin, de-
calcified for 7 days in cold 5% disodium EDTA,
embedded in gelatin, and cut as 20-Mm frozen sections.
The distribution of the beads was then observed by epi-
fluorescent UV microscopy.

Further tests were conducted to see if madreporite
oblation would lead to diminished functions. Specimens
(groups of 6) were anesthetized by a brief immersion
in 8% MgCl in tap water. The region of the madreporite
and stone canal (identified by the pigmentation pattern)
was then surgically destroyed and sealed with a small

Figure 7. Section just above Figure 6, showing the primary madreporite pore (PP) in the crevice of the
genital hursal slit. A, ampulla.

Figure 8. Section located 40 jim higher than Figure 7. The ampulla (A) is opening into the bottom of
the right axial sinus (RAS). opposite to the opening of the stone canal (SO, which contains long flagella.
The lower axial organ (AO) can now be seen in the left axial sinus (LAS).

Figure 9. Section located 60 ^m above Figure 8, showing typical form of the axial complex in this region.
nThe axial organ (AO) here is thin and contains only a few cell nuclei. It is applied closely to the flagellated
columnar cells of the stone canal (SC). LAS, left axial sinus; RAS. right axial sinus.

Figure 10. Section about 100 |im higher than Figure 9. At this level the axial complex lies between the
perivisceral coelom (PC) and the interradial muscle (IM). The axial organ (AO) gives off a pair of genital
hemal strands (GHS), surrounded by extensions of the left axial sinus (LAS). The right axial sinus (RAS) is
beginning to expand.

''>P?

-

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P'igure 11. Section about 170 ^m higher than Figure 10. In this region the nght axial sinus (RAS) has
expanded laterally and nearly surrounds the rest of the axial complex. It is separated by only a very thin
double peritoneal membrane from the extension of the perivisceral coelom (PC) that accompanies the large
interradial muscle (IM). GB, genital bursa; LAS, left axial sinus.

BRITTLESTAR WATER VASCULAR SYSTEM

103

cement plug. Control animals were treated similarly,
but a comparable wound was made through an oral
shield on the opposite side of the body from the mad-
reporite. All of the animals recovered from the imme-
diate effects of the anesthetic in about an hour. They
were then observed for 7 days to see if their tube feet
would extend and make normal stepping motions, and
if the specimens would right themselves when over-
turned. The animals also were periodically blotted and
weighed to see if weight variations could demonstrate
any changes in body fluid content. The cement plugs
were usually lost after about the second day as growing
tissue sealed the wound. Otherwise, all animals except
one, which fractured in two, appeared healthy through-
out the study period.

In a final set of observations, groups of three specimens
(tests and controls) previously operated on in the above
manner were placed in seawater to which sufficient dex-
tran (M.W. 5300) had been added to raise the osmotic
concentration 20 mosmoles/kg. The purpose of this ap-
proach was to try to counter any osmotic inflow by using
an osmolyte that should be neither permeable nor chem-
ically harmful to the integument. The animals were ob-
served and weighed as before until, on the third day, the
experiment was terminated.

Results

As previously noted madreporitic pores cannot be seen
in intact specimens of Ophioderma appressum. Exami-
nation of the serial sections revealed that they are small
and hidden by the outer edge of one of the oral shields.
It was found, however, that their position on the body
could usually be determined by the slightly greater amount
of pigmentation on that oral shield compared to the others,
or if the pigmentation were absent, by the dark shadow
of the ampullary complex showing through the translucent
oral shield (Figs. 1,2). From the oral perspective, the pores
lie just within the crevice of the central genital bursal slit
on the distal left side of the oral shield. Normally, water

currents could be seen passing into the peripheral slits of
the genital bursae and exiting through the central ones.
Thus, while the animals lie on the silty bay bottom, the
madreporitic pores are mainly bathed by seawater that
has passed through the genital bursae from the presumably
cleaner source at the edge of the disk. The water currents
within the genital bursae are maintained by cilia as well
as by rhythmical expansions and contractions of the whole
upper part of the disk.

Structure

The organization of the entire water vascular system is
shown in Figure 3, and a more detailed representation of
the complex madreporite-axial structure in Figure 4. Fig-
ures 5-18 show views of some of the serial sections from
which these two diagrams were developed (differences be-
tween the two specimens studied were negligible). To
avoid confusion in the following descriptions, the pho-
tomicrographs have been reversed in printing to compen-
sate for the normal optical inversion of the compound
microscope.

The primary opening into the system from the exterior
is a 10-15 nm diameter pore located in the folded edge
of the central genital bursal slit, just under the lip of the
oral shield (Fig. 7). Several undulations occur in the layer
of cuboidal cells that line the pore passage; raising the
possibility that more pores might develop in specimens
more fully grown then the two studied. A second pore of
nearly the same diameter is located about 150 ^m lateral
and slightly lower than the first (Fig. 6). Its duct points
towards the first and away from the stone canal. This
orientation suggests that the secondary pore may be a
rejection pathway, but there is no way of verifying that.

Both pores lead to a discrete lobe of the madreporite
"ampulla." This spacious chamber lies just under the sur-
face of the oral shield. It is lined with a distinctive cuboidal
epithelium, and it has several regions separated by sharp
angles (Figs. 5-8). After making tortuous turns, it opens
broadly into the lower end of the "right" axial sinus (Fig.

Figure 12. Section 70 Mm above Figure I I. The upper axial organ (UAO) has formed large cellular lobes
that hang down into the right axial sinus (RAS). The left axial sinus (LAS) penetrates the axial organ with
slit-like spaces (S) that continue through to the surrounding right axial sinus. The lower axial organ (LAO),
containing only scattered cell nuclei, still lies close to the stone canal (SO.

Figure 13. Next section above Figure 12. The stone canal (SC) here connects to the circumoral ring
canal (CRC). The cellular lobes of the axial organ and the slits between them connecting the two parts of
the axial sinus are quite evident.

Figure 14. Section 40 ^m above Figure 1 3. showing the top of the axial organ forming its cellular lobes.
Parts of the undulating circumoral ring canal (CRC) are visible on either side of it.

Figure 15. Section 60 nm above Figure 14, showing extensions from the upper axial organ (UAO) and
the right axial sinus (RAS) toward the circumoral hyponeural coelom (HC) and the oral hemal ring (not
visible). N. nerve ring.

Figure 16. A polian vesicle (PV) lying in the perivisceral coelom (PC) next to an interradial muscle
(IM). Note the heavy wall of the vesicle and its elastic membrane (arrow). GB, genital bursa.

104

OTC

J. C. FERGUSON

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Figure 17. The connection of an oral tube foot (TF) with its canal (OTC) from the circumoral ring
canal. There is a muscular restriction at the neck (arrow), hut no valve. N, nerve ring.

Figure 18. A transverse canal (TC) in an arm connecting to a tube foot (TF). It has a well-developed
valve. A strand of hemal tissue (H) connecting the tube foot to the radial hemal vessel lies nearby.

BRII IILSTAR WATER VASCULAR SYSTEM

105

8). Opposite this opening is the entrance to the lower end
of the stone canal (Fig. 8), which thus does not directly
connect with the ampulla, but rather with the right axial
sinus. On the far side of the stone canal (and attached
separately to it) is the "left" axial sinus, a somewhat
smaller chamber. It has no opening at the lower end, but
contains the axial organ (part of the hemal system),
broadly attached to the wall of the stone canal. Both parts
of the axial sinus are lined with a thin, simple squamous
peritoneum. The cells forming the stone canal stain
densely. They are cuboidal adjacent to the right axial sinus,
and columnar next to the axial organ. Long flagella extend
from the columnar cells and reach nearly across the 15-
20 nm width of the somewhat flattened lumen (Figs. 9-
1 2). The close proximity of the hemal tissue to the position
of the flagellated cells suggests that hemal fluid might
supply the high levels of energy nutrients that must be
needed by these cells to produce a current within the stone
canal.

The four structures (the two separate parts of the axial
sinus, the stone canal, and the axial organ) all rise in a
long arch towards the ring complex located high up in
the mouth frame. As they rise, they sweep over the large
interradial muscles (Figs. 10, 1 1). About one third of the
way up, the axial organ gives off the genital hemal strands
surrounded by extensions of the left axial sinus (Fig. 10).
These are thought to supply nutritive materials to the go-
nads (Walker, 1982; Byrne, 1988, 1989). Above that point,
the right axial sinus begins to expand and stretch out lat-
erally (Fig. 1 1 ). It comes to surround, almost completely,
the other three structures, and medially it forms a long
thin boundary with the perivisceral coelomic extension
that partially encases the interradial muscle. On the distal
side, it is separated from the perivisceral coelom by a thin
layer of connective tissue (Figs. 11-15).

After the right axial sinus begins its expansion, the left
axial sinus splits into a series of slit-like passages that enter
the axial organ, which now becomes highly cellular and
bulges out like a cauliflower (Figs. 1 2, 1 3). These passages
then open extensively into the right axial sinus, completing
the connection between the two portions of that cavity.
At its upper end, the axial organ becomes more solid again

(Fig. 14) and gives off an extension to the oral hemal ring
(Fig. 15). This extension is surrounded by a portion of
the right axial sinus, which appears to connect with the
hyponeural (perihemal) compartment that lies adjacent
to the nerve ring. This connection is not a spacious open-
ing, but a complex grouping of peritoneal cells and hemal
tissues that were difficult to resolve in the preparations
studied.

At the same level in which the two parts of the axial
sinus join, the top of the stone canal bends over and joins
the ring canal that encircles the mouth (Fig. 1 3). The ring
canal, like the other canals of the water vascular system,
is lined with squamous cells surrounded by an elastic
membrane, fibrous connective tissue, and a few muscle
cells (Figs. 13. 14). In the other four interradii, the ring
canal gives off canals that open into bulbous polian ves-
icles. These lie in the perivisceral coelom next to the in-
terradial muscles. Their walls possess a conspicuous elastic
membrane like that of the water canals (Fig. 16). In each
radius, the ring canal gives off three branches a radial
canal that descends and runs out the arm, and, well to
either side of it, canals that connect to upper and lower
oral tube feet that lie horizontally between the jaws, within
the mouth frame. There are no valves between the oral
tube feet and their connecting passages, although sphincter
muscles may be able to restrict the openings (Fig. 17).

Along the arms, transverse canals extend in pairs from
the radial canal. At the opening of each canal into a tube
foot, there is a well-developed valve that holds fluid within
the appendage (Fig. 18). Accessory elastic vesicles, such
as Woodley ( 1967) described as extending from the radial
canals of Amp/iiiim lililornns. were not seen. When the
tube foot retracts, it pulls up into a surrounding sheath
that is then closed over by two or three flattened spines.
When it extends, it slides out of this sheath as a unit and
then stretches out as a dexterous tentacle. Reiger and
Lombardi (1987) have reported on the ultrastructure of
the wall of the tube feet of Ophioderma brevispinum and
other species.

The radial canals lie in loose connective tissue in the
lower portion of the arm. Below them (above the nerve
cord) are extensions of the hemal tissues and hyponeural

Figure 19. An autoradiograph of an unstained radial section of the disk of an animal exposed to I4 C-
amino acids in seawater for 8 h. Note the considerable darkening (radioactivity) in the ampulla (A). A small
amount of label, perhaps from ingestion, is in the upper axial organ (UAO). The exposed epidermis (E) is
intensely labeled.

Figure 20. Autoradiograph of a section near that of Figure 18. Label is seen in the ampulla (A), lightly
in the stone canal (SC), and a bit more intensely in the upper axial organ (UAO).

Figure 21. Autoradiograph of a transverse section of an arm of the same specimen as Figures I 1 '. 20.
No radioactivity is seen in the radial canal (RC), but it is found clearly in the radial hemal vessel (H). Some
label may be in the lining of the tube foot (TF). but there is strong background from the heavily labeled
epidermis. N, radial nerve cord.

Figure 22. Autoradiograph of a radial section of arm base of the same specimen as others. Note the
high level of uptake in the radial hemal vessel (H). N. radial nerve cord.

106

J. C. FERGUSON

Figure 23. This and the next five figures show epifluorescent views of specimens exposed for 48 h to
fluorescent microbeads in seawater. Here a pore canal (C) extends towards the ampulla (arrow ). Numerous
beads are in the pore canal and epidermis (E); smaller numbers are in the cellular lining of the ampulla.

Figure 24. A view of the ampulla (A) just above the epidermis (E) of the oral shield. A few beads are
found in the ampullary chamber and some in its cellular lining.

BRITTLESTAR WATER VASCULAR SYSTEM

107

Table I

Weight variations (g) </Ophioderma oppression following destruction
of the madreporite area, and control animals with a comparable injury
to another site

Specimen Start 1 day 2 days 3 days 4 days 7 days

Matlreporite destroyed

1

1.14

1.12

1.11

1.07

1.07

1.09

2

1.01

1.01

1.02

1.02

1.07

1.04

3

1.29

1.28

1.29

1.26

1.32

1.28

4

1.49

1.50

1.57

1.50

1.57

1.55

5

0.61

0.61

0.60

0.62

0.66

0.63

6

1.10

1.09

(fragmented)

Controls

1

0.98

0.98

0.97

0.97

0.98

0.94

2

1.97

1.96

1.98

1.97

1.99

1.96

3

0.83

0.83

0.87

0.85

0.83

0.83

4

0.58

0.50

0.48

0.50

0.50

0.50

5

0.59

0.56

0.57

0.58

0.58

0.59

6

0.64

0.59

0.58

0.59

0.58

0.59

coelomic spaces. Hemal tissue extends laterally to each
tube foot (Fig. 18) where it appears to join, near the valve,
with the middle connective tissue layer of the appendage.

Tracer studies

Autoradiographs of animals placed for 8 h in ' 4 C-amino
acid mixture showed intense incorporation of the tracer
within all exposed epidermal cells including those of the
tube feet and their sheaths. This result was expected from
earlier studies (Ferguson, 1967; Fontaine and Chia, 1968).
The concern here was whether the tracer could demon-
strate seawater entry into the water vascular system. That
was the case, as notable radioactivity was found in the
lining cells of the madreporite ampulla (Figs. 19, 20). This
labeling diminished rapidly up the stone canal (Fig. 20).
[In animals fed labeled surf clams, radioactivity was found
abundantly in the hemal tissues, but not extensively in
the water vascular passages (Ferguson, 1985)]. No label
was visible within the radial canals, and amounts in the
lumen of tube feet were low and not readily distinguished

from background (Fig. 2 1 ). The radial hemal vessels ac-
cumulated considerable amounts of label (Figs. 21, 22),
and a small amount was seen in the upper, cellular portion
of the axial organ (Fig. 19). These accumulations could
have come from an oral route (cf. Ferguson, 1985).

Fluorescent microbeads were employed as a means
of more directly following the flow of seawater into the
water vascular passages. Unlike the labeled amino acids,
these beads should not easily penetrate membranes or
become rapidly depleted by cells as they take up nu-
trients from the fluid flowing by them. However, the
entry of beads into the body must overcome the ani-
mal's natural defenses against foreign particles. A very
large number of beads was used in the medium with
the expectation that a proportion of them would get
through if bulk fluids were indeed entering the body
through the pores. After 48 h of exposure, fluorescent
beads were found in many water vascular passages, but
not in great abundance (Figs. 23-28). Beads were seen
moderately distributed within the lining of the ampulla
(Figs. 23, 24). Some were found free in the radial canals
(Fig. 25) and in the tube feet (Fig. 27). More commonly,
beads were found engorged in clumps of coelomocytes.
These often accumulated in the lateral canals just before
the valves (Fig. 26), or within the lumen of tube feet
(Fig. 28). Many tube feet, however, could be seen with-
out any beads within them. Coelomocytes bearing many
beads collected in the hemal tissues of the arms, es-
pecially between the radial hemal vessels and the tube
feet, and beneath the radial canals (Fig. 25). A few
clumps of coelomocytes containing beads were found
in the perivisceral coelomic spaces of the arms. All ex-
posed epidermal areas were filled with beads, including
the areas just outside the madreporitic pores (Fig. 23)
and the sheaths of the tube feet (Figs. 27, 28).

Experiments

Because the tracer studies showed that seawater must
enter the madreporitic pores, at least in small quantities,
a study was made to see whether that uptake was es-
sential to the animals. It was found that surgical oblit-
eration of the madreporite, ampulla, and lower stone

Figure 25. Transverse section through the ambulacrum of an arm. Several discrete microbeads lie in
the radial canal (arrow). Below the arrow is part of a hemal strand extending towards a tube foot. It contains
many coelomocytes engorged with the beads. Most of the other fluorescence is natural.

Figure 26. A clump of coelomocytes engorged with beads lying in a transverse canal just before the
valve. Two or three coelomocytes containing beads are nearby, within the tube foot.

Figure 27. Transverse section through a tube foot lying in its sheath. A number of the fluorescent beads
are seen in the lumen (arrow). The epidermal tissue (E) of the tube foot and its sheath contains many beads
that were taken up directly from the seawater.

Figure 28. Transverse section through another tube foot. This one contains, in its lumen, many free
beads as well as coelomocytes engorged with beads.

108

J. C. FERGUSON

Table II

II t>/i,'/;; varuiliim* (g) l Ophioderma appressum in scauatcr raised 20
inouiMle.s/kH with de\nwi (5300 Jl/.H'J; specimen* mill inailrci'onlc.s
destroyed uiul control animal* with a comparable injury in another site

Specimen

Start

4 hours

1 day

2 days

3 days

Madri'poriie clem roved

1

1.09

1.04

1.05

1.06

1.12*

2

1.28

1.24

1.23

1.23

1.28*

3

0.65

0.61

0.60

0.60

0.69*

Controls

1

0.69

0.66

0.64

0.64

0.65*

2

0.45

0.43

0.41

0.40

0.42*

3

1.09

1 .05

1.01

1.02

1.04*

* Animals abnormal arched up and rigid.

canal had very little effect on tube foot function for at
least a week. For the first two or three days, tube foot
activity declined somewhat, especially in the oral tube
feet, but there was no consistent difference in observed
behavior when compared to controls. All tube feet could
extend and bend, and when animals were overturned,
righting movements involving the arms were unaffected.
The number of tube feet active at any one moment may
have diminished, but with the diversity of individual
behavioral responses demonstrated by the specimens,
that could not be quantified. Nor were there any con-
sistent variations in body weights that would reflect fluid
volume changes (Table I). Both test animals and con-
trols varied a few percent from day to day, but not sig-
nificantly.

If tube foot inflation and body fluid content are main-
tained by osmotic elevation, as by a potassium ion pump
in the tube feet (if. Prusch, 1977), the mechanism could
be sensitive to elevated colloidal osmotic pressure in
the medium. To test this possibility, animals with obliter-
ated madreporites, and controls, were placed in dishes
of seawater in which the osmotic levels had been ele-
vated a modest amount (20 mosmoles/kg) with dextran
(5300 M.W.). In both groups the immediate effect was a
small loss in weight (2 to 4%) over the first few hours and
then stability within a normal range (Table II). For the
next two days there was no diminishment of tube foot
function or other observable effects. On the third day.
animals in both groups showed some arching rigidity ot
their arms, rather similar to that seen previously in animals
placed in ionically altered seawaters. At that point the
experiment was terminated.

Discussion

This study has shown that Ophindcnna has a complex
water vascular system, but one in which the passages from

the exterior (the madreporite pores) appear to be of minor
importance compared to provisions for internal recircu-
lation of fluid via the axial sinus and its extensions. Al-
though it was shown that seawater routinely enters the
pores, it must do so only in small quantities. Under the
laboratory conditions employed, this uptake seemed to
provide little advantage. Perhaps under the stresses of the
natural environment uptake may be important in some
circumstances, but such conditions have yet to be dis-
covered. Fluid might also exit the pores when pressures
in the system become too great.

In contrast to the minimal madreporite of Ophioder-
ma, the madreporites of asteroids have many pores and
complex arrangements of ciliated gutters to keep them
free of suspended foreign particles (Ferguson and
Walker, 199 1 ). In one study on Echinaster graminicola,
seawater uptake through the madreporite was equal to
about 5.5% of the animal's body weight per day; and
more than half of it went into replacing the general
body fluid (Ferguson. 1989). The two small pores of
Ophioclcrnui. located very inconspicuously at the edge
of a genital bursal slit, clearly cannot allow much sea-
water into the system. Reduced inflow would alleviate
contamination from the silty environment in which the
animals normally live. Further, the genital bursa might
protect the pores from silt, as the asteroid gutters do.
Likewise, the complex form of the ampulla probably
plays a sanitary role, because many beads were seen
taken up by its cells.

The stone canal itself is fairly well developed. It is
smaller in diameter than those of asteroids, and it does
not have the internal ridges that make a larger diameter
tube more efficient as a ciliary pump. It also does not have
as much bony ossicle material, needed by a larger structure
for strength. However, for the much smaller body size of
Ophioflcmui. the stone canal seems proportionately
scaled. Its lumen is somewhat flattened, which allows the
flagellated cells to be very efficient in sweeping fluid
through the canal.

As noted, the stone canal does not connect directly
with the ampulla, but rather with the right axial sinus.
If fluid flows down this sinus into the stone canal, as it
appears to, it would be drawn from three sources: ( 1 )
the left axial sinus (and the genital perihemal vessels),
through the slits of the axial organ; (2) the circumoral
hyponeural (perihemal) coelom (and its connections
with the radial hyponeural coelomic spaces of the arms),
over the surface of the axial organ; and (3) through the
delicate peritoneal membranes separating much of the
axial sinus from the perivisceral coelom. I conclude,
then, that most of the fluid pumped by the stone canal
is coelomic not seawater flowing in through the two
madreporitic pores. As the fluid flows over the axial
organ it is probably purified, and it probably gives up

BRITTl FSTAR WATER VASCULAR SYSTEM

109

nutritive materials to form hemal fluid. [The transport
of nutritive material by hemal systems was previously
described (Ferguson, 1984, 1985)].

The fluid pumped by the stone canal then passes
through the water vascular canals, and most of it even-
tually travels out the arms. Some is diverted to the oral
tube feet and the polian vesicles, which among other
functions, probably collectively serve as a general reservoir
and pressure stabilizer for the system. In the arms, the
fluid passes through the valves into the tube feet only
when the hydrostatic pressure of the system surpasses that
maintained in the appendages. The tube feet also might
be kept inflated by osmotic inflow. Based on the obser-
vations on other animals by Robertson (1949), Binyon
(1976b), Prusch (1977), and Ferguson (1990b), the tube
feet likely contain a higher osmotic pressure than the sur-
rounding seawater. In the present experiments, however,
the tube feet failed to collapse when the external osmotic
pressure was increased with dextran, though by the third
day the treatment appeared to produce pervasive detri-
mental effects on the bodies of the animals. Although the
water vascular vessels can deliver fluid to the tube feet,
they may be equally valuable in providing a more general
circulatory flow by permeation to all the tissues of the
lower arms, and return via the hyponeural spaces. Ad-
ditional circulation is achieved by the perivisceral coe-
lomic passages.

When compared with the body cavities of asteroids,
the perivisceral coelom of Ophioderma is not large.
Within the arms it consists mostly of canals that ex-
pand into larger spaces between the vertebral-like os-
sicles. Asteroid perivisceral fluid is kept under low,
but positive, hydrostatic pressure (Ferguson, 1988).
That cannot be the case in Ophioderma, Distinct
"breathing" motions of the aboral disk alternately
stretch and compress the coelomic space, and that
movement pumps seawater in and out of the genital
bursae. The ventilation must produce negative coe-
lomic pressures that should lead to the accumulation
of fluid in the coelomic space by nitration. Pressure
from pumping by cilia in the genital bursae would have
the same effect. [Net negative coelomic pressures have
recently been described in sea urchins by Ellers and
Telford ( 1992), but these are produced by a different
mechanism.] It appears, then, that Ophioderma has
little need to take up seawater through its madreporite
either to support its tube feet or to maintain its peri-
visceral coelomic fluid, and it has a limited ability to
do so. Asteroids, on the other hand, often do tend to
lose large amounts of fluid from their bodies and must
replace it. For them, the madreporite system (together
with the Tiedemann's bodies) is a much more impor-
tant and well-developed mechanism.

Literature Cited

Binyon, J. 196-4. On the mode of functioning of the water vascular
system of Asterias nihcns L. ./ Mar. Bio/. Assoc. U. A" 44: 577-
588.

Binyon, J. 1966. Salinity tolerance and ionic regulation. Pp. 359-378
in Physiology of Echinodermata, R. A. Boolootian, ed. Wiley-Inter-
science. New York.

Binyon, J. 1976a. The permeability of the podial wall to water and
potassium ions. J. Mar Biol Assoc V. K 56: 639-647.

Binyon, J. 1976b. The effects of reduced salinity upon the starfish
Aslenas rubens L. together with a special consideration of the
integument and its permeability to water. Thalassia Jugoslav
12: 15-20.

Binyon. J. 1980. Osmotic and hydrostatic permeability of the integu-
ment of the starfish A stenas ruhens ./. Mar Biol Assoc. U. A 60:
627-630.

Binyon, J. 1984. A re-appraisal of the fluid loss resulting from the
operation of the water vascular system of the starfish. Asterias rubens
J. Mar Biol Assoc V K 64: 726.

Byrne, M. 1988. Evidence for endocytotic incorporation of nutrients
from the hemal sinus by the oocytes of the brittlestar Ophiolepix
paucispimi. Pp. 557-563 in Echinoderm Biology: Proceedings oj the
St.\th International Echinoderm Conference, l'ictoria/23-28 August
1987. R. D. Burke, P. Mladenov, P. Lambert and R. L. Parsley, eds.
Balkema. Rotterdam.

Byrne, M. 1989. Infrastructure of the ovary and oogenesis in the ovo-
viparous ophiuroid Ophiolepis nuticispina (Echinodermata). Biol.
Bull 176: 79-95.

Ellers, O., and M. Telford 1992. Causes and consequences of fluctuating
coelomic pressure in sea urchins. Biol. Bull 182: 424-434.

Ferguson, J. C. 1967. An autoradiographic study of the utilization
of free exogenous amino acids by starfishes. Biol. Bull 133: 317-
329.

Ferguson, J. C. 1984. Translocative functions of the enigmatic or-
gans of starfish the axial organ, hemal vessels. Tiedemann's
bodies, and rectal caeca: an autoradiographic study. Biol. Bull
166: 140-155.

Ferguson, J. C. 1985. Hemal transport of ingested nutrients by the
ophiuroid, Ophioderma brevispinum Pp. 663-626 in Proceedings of
the l-'itih International Echinoderm Conference Galway/24-29 Sep-
tember 1984. D. F. Keegan and B. D. S. O'Connor, eds. Balkema.
Rotterdam.

F'erguson, J. C. 1988. Madreporite and anus function in fluid vol-
ume regulation of a starfish (Echinaster graminicola). Pp. 603-
609 in Echmotlenn Biology: Proceedings of the Sixth International
Ecliiiwdcrm Conference. \'ictoria/23-28 August 19S7. R D.
Burke, P. Mladenov, P. Lambert and R. L. Parsley, eds. Balkema,
Rotterdam

Ferguson, J. C. 1989. Rate of water admission through the madreporite
of a starfish. / E.\p. Biol. 145: 147-156.

F'erguson, J. C. 1990a. Hyperosmotic properties of the fluids of the
perivisceral coelom and watervascular system of starfish kept under
stable conditions. Comp. Physio/. Biochem 95A: 245-248.

F'erguson, J. C. I990b. Sea water inflow through the madreponte and
internal body regions of a starfish (Leptasteriax hexactis) as dem-
onstrated with fluorescent microbeads. / E.\p. Zool 255: 262-271.

F'erguson, J. C. 1992. The function of the madreporite system in body
fluid volume maintenance by an intertidal starfish. Pisaster ochraceus.
Biol. Bull. 183: 482-489.

F'erguson, J. C. 1994. Madreponte inflow of seawater to maintain body
fluids in five species of starfish. Proceedings of the 8th International
Echinoderms Conference. Dijon. France. D. Bruno and A. Guille,
eds. Balkema, Rotterdam (in press).

110

J. C. FERGUSON

Ferguson, J. C., and C. VV. Walker. 1991. Cytology and function of
the madreponte systems of the starfish Henricia sanguinolenta and
Aslerias vulgaris. J Morphol 210: 1-11.

Fontaine, A. R., and F. S. Chia. 1968. Echinoderms: an autoradio-
graphic study of the assimilation of dissolved organic molecules. Sci-
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Hyman, L. 1955. The Invertebrates. II' Echinodermata. McGraw-Hill,
New York.

Nichols, D. 1966. Functional morphology of the water-vascular system.
Pp. 219-244 in Physiology of Echinodermala, R. A. Boolootian, ed.
Wiley-Interscience, New York.

Prusch, R. D. 1977. Solute secretion by the tube foot epithelium in
the starfish Asteriax forbesi. J Exp. Biol 68: 35-43.

Reiger, R. M., and J. Lombard!. 1987. Ultrastructure of coelomic lining
in echinoderm podia: significance for concepts in evolution of muscle
and peritoneal cells. Zoomorphology 107: 191-208.

Robertson, J. D. 1949. Ionic regulation in some marine invertebrates.
/ Exp. Biol. 26: 182-200.

Walker, C. W. 1982. Nutrition of gametes. Pp. 449-468 in Echi-
noderm Nutrition. M. Jangoux and J. M. Lawrence, eds. Balkema,
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Woodley, J. D. 1967. Problems in the ophiuroid water-vascular system.
Svmp. Zoo/. Soc. Lund. 20: 75-104.

CONTENTS

No. 1, FEBRUARY/MARCH 1995

RESEARCH NOTES

Fluck, Richard A.

Responses of the medaka fish egg (Oryzias latipes)
to the photolysis of microinjected nitrophenyl-

EGTA, a photolabile calcium chelator

Wittenberg, Jonathan B., and Jeffrey L. Stein
Hemoglobin in the symbiont-harboring gill of the
marine gastropod Alvinichoncha hessleri

BIOMINERALIZATION

Giles, R., S. Manne, S. Mann, D. E. Morse, G. D.
Stucky, and P. K. Hansma

Inorganic overgrowth of aragonite on molluscan
nacre examined by atomic force microscopy . . .

Thorn, Kurt, Robert M. Gerrato, and Mark L. Rivers

Elemental distributions in marine bivalve shells as
measured by synchrotron x-ray fluorescence ... 57
Gherardi, Francesca, and Paul M. Cassidy

Life history patterns of Discorsopagurus schmitti. a
hermit crab inhabiting polychaete tubes .... 68

NEUROBIOLOGY AND BEHAVIOR

Carlberg, Mats, Karin Alfredsson, Sven-Olle Nielsen,
and Peter A. V. Anderson

Taurine-like immunoreactivity in the motor nerve

net of the jellyfish Cyanea capillata 78

DEVELOPMENT AND REPRODUCTION

Bates, William R.

Direct development in the ascidian Molgula retor-
tiformis (Verriil, 1871) 16

Chang, Wen-Teh, and Robert J. Lauzon

Isolation of biologically functional RNA during
programmed death of a colonial ascidian 23

Hamel, Jean-Francois, and Annie Mercier

Prespawning behavior, spawning, and development

of the brooding starfish Leptasterias polaris 32

ECOLOGY AND EVOLUTION

Mead, Kristina S., and Mark W. Denny

The effects of hydrodynamic shear stress on fer-
tilization and early development of the purple sea
urchin Strongylocentrotus purpuratus 46

Miklosi, Adam, Jozsef Haller, and Vilmos Csanyi

The influence of opponent-related and outcome-
related memory on repeated aggressive encounters
in the paradise fish (Macropodus opercularis) .... 83

Young, Craig M., and Roland H. Emson

Rapid arm movements in stalked crinoids 89

PHYSIOLOGY

Ferguson, John C.

The structure and mode of function of the water
vascular system of a brittlestar, Qphioderma apressum 98

Volume 188

THE

Number 2

BIOLOGICAL
BULLETIN

995

APRIL, 1995

Published by the Marine Biological Laboratory

THE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

Associate Editors

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WILLIAM D. COHEN, Hunter College, City University of New York

DAVID EPEL, Hopkins Marine Station, Stanford University

J. MALCOLM SHICK, University of Maine, Orono

Editorial Board

PETER B. ARMSTRONG. University of California, Davis
THOMAS H. DIETZ, Louisiana State University
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WILLIAM F. GILLY, Hopkins Marine Station, Stanford

University

ROGER T. HANLON, Marine Biomcdical Institute,
University of Texas Medical Branch

MICHAEL LABARBERA, University of Chicago
CHARLES B. METZ. University of Miami

K. RANGA RAO. University of West Florida

BARUCH RINKEVICH, Israel Oceanographic &
l.imnological Research Ltd.

RICHARD STRAIHM ANN, Friday Harbor Laboratories,
University of Washington

STEVEN VOGEL, Duke University

J. HERBERT WAITE, LJniversity of Delaware

SARAH ANN WOODIN, University of South Carolina

RICHARD K.. ZIMMER-FAUST. University of South

Carolina

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Reference: Biitl. Bull 188: 111-116. (April,

Odor Plumes and Animal Navigation in Turbulent
Water Flow: A Field Study

RICHARD K. ZIMMER-FAUST 1 : \ CHRISTOPHER M. FINELLI",
N. DEAN PENTCHEFF 1 \ AND DAVID S. WETHEY 1 21

1 Department of Biological Sciences, 2 Marine Science Program, and* Belle (('. Barnch Institute

for Marine Biology and Coastal Research, University of South Carolina,

Columbia, South Carolina 2V208

Turbulence causes chemical stimuli to he highly variable
in time and space: hence the study of animal orientation
in odor plumes presents a formidable challenge. Through
combined chemical and physical measurements, we char-
acterized the transport of at tract ant released by clam prey
in a turbulent aquatic environment. Concurrently, we
quantified the locomotory responses of predatory crabs
successfully searching for sources of clam attractant. Our
results demonstrate that both rheotaxis and chemotaxis
are necessary for successful orientation. Perception of
chemical cues causes crabs to move in the upstream di-
rection, but feedback from attractant distributions directly
regulates movement across-stream in the plume. Orien-
tation mechanisms used by crabs differ from those em-
ployed by flying insects, the only other system in which
navigation relative to odor plumes has been coupled with
fluid dynamics. Insects respond to odors by moving up-
stream, but they do not use chemical distributions to de-
termine across-stream direction, whereas crabs do. Tur-
bulent eddy diflusivities in crab habitats are 100 to 1000
times lower than those of terrestrial grasslands and forests
occupied by insects. Insects must respond to plumes con-
sisting of highly dispersed, tiny filaments or parcels of odor.
Crabs rely more heavily on spatial aspects of chemical
stimulus distributions because their fluid dynamic envi-
ronment creates a more stable plume structure, thus per-
mitting chemotaxis.

The ability to detect chemical stimuli is nearly universal
among animals. Chemical signals are generated by lib-
erating stimulus molecules into a fluid, where they are
transported by advection and diffusion (eddy and molec-

Received 8 December 1994; accepted 25 January 1995.

ular) until detected and acted upon by a biological sensor.
Thus the physical process governing chemical transport
has a profound impact on the nature and success of che-
mosensory-mediated behavior ( 1, 2, 3, 4, 5, 6). Except at
microscopic scales, turbulence causes a stimulus pattern
whose concentration is highly variable in time and space
(3. 4. 5, 6. 7). As a result, the study of chemoreceptive
behavior presents a formidable challenge. It demands si-
multaneous measurements of stimulus release, fluid dy-
namics, and animal responses to perceived chemical cues.
Until now, however, these determinations have not been
combined in a single field study.

We experimentally establish a direct link between the
transport of chemical stimuli and animal navigation in a
natural, turbulent flow environment. Attraction of pred-
atory blue crabs (Callinectes sapidus) to odor released by
clam prey (Mercenaria mercenaria) was investigated. The
mechanisms directing predator to prey were identified for
crabs foraging naturally in estuarine tidal creeks, where
water flows unidirectionally for hours at a time. These
creeks were shallow enough to permit direct, noninvasive
observations of crab locomotory behavior.

Experiments were conducted in the North Inlet Estuary,
near Georgetown, South Carolina, USA (3220'N,
79 15' W). Flow records were obtained with an electro-
magnetic flow meter (Marsh-McBirney 523) equipped
with a two-dimensional sensor ( 1 cm diam), mounted on
a flat base, and submerged in the tidal creek. The base
was positioned flush with the sandy bottom, and the sensor
was placed 5 cm above the substrate. Sensor height was
chosen to match the elevation of a typical, adult crab
body. A data logger (Campbell CR10) was used to record
both horizontal and vertical flow velocities continuously
measured by the sensor at 1 Hz, between 26 June and 8

1 1 1

112

R K.. Z1MMER-FAUST ET AL

August 1993. Horizontal flow (downstream advection)
typically ranged from cm/s at slack tides, to 30 cm/s
during peak ebb and flood tides. We applied the eddy
correlation method to calculate shear velocities, at 1-min
intervals, using the correlation between the horizontal and
vertical flow velocities (8, 9). Shear velocity (;/*) is a mea-
sure of the strength and correlation of turbulent fluctua-
tions in flow speed near the substratum. Finally, we de-
termined the coefficients of turbulent mixing as the prod-
ucts of shear velocity, sensor height above the substratum,
and Von Karman's constant (9). These mixing coefficients
were remarkably low, ranging from 0.5 to 1.5 cirr/s. They
indicated that across-stream mixing occurred very slowly
in the tidal creek even though water flow was turbulent.

The dynamics of odor plumes were characterized by
measuring the transport of fluorescent dye (sodium flu-
orescein) and an electrochemical (dopamine) following
their combined release from a point source. Input con-
centrations of fluorescein and dopamine were l.Og/liter
and 2 mmol/1, respectively, with ascorbic acid added to
the mixture at 20 mmol/1 as an antioxidant. The mixture
was introduced through polyethylene tubing (0.5 mm ID)
at 6 ml/min.

Fluorescein provided a visual marker, and fluorometric
determinations were used to establish the time-averaged
distributions of dye at downstream and at across-stream
sites, relative to the release point. Water samples were
collected (at 1 ml/s) simultaneously over 1-min intervals
by syringes placed at each of 1 8 to 30 sites per trial. These
sites were distributed in a grid, with six sites placed across-
stream (0, 2, 4, 6, 8, 10 cm distant from the plume mid-
line) at three to five locations downstream of the release
point (5, 25. 50, and in some trials, 100 and 275 cm distant
from the source). The bell-shaped distribution of fluores-
cein concentration with gradual decay downstream (Fig.
1 ) is what a Gaussian plume model would predict (Pear-
son's product-moment correlation: /- : > 0.95; P < 0.001 ;
all replicate plume measurements). Concentration
dropped sharply at the plume's visible lateral edges
(Fig. 1).

Fluorometric measurements also provided an alter-
native method of calculating the mixing coefficient for
comparison with determinations made using the electro-
magnetic flow meter. Temporal changes in the across-
stream variance in fluorescein concentration were used
to estimate the mixing coefficient (9). Results from the
two methods matched well. For example, estimates
based on fluorometric determinations ranged from 0.5
to 1.2cnr/s during a time when estimates of 0.5 to
0.8 cm 2 /s were made from electromagnetic flow meter
records.

When measured at last temporal scales, chemical stim-
uli in odor plumes are patchily distributed due to tur-
bulence. Mean concentrations and time-averaged distri-

butions of fluorescein dye, therefore, may not be indicative
of the information available for crabs attempting to orient
towards an odor source (3, 4, 5, 6. 7). Because arthropod
chemoreceptors detect intermittent (or pulsed) chemical
stimuli applied at a maximum frequency of 4- 10 Hz (10.
1 1 ), we employed carbon fiber microelectrodes ( 150 /urn
diam) and a computer recording system (MedSystems
Corp. I VEC-1 Otto sample dopamine at 10 Hz (12). Elec-
trode recordings were made at the fluorescein sampling
sites (see above). Turbulent mixing caused the concen-
tration of dopamine sampled downstream of the source
to fluctuate strongly in time and space. Bursts of highly
concentrated chemical passed over the sensor, alternating
with periods of low or zero concentration (Fig. 1). The
plume's lateral edge, as defined by our high-speed dopa-
mine measurements, was positioned identically relative
to the edge detected both by our time-averaged fluorescein
measurements and by our visual observations. This lateral
edge, separating clean from chemical-laden water, was
very narrow (2-4 cm wide) compared to the body size of
an adult crab (10-15 cm carapace width). Thus a steep
concentration gradient was found across-stream, but not
downstream of chemical release.

We previously demonstrated that some crabs search
for and find intact live clams, and that these crabs are
responding to odor plumes created by the excurrent release
of attractant metabolites at low concentration (13). Once
a clam is found, however, it is chipped open by a crab
and attractants are released to form a plume of high con-
centration. High-concentration plumes then immediately
attract other crabs to the predation site. Hence, depending
on the situation, crabs may be exposed either to low or
high attractant concentrations, and crabs respond effec-
tively in each case.

Concurrently with hydrodynamic and chemical mea-
surements, we assessed crab orientation in odor plumes.
Our field studies focused on plumes characteristic of
chipped clams. We chose to work with chipped clams
because high attractant concentrations would better en-
sure an effective stimulus through the broad range of hy-
drodynamic conditions encountered by crabs in the field.
Stimulus plumes (dyed with fluorescein for visibility) were
created, either presenting a chipped clam or introducing
clam mantle fluid (membrane filtered to 0.22 ^m) at a
rate mimicking its release from a chipped clam. Each
stimulus plume was always paired with a control plume
that delivered fluorescein in filtered seawater. Both stim-
ulus and control solutions were introduced at 6 ml/min,
with inputs separated by 60 cm across-stream.

Free amino acid compositions of effluent leaking from
chipped clams (/; = 8 clams assayed), mantle fluid of intact
live clams (/; = 8 clams), and homogenized clam flesh
(n = : 7 clams) were all determined using a Beckman
6300 System Gold high-performance liquid chromato-

TURBULENT ODOR PLUMES AND ANIMAL NAVIGATION

50

25-

o

a
o
u

o - 1

Distance From Midline (cm)
-10 -5 5 10

50cm Y = 0cm X = SOcm Y=2cm X = 50cm Y = 4 cm X=50cm Y = 8 cm X = 50 cm Y=10cm

^ ou

_a

ou

c

^ 0.2 -
^

o

B 40 -

- 40

E

1|

|

c

u

5 0.1 -
X

820.

1

I ll

i u i

- 20

Q)

1 ll

O

D

"- nn -

III., I n

dLLllkU

uL.llkll

1

4M

i

Iliiijiiiv

. n

20 40 20 40 20 40 20 40 20 40 60

Time (s)

( = 25 cm Y = 0cm X = 25cm Y = 2cm X = 25 cm Y = 4 cm X = 25 cm Y = 6cm

^ 0.2 -

o>
E

c

'o> 0.1 -

Fluoresc

3
3

1,,

5 w

c
o

"5 40

o 20
O

60

40

20

20 40 20 40 20 40

Time (s)

X5cm Y-Ocm X = 5 cm Y = 1 cm X = 5 cm Y = 2 cm

20 40 60

D>

E

5 2 '

o

3

"- n -

fr

^ ou

3.

1

- ou

C

O

|40-

-40

1
o

c

020

ll

i i

i HI

-20

0)

UN

ii ,

i

8

kj 'M

y

m

i

ii i ,i

20 40 20 40 20 40 60

Time (s)

Plume Source

Figure I. Representative concentration distributions downstream from a point source in a tidal creek.
Histograms represent fluorescein concentrations (mg/1) in samples collected at 5 cm (bottom row), 25 cm
(middle row), and 50 cm (top row) downstream from the source and at 0, 2, 4, 6, 8, and 10 cm from the
midlmc of the plume. Note the scale difference between fluorescein concentrations at downstream locations.
The visible region of the fluorescein plume at each position is denoted by the shading. Panels to the right
of the histograms represent ftO-s records of instantaneous fluctuations in dopamine (tracer) concentration,
measured at 10 Hz with a carbon fiber electrode, at locations where the fluorescein was sampled. The left-
most panel in each row is the sample from the midline of the plume, and successive panels are samples
from 2, 4, 8, and 10cm from the midline (see tick marks on histogram axes for sampling sites). Highly
concentrated bursts of dopamine were common in all samples taken within the visible portion of the plume.

graph with a sodium ion-exchange column (4-mm ID
X 120mm; Beckman) for separation. In this system,
amino acids were monitored spectrophotometrically after
post-column reaction with ninhydrin. Compositions of
clam effluent and mantle fluid were almost identical
(Pearson's product-moment correlation: r 2 = 0.998; P
< 0.001; n = 18 amino acids chromatographed), indicat-
ing that mantle fluid was the source of effluent material.
Because taurine was by far the most abundant amino acid
in both clam effluent and mantle fluid (accounting for
>50% of the total amino acid composition), we used it
as a marker to measure the rates of fluid release from
chipped clams. In the laboratory, clams (n = 12) of various
sizes were chipped by using a metal rod to deliver a single

firm blow to the lateral shell margin. The resulting chip
was similar in size and shape to one produced by a blue
crab as it begins to feed. Each chipped clam was then
placed individually into a separate beaker of artificial sea-
water. The beakers were stirred, and they were maintained
at the same temperature and salinity as seawater in the
tidal creek from which clams were collected. Artificial
seawater was sampled from each beaker before placement
of the clam, and again at 30- to 60-s intervals for 15 min
after placement; HPLC analysis of this seawater indicated
that taurine (and mantle fluid) release was constant
throughout the trial period. The relation between taurine
release rate and clam size was then used to scale our de-
livery of mantle fluid in field experiments, simulating the

14

R k. ZIMMER-FAUST ET AL

40

X-N

H o
5

ce

o

"to

b

o>

O 40
<D

2! o

U = 4.2 cm/s

~w^^

U = 1 cm/s

**.

^ o
</>

-40

U = 25 cm/s

50 100 150 200 250

Distance Downstream from Source (cm)

Figure 2. Representative tracks of crabs following odor plumes. The crab symbols represent the positions
and orientations of individuals at 1-s intervals as they moved upstream toward the odor source. Naturally
foraging crabs normally walk sideways as well as forward. The visible region of the fluorescein/odor plume
is noted by the shading. The water velocity (U) at ? cm above the bottom was 4.2 cm/s (top panel). 10 cm/s
(center panel), and 25 cm/s (bottom panel). Distances downstream and across stream are in centimeters.

300

fluid input from an intermediate-sized chipped clam (6 cm
total shell length).

Quantitative observations of foraging crabs were made
noninvasively with a video camera (Sony TR8 1 ) mounted
4 m above the tidal creek. Video records of crabs re-
sponding to plumes were made during ebbing tides. A
3-m field of view was dictated by the resolution of the
video camera and the size of the crabs (10-15 cm carapace
width), whose positions could not be reliably quantified
in wider field images. A scale bar in the field of view was
employed to measure distance and to correct for distortion
due to perspective. In the laboratory, plume edges and
crab positions at 1-s intervals were traced onto acetate
sheets from video playbacks to a monitor. Both crab lo-

cation, in relation to the plume edge, and crab locomotory
kinematics were measured (Fig. 2).

Crab responses to the plumes were dramatic and un-
ambiguous: 29 crabs contacted the control plumes, but
only 4 of these crabs walked upstream towards the input
source. The near absence of positive responses to control
plumes demonstrated a lack of attractant effect by fluo-
rescein dye. In comparison, after contacting stimulus
plumes, 68 of 80 crabs walked upstream to the input
source. Crabs turned upstream within 1.5 s (0.3 SD) of
contacting an odor plume. Percentages of crabs respond-
ing positively to plumes from either chipped clams or
mantle fluid were nearly identical, being 86% (n = 52
crabs) or 82% (n = 28 crabs), respectively (G-test for ho-

TURBULENT ODOR PLUMES AND ANIMAL NAVIGATION

115

mogeneity: G 2 = 0.270; df = 1; P > 0.50). Our results
demonstrate an odor-conditioned rheotaxis that orients
crabs upstream. Previously, we reached an identical con-
clusion for blue crabs foraging in a laboratory flume. Crabs
walked upstream to find intact live clams in flowing water,
but they oriented indiscriminately and searched unsuc-
cessfully for clams in still water (13).

Oriented movements by crabs lateral to water flow are
controlled by chemotaxis. As crabs walked upstream to-
wards an attractant source, they frequently approached
the lateral edges of the plume. When crabs did reach the
edge, they nearly always turned directly back to the plume
(50 of 6 1 turns; G-test for goodness-of-fit. 1 : 1 hypothesized
ratio, G 2 == 14.97; df = 1; P < 0.001 ), without exhibiting
either casting or zigzagging (Fig. 2). Lateral movements
were initiated as crabs began to exit a plume and partially
contacted clean water. Fluorescein did not act as a visual
cue, because crabs displayed identical oriented responses
when tests were conducted in the dark (under infrared
illumination) and without fluorescein (13, and in prep.).
It took, on average, less than 1 s (0.8 0.2 s SD) for crabs
to renew upstream walking after they had begun moving
laterally towards the plume midline. Remarkably, we did
not observe walking speeds to change significantly as crabs
moved closer to attractant sources (analysis of covariance:
F = 0.60; df = 4,237; P = 0.66; walking speed: 12.8
0.4 cm/s SD), and we found no significant correlation
between walking speed and water flow velocity (Pearson's
product-moment correlation: /- : = 0.037; df = 1,64; P
= 0.12). We hypothesize that crabs perceive clam attrac-
tant as a binary cue (present/absent), both in their up-
stream movement and in their across-stream walking.
Because the plume edges were very sharp, when crabs
partially exited the plume, some pereiopods (legs or claws)
were outside the plume while others remained inside. A
comparison of simultaneous chemosensory inputs from
the appendages inside and outside the plume would pre-
sumably allow the crabs to determine the correct direction
and return to the plume. This binary response would
lead crabs to locations of higher concentration of clam
attractant.

Orientation mechanisms used by crabs in upstream
movement are similar to those of flying insects. However,
crabs differ from insects in their across-stream response.
Insects provide the only other system in which navigation
relative to odor plumes has been coupled with fluid dy-
namics. Flying insects locate a source of chemical attrac-
tant by moving upwind upon contacting a filamentous
trace of attractant odor (14, 15). After several seconds of
flying in clean air, insects shift to casting (regular reversals
of flight directed across-stream) until contact with another
odor trace causes a return to upwind flight (16). Flying
insects, therefore, do not use chemical concentration gra-
dients to determine either their upwind or across-wind

directions (16, 17, 18). The use of chemotaxis may be
impractical in their environment, where complex fluid
dynamics do not permit stable zones of high attractant
concentrations to exist. Crabs in contrast, consistently turn
back into the attractant plume rather than zigzagging after
losing the plume signal.

The difference between estuarine tidal creek flow and
atmospheric winds may explain why blue crabs and insects
use contrasting mechanisms for successful navigation to-
wards an odor source. The crop fields and forests used as
experimental models for insect flight are hydraulically
rough, with high advection. Eddy diffusivities in insect
habitats are 100 to 1000 times greater than those we re-
corded in estuarine tidal creeks (19, 20). Higher diffusiv-
ities yield plumes consisting of tiny, highly dispersed fila-
ments or parcels of odor. Wind direction changes fre-
quently, causing plumes to meander (3, 6). The dispersal
pattern of odor, coupled with the relatively fast flight speed
of insects, means that a flying insect has little chance to
detect more than the occasional pulse of passing odor.
Casting, zigzagging, and rapid behavioral modulation in
response to fine-scale changes in odor concentration may
be strategies appropriate for situations in which the entire
plume meanders away from the animal.

In contrast, the flow environment of estuarine tidal
creeks is markedly less turbulent, yielding relatively stable,
straight, and sharply delineated odor plumes. Plumes
cannot meander substantially, because flow is constrained
by water depth and by the sides of the creeks. A stable
plume structure permits direct binary comparisons of
chemical concentration inside and outside the plume, to
guide movement lateral to flow. The more direct plume-
following behavior and across-stream chemotactic re-
sponses shown by crabs reflect a strategy appropriate to
the plume structure characteristic of their environment.
Mechanisms of plume-following behavior, therefore, arise
in response to chemical stimulus distributions, as deter-
mined by the specific fluid dynamic environments in
which animals must naturally navigate.

Acknowledgments

We thank Dr. D. M. Allen, Director of the USC Baruch
Field Laboratory, for providing laboratory space and lo-
gistical support for field work. Dr. Y. Ishikawa, USC In-
stitute for Biological Research and Technology, performed
amino acid composition analyses. We also thank Dr.
S. A. Woodin, whose comments on earlier drafts greatly
improved this manuscript. This research was sponsored
by the National Science Foundation (IBN 92-22225) and
the University of South Carolina Research and Productive
Scholarship Fund.

Literature Cited

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116

R K. ZIMMER-FAUST KT AL

2. Bossert, \V. H., and E. O. Wilson. 1963. The analysis of olfactory'
communication among animals. / Theor. Biol 5: 443-469.

3. Murlis, J., and C. D. .(ones. 1981. Fine-scale structure of odour
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other attractant sources. Physiol. Entomol 6: 71-86.

4. Elkinton, J. S., R. T. Carde, and C. J. Mason. 1984. Evaluation
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5. Zimmer-Faust, R. K., J. M. Stanfill, and S. B. Collard, III. 1988. A
fast, multi-channel fluorometer for investigating aquatic chemore-
ception and odor trails. I. annul Oceant;r. 33: 1586-1595.

6. Murlis, J., J. S. Elkinton, and R. I. Carde. 1992. Odor plumes
and how insects use them. Anmi Rev Entomol. 37: 505-532.

7. Moore, P. A., and J. Atema. 1991. Spatial information in the three-
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408-418.

8. Schlichting, H. 1979. Boundary Layer Theory. McGraw-Hill. New
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9. Denny, M. W. 1988. Biolot<y and Mechanics <>/' ilie IIV/iv-
Sivepl Environment Princeton University Press. Princeton. NJ.
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10. Kaissling, K. E., C. Z. Straussfeld, and E. Rumbo. 1987.
Adaptation processes in insect olfactory receptors: mechanisms and
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I I. Gomez, G., R. Voigl, and .J. Alema. 1994. Frequency filter prop-
erties of lobster chemoreceptor cells determined with high-resolution
stimulus measurement. ./ Comp PliYsml A 17-1: 803-81 1.

1 2. Moore, P. A., G. A. Gerhardt, and J. Alema. 1989. High resolution
spatio-temporal analysis of aquatic chemical signals using micro-
electrochemical electrodes. Chem Senses 14: 829-840.

13. VVeissburg, M. J., and R. K. Zimmer-Faust. 1993. Life and death
in moving fluids: hydrodynamic effects on chemosensory-mediated
predation. Ecolivi 1-4: 1428-1443.

14. Mafra-Nelo, A., and R. T. Carde. 199-4. Fine-scale structure of
pheromone plumes modulates upwind orientation of flying moths.
Nut lire 3W: 142-144

15. Vickers, N. J., and T. C. Baker. 1992. Male lleloilus vircsccns
maintain upwind flight in response to experimentally pulsed filaments
of their sex pheromone (Lepidoptera: Nocturidae). ,/ Insect Behav.
5: 669-687.

16. Baker, T. C. 1986. Pheromone-modulated movements of flying
moths. Pp. 39-48, in Mechanisms of Insect Olfaeliim. T. L. Payne.
M. C. Birch, and C. E. Kennedy, eds. Clarendon Press. Oxford.

17. David, C. T.. J. S. Kennedy, and A. R. Ludlow. 1983. Finding of
a se\ pheromone source by gypsy moths released in the held, \nliire
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18. Arbas, E. A., M. A. \\ illis, and R. Kanazaki. 1993. Organization
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19. Shaw, R. H., J. Tavangar, and D. P. Ward. 1983. Structure of the
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20. Arya, S. P. 1988. Introduction to Micrometeorology Academic
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Reference: Biol Bull 188: 117-119. (April, 1995)

Evidence for Selection Against Heterozygotes:

Post-Settlement Excess of Allozyme Homozygosity

in a Cohort of the Chilean Oyster,

Ostrea chilensis Philippi, 1845

J. E. TORO AND A. M. VERGARA

Instituto de Biologia Manna, L'niversidad Austral de Chile. Casilla 567, I 'aldivia. Chile

Reports of heterozygote deficiencies in electrophoretic
survevs carried mil in marine bivalves abound in the lit-
erature (1-6), but the mechanism or mechanisms produc-
ing this phenomenon have not been well defined. H 'e report
that, in the Chilean oyster (Ostrea chilensis), heterozygote
deficiencies in a cohort obtained hy mass spawning in the
laboratory are not randomly distributed in lime among
genotypes. The eggs of the Chilean oyster are internally
fertilised, and the larvae, which are brooded within the
mantle cavitv. have limited dispersal capabilities because
of their extremely short pelagic stage (7). These features
could allow mechanisms such as inbreeding or Wahlwnl
effect to produce heterozygote deficiencies. However, we
observed no significant heteroiygote deficiencies in juve-
niles at 6 months of age; instead allozyme heterozygosiiy
decreased over time. Inbreeding. H 'ahlund effect, aneu-
ploidy. and null alleles are unlikely to be main causes of
the heterozygosity deficiency in this cohort; if they were,
the deficiency should be evident from the juvenile stage
and would not necessarily increase over time (2, 5. 8, 9.
10). We suggest that selection against heterozygotes is the
most probable cause of the increasing degree of hetero-
zvgote deficiency with age in this cohort ofO. chilensis, a
proposition that accords with data for other marine bivalve
species (2, 4. 1 1).

Populations of marine bivalves exhibit deficiency of
allozyme heterozygotes. This deficiency has been dem-
onstrated in laboratory studies of mussels and clams (2.
3), in studies using wild populations (8, 12) of Mytilus
edu/is. and in several studies of oysters (Crassoslrea vir-
ginica) (13-15). In the Chilean oyster (Ostrea chilensis) a

Received 10 May 1994; accepted 26 January 1995.

heterozygote deficiency was found in the carbonic an-
hydrase (C A) locus from a southern population (Melinka,
4353'S) ( 1 ). The time at which heterozygote deficiency
first appears in the population can help distinguish caus-
ative mechanisms ( 16). In laboratory studies with mussels,
an overall significant deficiency of heterozygotes was
found at the juvenile stage but not at the spat stage (4).

In the present study, we used a cohort of O. chilensis
settled on artificial collectors in the Quempillen hatchery,
Ancud, Chiloe (4552'S, 7346'W). The parental stock
was a cohort of O. chilensis collected during December
1987 from a natural spatfall in the wild population at
Quempillen estuary. The Chilean oyster becomes sexually
mature at the beginning of the second year of life with a
shell length of about 27 mm (7). After three years of
growth under uniform conditions, 800 randomly chosen
oysters were mass spawned in the laboratory. The tem-
perature and salinity used in the experiment were within
the range of those in the natural environment (10-18C
and 27-32 ppt). Although the use of mass spawning pre-
vents one from knowing how many individuals contribute
genes to the offspring obtained, the female contribution
can be estimated by keeping track of the number in each
brood of eyed larvae. Fecundity in O. chilensis ranges
between 10.000 and 1 15,000, with an average of 60,000
(7). The number of larvae released, more than 8.2 X 10 6 ,
indicates that at least 130 females contributed larvae. This
number of females may be an underestimation because
some of the eyed larvae released set within 5 min (7); thus
this cohort cannot be treated as a product of restricted
matings.

From an initial population size of 4050 randomly
tagged juveniles grown at the Hueihue location (4158'S,

117

18

J. E. TORO AND A. M. VERGARA

Table I

Helero:ygi>te Im/iu-iiey and D values tor lour loci at three stages of the
lite cvcle ot Ostrea chilensis tf>. IX. and 30 months of age)

Age

(months)

Locus

OH.

E.H.

D

OP)

6

LAP

0.410

0.398

0.053

NS

GPI

0.581

0.458

0.283

*

CA

0.645

0.617

0.046

NS

PGM

0.155

0.167

-0.072

NS

18

LAP

0.338

0.457

-0.260

*

GPI

0.373

0.389

-0.041

NS

CA

0.514

0.607

-0.153

*

PGM

0.247

0.383

-0.355

*

30

LAP

0.447

0.591

-0.243

*

GPI

0.268

0.292

-0.082

NS

CA

0.408

0.631

-0.353

#

PGM

0.231

0.374

-0.382

*

Genotype frequencies were investigated using random samples of 1 50
oysters taken from each class interval. Each locus was tested individually,
using the X 2 goodness of fit test with D as an index of heterozygote
deviation. Starch gel electrophoresis was used (18, 19) to score the loci
leucine aminopeptidase (LAP. EC 3.4. I.I), glucose phosphate isomerase
(GPI, EC 5.3.1.9), carbonic anhydrase (AC, EC 4.2.1.1), and phospho-
glucomutase (PGM, EC 2.5.7. 1 ).

O.H. = proportion of observed heterozygotes; E.H. = proportion of
expected heterozygotes; D = heterozygote deviation index denned as
(O.H. - E.H.l/E.H.; (P) = probability of the X 2 goodness of fit to the
Hardy Weinberg model (NS = nonsignificant; * = significant at alpha
= 0.05).

7330'W), the percentages of mortality at ages from 6 to
18 and 18 to 30 months were 25% and 17% respectively.
At each class interval, 1 50 oysters were sampled without
replacement. Neither significant deficiencies nor an excess
of heterozygotes was found in three of four loci in the 6-
month-old oysters; the exception was glucose phosphate
isomerase (GPI), which showed an excess of heterozygotes
(Table I). At 18 months, significant deficiencies of het-
erozygotes were found at LAP (D = -0.260), CA (D

-0.150), and PGM (D = -0.355) (Table I). In adult
oysters (30 months), negative values of D were present at
three of four analyzed loci, presenting significant values
at LAP (D = -0.243), AC (D = -0.353), and PGM (D

-0.382) (Table I). The data showed that between the
age of 6 and 18 months, three out of four loci studied
showed a genotype-dependent mortality. This differential
mortality produces a significant overall deficiency of het-
erozygosity in the cohort. One of the loci studied (GPI)
showed an excess of heterozygotes at 6 months and neither
an excess nor a deficiency of heterozygotes at 1 8 and 30
months; this result agrees with other studies carried out
on this locus in natural populations of bivalve molluscs
(I, 17, 18). High fecundity, external fertilization, and ex-
tensive larval dispersal characteristics common to most
of the bivalves molluscs make it unlikely that inbreeding

or the Wahlund effect could be the main cause of hetero-
zygote deficiencies. The reproductive features of O clii-
lensis favor mechanisms such as inbreeding or Wahlund
effect to act and produce heterozygote deficiencies. How-
ever, in accord with data for other marine bivalve species,
we suggest that selection against heterozygotes is the most
probable cause of the heterozygote deficiencies (2, 4, 1 1 ),
because the deficiency is not evident at the juvenile stage
but increases over time. We discarded inbreeding (which
would affect the whole genome), Wahlund effect, aneu-
ploidy, and null alleles as possible causes for the hetero-
zygosity deficiency in this cohort because a deficiency
produced by these factors should be evident from the ju-
venile stage and not necessarily increase over time (2, 5,
8,9, 10).

Acknowledgments

We thank Dr. J. B. Mitton and two anonymous re-
viewers for their valuable suggestions and advice for im-
proving our manuscript. This work was supported by the
Fondo Nacional de Desarrollo Cientifico y Tecnologico
(Fondecyt 91/0897) and by the Direccion de Investigation
y Desarrollo, U.A.Ch. (S-94-18).

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9. Gaffney, P. M. 1993. Heterosis and heterozygote deficiencies in
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HETEROZYGOTE DEFICIENCY IN OYSTERS

19

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Reference: Bi, >l. Bull 188: 120-127. (April. 1995)

Catch in the Primary Spines of the Sea Urchin

Eucidaris tribuloides: A Brief Review

and a New Interpretation

JOSE DEL CASTILLO*. DAVID S. SMITH**, ADA M. VIDAL*. AND CESAR SIERRA*

*lnstiliile of Newohiology, University of Puerto Rico M.S.C., Blvd. del 1'iille 201. Sun ./nan,

Puerto Rieo 00901; tint/ **Departnient of Zoology, University of Oxford,

South Parks Road. Oxford. OXl 3PS. United Kingdom

Abstract. Previous models of reversible catch in echi-
noid spines, as a property of muscle or of collagen, are
briefly reviewed and discussed. This brief review offers a
new interpretation of catch in primary spines of Eucidaris
tribuloides. viewing the collagen and small muscles of the
catch ligament working together as a variable-length ten-
don. In the model presented, changes in ligament length
when out of catch are accommodated by sliding of dis-
continuous, interdigitating and cross-link-stabilized col-
umns of collagen fibrils, the muscle layer external to the
ligament effecting spine movement. Catch is viewed as a
consequence of contraction of small muscles inserted on
the collagen columns within the ligament. Ligament
shortening tightens the profuse (en. 30,000/mrrr) and
highly ordered collagen insertion loops within the ster-
eoms of the spine base and test, and catch results from
the multiplicative effect of these friction sites in series.
New data are presented on novel structural cross-links
between collagen fibrils. The cross-links stabilize the liga-
ment columns. The central ligament in Eucidaris plays a
purely passive mechanical role in maintaining the align-
ment of the spine-test articulation. It contains no muscle
and neither contracts nor undergoes catch: its insertions
are simple, unlike the complex stereom insertions of the
main ligament.

Introduction

From the time that it was first recognized, the phenom-
enon of catch in sea urchin spines has attracted the interest
of investigators, but its basis has remained unclear. Two
seemingly contradictory theories have been proposed to

Received 5 January 1995; accepted 7 February 1995.

explain catch; but recent experimental observations allow
a new interpretation that reconciles the two discrepant
hypotheses.

Catch is an operational concept that can be defined in
this instance as a reversible, neurally controlled enhance-
ment of the passive mechanical resistance offered by the
spine test articulation (Fig. 1 ) to forces tending to change
the position of the spine. The sudden inducement of catch
freezes the primary spines in their respective positions,
whether normal to the test surface or angled from this
axis, thereby allowing the animal to maintain a fixed pos-
ture for long periods.

von Ui'.\kiil/'s catch muscle

At the turn of the last century. Count Jakob von
Uexkiill, a self-supporting German biologist noted for his
strong vitalist convictions, published a paper ( 1 900) titled
"The Physiology of the Sea-urchin Spine" in which he
reported that the voluntary and reflex movements of the
spine are powered by a thin layer of muscle fibers that
surrounds the thick articular capsule. In addition, he
found that the integrity of this capsule, which is also
known as the spine ligament or catch apparatus, is essen-
tial for the development of catch.

Von Uexkull described the breakage of the capsule by
forcible displacement of the spine while in catch: spines
treated in this manner failed to show catch, but they re-
tained the ability to perform voluntary and reflex move-
ments because the thin muscle layer was not disrupted.
Accordingly, von Uexkull called this muscle layer Be-
wegungsmuskulatur (motion-supporting muscle) as op-
posed to the articular capsule, which he believed also to
be a muscle, the Sperrmuskulatur (catch or holding mus-
cle). As we shall see below, this was an inspired guess that

CATCH IN SEA URCHIN SPINES

121

Figure 1. The spine-test articulation of Eucidaris. In this Chlorox-
digested preparation a small area ofligament remains, maintaining the
ball-and-socket arrangement. 18

defied contemporary evidence, because the latter tissue
had been studied by 19th century microscopists (Prouho.
1887; Hamann, 1887) and was recognized by them as
being primarily a connective tissue.

Takahaslu's mutable connective tissue

The problem of catch in echinoderm spines was studied
again in the 1960s by Takahashi (1966, 1967a, b, c), who
confirmed von Uexkiill's results while disagreeing with
him on the nature of the ligament. In the discussion of
his landmark paper (1967b) on "Responses to stimuli,"
Takahashi gave an account of the experimental results
that led him to propose a new hypothesis to explain catch.

Because at that time the ligament was still regarded as
a muscle, Takahashi first attempted to record its contrac-
tion following the application of chemical or electrical
stimuli. He was not successful. Yet Takahashi was greatly
impressed by the effects of the same chemical stimuli on
the rate of elongation of ligaments subjected to a constant
load (isotonic recording; creep test). In his words "the
effects were clear, sometimes even dramatic, and they
varied according to the kind of drug applied." Lengthening
was retarded by acetylcholine. while adrenaline exerted
an accelerating effect.

These observations led Takahashi to seek the identity
of the structural element responsible for the ligament ex-
tension under constant load, and he saw a plausible can-
didate in the collagen. He accounted for his results on the
premise that the mechanical consistency of collagen can
switch reversibly between two extreme conditions or
states: one pliant and extensible and the other stiff and
inextensible. This hypothesis was attractive because it ex-
plained a variety of experimental observations and was

accepted by most workers including ourselves (Morales
et al.. 1989, 1993). This view gave rise, more or less di-
rectly, to the concepts of "connective tissue catch" (Riiegg,
1971), "mutable connective tissue" (Eylers. 1982) and
"variable tensility" (Wilkie, 1984).

Takahashi's observations had a great impact on the
study of echinoderm connective tissue, and it is now ac-
cepted that the members of each of the five extant classes
of the phylum possess some connective tissue with prop-
erties that differ significantly from those of vertebrate col-
lagen (Motokawa, 1984, 1985). In this context, we stress
that the present account deals only with the primary spine
ligament of Eucidaris tribuloides. and while we do not
extrapolate our conclusions to other echinoderms, neither
do we suggest that our model of catch is restricted to this
echinoid.

The Ligament as a Myotendinous Organ
Muscle fibers

The fine structure of the ligament was first studied by
Smith et al (1981) and Hidaka and Takahashi (1983),
who noted the presence of muscle fibers in the spaces
between the cylinders or columns of collagen fibrils that
occupy most of the volume. As described by the above
authors, the muscle cells are slender (only about 0. 1- 1 nm
in diameter) and unstriated, and they include large para-
myosin filaments in their contractile array. They make
only a small contribution to the volume of the ligament:
about 1.5% of the cross-sectional area in our micrographs
of Eucidaris. We have determined that they insert directly
onto the collagen columns. Although we lack information
about their length and arrangement, the almost exact
alignment of the muscle fibers and the collagen columns
in transverse sections suggests that the two are virtually
parallel, and we view the muscle as probably extending
between adjacent collagen columns.

In addition. Hidaka and Takahashi suggested that
changes in length of the ligament might reflect sliding
within the array of collagen fibrils, and that catch could
be accounted for by the formation of cross-links between
the sliding elements. This suggestion stimulated work and
speculation on the nature of the proposed cross-links,
which were pictured variously as simple divalent cations,
notably calcium (Hidaka, 1983; Diab and Gilly, 1984),
and proteoglycans binding together the collagen fibrils
(Trotter and Koob, 1989).

Insertion of the muscle fibers

Working on Anthoddaris, Hidaka and Takahashi
(1983) noted that the muscle fiber surfaces were very
closely apposed to the peripheral fibrils of the collagen
columns and, while favoring the view that the muscles
are long, running from insertions on collagen near the

122

J. DEL CASTILLO ET AL.

spine base and test, they suggested that the muscle cells
might be relatively short and serve as cross-links between
the collagen columns. They further described the fine
structure of the ligament stretched to three times its resting
length, noting "empty spaces" in the collagen array. They
attributed this pattern to the slippage of collagen fibrils
relative to one another. Thus they regard the individual
collagen fibrils as the units responsible for sliding during
forced ligament elongation. In contrast, we view the col-
umns, rather than individual fibrils, as the functional units
of the ligament length change accomplished by sliding.
Each column is separated from its neighbors by 'matrix
spaces,' but there is no fine structural evidence of cross-
links between columns; i.e.. between the peripheral fibrils
of adjacent columns. We suggest that the linkage between
columns is effected by the muscle fibers.

We offer a rather different interpretation of Hidaka and
Takahashi's micrographs: namely, that during irreversible,
non-physiological stretching, peripheral portions of dis-
continuous collagen columns are torn away at the region
where the muscle fibers are firmly inserted onto the col-
umns. Before describing our reinterpretation of Hidaka
and Takahashi's experimental findings, however, we
should introduce a further piece of evidence concerning
the collagen columns in Eucidaris.

Struct lire oj the collagen columns

Other than the presence of transient links invoked in
previous models of catch, the columns have been regarded
as groups of mechanically independent fibrils. Indeed Hi-
daka and Takahashi's model is based on this assumption.
But, in Eucidaris (Figs. 2, 3) we have observed a novel
feature in conventionally prepared material 1 , the fibrils
of each column are profusely cross-linked by asymmetrical
junctions and by apparently different, simpler, and sym-
metrical bridges; a single large-diameter fibril profile often
shows multiple links with its neighbors. The apparent sta-
bility of the collagen columns seen in the transverse plane
is also suggested by the regularity of organization of the
columns seen in longitudinal sections (Fig. 6). Not only
are individual fibrils precisely parallel, but some degree
of register is often seen in the striation pattern of adjacent
fibrils. As previously noted by Scott (1988) in the holo-
thurian body wall and in vertebrate tendons, and by Trot-
ter and Koob ( 1989) in Eucidaris, proteoglycan strands

1 Material illustrated in Figures 2-5 and 10 was conventionally fixed
(2.5% glularaldehyde, 0.05 A/cacodylate butler pH 7.4 with 14% sucrose),
treated with !%OsO 4 and embedded in Araldite. Contrast was enhanced
on the grid hy treatment with lead citrate followed by unbuffered 1%
RMnO 4 . Proteoglycans (Fig. 6) were visualized by the method of Scotl
(1980, 1 988); low contrast enhancement of the collagen was obtained
by treating sections with lead citrate alone. Material for SEM examination
was fixed as for thin sectioning, but without OsO 4 treatment, and critical
point dried. Details of preparation of frozen-fractured material (Fig. 9)
are given in Smith cl til ( 1990).

form a regular meshwork between the fibrils. In Eucidaris
the regularity of their placing with respect to the fibril
striations is noteworthy (Fig. 6). although their function
remains undetermined. Proteoglycans are visualized only
after special tissue preparation (Scott, 1980, 1988), and
it is unlikely that the cross-bridges seen in conventionally
prepared material, mentioned above, are related to the
proteoglycan moieties of the columns. Although the na-
ture of these bridges remains unknown and neither type
matches the fine proteoglycan strands described by Scott
in tendon, by Trotter and Koob in Eucidaris ligament,
and shown here in Figure 6 we regard this elaborate
system as likely to give added mechanical stability to each
column as a structural unit.

Trotter and Kooh 's mode/

Trotter and Koob (1989) reported a model of the liga-
ment in which the collagen fibrils are the discontinuous
fiber phase of a fiber-reinforced composite material. Their
measurements of single isolated collagen fibrils revealed
that, although varying in length and radius by more than
an order of magnitude, they have a high and constant
length/radius ratio, which was interpreted as indicating
that the non-fibrillar material must act to transfer stress
between fibrils. Trotter and Koob suggested that proteo-
glycan "may be an important component of the stress-
transfer matrix," and illustrated the regular disposition of
this material with respect to the collagen band pattern.
We repeated this, with similar results (Fig. 6). But such
proteoglycan components seem to be commonly asso-
ciated with collagen, including that of vertebrate tendon
(Scott 1980, 1988).

Nature oj the sliding elements in the collagen array

We envisage ligament length change as being accom-
plished by a sliding movement between stabilized, dis-
continuous, and interdigitating columns. Adopting this
view, we see Hidaka and Takahashi's observations in a
different light. First, we noted, in Eucidaris, a very close
apposition of muscle cell surface to column periphery, as
Hidaka and Takahashi reported in Anthocidaris: i.e.. a
gap of only about 10 nm separates the muscle plasma
membrane from the outermost collagen fibrils, and this
membrane is often contoured to match the fibrillar sur-
faces (Figs. 4. 5). Whereas Hidaka and Takahashi favored
the view that the muscle fibers run from insertions near
the spine base and the test, the high frequency with which

Figure 2. TransNcrse section of collagen fibrils. Note the frequent
inter-fibrillar cross-links, shown further in the next figure. 60.000

Figure 3. The collagen filaments of the ligament are linked by fre-
quent asymmetrical (arrows) and symmetrical (arrowheads) cross-bridges,
xl 10,000

CATCH IN SEA URCHIN SPINES

123

j

X "'

ACS

,'

.

.-*W

g*

r ;;i :' '

J%,

.

^ '"^

!

- ,. . ,

Figures 4, 5. Illustration of the close apposition of muscle hlx-rsand collagen lihnls in the mam ligament
of Eucularis. A gap of about 10 nm separates the liber plasma membrane from the collagen surface, and
the membrane is often indented around the hbrilar contours. Figure 4, X90,000; Figure 5, X 120,000

Figure 6. Longitudinal section of the main ligament in Eucidaria. Proteoglycan is visualized by staining
with cuprolinic blue. Note the precisely parallel disposition of the fibrils and areas of alignment of collagen
banding. / 72,000

124

J. DEL CASTILLO /// II.

Figure 7. SEM of the insertion cavity and the central ligament on the test of l-'.uciilan \ x90

Figure 8. As in Figure 7, hut with insertion of the central ligament exposed. The ligament ramifies into

slender processes (arrows), which loop through stereom traheculae. 130

Figure 9. SEM of frozen-fractured main ligament insertion on the i'.iuitlun\ test. Note collagen straps

(c) looping through stereom traheculae and tightly appressed to the stereom struts. (From Smith el til ,

1440). 1,500

CATCH IN SEA URCHIN SPINES

125

we have observed muscle insertions on thin sections of
the ligament led us to the alternative view that the muscle
fibers are very numerous and relatively short, linking ad-
jacent collagen columns throughout the ligament. A reex-
amination of their figures (i.e.. Hidaka and Takahashi,
1983, Figs. 8, 9) suggests that stretching somewhat distorts
but does not obscure the arrangement of collagen columns
and that the 'holes' appearing in the transversely sectioned
array are not random but represent lenticular gaps where
bundles of fibrils have been torn apart. In addition we
regard the muscle fibers as very strong, as shown, for ex-
ample, by the presence of highly stretched but essentially
intact muscle fibers in pictures published by Hidaka and
Takahashi (1983). The linkage between muscle fibers and
columns must be strong if our model is correct.

Although there is no fine structural evidence of discon-
tinuity, it seems likely that the columns taper at their
ends. Profiles of 'tiny' columns are dispersed in transverse
sections, probably columns near their ends (see Fig. 3).

The Ligament Contracts

In view of the presence of contractile cells, one should
expect that cholinergic agonists would induce some me-
chanical effect on the ligament. The very modest contri-
bution of muscle to the volume of the ligament seemed
to rule out a leading role for them in ligament mechanics
analogous to that of muscle in the molluscan catch mech-
anism. Indeed, the only functional alternative that Smith
el a/. (1981) suggested was the relatively minor task of
returning an extended sector of the ligament to its 'normal'
position.

To obtain further information on the physiological
properties of the ligament, we reinvestigated its responses
to electrical and chemical stimuli. We found that the liga-
ment behaves as an excitable motile tissue, shortening
and developing a mechanical force following the appli-
cation of either type of stimulus (Vidal el al.. 1983). The
most probable explanation of the discrepancy, in identical
experiments, between our positive results and the negative
ones of Takahashi is that prior to stimulating the ligament,
we treated it briefly with tyramine ( 1 mM. 2-5 min). This
compound, like its close analog octopamine, exerts a lytic
effect on catch and a relaxing effect on contraction (Mo-
rales el al., 1989). The use of tyramine allowed us to work
with a fully relaxed preparation in every experiment. In
addition, we applied a force of 2 to 3 g, which tends to
separate the two calcareous moieties of the spine-test joint.
By comparing the kinetics of the catch with the contrac-
ture induced by cholinergic agonists on the same prepa-

ration, we concluded that these phenomena are two sides
of the same coin. In other words, we believe that catch is
simply the expression of the shortening of the ligament.

Mechanism of Catch

We must now consider how the contraction of the lig-
ament opposes, or counteracts altogether, the passive
movements of the spine. An answer to this question must
explain how the force generated by the scant and slender
muscle fibers can overpower the stresses generated, often
with considerable mechanical advantage, by the external
forces acting on the shaft of the spine. The fine structure
of the essentially simple but highly ordered insertions of
the ligament onto the stereom in Eucidaris was described
by Smith el al. ( 1990) and revealed an order first hinted
at in the light micrographs of Takahashi (1966). Within
the stereom, the collagen columns divide into a series of
successive, parallel slender straps, passing reflexively across
struts or microbeams that border spaces considerably
wider than the straps they accommodate. In most micro-
graphs obtained by Smith and co-workers, the straps ap-
pear to be tightly cinched to the struts (see Fig. 9), but
they are sometimes seen lying free within the lacunae,
suggesting that they are not "glued" immovably to the ster-
eom microbeams. The lacunae are sufficiently wide to
permit some movement of the straps when disengaged.

An answer to the main question posed above may be
found in the frictional resistance generated at the ligament
insertions by minute but crucial movement of the straps
over the struts. As the friction between two sliding surfaces
is proportional to the force that keeps them together, the
resistance between straps and struts will be modulated by
the muscle fibers in parallel with the collagen columns.
In this model, shortening of the ligament that initiates
catch will increase the force that presses the straps upon
the struts, thereby increasing the friction between these
two structures.

Our model of catch is shown in Figure 1 1. In the ab-
sence of cholinergic stimulation, the muscle fibers will be
relaxed and, therefore, the ligament will be slack. The
straps will rest loosely on the struts, and the spine-test
joint can be moved passively without offering significant
resistance. As muscle contraction starts to tighten the
ligament, a very small change in the position of the straps
is envisaged as introducing frictional resistance at the sites
where they appressed the struts within the stereom. The
friction between the surfaces of both structures, according
to this model, will absorb the energy applied by external
forces. The model further emphasizes that, rather than

Figure 10. Transverse section of Eucitlaris central ligament. Note that the collagen forms a continuous
array largely filling the field. Groups of microfilaments are present (arrows) but nerve processes and muscle
cells are absent, x 30,000

126

J. DEL CASTILLO /:T .11.

acting as a work-generating device, the function of the
muscle fibers of the ligament seems to be (like the braking
pedal of a car) that of controlling an energy-absorbing or
energy-dissipating system, similar in design to an auto-
motive friction brake, engineered to take advantage of the
roughly 30,000 bands or straps underlying each square
millimeter of the insertion surfaces both at the spine base
and test. The resistance generated at single strap-strut
contacts will be greatly amplified by the multiplicative
effect of friction sites in series.

The Central Ligament

A final piece of evidence in support of the above model
is provided by comparing the structure and function of
the main ligament and a supplementary structure, the
central ligament, that is present in many echinoid spines
including those ofEucidaris. The central ligament extends
across the midpoint of, and inserts into cylindrical cavities
in, each surface of the spine-test articulation (Cuenot,
1948; Hyman, 1955). Motokawa (1983) described this
structure in Diaclcma xetosum: it is relatively robust, about
0.5 mm in diameter, and responsible for maintaining the
attachment between spine and test, even when the joint
is dislocated by extreme spine declination (Takahashi,
1967c). In our observations on Eitcidaris (Fig. 7), the cor-
responding structure is <0. 1 mm in diameter, consider-
ably smaller in relation to the articulation than in Dia-
denui. The central ligament in Eucidaris differs strikingly
in both fine structure and stereom insertion from the main
ligament. First, the collagen fibrils form a continuous and
compact block that is not divided into discrete columns
(Fig. 10). Second, it contains neither muscle cells nor
granule-containing neurites. In common with the main
ligament, collagen straps do enter the stereom. but they
loop irregularly through cavities of the unmodified, tetra-
gonal, stereom fabric (Fig. 8), and the elaborate system
of struts and straps of the main ligament is absent. Fur-
thermore, we were unable to detect any mechanical re-
sponse to acetylcholine in the central ligament in contrast
to the contracture and catch elicited in the main ligament.
We view the central ligament in Euddaris as a physio-
logically inactive link, presumably safeguarding the align-
ment of the articulation during spine movement.

The structural peculiarities of the central ligament are
consistent with our view of the way in which the main
ligament achieves the catch state. Without muscle fibers,
the central ligament cannot shorten. Because the central
ligament does not undergo length change and is not in-
volved in catch, the collagen is arranged for maximal ten-
sile strength, not to accommodate an intra-ligament slid-
ing movement. Rather than being arranged in interdigi-
tating columns, as in the main ligament, the collagen fibrils
are disposed as a continuous block virtually filling the
structure. In the main ligament, columns of collagen fibrils

Q)

E

Ca 4pm

1

ca. 2mm

ca.4|jm

Figure II. Schematic diagram of model discussed in the text. R
relaxed ligament; C: contracted ligament. Approximate dimensions of
the spine and test stereom, and the ligament, included in the diagram
are indicated; note the great difference in scale. Stippled circles represent
transverse profiles of stereom struts: only three struts are shown in each
stereom (of the five or six actually present in each row). In R the collagen
straps are represented as looping loosely between the struts; in C' they
are tightly applied to the struts. In R relaxed muscle fibers inserting on
collagen cylinders of the main ligament are represented by wide-spaced
dotted lines (. . . .); in (' contracted fibers are represented by close-
spaced dotted lines ( ).

are stabilized by cross-links; we suggest that similar links
present in the central ligament stabilize it for its purely
passive, mechanical role. Furthermore, the central liga-
ment, stressed only by spine movement, is well-served by
an unspecialized anchorage, contrasting with the precise
arrays of collagen straps and stereom struts of the main
ligament, discussed above.

Our findings in liiicidiin.'i differ in important respects
from those of Motokawa (1983) in Dicidcma. He found
the central ligament physiologically similar to the main
ligament, its viscosity was increased by acetylcholine and
decreased by epinephrine. He suggested that the central
ligament is mechanically and structurally similar to the

CATCH IN SEA URCHIN SPINES

127

main ligament, except for the apparent absence of muscle
fibers in the former. Moreover, in Diadcma the collagen
fibrils are grouped in columns in both main and central
ligaments. Other than noting the very different functions
of spines in Eucidarix and Diadcma. we can at present
only draw attention to these discrepancies, not account
for them.

Conclusions

In the model of catch we have proposed, we view evo-
lutionary experimentation as providing a solution to the
problem of minimizing muscle tissue mass, while achiev-
ing maximal efficiency. Indeed, a catch apparatus that
would meet the mechanical needs of the spine, but made
up of conventionally arranged muscle fibers, might be too
'expensive' for the very limited energetic resources of the
sea urchin (Bianconcini el a/.. 1985). The layer of con-
ventional muscles external to the ligament is responsible
for moving the spine, whereas the muscle of the catch
system was diverted from power generation to the regu-
lation of the energy-absorbing function of the ligament.
In a sense, von Uexkiill and Takahashi were both correct
in seeing catch as a property, respectively, of muscle and
collagen. The spine ligament, with its catch capacity, may
be regarded as a myotendinous organ that combines in a
uniquely efficient manner the contractile properties of
muscle with the tensile strength of collagen fibrils, to pro-
duce a variable-length tendon.

Acknowledgments

We are grateful to Dr. Mark Miller and Dr. Richard
Orkand for their critical reading of the manuscript, and
to Mr. Faustino McKenzie for collecting and maintaining
the sea urchins. TEM and SEM preparations were made
with the assistance of Mrs. Barbara Luke. Supported by
NIH Grants Nos. NS-07464 and NS- 14938, and HRD
93531 30 (NSF-RIMI).

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Reference: .Bio/ Bull 188: 128-135. (April, 1995)

Protein-Membrane Interaction Is Essential to Normal
Assembly of the Microsporidian Spore Invasion Tube

EARL WEIDNER 1 -, S. B. MANALE 1 2 , S. K. HALONEN 1 \ AND J. W. LYNN

Department of Zoology ami Physiology, Louisiana Stale University, Baton Rouge. Louisiana 70803;

-Marine Biological Laboratory. Woods Hole, Massachusetts 02543: and ^Department of Neurology,

Albert Einstein College of Medicine. Bronx. New York 10461

Abstract. Changes in the protein-membrane interaction
during assembly of the microsporidian spore invasion
tubes were followed by electron microscopy, by video im-
aging with differential interference contrast (DIC), and by
the fluorescent probes 4',6-diamidino-2-phenylindolc
(DAPI) and 9-diethylamino-5H-benzo[n]phenoxazine-5-
one (Nile red). Microsporidian spore invasion tubes form
by the eversion of polar filament protein (PFP) and pre-
sumptive extrusion apparatus (EAP) membrane. Both of
these components are essential for formation of the in-
vasion tube. The results indicate that the behavior of the
EAP membrane is greatly affected by the position and
chemical state of the PFP at the eversion area that con-
stitutes the advancing tube terminal assembly site (TAS).
Visual evidence indicates that the EAP membrane is the
vehicle for PFP and that this membrane also provides the
envelope that surrounds the sporoplasm after its passage
through the invasion tube.

Introduction

Microsporidians are a large group of intracellular par-
asites widely distributed within many animal groups
(Canning and Lom, 1986). Some species, as opportunistic
parasites, have developed a significant presence in AIDS
patients (Ornstein et ai. 1990; Canning and Hollister,
1991; Cali et ai. 1993). The spore stage is characterized
by an extrusion apparatus (EAP) with two principal as-
semblages, a polar filament protein (PFP) coil, and an
envelope consisting of an extensive pleated system of

Received 24 October 1994; accepted IS January 1995.

Abbreviations Nile red. 9-diethylamino-5H-benzo[]phenoxazine-5-
one; DAPI, 4',6-diamidmo-2-phenylindole; DIC, differential interference
contrast; EAP, extrusion apparatus; PFP, polar filament protein; TAS,
terminal assembly site.

membranes (Cali, 1991). The infective microsporidian
spore is remarkable because it discharges its entire contents
(excluding the plasma membrane) through a 100-nm-wide
tube and into a target cell with the time frame of 100-
150 ms (Lom and Vavra, 1963; Undeen, 1990; Frixione
el a/., 1992). During spore discharge, the invasion tube
cinemalographically resembles an extruding cnidarian
nematocyst (Blanquet. 1983), a discharging capsule from
a myxosporean spore (Lom and Noble, 1984; El-Matbouli
el a/.. 1992), or a discharging toxicyst from the ciliate
Loxop/ivl/um (Hausmann, 1978). In all of these extreme
forms of exocytosis, the extrusion apparatus is confined
to an organelle with an outer wall; but. with microspo-
ridians, the EAP is segregated from the spore cytoplasm
by a membrane, rather than a walled structure (Weidner
et ai. 1984).

The primary components of the microsporidian extru-
sion apparatus, the PFP and the EAP membrane, display
a significant interactive change during invasion tube dis-
charge. In the presence of PFP, the EAP membrane ini-
tially develops into a tubular shape; but with removal of
the PFP, the membrane develops a saccular shape. The
PFP is a low molecular weight peptide (recently, resolved
to two elements) that assembles into stable polymers.
Polymeric PFP is stable in sodium dodecyl sulfate but
dissociates in dithiothreitol after urea pretreatment
(Weidner, 1976). The fluidity of polymerized PFP is
modulated by Ca : * and by pH yielding unstable mono-
layers (Weidner, 1976. 1982).

Here, we report that the EAP membrane is essential
for the formation of the invasion tube because it affects
the assembly of PFP. Major modulations in the interac-
tions between the PFP and the EAP membrane occur at
the advancing tube tip terminal assembly site (TAS).
Video imaging with differential interference contrast

128

INVASION TUBE ASSEMBLY

129

(DIC) and fluorescent probes were used in this study. 4',6-
diamidino-2-phenylindole (DAPI), a binder of certain
polyanionic substrates and double-stranded DNA (Bonne
el al., 1985; Meszaros el at., 1987), was used to identify
the nuclear and cytosol positions during invasion tube
discharge. A lipophilic generalized membrane probe. 9-
diethylamino-5H-benzo[a]phenoxazine-5-one (Nile red)
(Greenspan et al.. 1985: Greenspan and Fowler, 1985),
was used to monitor the membrane because it provides
an intense fluorescence and did not affect spore discharge.
A preliminary study using fluorescent probes to monitor
spore discharge has been previously published (Weidner
ct ill.. 1994).

Materials and Methods
Microsporidian spores

Spragiiea lophii spores were recovered from the central
nervous system of the monkfish, Lophins americanns.
Monkfish were provided by the Marine Resources de-
partment of the Marine Biological Laboratory in Woods
Hole, Massachusetts. Spores were purified by a wash cycle
described elsewhere (Weidner, 1976).

Spore activation

Isolated spores previously maintained in 0.05 M
Hepes were placed in medium adjusted to pH 7 with
10~ 5 A/ Ca 2+ for 30 min and then transferred into ac-
tivation medium consisting of 2.0% mucin (human or
porcine type 1 from Sigma) and Hepes buffer adjusted
topH 9.5.

Fluorescence

S. lophii spores were placed in incubation medium con-
taining 0.1 Mg/ml 4',6-diamidino-2-phenylindole (DAPI)
or 0.1 Mg/ml 9-diethylamino-5H-benzo[rt]phenoxazine-5-
one (Nile red) in 0.05 M Hepes buffer for 30-45 min.
Spores were then transferred to activation medium and
examined with a Nikon Microphot FXA or Zeiss Axio-
phot optical system. The DAPI imaging was carried out
with a 365-nm excitation frequency and with a 400-nm
highpass barrier filter. Nile red imaging was carried out
using an FITC Filter set (Nikon) with a 480-nm excitation
frequency and a 510-nm highpass barrier filter. Images
were examined with 60X or 100X Fluor or Planapochro-
matic oil immersion lenses.

I "lit CO

Images of microsporidian spore discharge were re-
corded using a customized Nikon Optiphot-2 adapted
to an Odyssey XI 1 laser confocal unit (Noran Instru-
ments, Inc.). Video recordings were recovered with a

Nikon 60x oil immersion lens (Nikon Inc., Melville,
NY). Confocal fluorescence measurements were re-
corded using a 488-nm excitation wavelength and a
515-nm highpass barrier filter. Image recordings were
saved on an optical memory disk (Panasonic 2028F),
digitalized, and analyzed with Intervision (Noran In-
struments, Inc.). Photographs were taken using a 35-
mm camera to capture images from a 23-in silicon
graphics monitor.

Elcclron microscopy

Negative-stained material was prepared by applying
discharged spores onto Formvar-coated grids. Grids were
stained with 2% phosphotungstic acid adjusted to pH 6.9.
For topographical images of discharged spores, platinum-
carbon replicas were prepared as described earlier (Koo
el al.. 1973). Material prepared for sectioning was fixed
in 1% glutaraldehyde in 0.1 M cacodylate buffer, post-
fixed in osmium, and processed by standard procedures
(Weidner, 1976).

Results

Transmission electron microscopy oj microsporidian
spore invasion tnhes

Polar filament protein (PFP) and membrane assembly
were the main part of the extrusion apparatus of S lophii
spores. Before filament extrusion and tube formation,
paracrystalline-like proteins organized as a coil at the polar
end and extended to a subsurface spore aperture (Fig.
I A). In unfired spores, a single membrane surrounded the
filament coil and extended as a system of pleats along
with the ascending filament near the polar aperture.
However, with spore discharge, PFP emerged from the
aperture and became positionally reversed in reference to
the filament membrane (Fig. I A, B, and C). The dis-
charged membrane was a double cylinder at the distal
region of the emerging tube with PFP apparent within
the membrane cylinders (Fig. IB and C). However, the
extruded tubes showed significant amounts of PFP on the
membrane exterior assembled as a stable outer envelope
(Fig. 1C). Platinum-carbon replicas of discharged tubes
show a distinction between empty, incompletely extruded,
and fully extruded invasion tubes. The incompletely ex-
truded tubes (50 ^m or less) had internal cylinders of ma-
terial (Fig. 1 D), whereas the fully extruded invasion tubes
(measuring 65-70 /urn) were devoid of internal cylinders
(Fig. ID).

DAPI anil DIC' inutxiii.i; of discharging S. lophii spores

The DNA fluorophore, DAPI, labeled S. lophii nuclei
without significantly affecting spore activation and tube
discharge when incubations were for less than 1 h with

130

E. WEIDNHR ET II

' ii' *

-,'*.-, , ^ Jl . , ^ibWvaA^o- -

B

v -

Figure I. Electron micrographs of microsporidian extrusion apparatus. (-\) I'nfired spore of Spraguea
loplui with ascending part of polar filament protein (PFP) surrounded by membrane pleats. (B) Partially
discharged spore is shown in section: the double cylinders of membrane are apparent (M). as is the exteriorized
PFP (S). (C| Negative-stained invasion tube shows outer protein envelope (arrow) and an inner membrane
cylinder bearing what appears to be PFP material. (D) The platinum-carbon replica distinguishes between
extruded invasion tubes with inner cylinders of membrane (incomplete), and completely extruded tubes
that appear empty.

5 pM of label. However, extended (overnight) incuba-
tions with 50 \iM DAPI affected tube extrusion and
sporoplasmic sac emergence at the end of the tube.
When D1C was coupled with DAPI fluorescence, nu-
clear movement was observed in association with in-
vasion tube formation and sporoplasmic sac emergence
(Fig. 2A-F). Sporoplasmic sacs did not appear at the
tube ends until the tubes had reached a maximum
length of 10 nm. The spore nucleus did not begin to
traverse the tube until 90-95% of the discharged tube
had emerged from the spore. However, in 1-2% of the
discharged spores with extruded sporoplasmic sacs, the
nuclei were still within the invasion tube (Fig. 2D).
The failure rate of sporoplasmic sac emergence or of
sporoplasm extrusion was increased with extended
spore incubations (6-12 h) in higher concentrations of
DAPI (S

of microsporidian spore invasion lithe
with A';7f m/

Intense Nile red fluorescence was apparent during
membrane discharge through invasion tubes, but the in-
tensity diminished rapidly after the sporoplasmic sacs had
emerged (Fig. 3A and B). Similarly, discharged tubes pre-
washed in SDS detergent showed little fluorescence. Be-
cause of the intensity of Nile red fluorescence for mem-
brane, this dye was used to monitor the movement of
membrane down the invasion tube during spore extrusion
(Fig. 4A-F). Video analyses showed that at the onset of
spore discharge, the Nile red fluorescence was highest at
the distal end of the emerging tube (Fig. 5A-F). Although
the fluorescence was strongest along the distal 1 5 ^m of
invasion tubes 50-55 /JITI in length, fully extruded inva-
sion tubes (70 ^m) exhibited an even fluorescence

INVASION TUBE ASSEMBLY

131

Figure 2. Light optics showing discharging SpraKiiea laphii spores prelaheled with DAPI and examined
with DIG imaging. (A) The discharged spore invasion tube has emerged (arrow) with the nucleus (DAPI-
hlue stain) at the tube tip. Note the fully emerged sporoplasm (sp) from another spore. (B and C) Sporoplasmic
sac emerging at the invasion tube tip (arrows). (D) The sporoplasmic sac has fully emerged, but the nucleus
(arrow) is still within the invasion tube. (E) Nucleus is emerging into the sporoplasmic sac at the invasion
tube tip. (F) Fully emerged sporoplasm with nuclear components (arrows).

throughout the tube (Fig. 5G). Ultrastructural observa-
tions indicate that discharged tubes 40-50 pm in length
had double membrane cylinders at the distal ends; how-
ever, fully extruded invasion tubes did not exhibit double
membranes. Tube fluorescence was reduced significantly
with the emergence of the sporoplasmic sac membrane
at the tube ends (Figs. 4F. 5G and H). Ultrastructural
observations of discharged spores with extruded sporo-

plasmic sacs revealed discharged tubes without mem-
branes.

DIC imaging ofmicrosporidian invasion tiihc.\
immediately prior to sporoplasmic sac emergence

Ultrastructural studies indicate that the invasion tube
tips had double membrane cylinders at the emerging tube

Figure 3. Light optics showing discharging Spraxuea laphii prelabeled with Nile red. Note the intensity
of the fluorescence in the incompletely discharged tubes, indicating the presence of the membrane cylinders
extruding from the discharge apparatus (A and B). Arrow shows a completely discharged invasion tube that
has been cleared of the membrane cylinders; fluorescence is nearly absent. The spherical body at the tip of
this invasion tube is the sporoplasmic sac (arrowhead).

132

E. WEIDNER AT AL

Figure 4. Video sequence showing Nile-red-labeled Spru.viicu lop/iu spore undergoing tube discharge.
(A-E) The fluorescence is higher at the tube tip; ultrastructural images show this area to have two membrane
cylinders. (F)Tube is completely assembled and the membrane has emerged into a sporoplasmic sac. Arrows
show the end of the extruding tube (A-E) and sporoplasm (F).

ends. With the completion of tube assembly (65-70
membrane sacs emerged followed by a flow of sporoplasm
into the sacs. We used DIC imaging to investigate the
nature of the tube tip immediately before sporoplasmic
sac emergence. Assuming that the inner membrane cyl-
inder moved by an everting motion over the surface of
the external cylinder of membrane at the tube tip, a pre-
dictable funnel-shaped bladder would be expected before
full sporoplasmic emergence. Such a funnel-shaped
emergence was observed milliseconds before the appear-
ance of the full sporoplasmic sac (Fig. 6A-C). The timing
for this event was significantly increased by preincubating
spores in 50 /iM DAPI overnight. This did not affect spore
activation, but produced either a block or a delay in spo-
roplasmic sac emergence at the tube end.

Discussion

Interactions ol invasion tube /naicin tun/ membrane
assembly

Nile red video data indicate a strong fluorescence for
membrane along the length of the invasion tube through-
out tube assembly; however, this fluorescence moves to
the assembled tube end with the emergence of the spo-
roplasmic sac. Ultrastructural observations indicate that
tube fluorescence is due to the double membrane cylinders
that extend the length of forming tubes. Before the PFP
begins the eversion process with membrane at the ad-
vancing tube terminal assembly site (TAS), the protein is
believed to be monomeric and confined to the inside of
the tube membrane. However, when the PFP begins ever-
sion with membrane at the TAS, evidence indicates that
the protein transforms into a stable polymer (Weidner,
1976). Ultrastructural observations of the TAS indicate
that the PFP polymer and adjoining membrane are con-
tinuous with the inner tube monomeric PFP and inner
membrane at the TAS. At the opposite end of the invasion
tube from the TAS, the polymerized PFP and the external
cylinder of membrane are attached to the polar aperture
of the spore. This membrane has a short-term attachment
restricted to the period of tube assembly as indicated by
ultrastructural and fluorescent probe studies showing that

this membrane is liberated and discharged during spo-
roplasmic sac formation at the end of the invasion tube.
Video analysis of discharging spores shows invasion
tubes growing exclusively at the TAS. This is most obvious
in discharging tubes that follow a tortuous path, since all
points on the formed tube remain fixed in position while
the TAS movement continues by eversion (Weidner.
1982). Furthermore, when video recording data of tube
discharge were analyzed with digital sequential frame
subtractions (30-ms intervals), the only apparent change
was at the discharging tube tip, indicating membrane
movement exclusively at the TAS. Finally, DIC imaging
clearly indicates tube growth at the TAS by eversion, es-
pecially after completion of tube assembly when the
membrane at the TAS begins eversion by forming a fun-
nel-shaped bladder (as modeled in Fig. 8).

Membrane behavior in the microsporidian invasion tube
is atleclecl by 1'1-P

When membrane assemblages move on or within a cell,
the shape the membrane assumes depends on the inter-
actions between the membrane and the associated proteins
(Cunningham and Edelman, 1990). The question of
whether this general rule applies to specialized organelles
that explosively discharge membrane by eversion lias
arisen because a tubular shape was thought to be an in-
herent property of this membrane configuration. How-
ever, specific examples indicate that this is not the case.
Certain kinds of nematocysts (rhopalonemes of siphono-
phores and spirocysts of anemones) evert a saccular
membrane rather than a slender tubular one (Hyman,
1940). Although the end result of microsporidian spore
discharge is membrane that is saccular in shape, the initial
process begins with the eversion of membrane in a slender
tube. Assembly of the microsporidian spore invasion tube
continues until all the PFP has emerged to complete tube
assembly. After all the PFP has emerged at the TAS,
membrane continues to exit the tube. Since this mem-
brane is without associated PFP, it shows an altered be-
havior by forming a saccular shape at the TAS.

Evidence indicates that the PFP effect on membrane
depends on its position and chemical state within the ex-

INVASION TUBE ASSEMBLY

133

Figure 5. Digital sequential frame subtractions showing Nile-red-labeled Spiu^iicti lophn spore undergoing
tube discharge and sporoplasmic sac emergence. Each image represents a 30-ms shift in the video. (A-F)
The invasion tube is emerging, with the highest level of membrane fluorescence at the tube tip (arrows). (G
and H) Fluorescence is greatest in the extruded sporoplasmic sac (arrows), while the remaining tube has lost
much of the fluorescence.

trusion tube. During tube assembly, the PFP first appears
fluid and has an apparent adhesive affinity for membrane,
as indicated by the sharpness by which the PFP everts
with its adjoining membrane at the TAS. The sharpness
of eversion at the TAS is believed to give the tube some
capacity to pierce through substrate (Lom and Vavra.

1963). an ability that would be essential to penetration of
host-cell membrane. Although the fluidity of the PFP
within the invasion tubes is not clear, isolated PFP poly-
mer displays increased fluidity when subjected to 10 4 Af
Ca :+ ; moreover, discharged PFP tubes show some ten-
dency to fuse or branch when exposed to medium with

134

E WEIDNER AT AL

Figure 6. DIC imaging of microsporidian spore invasion tubes im-
mediately before sporoplasmic sac emergence. These three images show
a characteristic funnel-shaped bladder emerging from the tube tip mil-
liseconds before the full sporoplasmic sac emerges. Such a shape is pre-
dicted when an inner cylinder of membrane is everting and sliding over
an outer envelope. Tube thickness is 0.1-0.13 pm.

calcium (Weidner, 1982). Also, isolated PFP displays sig-
nificant fluidity under //; vitro conditions, although it will
not form discrete tubular profiles (Weidner, 1976). When
the PFP emerges from discharging tubes at the TAS, the
PFP is clearly a polymer, and its stability is obvious be-
cause it resists shape change across the pH range and be-
cause it resists dissociation in 1 % SDS. On the other hand,
internal tube PFP that has yet to reach the TAS appears
to dissociate in SDS (Weidner. 1976), indicating that it is
not a polymer, but monomeric. When monomeric PFP
approaches the TAS. it apparently retains an adherence
for membrane since these two elements always exteriorize
together at the TAS regardless of the physical conditions
of the external medium. Several observations indicate that
polymerized PFP has little or no membrane adhesiveness.
Ultrastructural observations indicate that a substantial
space is present between exteriorized PFP polymer and
the adjoining membrane within the invasion tube. Also,
after all the internal PFP has exited the tube, the remaining
membrane of the tube is liberated from the attachment
point at the polar aperture and passes to the sac at the
end of the tube. Therefore, it appears that monomeric
PFP has a strong affinity for membrane until it is exter-
iorized from the invasion tube, where it becomes a poly-
mer and loses some of its membrane affinity. In a sense,
PFP is a morphoregulatory molecule with the membrane
behavior being regulated by its association with protein
(see Cunningham and Edelman, 1990).

I'lasma membrane origins during microsporidian
spore diseharge

One remarkable feature of microsporidian spore dis-
charge is the discard of the plasma membrane with the
spore ghost while all of the sporoplasm (nucleus and cy-

Kigure 7. Model illustrating the emergence of the extrusion apparatus
(EAP) from the spore. The membrane-bound polar filament everts, with
membrane sliding on the membrane, as the inner cylinder delivers the
polar filament protein (PFP) (dots within the apparatus) to the tube tip.
and the protein stabilizes into outside polymer. Ultimately, when all the
protein has been assembled, the remaining membrane continues to slide
out and contributes to the sporoplasmic sac at the end of the tube. The
last diagram in the sequence shows the empty tube, now represented
only by the PFP polymer, and the emerging sporoplasmic sac. The spo-
roplasm characteristically enters the sporoplasmic sac milliseconds after
its emergence.

toplasm) exits through a tube and into a new membrane
sac. This discharged sac is thought to develop by the ever-
sion of the extrusion apparatus (EAP) membrane. The
EAP membrane is the only probable source for the spo-
roplasmic sac membrane (Weidner et at.. 1984). The
model proposed here illustrates how the sporoplasm nu-
cleus and cytoplasm are transferred out of the spore by
membrane eversion of the EAP (Figs. 7 and 8). The
movement of sporoplasm into the sporoplasmic sac gen-
erally appears to occur concurrently. Whether any spo-

Figure 8. Scheme illustrating how the inner cylinder of membrane
slides over the outer cylinder during the emergence of the sporoplasmic
sac al the end of the discharged invasion tube. Such an event happens
only when the discharged tube is fully assembled. This scheme is primarily
based on the DIC images of the tube tip (see Fig. 6) in addition to the
Ultrastructural observations.

INVASION TUBE ASSEMBLY

135

roplasm moves into the sporoplasmic sac without mem-
brane is not clear at this time. The following observations
with DAPI and DIG imaging indicate that the EAP mem-
brane system is exterior to the nuclear and cytosolic com-
ponents of the spore. First, the nucleus and cytosol do
not enter the invasion tube until it is fully assembled.
Because the invasion tube forms by everting membrane
from the EAP, its membrane system segregates it from
the sporoplasm within the spore. Thus, the EAP mem-
brane probably has to turn itself inside out before the
sporoplasm can move into the EAP compartment. Sec-
ond, discharged, empty EAP membranous sacs are oc-
casionally observed attached to the ends of invasion tubes
while the DAPI stained nucleus remains within the tube.
This condition would be impossible unless the EAP and
sporoplasm were initially segregated within the spore. The
most logical explanation is that the EAP membrane pro-
vides the new plasma membrane for the discharged spo-
roplasm through an everting process.

Figure 8 illustrates our model for EAP eversion into
the plasma membrane sac. It is based on DIC images of
the TAS immediately preceding full emergence of the
membrane sac. The funnel-shaped bladder that com-
monly appears at the end of the tube is believed to be
derived from the inner cylinder of the EAP membrane
moving in relation to the outer envelope. Prolonged in-
cubations of activated spores with DAPI slowed the rate
of membrane emergence at the ends of assembled tubes
and made it easier to observe sac formation. Extended
DAPI incubations are thought to affect the membrane
movement by perturbing the Ca 2+ levels within the tube.
PFP assembly and disassembly are affected by Ca :f
(Weidner, 1982) and, because DAPI can affect the kinetics
of Ca 2t uptake or release in molecular systems (Meszaros
et al.. 1987), the changes in membrane behavior caused
by prolonged incubations in DAPI may be due either to
direct interference with the PFP or to alterations in Ca 2+
uptake that change the PFP polymerization.

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Reference: Blot Bull, 188: 136-14? I April. 1945)

Microtubule Arrays During Ooplasmic Segregation
in the Medaka Fish Egg (Oryzias latipes)

V. C. ABRAHAM 1 , A. L. MILLER 2 . AND R. A. FLUCK 1 ,*

^Biology Department. Franklin and Marshall College. Lancaster, Pennsylvania 17604-3003: ami
2 , Marine Biological Laboratory. Woods Hole. Massachusetts (12543

Abstract. We used indirect immunofluorescence to
study microtuhule arrays in the medaka egg between fer-
tilization (normalized time. T,,, = 0) and the first cleavage
(T n = 1.0). Eggs were fixed at various times after fertil-
ization and examined with conventional fluorescence mi-
croscopy, laser scanning confocal microscopy, and three-
dimensional fluorescence microscopy. Soon after the eggs
were fertilized (T,, = 0.02). we saw microtiibules oriented
perpendicular to the plane of the plasma membrane but
none parallel to the plasma membrane. Later ( T,, = 0.08),
we saw an array of microtubules oriented more or less
parallel to the plasma membrane but having no apparent
preferred orientation with respect to the animal-vegetal
axis of the egg. In the interpolar regions of the egg, this
network increased in density by T,, = 0.24 and remained
a constant feature of the ooplasm until the first cleavage.
From T n = 0.30 to 0.76 the polar regions of the egg con-
tained dense arrays of organized microtubules. At the
animal pole, microtubules radiated from a site near the
pronuclei; while at the vegetal pole, an array of parallel
microtubules was present. Injection of the weak (K.,,
= 1.5 nl\I) calcium buffer 5,5'-dibromo-BAPTA disrupted
the radial pattern of microtubules near the animal pole
but had no apparent effect on the parallel array of micro-
tubules near the vegetal pole. Because this buffer has pre-
viously been shown to suppress a zone of elevated cytosolic
calcium at the animal pole and to disrupt ooplasmic seg-
regation in this egg, the results of the present study ( I )
are consistent with a model in which microtubules are
required for ooplasmic segregation in the medaka egg,
and (2) suggest that the normal function of a microtubule-
organizing center at the animal pole of the egg requires a
zone of elevated calcium.

Received 2(1 June 1994; accepted II Januan, 1995.
* To whom correspondence should he addressed

Introduction

Ooplasmic segregation in the medaka egg consists of
the approximately simultaneous streaming of ooplasm
toward the animal pole, the saltatory movement of parcels
toward the animal pole and vegetal pole, and the move-
ment of oil droplets toward the vegetal pole (Abraham el
ul.. 1 993a; Webb and Fluck. 1993). These movements are
roughly simultaneous and are essentially completed dur-
ing the first cell cycle. Streaming of ooplasm toward the
animal pole is inhibited by eytochalasin D and thus pre-
sumably requires microfilaments (Webb and Fluck. 1993),
whereas saltatory movements and the movement of oil
droplets toward the vegetal pole are both inhibited by
microtubule poisons and thus presumably require micro-
tubules (Abraham el a/.. 1993a). All of these movements
can be easily observed in the optically clear medaka egg,
in which a thin (^15 ^m thick) peripheral layer of oo-
plasm surrounds a large, central yolk vacuole.

In a number of diverse animals, including annelids
(Astrow el ai. 1989). ctenophores (Houliston ct al.. 1993).
echmoderms (Harris el al., 1980), ascidians (Sawada and
Schatten. 1988). and amphibians (Elinson and Rowning,
1 988: Houliston and Elinson. 1 99 1 ; Schroeder and Card.
1992; Elinson and Palacek. 1993). a network of micro-
tubules that forms during the first cell cycle is required
for the movement of specific components of the ooplasm.
including the pronuclei. In the present study, we describe
the development of such an array of microtubules during
the first cell cycle of the medaka egg. Previous studies of
microtubules in teleost embryos either have provided very
little information (Beams el al.. 1985) or have been done
on later stages of development (Strahle and Jesuthasan,
1993; Solnica-Kre/el and Driever. 1994).

We also pursued the hypothesis that gradients of cy-
tosolic free Ca :< organize the multimolecular assemblies

136

MICROTUBULES IN THE MEDAKA EGG

137

involved in ooplasmic segregation in the medaka egg.
Zones of elevated calcium are present at the animal and
vegetal poles of the medaka egg during segregation (Fluck
el a/., 1992). Moreover, injection of 5.5'-dibromo-BAPTA
(hereafter referred to as dihromo-BAPTA), a weak calcium
buffer that dissipates cytosolic calcium gradients (Pethig
ct nl.. 1989; Speksnijder et at.. 1989; Fluck ct <//., 1992),
inhibits ooplasmic segregation in this egg (Fluck ct ill .
1994). In the present study we monitored the effects of
this buffer on the spatiotemporal pattern of microtubules
and on pronuclear movements. A preliminary account of
these findings has been published (Abraham ct ill.. 1993b).

Materials and Methods

Procedures

Methods for removing gonads from breeding medaka
and for the in vitro fertilization of eggs have been described
previously (Abraham ct <//.. 1993a). Some eggs were in-
cubated in 100 n\l colchicine (dissolved in buffered saline
solution: 1 1 1 m.U NaCl; 5.37 mM KC1; 1.0 m.M CaCl : :
0.6 mM MgSO 4 ; 5 mM HEPES, pH 7.3) for 1 h at room
temperature (20-25C) before they were fertilized.

Using a high-pressure microinjection system, we in-
jected enough 50 mM dibromo-BAPTA (tetrapotassium
salt: containing 5 mM HEPES, pH 7.2, and sufficient
CaCl 2 to set [Ca 2+ ] fri , c at 100 n.\f) to raise the cytosolic
concentration to 2.7 mM (Fluck ct ai, 1992). Injections
were made within 10 arc toward the vegetal pole from
the equator within 6 min after fertilization. Control eggs
received a comparable volume (ca. 1.5 nl) of 150m/U
KG. 5 m.U HEPES. pH 7.2.

The procedures for indirect immunofluorescence were
those developed by Card (1991) for the study of micro-
tubules in Xcnopus lucvis eggs. At regular intervals after
fertilization, eggs were transferred to fixative solution at
room temperature (3.7% formaldehyde, 0.25% glutaral-
dehyde, 0.2% Triton X-100. 5 nWEGTA, 1 mA/MgCU,
80 mM potassium PIPES. pH 6.8). After 4 h. the eggs
were dechorionated with fine forceps and post-fixed in
absolute methanol (-20C) overnight. The eggs were then
washed with phosphate-buffered saline (PBS: 128 m.U
NaCl; 2 mM KC1; 8 mMNaH : PO 4 ; 2 m.M KH : PO 4 , pH
7.2) and incubated in 100 m.M sodium borohydride (in
PBS) for 6 h. The eggs were then washed with cold Tris-
buftered saline (TBS: 155 mA/NaCl; 10 mM Tris-CI. pH
7.4; 0. 1 % Nonidet P-40); transferred to Lab-Tek chamber
slides (Thomas Scientific, Swedesboro. New Jersey); in-
cubated with a monoclonal mouse anti--tubulin anti-
body (ICN, DM1 A; diluted 1:250 with TBS containing
2% bovine serum albumin); washed with TBS for 24 h;
incubated with the secondary antibody (rhodamine-con-
jugated goat anti-mouse IgG. diluted 1:25 with TBS con-
taining 2%> bovine serum albumin): and washed for 24 h

with TBS. We performed two kinds of antibody controls:
omission of the primary antibody and omission of both
the primary and the secondary antibodies. To stain the
nuclei, we incubated the eggs for 30 min in a solution of
Hoechst 33258 ( 10 /ug ml ') in TBS.

The eggs were mounted between a coverglass and a
microscope slide as described previously (Abraham ct til .
1993a) and examined by one of three methods. Conven-
tional epifluorescence microscopy was performed with a
Nikon Optiphot microscope coupled to a Dage-MTI SIT
camera and video monitor; in later replicates, the SIT
camera was coupled to a Dage-MTI DSP-2000 image
processor. In either case, the image on the monitor was
photographed through a Ronchi grating (Rolyn Optics
Co.. Covina. California: Inoue, 1981). Some eggs were
examined with a laser scanning confocal microscope (Zeiss
LSCM 410) at the Marine Biological Laboratory.

We also acquired and processed images at the Science
and Technology Center at Carnegie-Mellon University.
Images at each focal plane were acquired on a multimode
microscope (Biological Detection Systems, Inc., Pitts-
burgh. Pennsylvania) equipped with a 576 X 384 Thomp-
son chip, cooled CCD camera (Photometries Ltd.. Tucson,
Arizona). Three-dimensional fluorescence microscopy
used the nearest neighbor algorithm to correct for out-of-
focus fluorescence (Biological Detection Systems, Inc.)
The final three-dimensional representation was generated
with the program ANALYZE (Biomedical Imaging Re-
source, Mayo Foundation, Rochester, Minnesota) on the
SGI. Inc.. ONYX workstation.

The results summarized herein represent 1 1 replicate
experiments in which we fixed eggs at various intervals
after fertilization and three replicate experiments in which
we examined the effects of dibromo-BAPTA on micro-
tubules. In all, we have examined a total of 1 1 7 eggs from
17 females, including 20 eggs into which we injected di-
bromo-BAPTA and 8 eggs into which we injected KC1.

Chemicals

Formaldehyde and glutaraldehyde were obtained from
Electron Microscopy Sciences (Fort Washington, Penn-
sylvania): Triton X-100. Nonidet P-40, sodium borohy-
dride. bovine serum albumin. Hoechst 33258, and col-
chicine from Sigma (St. Louis. Missouri); 5.5'-dibromo-
BAPTA from Molecular Probes (Eugene. Oregon); anti-
rt-tubulin antibody from ICN (Costa Mesa, California):
and rhodamine-conjugated goat anti-mouse IgG from
Organon Teknika (Malvern, Pennsylvania).

Results

The various events that constitute ooplasmic segrega-
tion in the fertilized medaka egg have already been de-
scribed in detail (Abraham ct ii/.. 1993a) and are only

138

V. C. ABRAHAM 1:1 II.

summarized below. To indicate the relative temporal po-
sition of these events, we have used a normalized time
(7",,) scale in which the time between fertilization and the
beginning of cytokinesis is 1 unit. In the range of room
temperatures in which the experiments were done (20-
25C). 1 unit of normalized time corresponds to 75-
1 10 min. Fertilization in the medaka egg is followed by
a cortical granule reaction and a contraction, during which
the ooplasm and its contents appear to be pulled toward
the animal pole. The second meiotic division is completed
by T,, = 0. 1 2 and is followed closely by a second animal-
pole-directed contraction at T n ~ 0. 1 5. This second con-
traction heralds the beginning of ooplasmic segregation
in the form of streaming of ooplasm toward the animal
pole, saltatory movements of parcels toward both polar
regions, and movement of oil droplets (a class of ooplasmic
inclusions) of various sizes toward the vegetal pole. At T n
= 1 .0. the blastodisc divides into two blastomeres. By this
time the oil droplets have formed a crude ring around the
vegetal pole.

Microtubule network dyiuimic.\ during ooplasmic
segregation

We identified three regions of the medaka egg on the
basis of the spatiotemporal pattern of microtubules in
them (Figs. 1-3): (1) a region within 30 ( = 300//m) arc
of the animal pole: (2) a region within 60 (sa600^m)
arc on both sides of the equator: and (3) a region within
30 ( = 300 ^m) arc of the vegetal pole. In all regions of
the egg except the animal pole, all the microtubules were
within 2-3 ftm of the surface of the egg and were visible
in a single optical section.

T n = 0.02. O.OS. ami 0.16. The earliest time at which
we fixed eggs was T n = 0.02; that is, immediately after
the contraction that follows the cortical granule reaction.
Though we searched the ooplasm everywhere on these
eggs (and all of the ooplasm is peripheral, see Introduc-
tion), we saw no microtubules parallel to the surface of
the egg but saw instead a punctate pattern of fluorescence
(Fig. 1 A). Analysis of these eggs by three-dimensional flu-
orescence microscopy suggests that the sources of fluo-
rescence were microtubules oriented perpendicular to the
surface of the egg (Fig. IB). This punctate pattern was a
prominent feature of eggs fixed at 7",, = 0.02 and 0.08 and
was less prominent at later stages.

In eggs fixed at T,, = 0.08, we saw a very sparse network
of microtubules oriented parallel to the surface of the egg
but having no apparent preferred orientation with respect
to the animal-vegetal axis of the egg. This network was
roughly confined to the animal hemisphere, and we saw
no microtubules in most of the vegetal hemisphere (Fig.
1C, D).

In eggs fixed at T,, = 0. 16 by which time ooplasmic
segregation has begun in the form of streaming, saltatory

movements, and oil droplet movement a network of
microtubules was present throughout the ooplasm, and
the network in the equatorial region was denser than at
7",, =t 0.08 (Fig. IE). The network did not yet appear to
have a preferred orientation.

To test for autofluorescence. we examined fixed eggs
that were incubated with no antibodies and that were pro-
cessed for viewing up to the first wash with TBS. We saw
no fluorescence at all in these eggs (data not shown). We
also examined eggs that were incubated with the secondary
antibody but not with the primary antibody. In these eggs.
we saw difluse fluorescence but no linear elements or
punctate sources of fluorescence (Fig. IF). In eggs incu-
bated with 100 JJ.M colchicine before fixation and incu-
bated with both the primary and secondary antibodies,
we saw diffuse fluorescence but no microtubules or any
punctate sources of fluorescence (data not shown).

The pattern of microtubules in eggs fixed at T n = 0. 16
is summarized in Fig. 3A.

T n = 0.24. 0.3, 0.4. 0.76. In ooplasm near the equator,
the network of microtubules was denser than at T,, = 0.16
(Fig. 2A; compare with Fig. IE) but still showed no pre-
ferred orientation with respect to the animal-vegetal axis.
In contrast, oriented microtubule networks were present
near the vegetal pole and animal pole by T,, = 0.24 and
T,, = 0.3. respectively. Near the vegetal pole, an array of
(mostly) parallel microtubules was present (Fig. 2B). This
"vegetal mat" extended about 30 arc (300 A<m) in all
directions from the vegetal pole and covered at least 30%
of the area of the vegetal hemisphere. The orientation of
microtubules was uniform throughout the vegetal mat.
Beyond the edges of the mat, the network of microtubules
abruptly lost its orientation and became the network
characteristic of interpolar ooplasm. Using the z-section-
ing function of the LSCM 410, we determined that the
thickness of the microtubule network in the vegetal mat
( = 2.7 ^/m) was the same as the thickness of the unoriented
microtubule network just outside the mat. The two net-
works appeared to be continuous with each other and
were in the same optical section, suggesting that the two
networks are located at the same depth in the ooplasm.

Near the animal pole, microtubules were oriented more
or less along meridian lines and appeared to radiate from
a microtubule-organizing center near the male and female
pronuclei, which were 38.6 6.7 i/rn (X SD, /; = 5)
below the surface of the blastodisc at T,, = 0.76 (Fig. 2D-
I). This network of oriented microtubules extended ap-
proximately 30 arc (300 /urn) in all directions from the
animal pole.

The pattern of microtubule distribution in eggs fixed
at T,, = 0.30-0.76 is summarized in Fig. 3B.

T n = 1.0. In these eggs, the blastodisc has begun to
divide into two blastomeres. and essentially all oil droplets
have aggregated in a crude ring near the vegetal pole. Each

M1CROTUBULES IN THE MEDAKA EGG

139

Figure 1. Formation of a network of microtubules in the fertilized egg. A, C, D, E, and F were obtained
by conventional epifluorescence microscopy and B by three-dimensional fluorescence microscopy. Scale
bars, lOfim. A, C, D, and E are printed at the same magnification, B at another, and F at another. (A)
Ooplasm near the equator of an egg fixed at T n = 0.02. Characteristic of this stage of development are a
punctate pattern of fluorescence throughout the ooplasm and the absence of unambiguous microtubular
elements oriented parallel to the plasma membrane. The dark structures (arrowheads) are ooplasmic inclusions.
(B) Three-dimensional rendering of optical sections of ooplasm near the equator at T n = 0.024. Using a
lOOx objective lens, we made 33 optical sections at steps of 0.158 nm. beginning at the surface of the egg.
Visible in this reconstruction are both the punctate pattern at the surface of the egg (the top of the image)
and linear elements that are oriented perpendicular to the surface of the egg (arrowheads). Two edges of the
reconstruction are marked by a white dashed line. (C) Ooplasm near the animal pole of an egg fixed at ?
= 0.08. Note the presence of unambiguous microtubules in the field (arrowheads). (D) Ooplasm near the
vegetal pole of an egg fixed at /" = 0.08. This image was obtained from the same egg as Fig. 2C. No
unambiguous microtubules oriented parallel to the plasma membrane can be seen. (E) Ooplasm near the
equator of an egg fixed at T n = 0.16. The animal-vegetal axis runs from lower left to upper right. This
unoriented network is denser than that found at the equator of an egg fixed at T n = 0.08 (not shown). (F)
In eggs not incubated with the primary antibody after fixation (T,, = 0.4), we saw only weak and diffuse
fluorescence but saw neither any filamentous structures nor a punctate pattern of fluorescence. These images
were obtained by standard epifluorescence microscopy.

Figure 2. Microtuhule arrays in eggs fixed at T a = 0.4-1.0. All images were obtained by conventional
epifluorescence microscopy. Scale bars: A, 10 ^m; D, 25 ^m. A-C were printed at one magnification. D-I
at another. (A) Equatorial ooplasm of an egg fixed at T n = 0.4. The animal-vegetal axis runs from lower left
to upper right. The microtubule network here is denser than in eggs fixed at T n = 0. 16 (compare with Fig.
1C) and still has no apparent preferred orientation. (B) The vegetal mat of parallel microtubules in an egg
fixed at T n = 0.4. Practically all the microtubules share a single orientation in this mat, which we saw at T n
= 0.24-0.76. (C) Ooplasm at the vegetal pole of an egg fixed at T n = 1.0. The parallel organization of
microtubules near the vegetal pole is gone. (D and E) The same microscopic field of a double-stained egg

140

M1CROTUBULES IN THE MEDAKA EGG

141

T n = 0.16

B

T n = 0.4

MTOC

Oil
Droplet

Vegetal Mat

Blastomere
MTOCs

T n =1.0

Unoriented
Vegetal MTs

Equator =

Figure 3. Summary of the spatial distribution of microtubules in medaka during the first cell cycle. The
diagrams were drawn approximately to scale with respect to the size of the microtubule-organizing center,
the vegetal mat, and oil droplets; but the thickness of individual microtubules has been greatly exaggerated
to demonstrate the arrays clearly. The diameter of the egg is about 1 140 ^m. (A) Microtubules in an egg
fixed at /" = 0. 16. An unoriented network of microtubules, denser near the animal pole, is present throughout
the egg. (B) Microtubules in an egg fixed at T = 0.4. An array of microtubules that radiate from a zone in
the blastodisc near the pronuclei forms by T n = 0.3 and persists until at least T n = 0.76, while at the vegetal
pole, a mat of parallel microtubules forms by T n = 0.24 and persists until at least T n = 0.76. Microtubules
in interpolar ooplasm have no preferred orientation at any time. Note that most of the oil droplets from
the animal hemisphere have moved into the equatorial region by this time. (C) Microtubules in an egg fixed
at T n = 1 .0. Two microtubule-organizing centers, one at each pole of the dividing blastodisc, are present.
The density of microtubules in interpolar ooplasm has decreased, and the parallel organization of microtubules
at the vegetal pole has been lost.

Animal
Hemisphere

Vegetal
Hemisphere

of these blastomeres had a microtubule-organizing center
(data not shown) similar to the one present in the blas-
todisc from T n = 0.30 to at least T,, = 0.76. The parallel
organization of the microtubule network near the vegetal
pole was lost by this stage (Fig. 2C). The pattern of mi-
crotubules in eggs fixed at T,, 1.0 is summarized in
Fig. 3C.

Microtubule networks in eggs injected
with 5,5'-dibromo-BAPTA

The effects of injected KC1 and dibromo-BAPTA on
microtubule networks are summarized in Figures 4 and
5. The microtubule networks in eggs into which we in-
jected KC1 were indistinguishable from those found in
uninjected eggs fixed at the same time (T,, = 0.76 [from
57 min to 84 min after fertilization, depending on the
temperature]; Fig. 4A [compare with Fig. 2B], 5A [com-
pare with Fig. 2A]). The parallel organization of micro-
tubules in the vegetal mat was not disrupted by dibromo-
BAPTA (Fig. 4B). However, the injection of dibromo-
BAPTA had profound effects on the microtubule networks
near the animal pole, where the radiating pattern of mi-

crotubules near the animal pole was disrupted (Fig. 4C,
D). The only apparent effect of dibromo-BAPTA on the
microtubule arrays in the equatorial region was a decrease
in the density of microtubules near the injection site (Fig.
5B, C).

Dibromo-BAPTA also inhibited the movement of the
pronuclei. At T n ^ 0.5, the distance between the male
and female pronuclei was as follows: uninjected control
eggs, 1.8 3.0 /^m (X SD, n = 10 eggs); eggs into which
we injected KC1, 1.7 3.4 ^m (/; = 4 eggs); eggs into
which we injected dibromo-BAPTA, 40.4 28.6 ^m (n
= 5 eggs). A one-way analysis of variance and post-hoc
Student's /-tests revealed that the BAPTA-treated eggs dif-
fered significantly (P = 0.038) from the other two treat-
ments.

Discussion

The pattern of microtubules in the fertilized medaka
egg varied both temporally and spatially during the first
cell cycle. Temporal changes included an increase in the
density of microtubules throughout the ooplasm and
changes in the degree of orientation of microtubules in

(T n = 0.4) is shown, using either a filter to show the fusing Hoechst-stained pronuclei (D) or one to show
rhodamine-stained microtubules (E). Microtubules radiate from a region near the pronuclei, roughly following
meridian lines. (F-I) These are photographs of four quadrants around, and just outside, the region containing
the pronuclei. Note the strong orientation of microtubules along meridian lines. The pronuclei are to lower
right in F, lower left in G, upper right in H, upper left in I.

142

V. C. ABRAHAM ET AL

Figure 4. The effect of dibromo-BAPTA on microtubule arrays in polar ooplasm. All eggs were fixed
at /" = 0.50. A and B are confocal images obtained using the Zeiss laser scanning confocal microscope; C
and D were obtained by standard epifluorescence microscopy. Scale bar = 10 ^m; A and B are at the same
magnification, C and D at another. (A and B) Injection of neither KC1 (A) nor dibromo-BAPTA (B; fixed
32 min after fertilization and about 26 min after injection of dibromo-BAPTA) had any apparent effect on
the parallel array of microtubules in the vegetal mat. (C) Microtubules are oriented along meridian lines
near the animal pole of this control (uninjected) egg. The pronuclei are to the right of the region shown in
this photograph. (D) Injection of dibromo-BAPTA disrupted the radial pattern of microtubules in the blas-
todisc. This egg was fixed about 52 min after fertilization and about 46 min after the injection of dibromo-
BAPTA. Note the absence of radially oriented microtubules in this photograph, which is of the same region
of the egg as the one shown in Fig. 4C.

the polar regions of the egg. The striking feature of the
region near the animal pole was the development of an
array of microtubules that radiated from near the pro-
nuclei to approximately 30 arc from the animal pole.
Though microtubules were initially (T,, = 0.08) present
near the animal pole as a sparse unoriented network, a
dense network of radially oriented microtubules had
formed by T n = 0.3. Such radial arrays of microtubules

are present in fertilized eggs of a number of animals, in-
cluding annelids (Astrow el at., 1 989). ctenophores (Hou-
liston el a/., 1993), echinoderms (Harris el al, 1980), as-
cidians (Sawada and Schatten. 1988), and amphibians
(Houliston and Elinson, 1991; Schroeder and Card, 1992;
Elinson and Palacek, 1993), and are associated with the
sperm aster and sometimes with the female pronucleus
as well.

MICROTUBULES IN THE MEDAKA EGG

143

Figure 5. The effects of K.C1 and dibromo-BAPTA on microtubule
arrays in equatorial ooplasm. All eggs were fixed at T n = 0.76 (about
60 min after fertilization and about 54 min after injection of dibromo-
BAPTA), and all images were obtained using the Zeiss laser scanning
confocal microscope. Scale bar. 10 ^m. (A) In eggs into which we injected
K.C1. the array of microtubules was similar to uninjected controls (not
shown). The animal-vegetal axis runs from left to right. (B and C) Injection
of dibromo-BAPTA appeared to decrease the density of microtubules
near the injection site (B: the animal-vegetal axis runs from right to left)
relative to the density observed at the antipode of the injection site (C;
the animal-vegetal axis runs from bottom to top).

The mat of parallel microtubules at the vegetal pole of
the medaka egg is reminiscent of the one found at the
vegetal pole of amphibian eggs, where it is involved in the
cortical rotation that forms the gray crescent and thus
determines the dorsal-ventral axis of the embrvo (Manes

el a/.. 1978; Scharf and Gerhart, 1983; Vincent et al.,
1987; Gerhart et al.. 1989; Elinson and Rowning, 1988:
Elinson and Palacek, 1993). A similar array of parallel
microtubules is also present at the vegetal pole of the ze-
brafish (Danio rerio) egg (see Fig. 9C in Strahle and Je-
suthasan, 1993). Although there is no evidence for the
occurrence of a cortical rotation in the medaka egg, em-
bryos of some primitive fishes that undergo holoblastic
cleavage (species in the Superorder Chondrostei within
the Actinopterygii, for example the sturgeon. Acipenser
giildenstlidti) do form a gray crescent and thus perhaps
do undergo a cortical rotation (reviewed by Bolker, 1993;
see also Clavert. 1962: Ginsburg and Dettlaff. 1991). In
amphibian eggs, the plane of the first cleavage is parallel
to the microtubules near the vegetal pole; and in both
amphibians and primitive fishes, the plane of the first
cleavage bisects the gray crescent along a meridian (Bolker.
1993). We are currently pursuing the question of the re-
lationship between the orientation of microtubules in the
vegetal mat and that of the embryonic axes in the medaka
egg. In both Xenupux laevis and the medaka. the parallel
orientation of the microtubules in the vegetal pole region
is transient. In .V. laevix. this mat begins to form at T,,
= 0.55 and disappears after T n = 0.8 (Elinson and Pa-
lacek. 1993). In medaka eggs, it is present by T n = 0.24
and is still present at T,, = 0.76; but by the beginning of
the first cleavage, the network has lost its parallel orga-
nization.

In sharp contrast to the strongly oriented networks of
microtubules in polar ooplasm, interpolar ooplasm con-
tained a network of crisscrossed microtubules having no
apparent preferred orientation. Microtubules were present
here by T n = 0.08 and subsequently increased in density
through T n = 0.24. The lack of orientation of microtubules
in this region of the medaka eggs contrasts with the sit-
uation in the zebrafish egg. in which microtubules are
oriented along the animal-vegetal axis during both the
first cell cycle and epiboly (Strahle and Jesuthasan, 1993;
Solnica-Krezel and Driever. 1994).

Because microtubule poisons inhibit the movement of
oil droplets toward the vegetal pole of the medaka egg. it
has been suggested that these droplets are transported
along microtubules by microtubule-based motors (Abra-
ham et al.. 1993a). Further, because oil droplets move
toward the vegetal pole roughly along meridians, we pre-
dicted that a network of microtubules, also oriented more
or less along meridians, would extend from the animal
pole to near the vegetal pole. Although such a pattern did
develop near the animal pole by T,, = 0.3. a time when
oil droplets have begun to move toward the vegetal pole,
we saw no such preferred orientation of microtubules in
most of the interpolar ooplasm, suggesting that oil droplets
move toward the vegetal pole along a biochemically dis-

144

V. C. ABRAHAM ET AL

tincUGard. 1991; Wolff tf a/., 1992) subset of interpolar

microtubules that are oriented along meridian lines.

The movement of most oil droplets toward the vegetal
pole of the medaka egg begins at T,, = 0. 1 5-0.30 (Catalone
and Fluck. 1994). The results of the present study, which
show that microtubules are present before oil droplets
begin to move toward the vegetal pole, suggest that this
movement is triggered by some factor other than the ap-
pearance of microtubules. We have also shown that even
though the movement of oil droplets is "frozen" at the
antipode of the site at which dibromo-BAPTA is injected
into the egg (Fluck el at., 1994), many microtubules are
present in this region of the ooplasm. Factors that could
initiate the movement of oil droplets toward the vegetal
pole include the formation of an appropriate subtype of
microtubules, the activation of a microtubule-based motor
such as kinesin (Brady et al.. 1990; Schnapp et al . 1992),
fulfillment of a requirement that a minimum number of
microtubules be associated with an oil droplet before it
begins to move, or a gel-sol transition of the ooplasm
(Janson and Taylor, 1993) that frees the droplets and en-
ables them to move toward the vegetal pole.

We have also demonstrated in the present study that
injection of dibromo-BAPTA into medaka eggs affects
the pattern of microtubules in the egg. This relatively weak
calcium buffer (K D = 1.5 M; Pethig el al.. 1989) is be-
lieved to act as a shuttle buffer, one that binds Ca :+ at the
high end of a [Ca 2+ ] gradient and releases it at the low
end; in other words, the buffer facilitates the diffusion of
Ca :+ within the cell (Speksnijder et al.. 1989). The time
between injection of dibromo-BAPTA and fixation of the
eggs in the present study was sufficient for the buffer to
diffuse to both the animal pole and vegetal pole, to dis-
sipate cytosolic Ca :4 gradients near the poles, and to
inhibit the formation of the blastodisc and the move-
ment of oil droplets toward the vegetal pole (Fluck et al..
1992, 1994).

Our results suggest that at least some of the effects of
dibromo-BAPTA on ooplasmic segregation in the medaka
egg are caused by its disruption of microtubule formation
and organization, which are controlled by a number of
calcium-binding regulatory proteins (Weisenberg, 1972;
Schliwa et al.. 1981; Keith el al.. 1983; Lieuvin et a/.,
1994). Facilitated diffusion theory predicts that calcium
buffer concentrations similar to those found effective in
the present study act by dissipating zones of cytosolic
[Ca 2+ ]f ree in the micromolar range (Speksnijder el al..
1989). An example of a protein that is responsive to
changes in [Ca :+ ], ree in this range is calmodulin (Cheung.
1980), which regulates the activity of proteins that interact
with microtubules (Ishikawa el a/.. 1992).

The inhibition of pronuclear movements by dibromo-
BAPTA is consistent with the explanation that the buffer
disrupts the organization of microtubules near the animal

pole. In a number of species, including the medaka, the
movement of the pronuclei is inhibited by microtubule
poisons (Hiramoto et al.. 1984; Sawada and Schatten,
1989; Abraham et al.. I993a). suggesting that microtu-
bules are necessary for these movements.

The parallel array of microtubules near the vegetal pole
was apparently resistant to the effects of the buffer, sug-
gesting that microtubules in this region of the egg differ
in some way from those in other regions of the egg. This
possible difference could be probed by microinjecting a
microtubule poison, for example demecolcine, directly
into the vegetal ooplasm.

The suggestion that the medaka egg has two indepen-
dent microtubule networks is consistent with the situation
in A laevis. in which two independent networks of mi-
crotubules are present during the first cell cycle: one is
near the animal pole and is associated with the pronuclei,
while the other is the parallel array of microtubules near
the vegetal pole (Elinson and Rowning. 1988; Houliston
and Elinson. 1991; Elinson and Palacek. 1993; Sardet et
al.. 1994). Moreover, in both amphibian eggs (Houliston
and Elinson. 1991; Elinson and Palacek, 1993) and me-
daka eggs (Webb and Fluck, unpub. obs.), vegetal cortical
arrays of microtubules form in parthenogenetically acti-
vated eggs. Using the "Colcemid-UV" method (Hiramoto
et al.. 1984: Webb and Fluck. 1993), we are currently
pursuing the question of the existence of independent ar-
rays of microtubules in the animal pole region, vegetal
pole region, and interpolar ooplasm. Our data at this time
do not permit us to say whether microtubules are contin-
uous from one region of the egg to another.

Acknowledgments

Supported by NSF DCB-9017210 and NSF MCB-
9316125 to RAF, NSF BIR 9211855 to Lionel F. Jaffe
and ALM, and grants from Franklin & Marshall College's
Hackman Scholar Program and Harry W. and Mary B.
Huffnagle Fund to VCA. Louis Kerr provided technical
assistance in the use of the laser scanning confocal mi-
croscope in the Central Microscope Facility at the Marine
Biological Laboratory. Tamika Webb assisted in the
preparation and examination of one batch of eggs, and
Bob Golder assisted in the preparation of Figure 3. The
research at Carnegie-Mellon University was supported by
National Science Foundation and Technology Center
grant DIR-89201 18. We thank Robbin L. DeBiasio for
image acquisition, data processing, and 3-D representa-
tion; and Gregory M. LaRocca for technical assistance
and image acquisition.

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Reference: Biol Bull 188: 146-156. (April. 1495)

Microtubule-Based Movements During Ooplasmic
Segregation in the Medaka Fish Egg (Oryzias latipes)

TAMIKA A. WEBB, WENDY J. KOWALSKI, AND RICHARD A. FLUCK*

Franklin and Marshall College, Department of Biology, Lancaster, Pennsylvania 17604

Abstract. We used time-lapse video microscopy to
monitor the effects of cytochalasin D (CCD) and demc-
colcme on cytoplasmic streaming toward the animal pole
of the medaka egg, the formation of the blastodisc at the
animal pole, the movement of oil droplets in the cyto-
plasm toward the vegetal pole, and the saltatory move-
ment of small cytoplasmic parcels toward the animal pole
and vegetal pole. Cytochalasin D inhibited both cyto-
plasmic streaming toward the animal pole and the for-
mation of the blastodisc, suggesting a role for microfila-
ments in these processes. However. CCD had no apparent
effect on saltatory movement or on the movement of oil
droplets toward the vegetal pole. Thus, the segregation of
oil droplets toward the vegetal pole is not the result of the
bulk movement of ooplasm toward the animal pole.

In eggs treated with demecolcine, oil droplets did not
move toward the vegetal pole but instead floated to the
uppermost portion of the egg, and saltatory movement
was absent, suggesting that microtubules are required for
these movements. The effects of demecolcine on oil drop-
let movement and saltatory movement could be reversed
by irradiating the eggs with UV light (360 nm). Using
indirect immunofluorescence, we showed that irradiation
of demecolcine-treated eggs with UV light regenerated
microtubules within the irradiated region.

The specificity of the mechanism responsible for the
vegetal poleward movement of oil droplets was assessed
by microinjecting droplets of five other fluids mineral
oil, silicone oil, vegetable oil, and two tluorinated aliphatic
compounds into the ooplasm. None of these fluids seg-
regated with the endogenous oil droplets. These results
suggest that a specific mechanism, probably involving
microtubules, is responsible for the segregation of oil
droplets to the vegetal pole.

Received 19 July 1 994; accepted II Januan 1995.
* To whom correspondence should be addressed.

Introduction

The optical clarity, size (diameter = 1.2 mm), and year-
round availability of the medaka fish egg make it a fa-
vorable system in which to study ooplasmic segregation.
Ooplasmic segregation in this egg consists of the bulk flow
of ooplasm toward the animal pole of the egg, the move-
ment of oil droplets toward the vegetal pole, and the sal-
tatory movement of small ooplasmic inclusions toward
the animal and vegetal poles of the egg (Iwamatsu, 1973;
Abraham el aL. I993a). all of which take place more or
less simultaneously. Formation of the blastodisc at the
animal pole of zebrafish (Dcinio rerio) and loach (Mis-
gumus fossilis) eggs is inhibited by cytochalasin cyto-
chalasin B (CCB) in zebrafish (Katow, 1983) and cyto-
chalasin D (CCD) in loach (Ivanenkov el al.. 1987). This
inhibition suggests that microfilaments are involved in
the movement of ooplasm toward the animal pole of these
eggs. However, there are no reports of the effects of CCD
on streaming or on formation of the blastodisc in the
medaka egg. Thus, one objective of the present study was
to monitor the effects of CCD on these phenomena in the
medaka egg.

The possible involvement of microtubules in segrega-
tion in the medaka egg has been explored more fully
(Abraham el al., 1993a. 1993b). A network of microtu-
bules is organized during the period of ooplasmic segre-
gation in the medaka (Abraham et al.. 1993b), and this
network is absent from eggs incubated with a microtubule
poison (Abraham et al., 1993b). Moreover, three micro-
tubule poisons colchicine, demecolcine, and nocoda-
zole inhibit the normal movement of oil droplets toward
the vegetal pole, eliminate the saltatory motion of small
inclusions, and slow the growth of the blastodisc: eggs
treated with /i-lumicolchicine, an inactive derivative of
colchicine. segregated normally (Abraham et al., 1993a).
The second objective of the present study was to determine

146

OOPLASMIC SEGREGATION IN MEDAKA EGGS

147

whether the inhibitory effects of one of these poisons (de-
mecolcine) on microtubules and ooplasmic segregation
could be reversed by illuminating the eggs with ultraviolet
light (360 nm), which photolyzes demecolcine, converting
it to lumidemecolcine, a molecule that is not a microtu-
bule poison (Aronson and Inoue, 1970). This "Colcemid-
UV method" (Colcemid is a trade name for demecolcine)
has been used by a number of investigators to study mi-
crotubule-mediated movements (Sluder, 1976; Hiramoto
el a!., 1984; Hamaguchi and Hiramoto, 1986; Ladrach
and La Fountain, 1986).

The suggestion that microtubules are involved in the
segregation of oil droplets toward the vegetal pole is in-
consistent with the hypothesis that this segregation is
caused simply by the bulk flow of ooplasm in the opposite
direction, that is, toward the animal pole (Sakai, 1965).
This hypothesis can be tested directly by determining
whether oil droplets segregate toward the vegetal pole
when there is no bulk flow of ooplasm toward the animal
pole. Thus, another objective of the present study was to
monitor the effects of CCD on the segregation of oil drop-
lets toward the vegetal pole.

Sakai's hypothesis was based in part on the observation
that when droplets of three oils vegetable oil, liver oil,
and mineral oil were injected into the ooplasm of me-
daka eggs, they segregated toward the vegetal pole along
with endogenous droplets (Sakai, 1965). However, it is
possible that the injected droplets simply floated toward
the vegetal pole, because the eggs in the experiments were
all oriented with their vegetal pole uppermost. Thus, an-
other objective of the present study was to monitor the
movement of injected droplets of five fluids that differ
widely in their chemical and physical properties; the eggs
in the present study were oriented with either their animal
pole or their vegetal pole uppermost.

A preliminary account of these findings has been pub-
lished (Webb and Fluck, 1993).

Materials and Methods

Biological material

The dissection of gonads from breeding medaka, the
preparation of gametes, and the in vitro fertilization of
eggs have been previously described (Yamamoto, 1967;
Abraham el al., 1993a). To indicate the relative temporal
position of these events, we used a normalized time (T n )
scale in which the time between fertilization and the be-
ginning of cytokinesis is 1 unit.

Effects of cytochalasin D on segregation

Stock solutions of CCD (Sigma) in dimethylsulfoxide
(DMSO, Fisher) were diluted 100-fold to make working
solutions. Four eggs were placed in a small Syracuse watch

glass containing 500 /ul of either 1% DMSO in buffered
saline solution (BSS: 1 1 1 mA/ NaCl; 5.37 mA/ KC1;
1 .0 mA/ CaCl 2 ; 0.6 mA/ MgSO 4 ; 5 mA/ HEPES, pH 7.3)
or 10 Mg ml' 1 CCD/1% DMSO in BSS and incubated for
1 h at room temperature ( 24-25 C). The eggs were fer-
tilized by pipetting 50 n\ of a fresh sperm suspension, pre-
pared by mincing testis in BSS, onto the eggs and stirring
quickly. Fertilized eggs were transferred to a microscope
slide on which a cover glass was supported by four pillars
of petroleum jelly (Abraham el al.. 1993a). The volume
of the blastodisc was measured with an image analysis
program (Microcomp Planar Morphometry, Southern
Micro Instruments; Abraham el al., 1993a). To monitor
cytoplasmic streaming, we oriented the eggs with their
animal pole-vegetal pole axis parallel to the surface of
the slide, placed the slide on the stage of a compound
microscope (Nikon Optiphot), and used time-lapse video
microscopy via a 40x phase-contrast objective and a
Dage/MTI Newvicon camera to record ooplasmic move-
ments. On playback, the positions of five particles in each
egg at r n as 0.35 were mapped onto acetate sheets (Abra-
ham el al., 1993a). At the end of recording, the eggs were
washed twice with embryo reading medium ( 1 7 mA/
NaCl; 0.4 mA/ KC1; 0.3 mM CaCU; 0.67 mA/ MgSO 4 ;
0.001 g/1 methylene blue) and transferred to a petri dish
containing 3 ml of embryo rearing medium. Subsequent
development of the eggs was monitored for several days.
A total of 1 1 1 eggs from six females were used in these
experiments; we monitored ooplasmic segregation closely
in 30 eggs.

Effects of demecolcine on segregation

Working solutions of demecolcine (7V-deacetyl-A r -
methyl-colchicine; Sigma) were prepared by diluting (at
least 100X) an aqueous stock solution of 0.35 mA/ de-
mecolcine. Eggs were placed in BSS containing the ap-
propriate concentration of demecolcine and incubated for
1 h at room temperature in dim light. Typically three eggs
were incubated in 375 /ul or four eggs in 500 ^1 of medium.
To fertilize the eggs, we minced a portion of testis in 200 ^1
of BSS and pipetted 50 ^1 of sperm suspension onto the
eggs in the appropriate (drug-containing) medium. Eggs
were then transferred to a coverglass-slide assembly for
microscopy. To monitor ooplasmic segregation in these
eggs, we transilluminated the egg with light from a quartz-
halogen lamp, using a heat filter (KG5) and a 486 DF32
filter (Omega Optical). Movements were recorded by time-
lapse video microscopy with a Dage/MTI SIT camera.

In some experiments, we treated eggs with both CCD
(10 ng mP 1 ) and demecolcine (0.35

Irradiating eggs with ultraviolet light

To irradiate eggs with UV light, we used an Osram
100 W mercury arc lamp. The light from the lamp was

148

T. A. WEBB ET AL

passed through a custom filter cube (Omega Optical) con-
taining a 360 DF 40 filter and a DC 405 dichroic mirror.
An octagonal diaphragm was used to control the size of
the light beam. To reduce the light intensity in some ex-
periments, we used either an ND16 filter (Nikon, which
reduced the intensity = 94%) or an ND 1 .5 filter (Omega
Optical, which reduced the intensity ~ 97%). The light
was projected onto the egg via one of three objective lenses
(all from Nikon): Plan 4X, N.A. =0.1; Fluor/Ph 2 DI.
10X; N.A. = 0.5; Fluor/Ph 3 DL 20x, N.A. = 0.75. Spe-
cific regions of the egg animal pole, vegetal pole, equa-
torial region were illuminated either en lace or en projil.
In most of the experiments described herein, we used the
10X objective lens and irradiated an octagonal region
having a diameter of 475 /urn and an area of 1 .9 X 10 5 //m 2 .
Light intensity was measured with a UVX radiometer
with a long wave sensor (UVP, Inc.). Given a light inten-
sity of 523 juW cm" 2 and assuming that all the UV light
was of wavelength 360 nm. we calculated an incident light
intensity of 4.9 X lO 9 quanta s ' /um 2 (10x objective
lens; no neutral density filter). To monitor the subsequent
development of eggs, we washed them twice with embryo
rearing medium and transferred them to a petri dish con-
taining 3 ml of embryo rearing medium. A total of 292
eggs from 18 females were used in these experiments; we
monitored ooplasmic segregation closely in 96 eggs.

Indirect immunpfluorescence

The methods for fixing eggs and performing indirect
immunofluorescence assays for alpha-tubulin have been
described previously (Gard. 1991; Abraham ct ai, 1993b).
Briefly, the eggs were fixed for 4 h in a mixture of form-
aldehyde (Electron Microscopy Sciences) and glutaral-
dehyde (Electron Microscopy Sciences) at room temper-
ature and then in cold (-20C) methanol; treated with
sodium borohydride; incubated with a monoclonal mouse
anti-alpha-tubulin antibody (ICN, DM1 A; diluted 1:250
with TBS ( 1 55 mAl NaCl; 10 m/U Tris-Cl, pH 7.4; 0. 1%
Nonidet P-40) containing 2% bovine serum albumin; and
incubated with a secondary antibody (Organon Teknika,
rhodamine-conjugated goat anti-mouse IgG, diluted 1:25
with TBS containing 2% bovine serum albumin). Control
eggs were not incubated with the primary antibody. Eggs
that were in a solution of demecolcine and being irradiated
with UV light were fixed on the stage of the microscope
for 5 min before being transferred to a vial of fixative. To
stain nuclei, we incubated the eggs for 30 min in a solution
of Hoechst 33258 (Sigma, 10 M g ml ') in TBS. A total of
68 eggs from 1 1 females were used in these experiments.

Microinjecting fluids into meilaka eggs

Using methods described previously (Fluck el ai, 1991,
1992, 1994), droplets of fluid were microinjected into the

ooplasm between 35 and 90 arc from the animal pole.
Of the 25 eggs used in these experiments. 21 were par-
thenogenetically activated by the injection needle, and
four others were fertilized after injection. Similar results
were obtained with parthenogenetically activated and
fertilized eggs. Droplets of five fluids were injected:
mineral oil, density = 0.84 g ml" 1 ; vegetable oil, density
= 0.9 g ml '; siliconc fluid (Sigma), density = l.OSgml ';
and two fluorinated aliphatic compounds, FC-77 and FC-
3275 (3M), density = 1.78 g ml~'. Although droplets of
silicone fluid and the fluorinated compounds could be
distinguished visually from endogenous oil droplets, we
stained the vegetable oil and mineral oil with either Sudan
black B or Sudan III to identify them.

To monitor the movement of the injected droplets, we
used one of two techniques. In the first, we placed an egg
on a small mound of Dow Corning high vacuum grease
in the bottom of a rectangular plastic spectrophotometer
cuvette and used two video cameras and two time-lapse
videocassette recorders to record the movement of the
droplets simultaneously from two perspectives: a polar
view and a side view. In the second technique, we glued
the egg to the bottom of a petri dish (diam.. 35 mm) with
Cel-Tak (Bio-Polymers, Inc.) and placed a right-angle
prism (Melles-Griot) next to the egg. The petri dish was
then placed on the stage of an inverted microscope (Nikon
Diaphot) and viewed either directly (polar view) or in-
directly via the prism (side view). To view the egg indi-
rectly, we illuminated it from the side, using a fiber optic
cable.

Results

Kllccts ofcytochulasin D and demecolcine on ooplasmic
segregation am/ development

Eggs treated with 1% DMSO formed a blastodisc (vol-
ume at 7 n = 0.85-1.0 = 15.4 2.0 nl, X SD, N = 1
eggs; Fig. 1 A), displayed cytoplasmic streaming (speed of
parcels toward the animal pole = 9.00 5.52 ^m min ',
X SEM, N -- 7 eggs), and developed normally. In
contrast, eggs treated with CCD (lO^gml ') had
essentially no cytoplasmic streaming (velocity = 0.66
0.84 yum min ', X SEM. N = 5 eggs), did not form
a blastodisc (Fig. IB), and did not undergo cell division.
CCD had no apparent effect on the segregation of oil
droplets toward the vegetal pole (Fig. IB) or on the sal-
tatory movement of particles toward either the animal or
vegetal pole.

The effects of demecolcine on the eggs were those pre-
viously described by Abraham el a/. ( 1993a): ( 1 ) oil drop-
lets did not segregate to the vegetal pole but instead floated
to the portion of the egg that was uppermost during the
experiment equator (Fig. 1C), animal pole (Fig. 2C and
D), or vegetal pole (not shown); (2) a smaller-than-normal

OOPLASMIC SEGREGATION IN MEDAK.A EGGS

149

B

Figure I . The effects of CCD and demecolcine on formation of the
hlastodisc and movement of oil droplets. The animal pole is near the
bottom and the vegetal pole near the top of each figure. (A) /" = 1.24.
DMSO (1% in BSS) had no apparent effect on the formation of the
blastodisc at the animal pole of the egg, the movement of oil droplets
toward the vegetal pole, or cell division (note the cleavage furrow, ar-
rowhead). (B) T, = 1.34. CCD ( in M g ml" 1 ) inhibited formation of the
blastodisc but had no apparent effect on the segregation of oil droplets
toward the vegetal pole. (C) T a = 1 .0. This egg was treated with 1 .

blastodisc formed (Fig. 1C); (3) saltatory motion was ab-
sent; and (4) the eggs did not cleave.

In eggs treated with both CCD and demecolcine, we
saw no saltatory movement, no cytoplasmic streaming,
and no movement of oil droplets toward the vegetal pole
(Fig. ID). These eggs also did not cleave (Table I).

Reversal oj the inhibitory effects of demecolcine on
ooplasmic segregation and embryonic development

When demecolcine-treated eggs were irradiated with
UV light, the inhibitory effects of demecolcine were re-
versed: oil droplet movement and saltatory movement
were restored; and the eggs subsequently cleaved, formed
an embryonic axis, and hatched. The effects of UV irra-
diation were less pronounced at higher concentrations of
demecolcine. For example, of 1 1 eggs treated with
0.35 nM demecolcine and irradiated en face at the animal
pole (2.8 X 10 8 quanta s~ ' ^m" 2 ), vegetal pole, or equator,
all 1 1 cleaved and developed an embryonic axis; while of
three eggs treated with 3.5 fiM demecolcine and irradiated
under similar conditions, all three cleaved but none de-
veloped an embryonic axis. The effects of UV irradiation
were also less pronounced when we irradiated the eggs en
profit instead of en face. For example, of five eggs treated
with 0.35 nM or 1.0 ^M demecolcine and irradiated en
face near the equator (2. 8 X 10 8 quanta s~' urn' 2 ), all five
cleaved and developed an embryonic axis. However, of
three eggs treated similarly but irradiated en profil at the
equator, none cleaved.

Apparently normal oil droplet movement was restored
when we irradiated the animal pole or equatorial region
en face, with oil droplets near the animal pole or equator
moving away from the animal pole and toward the vegetal
pole more or less along meridian lines (Fig. 2A-F). Under
appropriate experimental conditions (1.0/j.U demecol-
cine; irradiation with low intensities of UV light), this
reversal of the effects of demecolcine on oil droplet move-
ment was restricted to the portion of the egg that was
irradiated. In such eggs, oil droplets outside the irradiated
region floated to the top of the egg and accumulated at
the edge of the irradiated region, while droplets within
the irradiated region moved out of the irradiated region
and toward the vegetal pole (Fig. 2G and H).

When we irradiated an equatorial region en profil, oil
droplets in the irradiated region moved toward the vegetal
pole, while oil droplets in the rest of the egg either floated

demecolcine. Note the small blastodisc at the animal pole (compare with
Figure I A I and that most oil droplets did not move toward the vegetal
pole but floated to the top of the egg. (D) 7" n = 0.73. This egg was treated
with both CCD and demecolcine (0.35 fiM). Note the absence of a blas-
todisc at the animal pole and that the oil droplets have floated to the
top of the egg. Scale bar. 250 ^m.

150

T. A. WEBB ET AL

to the top of the egg or did not move at all (Fig. 21 and
J). In eggs irradiated en profit at the equator, we also saw
a small accumulation of ooplasm at the animal-pole side
of the irradiated region (Fig. 21 and J).

The effects of UV irradiation on oil droplet movement
and other phenomena clearly extended beyond the irra-
diated region when we used a lower concentration of de-
mecolcine or a higher intensity of UV light. For example,
eggs irradiated en face with a high intensity of UV light
near their equator often underwent cleavage and formed
an embryonic axis. In addition, oil droplets in such eggs
moved beyond the edge of the irradiated region (Fig. 2K).

The movements of droplets within the irradiated re-
gions were very similar to those in control (not treated
with demecolcine) eggs; that, is roughly along meridian
lines and away from the animal pole (Fig. 3).

Segregation appeared normal in medaka eggs that were
irradiated with UV light intensities < about 530 ^W cirT :
(4.9 X 10 9 quanta s~" ^m~ : ) but not treated with
demecolcine (not shown). However, in preliminary
experiments, we found that higher light intensities
(>1100MWcm" : , 1.0 X 10"' quanta s 1 nm~ 2 ) inhibited
both the growth of the blastodisc and the movements of
oil droplets.

Microtiibule regeneration hy ultraviolet irradiation

We saw no microtubules in eggs that were treated with
demecolcine but not irradiated with UV light before fix-
ation (Fig. 4A). Moreover, we saw no or very few micro-
tubules outside the irradiated region of eggs that were ir-
radiated with UV light (Fig. 4B and C). However, the U V-
irradiated regions contained microtubules that in number
and orientation were very similar to those in control eggs
that had not been treated with demecolcine (Fig. 5).

The movement oj droplets of injected fluids

Droplets of mineral oil were injected into five eggs,
which were oriented with their animal pole uppermost.

In two of them, the injected droplets fused with native
droplets, and the hybrid droplets moved toward the vegetal
pole. In two eggs in which the injected droplets were not
observed to fuse with native droplets, the injected droplets
moved up, toward the animal pole (Fig. 6A); and in an-
other egg, the droplet of mineral oil did not move at all,
even though nearby native droplets did move toward the
vegetal pole. The eggs into which we injected FC-77 (four
eggs), FC-3275 (one egg), and silicone oil (four eggs), were
oriented with their vegetal pole uppermost. In all these
eggs, the droplets of injected fluids moved toward the ani-
mal pole and came to rest in the blastodisc (Fig. 6B-D).

We injected vegetable oil into 1 1 eggs and oriented them
with their animal pole uppermost. In eight of these eggs,
the injected droplet fused with endogenous oil droplets,
and these hybrid droplets segregated toward the vegetal
pole. However, in one egg in which the injected droplet
was not observed to fuse with endogenous droplets, the
injected droplet moved up, toward the animal pole, and
came to rest near the blastodisc, while nearby endogenous
droplets moved by it in the opposite direction on their
way to the vegetal pole. In two cases in which we injected
vegetable oil, the injected droplets appeared to follow
larger endogenous droplets that they were touching.

In the case of every fluid vegetable oil, mineral oil,
FC77, FC3275, and silicone oil the movements of
nearby or immediately adjacent endogenous oil droplets
appeared to be unaffected by the presence of the injected
fluid. In other words, endogenous droplets, moving toward
the vegetal pole, passed by the droplets of injected fluid,
which were either stationary or moving toward the animal
pole.

Discussion

The inhibition of cytoplasmic streaming and formation
of the blastodisc by CCD in medaka eggs is consistent
with data obtained from other teleost eggs (Katow, 1983;
Ivanenkov el a/., 1987) and from eggs of other organisms,
including ascidians (Sawada and Osanai, 1981; Jeffery,

Table I

Cytoplasmic streaming, saltatory movement, oil droplet movement, and cleavage in the presence of CCD (10 fig ml~'),
demecolcine (0.35 nM), and CCD/demecoldne

Cytoplasmic

Oil droplet movement

Saltatory

Treatment

streaming

toward vegetal pole

movement

Cleavage

Cytochalasm D

No

Yes

Yes a

No

Demecolcine

Yes

No h

No

No

Cytochalasm D and

No

No

No

No

demecolcine

a Saltatory movement toward both Ihe animal and vegetal poles of the egg.
b Oil droplets floated to the top of the egg.

o V 0:^ M

Figure 2. Ultraviolet light reverses the effect of demecolcine on oil droplet movement. (A) and (B) show
oil droplets near the animal pole of a control egg (not treated with demecolcine) at T n = 0.3 and 0.5,
respectively. Note that many oil droplets have left this region of the egg by T n = 0.5. In (C), where T n
= 0.14, and (D). where 7 n = 0.65, oil droplets have floated to the top and accumulated at the animal pole
of an egg treated with 0.35 pM demecolcine and oriented with animal pole uppermost. An egg that was
treated with 1 .0 ^M demecolcine and irradiated with UV light (7.6 x 10* quanta s" 1 /im" 2 ) en face at the

animal pole is shown at T n = 0.3 in (E) and 7 n = 0.5 in (F). The edges of the diaphragm are shown ().
Note that the oil droplets have moved away from the animal pole. The egg shown in (G) at T a = 0.75 was
incubated in 1.0 //A/ demecolcine and irradiated en lace a\ its animal pole (2.8 / 10* quanta s~' Mm" 2 ). The
ring of oil droplets is the result of (1) oil droplets outside the irradiated region floating to the top of the egg
(toward the viewer) and accumulating at the edge of the irradiated region, and (2) oil droplets within the
irradiated region moving to the edge of the irradiated region. This embryo developed normally and hatched.
The egg shown in (H) at T D = 0.67, was incubated in 1.0 nAt demecolcine and illuminated en face at its
equator (2.8 X 10 8 quanta s" 1 Mm" 2 ). As in Figure 2G, note the absence of oil droplets from the irradiated
region and the presence of a ring of oil droplets at the edge of the irradiated region. This egg cleaved and
formed an embryonic axis, but morphogenesis was very abnormal and the embryo did not hatch. (I) at /"
= 1.1, shows an egg that was treated with 1 .0 pM demecolcine and irradiated en />/<>/;/ near its equator (2.8
x 10 8 quanta s~' Mm' 2 )- Note the accumulation of most oil droplets at the top of the egg (toward the viewer
and out of focus) and the formation of a smaller than normal blastodisc at the animal pole. Three oil droplets

151

152 T. A. WEBB ET AL.

V

Figure 3. Oil droplets within UV-irradiated regions move toward the vegetal pole. A star marks the
starting position of each droplet, and black circles mark the positions at 2-min intervals. The eggs in (A)
and (B) were untreated controls shown from either the animal pole (A) or the equator (B); the eggs shown
in (C) and (D) were treated with 1.0 ^.1/demecolcine and irradiated en luce (3. 3 10 s quanta s~' nm~-) at
either the animal pole (C) or equator (D). Oil droplets near the animal pole (near the center of the figure)
in (A) and (C) moved away from that pole more or less along meridian lines; droplets near the equator in
(B) and (D) moved toward the vegetal pole (to the right). Scale bars: (A) and (B), 200 ^m: (C) and (D).
100 ^m. (A) and (B) were printed at one magnification and (C) and (D) at another.

1984) and an oligochaete (Shimizu. 1982). The inhibition ing. This suggestion should be pursued by staining eggs

suggests that the formation of the blastodisc in the medaka with fluorescently labeled phalloidin during segregation,

egg is the result of the streaming of ooplasm to the animal The solation-contraction coupling hypothesis (Janson

pole and that microfilaments are involved in this stream- and Taylor, 1993) provides a model for how this stream-

Figure 2. (Continued) (arrowheads) were observed to move through the irradiated region toward the
vegetal pole. The small accumulation of ooplasm at the animal pole side of the irradiated region (arrow) is
shown at higher magnification in (J). The egg in (K), where 7~ n = 0.60, was treated with 1.0 M^/demecolcine
and irradiated at the animal pole enfacev/ilh a high intensity of UV light (5.0 x 10 9 quanta s~' ^m" 2 ). Note
that oil droplets have moved beyond the edges of the irradiated region. Scale bar, 100 ^m. (A-F) are printed
at the same magnification, (G-I) at another, and (J) at another.

OOPLASMIC SEGREGATION IN MEDAKA EGGS

153

Figure 4. Microtubules in eggs treated with demecolcine. All three
photographs were taken near the equator of the egg. The egg shown in
(A) was treated with 3.5 n.\l demecolcine but was not irradiated with
UV light. Note the absence of microtubules. Of 1 7 such eggs we observed,
we found no microtubules in 15' and a few scattered ones in 2. (B) and
(C) show ooplasm near the equator in eggs that were treated with 3.5 nM
demecolcine and irradiated with UV light (5.2 x 10 8 quanta s~' jjirT 2 )
en face at the animal pole (B) or en profit at the vegetal pole (C). Note
the apparent absence of microtubules in (B), a typical field, and the
relatively few even in (C). which had the most microtubules that we
observed outside an irradiated region (compare with Fig. 5). Scale bar.
lO/urn.

ing, which is likely driven by the interaction of actin and
myosin. could be organized and driven by the zone of
elevated cytosolic Ca- + present at the animal pole
throughout the period of segregation (Fluck el ai. 1992).
Dissipation of this zone of elevated Ca 2+ inhibits forma-
tion of the blastodisc (Fluck ct ai, 1992. 1994).

The experiments with CCD directly test the hypothesis
that the movement of oil droplets toward the vegetal pole
is caused by the movement of ooplasm in the opposite
direction toward the animal pole. The results are not
consistent with this hypothesis, because oil droplets moved

toward the vegetal pole even when we did not observe
any cytoplasm streaming to the animal pole. Other data
from the present study also conflict with the hypothesis.
Specifically, in demecolcine-treated eggs oriented with
their animal pole uppermost, oil droplets collected at the
animal pole even as ooplasm was also streaming in that
direction.

Moreover, the failure of injected droplets of several
other fluids fluorinated aliphatic compounds, silicone
fluid, mineral oil, and vegetable oil consistently to move
away from the animal pole and toward the vegetal pole
during segregation is inconsistent with the hypothesis. This
failure was probably not due to a toxic effect of the fluids
on nearby cell components, because the movements of
adjacent endogenous droplets appeared to be unaffected
by proximity to a droplet of injected fluid. The behavior
of the injected droplets also suggests a specificity of the
mechanism responsible for the movement of endogenous
oil droplets.

We have previously suggested that microtubules are
required for the segregation of oil droplets to the vegetal
pole of the medaka egg (Abraham el ai. 1993a). In the
present study, we explored the connection between mi-
crotubules and oil droplet movement further by using the
"Colcemid-UV" method to photolyze demecolcine in
specific regions of the egg and thus presumably create
conditions that might support microtubule polymeriza-
tion (Dustin, 1984, ch. 5; Bray, 1992, p. 207). We found
that irradiation of the egg with UV light regenerated both
microtubules and apparently normal oil droplet move-
ment within the irradiated region of the egg. These results
are consistent with a role for microtubules in the move-
ment of oil droplets. To determine whether the micro-
tubule-based motor protein, kinesin, has any role in oil
droplet movement, we are planning experiments in which
we will microinject anti-kinesin antibodies (Ingold ct ai.
1988) into medaka eggs.

Microtubule poisons also disrupt intermediate filaments
in a number of cells (Knapp et ai, 1983; Hunt and Davis,
1990; Pasdar et ai. 1992), so it is possible that some of
the effects we observed were caused by such disruption
in the medaka egg. We are investigating whether inter-
mediate filaments are present in the medaka egg and can
be disrupted by microtubule poisons.

Although the effects of UV irradiation on oil droplet
movement could be localized under appropriate experi-
mental conditions, the effects of irradiation sometimes
clearly extended beyond the irradiated region. For ex-
ample, eggs that were irradiated en face at their equator
usually cleaved and formed an embryonic axis. These re-
sults suggest that UV irradiation at the equator lowered
the concentration of demecolcine in the animal pole re-
gion (where -cell division occurs) enough to permit the
polymerization of tubulin. Moreover, oil droplets often

154

T. A. WEBB l-:i I/

Figure 5. LI V light reverses the effect of demecolcine on tubulin poh men/ation. The eggs shown in (A).
(C). and (E) were neither treated with demecolcine nor irradiated with UV light: those shown in (Bl. (D).
and (F) were Healed with 3.5 ji.U demecolcine and irradiated with UV light (5.2 X 10" quanta s~' ^nr : )
either iv; /</. v at the animal pole (B) or equator (D) or en frulil at the vegetal pole (El. All eggs were fixed
at T n = 0.3. Near the animal pole, in (A) and (B), and the equator, in (C) and (D). a dense network of
microtuhules basing no apparent preferred orientation is present, hut near the \egetal pole, in (E) and (F),
most of the microtuhules are parallel to each other. /:"/; />/<>/;/ and en /</ic irradiation ga\e identical results
in all three regions ol the egg. Scale har, 10 ^m.

moved beyond the edge of the UV-irradiated region, sug-
gesting that microtubules that formed in the irradiated
region subsequently extended beyond this region. Irra-
diation of the egg 01 lace likely photolyzed demecolcine
in the ooplasm on both the near side of the egg and its
antipode as well as in the portion of the yolk vacuole that
is in the light path. As molecules of demecolcine were
inactivated within the irradiated region and the product
diffused away from it. molecules of demecolcine in ad-
jacent, unirradiated regions diffused into the irradiated
region. In other words, the irradiated region acted as a
demecolcine sink, and eventually the concentration of
demecolcine decreased throughout the egg. The spatio-
temporal pattern of the change in demecolcine concen-
tration in an irradiated egg presumably depends on at

least three variables: (a) the initial concentration of de-
mecolcine in the egg. (b) the intensity of the UV light,
and (c) the volume of the irradiated region. Our results
demonstrate that all three variables affected the extent to
which UV irradiation reversed the effects of demecolcine
on oil droplet movement, cleavage, and subsequent de-
velopment.

Irradiation of control medaka eggs with UV light
(360 nm) at intensities adequate to regenerate microtu-
bules and normal movement of oil droplets in poisoned
eggs had no apparent effect on ooplasmic segregation.
These results are consistent with those of other studies in
which UV light has been used to modify amphibian de-
velopment (Grant and Wacaster, 1972; Scharf and Ger-
hart. 1980; Holwill ci <//.. 1987). In all these studies, the

OOPLASMIC SEGREGATION IN MEDAKA EGGS

155

'

B

\

Figure 6. Behavior of injected droplets of fluids. Droplets of fluids
were injected into unfertilized eggs, which were parthenogenetically ac-
tivated by the injection. The egg in (A) was oriented with its animal pole
uppermost; all others had vegetal pole uppermost. In all four eggs, the
droplets of injected fluid (arrowheads) moved toward the animal pole
of the egg and came to rest in the blastodisc at the boundary between
the blastodisc and the yolk vacuole. (A) Mineral oil stained with Sudan
black B, (B) FC-77, (C) FC-3275. (D) sihcone fluid. Scale bar. 250 pm.

predominant wavelength emitted by the lamps was about
254 nm. Moreover, an action spectrum of the effects of
UV irradiation on the inactivation of components re-
quired for neural induction in the amphibian egg showed
a peak at 280 nm and a decline toward both 250 and
320 nm (Youn and Malacinski, 1980). However, irradia-
tion of cells with high-intensity near-UV light (360 nm,
2.5 mW cm : , about 5-fold higher than that used in the
present study) disrupts f-actin in epithelial cells (Rafferty
el (//.. 1993). Although we saw an inhibition of segregation
at such a high UV light intensity in preliminary experi-
ments, UV light intensities sufficient to regenerate micro-
tubules and normal oil droplet movement had no appar-
ent effect on ooplasmic segregation in the medaka egg.

That we could regenerate microtubules and oil droplet
movement near the animal pole by irradiating this region
en face is not surprising, given the apparent presence of
a microtubule-organizing center there (Abraham et a!..
1993b). More interesting is the observation that we could
regenerate microtubules and oil droplet movement near
the equator region and could regenerate microtubules at
the vegetal pole. This result is consistent with Sakai's ob-
servation ( 1 964) that oil droplets will segregate toward the
vegetal pole in vegetal halves of eggs that have been sur-
gically separated from their animal halves. This result
suggests the presence of microtubule-organizing centers
outside the animal pole region of the medaka egg. In Xen-
opus laevis eggs, the biochemical basis for such an ability
may be the presence of 7-tubulin in cortical cytoplasm
in the vegetal hemisphere of the egg (Gard. 1994). We are
currently pursuing this question.

Finally, we note that when we irradiated an equatorial
region of an egg en pro ft I. ooplasm accumulated at the
animal pole side of the irradiated region, suggesting that
microtubules are involved not only in the movement of
oil droplets toward the vegetal pole but also in the move-
ment of components of the ooplasm toward the animal
pole. Though the growth of the medaka blastodisc is
slowed by microtubule poisons (Abraham et a/.. 1993a),
no specific effect of these poisons on the movement of
the ooplasm or its constituents toward the animal pole
has been reported. However, here we have described the
saltatory movement of subcellular parcels toward the ani-
mal pole and its inhibition by demecolcine. This move-
ment was especially apparent in eggs treated with CCD.
which displayed no cytoplasmic streaming; this streaming
obscures the component of saltatory movement directed
toward the animal pole. Ooplasm in and near these ir-
radiated interpolar regions appeared to segregate along
the same axis as that of the entire egg: oil droplets moved
toward the vegetal pole and certain other components of
the ooplasm toward the animal pole. These results suggest
that a local mechanism, operating with a predetermined
polarity, is responsible for the segregation of ooplasm in

156

T. A. WEBB ET .11.

the medaka egg. This suggestion is consistent with results
obtained by Gilkey ( 1983), who achieved local activation
of medaka eggs by injecting them with Ca/EGTA mix-
tures. He reported that oil droplets in such eggs moved
to the vegetal-pole side of the activated region and a small
amount of ooplasm accumulated at the animal pole side.
We are seeking to determine whether these localized re-
gions of segregation in the medaka egg are accompanied
by polar zones of elevated cytosolic [Ca 24 ] like those pres-
ent at the poles of the egg during segregation (Fluck el
a/., 1992).

Acknowledgments

Supported by NSF DCB-9017210 and NSF MCB-
9316125 to RAF, Franklin and Marshall College's Hack-
man Scholar Program, and a grant from GTE to Franklin
and Marshall College. We thank Shinya Inoue for sug-
gesting the use of the Colcemid-UV method and for his
advice in the selection of fluids for microinjection into
the eggs, Vivek Abraham for helpful discussions. Richard
Elinson for telling us about the paper by Youn and Ma-
lacinski, and Valerie Stone for improving the text of the
manuscript.

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Reference: fi/W. Bull 188: 157-165. (April. 1995)

Expression of the Engrailed Gene Reveals Nine

Putative Segment-Anlagen in the Embryonic Pleon

of the Freshwater Crayfish Cherax destructor

(Crustacea, Malacostraca, Decapoda)

GERHARD SCHOLTZ*

School of Biological Science, The University of New Soul/i Wales.
P.O. Box I, Kensington, NSW' 2033, Australia

Abstract. Segment formation in the embryonic pleon
of the freshwater crayfish Cherax destructor was analyzed
by using the monoclonal antibody mAb 4D9 against the
product of the segment-polarity gene engrailed. As in other
body regions, engrailed is expressed in transverse stripes
in the posterior portion of segments in the pleon. Nine
engrailed stripes are formed in the pleon. The anterior six
stripes correspond to the six pleon segments of adult eu-
malacostracan crustaceans. The uropods are clearly the
appendages of the sixth pleon segment. The seventh en-
grailed stripe marks the anlage of a seventh ganglion.
Stripes eight and nine are transient and disappear before
morphogenesis begins. The engrailed stripes seven to nine
are interpreted as vestiges of ancestral segments. The sev-
enth segment anlage is thus a recapitulation of the seventh
pleonic segment, which is retained in recent adult leptos-
tracans and is considered to be part of the malacostracan
ground plan. The stripes eight and nine might point still
further back into the phylogeny of crustaceans or even
mandibulates. The use of rhodamine-labeled phalloidin
reveals that the terminal ganglion of adult crayfish is the
fusion product of the anlagen of the sixth and seventh
pleonic ganglia and an eighth hemiganglion that is devoid
of engrailed expression.

Introduction

The Malacostraca constitutes a monophyletic taxon
well defined by several apomorphic characters such as a

Received 15 November 1994; accepted 18 January 1995.
* Present address: Institut fur Zoologie. Freie Universitat Berlin.
Konigin-Luise-Str. 1-3, D-14195 Berlin, Germany.

characteristic tagmosis. the position of the gonopores, the
subdivision of the stomach into specific functional units,
and a ring of 19 embryonic ectoteloblasts (for review and
discussion of different views see Dahl. 1992; Wagele,
1992). Within the Malacostraca. the Leptostraca possess
a pleon (abdomen) consisting of seven segments and a
telson. In contrast to all anterior segments, the seventh
pleonic segment is limbless. The other malacostracan
groups, unified as the Eumalacostraca (sensu Grobben,
1892). have only six pleomeres. all equipped with limbs.
The posteriormost limbs are the uropods which, together
with the flattened telson, form the tail fan. The general
view is that the pleon of the Leptostraca represents the
plesiomorphic condition, and the loss of a pleon segment
and the evolution of the tail fan are considered to be de-
rived characters of the Eumalacostraca (e.g., Lauterbach,
1975; Hessler, 1983). However, there has been some con-
troversy over which pleomere has been lost in the course
of evolution and to which segment the uropods belong.
On the basis of anatomical and paleontological data.
Siewing (1956. 1963) argued that the uropods might be
the appendages of the seventh pleomere and that the sixth
(penultimate) pleomere has been lost in most eumala-
costracans. Based on her embryological studies in mysi-
daceans. Manton (1928a. b). in contrast, suggested that
the uropods belong to the sixth pleomere and that the
original seventh is fused to the sixth pleomere. Although
it was shown in the meantime that paleontological data
do not support Siewing's suggestions (Dahl. 1983) and
although Manton's view has been adopted by many car-
cinologists such as Lauterbach (1975), Hessler (1983). and
Dahl ( 1992) the problem is still far from being settled

157

158

G. SCHOLTZ

(see, for instance, the discussion after the 1983 paper by
Hessler) and a different approach seemed to be required
to clarify this issue.

Within the arthropods, insects and crustaceans have
the segment-polarity gene engrailed already expressed in
transverse stripes at the posterior margin of embryonic
segments before morphogenesis takes place (e.g.. Patel et
al. 1989; Manzanares ct at.. 1993; Scholtz et al. 1993,
1994; Patel, 1994). Therefore, it is a suitable marker for
the analysis of the terminal regions of arthropods where
segmentation is obscured by morphological rearrange-
ments and the loss of segmental structures in the adult.
Several studies have used engrailed to analyze the seg-
mentation of the head in insects and crustaceans (e.g..
Fleig, 1994; Schmidt-Ott et al.. 1994; Scholtz, 1995), but
the caudal segments have been analyzed only in insects
(e.g., Kuhn et al., 1992; Schmidt-Ott et al.. 1994).

In the present investigation. I used the anii-engrailed
antibody mAb 4D9 (Patel et al., 1989) to analyze the seg-
mentation of the embryonic pleon of a eumalacostracan,
the Australian freshwater crayfish Cherax destructor. I
found that, posterior to the six pleomeres typical for adult
eumalacostracans, three additional vestigial segment an-
lagen occur in front of the telson. Furthermore, a true
seventh pleonic ganglion and an eighth partial ganglion
are formed embryologically. These fuse with the sixth
pleonic ganglion anlage to form the terminal ganglion of
the adult animal. I interpret these findings as recapitula-
tions of ancestral characters, and they shed new light on
the evolutionary transformation of crustacean segmen-
tation patterns.

Material and Methods

The rearing and maintenance of embryos of the Aus-
tralian freshwater crayfish Chera\ destructor were de-
scribed by Sandeman and Sandeman (1991). Their paper
also defines the embryonic stages in percent of develop-
ment and the postembryonic stages (e.g., PO I) that are
used in the present investigation. Immunocytochemistry
and fluorescent staining were described in detail in Scholtz
et al. (1994) and Scholtz (1995).

Results

A short summary oj 'previous investigations

The cell lineage in the ectoderm of the germ band of
Cherax was described in a previous study (Scholtz, 1 992).
As in most other malacostracans, the largest part of the
germ band is formed by stem cells in the posterior growth
zone, the ectoteloblasts. The ectoteloblasts produce trans-
verse cell rows in an anterior direction by highly unequal
divisions. Thirteen of these rows are formed (el to eXIV)
before the ectoteloblasts divide equally into the fourteenth

and fifteenth rows (eXIV and eXV). Each row (el to eXI V)
cleaves twice, forming four regularly arranged descendant
rows. The intersegmental furrow, separating two adjacent
segments, is formed within the descendants of one original
ectoderm row. In contrast to all other rows, row fifteen
cleaves rapidly several times, forming a field of cells in a
grid-like arrangement.

In Drosoplula. the segment-polarity gene engrailed
plays a crucial role in specifying the fate of the cells in
the posterior part (compartment) of segments and in es-
tablishing the segmental boundaries (Lawrence, 1992).
The expression pattern of engrailed K very similar in dif-
ferent insect and crustacean species. Therefore, a con-
served function of the engrailed gene throughout the ar-
thropods has been suggested (Patel. 1994). The basic
modes of pleonic engrailed stripe formation described in
the following correspond to those reported for other body
regions of Cherax (Scholtz et al.. 1993; Scholtz. 1995)
and for other crustacean species ( Patel et al.. 1989; Scholtz
et al.. 1993. 1994). In the post-naupliar germ band of
Cherax and other malacostracans, engrailed is expressed
in the anterior descendants of each ectoderm row, and
the intersegmental furrow is formed posterior to the en-
grailed stripes.

The formation <>t engrailed stripes

Nine engrailed stripes are formed in the embryonic
pleon of C. destructor (Fig. 1). The first pleonic stripe
appears in ectoderm row elX and the sixth stripe in row
eXIV. Stripes seven to nine are formed within the deriv-
atives of row eXV.

The stripes appear in a strictly anteroposterior sequence
(Fig. 1). At about 40% to 42% development the first en-
grai/ed stripe is formed and indicates the posterior margin
of the prospective first pleomere (Fig. 1A). The ninth
pleonic engrailed stripe appears at about 65%> develop-
ment (Fig. ID, E). The formation of each individual stripe
starts close to the midline and proceeds laterally (Fig. 1 D).
The initial distance between the last two engrailed stripes
of any stage is one row of engrailed negative cells. This is
also true for stripes seven to nine (Fig. IE). Stripes one
to seven are associated with the complex metamerically
repeated cleavage pattern in the post-naupliar germ bands
of Cherax. The cell division pattern in the area of stripes
eight and nine, although not analyzed in detail, is some-
what different (Scholtz, 1992).

Initially, all stripes are one cell wide (Fig. 1 ). Also, at
least pleonic stripes one to eight (not confirmed for pleonic
stripe nine) pass through a transient widening phase
caused by divisions of the engrailed-posilive cells (Fig.
IB, C). The widening phase is followed by narrowing to
a one-cell width again due to the loss of engrailed expres-
sion in posterior cells in the stripe (Fig. 1 B, C). After nar-

EMBRYONIC PLEON OF CRA^i I ISM

159

V

te

Figure 1. Formation of engrailed stripes: st. stomodeum; cp. caudal papilla; te. telson anlage: up. uropods.
(A) Germ band at 40% to 42% development. All engrailed stripes of head and thorax are formed. The
engrailed stripe marking the first pleonic segment appears (arrow) on the ventral side of the ventrally Hexed
caudal papilla. (B) Posterior part of the caudal papilla of an embryo at 45% development (ventral view).
The engrailed stripes up to the third pleonic segment ( 1, 2. 3) are formed. The posteriormost stripe (3) is
one cell wide. The next anterior stripe (2) is in the narrowing phase after the initial widening. The stripe of
the first pleonic segment ( 1 ) is in the phase of secondary widening in correlation with the formation of the
intersegmental furrow and the limb buds (for details see Scholtz cm/.. 1993). (C) Posterior part of the caudal
papilla of an embryo at about 60% development (ventral view); engrailed stripes seven and eight are formed.
Stripe eight is in the first widening phase; stripe seven has already narrowed to one cell width. The uropods
begin to appear in the area of the sixth engrailed stripe (sixth pleonic segment). Nole the buds of the first
pleopods (arrow), which are lacking in the adults of Cherax (comp. Fig. ID). (D) Posterior part of the caudal
papilla of an embryo at about 65% development (ventral view). The ninth engrailed stripe appears following
a mediolateral sequence of engrailed expression. The ninth stripe is the posteriormost area of engrailed
expression, the posterior margin of the telson anlage does not express engrailed. The arrow points to the
buds of the first pleopods. (E) Closeup of the same preparation as in (D); 7 to 9 seventh to ninth pleonic
engrailed stripes. (F) Posterior part of the caudal papilla of an embryo at 70'v development (ventral view).
Neuronal engrailed expression begins in the area of the seventh pleonic stripe (7). The cells of engrailed
stripe eight begin to cease engrailed expression; stripe nine has disappeared.

160

G. SCHOLTZ

rowing, stripes one to seven widen during the morpho-
genesis of segmental structures such as intersegmental
furrows, ganglia, and limb buds (Fig. 1 ). Stripes eight and
nine disappear before widening takes place (Fig. IF).

Stripes one to six surround the caudal papilla and form
complete circles of about 40 etwui/ecl-positi\e cells (Fig.
1, 2A). Thus, engrailed is expressed in the midventral
neurogenic region, in the lateral limb budding area, and
in the dorsal portion of the forming segments one to six.
Stripes seven to nine are restricted to the medioventral
part, which includes the neurogenic region (Fig. ID, E).
They consist of seven to nine engrailed expressing cells.

Stripes one to seven show a twofold engrailed expression
in the embryonic epidermis and in the forming ganglia
(Figs. 1, 2). In the stages examined, the superficial epi-
dermal engrailed expression continues in the forming
limbs and the dorsal portions of the segments (Fig. 2). In
the neurogenic area of advanced stages, engrailed expres-
sion is restricted to individual neuronal precursors and
neurons, whereas the superficial engrailed expression has
disappeared (Figs. 1, 2). In contrast to this, stripes eight
and nine appear only transiently in the embryonic epi-
dermis (Figs. 1, 2).

Morphogenesis

In all other body regions, the intersegmental furrows
are formed immediately posterior to the engrailed stripes

(Fig. 1 ). This is also true for pleonic stripes one to six, but
stripes seven to nine are not related to the formation of
intersegmental furrows (Figs. ID, E. 2). In segments one
to six, limb buds are formed whose posterior portions are
also engrailed positive (Figs. 1, 2). No limb buds occur
in segments seven to nine, and in the corresponding re-
gions the cells do not show engrailed expression (Figs. 1,
2). Interestingly enough, embryonic limb buds are formed
in the first pleonic segment (Fig. 1C, D) where the adult
('. iteMnielor. like all parastacid crayfish species, is devoid
of appendages in both sexes (Hobbs, 1988). The limbs
associated with the sixth pleonic engrailed stripe are clearly
the posteriormost appendages on the germ band (Figs.
1C, D, E. 2). On the basis of their shape in advanced
developmental stages, they can be identified to be the uro-
pods (Figs. 1.2).

Neurogenesis

In the pleonic segments one to seven, engrailed is ex-
pressed in cells of the forming ganglia (Figs. 1,2). Thereby,
the arrangement of engrailed-positive cells (neurons ?) is
very similar between the seventh and more anterior seg-
ments (Fig. 2). The engrailed expression in stripes eight
and nine disappears at about 70% to 75% development
and is not correlated with neurogenesis. The staining of
the embryonic central nervous system with rhodamine-

Figure 2. tarly neuronal engrailed expression in the pleon: up. uropods. (A) Posterior part of the caudal
papilla of an embryo at about 75% development (ventral view; brightfield micrograph). The pattern of
I'li^ruiU'J expression in neuronal precursors and early neurons in the seventh pleonic ganglion anlage is very
similar to lhat of more anterior pleonic ganglion anlagen, indicating a serial homology between all seven
pleonic ganglia. The resemblance concerns, in particular, a median engrailed-positive cell (arrowheads) and
a set of three inlenscK stained cells at the posterior margin of the engrailed-positive area (compare Fig. 2C).
Note the dorsal engrailed expression in pleomeres one to six. (B) Same preparation as in (A), with Nomarski
optics. This technique shows the buds of the pleonic limbs. The uropods are beginning to get their characteristic
shape and are clearly connected with the sixth pleomere. (C) Closeup of another preparation (Nomarski
optics). The serially homologous group of three intensely stained cells in ganglion anlage seven and more
anterior ganglion anlagen is marked by arrowheads.

FMBRYONIC PI EON OF CRAYFISH

161

labeled phalloidin reveals that a true seventh embryonic
ganglion is formed in addition to the anterior six pleonic
ganglion anlagen (Fig. 3). All pleonic ganglion anlagen
one to seven share the occurrence of two main commis-
sures and a characteristic median Y-shaped neuron that
might correspond to the neuron S described by Whiting-
ton el at. (1993) (Fig. 3 A. B). Posterior to the seventh, an
eighth ganglion anlage is formed. It possesses only one
commissure, and the characteristic median cell is lacking
(Fig. 3B). Furthermore, no neuronal engrailed expression
occurs. From this eighth hemiganglion, two nerves run
posteriorly towards the embryonic telson region (Fig. 3B).
During further ontogenesis the anlagen of ganglia six,
seven, and eight fuse and become a morphological unit
that forms the terminal ganglion of the adult animals (Fig.
3C). Thereby the commissures remain separated. The co-
alescence of these ganglion anlagen is clearly visible from
about 80% development. However, engrailed expression
still indicates the composed origin of the terminal ganglion
in postembryonic stages (e.g., PO I) (Fig. 4).

Discussion

Development oj the terminal ganglion

The present investigation shows that the terminal gan-
glion of the adult parastacoid crayfish Cherax destructor
is the fusion product of three embryonic ganglion anla-
gen the sixth and seventh pleonic ganglia and a partial
eighth ganglion. This developmental pattern corresponds
to that described for the astacoid crayfish Procambarus
clarkii (Dumont and Wine, 1987). Furthermore, these
embryological data are consistent with results from neu-
roanatomical and immunohistochemical studies analyz-
ing the terminal ganglia of the adults of several freshwater
crayfish species (Stoll, 1925; Kondoh and Hisada, 1986;
Audehn et at., 1993) and of the lobster Homams gam-
mams (Winlow and Laverack, 1972). The embryonic
morphology and the pattern of engrailed expression of
the seventh pleonic ganglion resemble to a high degree
those of the anterior pleonic ganglia. This suggests that it
represents a true segmental ganglion homologous with
the anterior segmental ganglia. The eighth pleonic gan-
glion anlage shows only one commissure. Furthermore,
this ganglion lacks any engrailed expression which char-
acterizes the posterior part of forming ganglia, although
an epidermal eighth pleonic engrailed stripe is formed.
Taken together, this suggests that the eighth pleonic gan-
glion of the embryo might be the anterior part of a true
segmental ganglion anlage. On the basis of the occurrence
of specifically arranged identified neurons. Audehn et al.
(1993) came to similar conclusions.

The formation of an embryonic anlage of a seventh
pleonic ganglion that later fuses with the sixth ganglion
has been reported from representatives of most higher

malacostracan taxa including Leptostraca (Claus, 1888;
Manton, 1928b, 1934), Hoplocarida (Shiino, 1942), Syn-
carida (Hickman, 1937), Mysidacea (Manton, I928a, b),
Tanaidacea (Scholl, 1963), and Isopoda (Stromberg,
1967). No seventh pleonic ganglion occurs in the embryo
of the amphipod (.iammarus pu/e.\", however, for a short
time during development, the anlage of the sixth pleonic
ganglion is subdivided into two distinct adjacent areas
a phenomenon interpreted as a vestigial formation of a
seventh pleonic ganglion (Weygoldt. 1958). None of these
authors has mentioned the partial eighth pleonic ganglion,
but this might be due to the techniques used (no whole-
mounts or horizontal sections). Adult eumalacostracans
possess only six pleonic ganglia (see Hanstrom, 1928),
and this is also true for leptostracans with a seventh pleo-
mere (Claus, 1888; Manton, 1928a). Against this back-
ground, I conclude that the composite nature of the ter-
minal (sixth) ganglion and the pattern of its formation by
fusion during embryogenesis is part of the malacostracan
ground plan. Thus, the fusion of the terminal ganglion is
apparently not correlated with the evolution of the uro-
pods and their complex function in eumalacostracans.

Origination of the uropods from the sixth pleomere

The anterior six engrailed stripes in the embryonic
pleon of Cherax mark the posterior border of the six pleo-
meres that persist in the adult. Engrailed is expressed in
the ventral region comprising the ganglion primordia and
the limb buds as well as the lateral and dorsal sides of
each segment. The seventh to ninth pleonic engrailed
stripes are restricted to the ventral side of the embryo.
However, from the mode of formation and the distance
between them, they correspond to the anterior stripes.
Thus I conclude that all pleonic engrailed stripes are serial
homologues and that stripes seven to nine also indicate
segment anlagen.

The pattern of the sixth engrailed stripe clearly reveals
that the uropods of Clierax are the limbs of the sixth pleo-
mere, which is also true for the mysid Neomysis integer
(unpub. obs.). This result confirms some of the suggestions
of Manton (1928a, b), and since there is good evidence
that the tail fans of all eumalacostracan groups are ho-
mologous (Hessler, 1983; Wa'gele, 1994). the findings pre-
sented here might also be valid for eumalacostracans in
general. Therefore. Siewing's (1956, 1963) hypothesis that
the uropods originate from the seventh pleon segment
cannot be maintained. The present results also contradict
the assumption that the caudal rami of the telson of lep-
tostracans are homologous with the eumalacostracan
uropods (Bowman, 1971; for further arguments against
this view see Schminke, 1976). Furthermore, the telson
of decapods does not correspond to the ancestral seventh
pleomere, as was suggested by Kondoh and Hisada (1986).

162

G. SCHOLTZ

Figure 3. Gangliogenesis as seen with rhodamine-labeled phalloidin: st. stomatodeum; pr. proctodeum;
pg2, second pleonic ganglion; pg5. fifth pleonic ganglion; tg. terminal ganglion. (A) The nerve cord of an
embryo at 7y, to SO', development. The black arrow points to the eighth thoracic ganglion anlage; the
white arrow indicates the eighth pleonic (hemi)ganglion anlage. (B) Closeup of posterior ganglion anlagen.
The seventh ganglion anlage (asterisk) shows the same pattern as those of more anterior segments (t'.# . the
sixth ganglion anlage; star). The eighth ganglion anlage (large white arrow) consists of only one commissure.
The black arrow shows the eighth thoracic ganglion anlage; the small white arrows point to the median Y-
shaped neurons. (O Advanced stage (85% to 90% development) showing that the sixth (star), seventh (asterisk),
and eighth (white arrow) ganglion anlagen are embedded in a morphological unit, forming one large terminal
ganglion.

The additional engrailed stripes seven to nine lie clearly
in front of the telson anlage, which is characterized by the
proctodeum and which lacks engrailed expression. Against
this background, the seemingly "seven-segmented" pleon
with uropods originating from the "seventh" pleomere of
some lophogastrids is not plesiomorphic, as considered
by Manton (1928b), Siewing (1956). and Lauterbach
( 1975), but is a derived feature. There is ontogenetic and
phylogenetic evidence for this suggestion. Manton's
(1928b) hypothesis that the uropods migrate posteriorly
before the border between the last two segments is formed
is not consistent with the finding that the intersegmental
furrows are formed before limb buds appear (Scholtz,
1990; present investigation). We know from Drosophila
genetics that the establishment of the segmental border is
the prerequisite for the subsequent differentiation of seg-
ments (Lawrence, 1992). Therefore, the "seventh pleonic
segment" of lophogastrids is apparently the result of a
secondary nonsegmental(?) subdivision of the terminal
eumalacostracan segment, as was suggested earlier by
Claus (1888). With respect to the position of lophogastrids
in the eumalacostracan phylogenetic tree (e.g.. Siewing,
1956; Richter, 1993), it is more likely that a subdivision
of the terminal segment occurred only once in the lopho-
gastrid line than that the seventh pleomere has been lost
independently in several eumalacostracan lines.

The seventh pleonic engrailed stripe in the embryo
otCherax is an obvious example of the recapitulation
of ancestral conditions (see Sudhaus and Rehfeld, 1992).
It demarcates the posterior border of an additional
(seventh) pleonic segment that is missing in adult cray-
fish and other eumalacostracans but is present in adult
Leptostraca, the sister-group of the Eumalacostraca.
There is no reason to assume that pedomorphosis led
to the occurrence of the seventh segment in adult lep-
tostracans and to the loss of the highly complex eu-
malacostracan tail-fan (Messier. 1983; Fault'/ al.. 1985)
in this group.

Interestingly, in the crayfish as in other eumalacos-
tracans, the corresponding seventh pleonic ganglion
persists and is fused with the sixth to form a morpho-
logical and functional unit (see above). In addition,
some authors report that the embryos of several ma-
lacostracan species contain terminal mesodermal so-
mites that might be related to a vestigial seventh pleonic
segment and that also fuse with the sixth pleonic somites
(e.g.. Manton, 1928a; Shiino. 1942). Those processes
can be characterized as fusions, but fusion does not
seem to be the appropriate description for events in the
superficial segmental parts. The pleonic engrailed stripe
seven (like stripes eight and nine) is more like a transient
segment anlage that is not involved in morphogenesis

EMBRYONIC PLEON OF CRAYFISH

163

Figure 4. Expression of engrailed in the pleonic nerve cord of the first postembryonic stage (PO I): pgl.
first pleonic ganglion: pg4, fourth pleonic ganglion; pg5. fifth pleonic ganglion; tg. terminal ganglion. (A)
The six ganglia of the pleon of the first stage after hatching (PO I) (ventral aspect). (B) Higher magnification
of the same preparation, showing the ganglia of the fourth and fifth pleomeres. They exhibit a serially
repeated pattern of two areas of engrailed expression. An anterior region of cells (neurons, glia, or their
precursors) with relatively small nuclei (asterisks) and a posterior region with large engrailed-positive cells
(arrows). (C) The terminal ganglion (same preparation as in (A)). The pattern of engrailed expression reveals
the composed nature of this ganglion. The asterisks indicate the anterior segmental engrailed area and the
arrows point to the posterior segmental engrailed area (compare Fig. 4B). The pattern of engrailed expression
in the seventh ganglion anlage (7) is somewhat reduced when compared with that of the sixth ganglion an-
lage (6).

and that disappears during further development. From
the outset, the morphological border between the ter-
minal segment and the telson lies behind the sixth
pleonic engrailed stripe.

Phylogemtic significance of pleonic engrailed stripes
eight and nine

The eighth and ninth pleonic engrailed stripes are also
considered to indicate vestigial segments that recapitulate
ancestral conditions. This suggestion is based on the sim-
ilar appearance of stripes seven to nine and on the fact
that many non-malacostracan crustaceans possess more
segments than malacostracans. However, it is difficult to
say how far back stripes eight and nine point in phylogeny
and in which ancestral lineage these segments have been
lost in the adults. The question of whether segmentation
and tagmatization of the Malacostraca are primitive or
are derived within the Crustacea has been debated, and
the many attempts to reconstruct the crustacean stem
species have yielded very different results concerning tag-
mosis and segment number. The proposals reach from
short animals with only a few segments (Miiller, 1864) to
forms with many segments (Hessler and Newman. 1975:
Lauterbach, 1986), and from forms with a more or less

homonomously segmented trunk (Hessler and Newman,
1975; Schram, 1982; Walossek, 1993) to animals with a
distinct subdivision of the trunk into thorax and a limbless
abdomen (Lauterbach, 1986; Fryer, 1992). But until the
phylogenetic relationships between the higher crustacean
taxa are resolved see Siewing (1963), Schram (1986),
and Wilson (1992) for various proposals the recon-
struction of a crustacean ground plan (sensit Hennig.
1966) will be pure speculation.

Nevertheless, the present findings permit some tentative
conclusions. The occurrence of additional segment rem-
nants in the embryonic pleon of Cherax argues against
an original number of 15 trunk segments in crustaceans
or even mandibulates as suggested by Walossek (1993);
the number of trunk segments in the crustacean stem spe-
cies must have been higher. Therefore, the additional en-
gniiled stripes in the pleon ofChera.\ rather argue in favor
of Lauterbach's (1975) hypothesis of a loss of posterior
segments in the ancestral lineage of malacostracans. The
restriction of these additional stripes to the neural region
and the entire lack of limb anlagen furthermore support
the suggestion that these segments are vestiges of the
limbless abdomen postulated by Lauterbach (1986) and
Fryer (1992) for the crustacean stem species.

164

G. SCHOLTZ

Acknowledgments

I thank David Sandeman and Renate Sandeman for
their helpful advice as well as for the opportunity to use
their crayfish culture and to work in their laboratory. The
anti-engruili'd antibody was a generous gift from Nipam
Patel. I am grateful to Stefan Richter, Wolfgang Dohle,
and David Sandeman for critically reading the manu-
script. This work was supported by grants from the Uni-
versity of New South Wales (Visiting Fellowship) and from
the Deutsche Forschungsgemeinschaft (Scho 443/3-1 ).

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Reference: Binl Bull 188: 166-178. (April,

The Tidal Rhythm of Emergence, and the Seasonal
Variation of This Synchrony, in an Intertidal Midge

MASAYUKI SAIGLJSA 1 AND TADASHI AKIYAMA 2

1 Department of Biology, Faculty oj Science. Okayama University. Tsushima 2-1-1. Okayanui
700. Japan- and 2 Ushimado Marine Laboratory, Okayama University, Oku-Ushimado, Okayama 701-43. Japan

Abstract. The emergence of an intertial midge was in-
vestigated at a site on the coast of the Inland Sea of Japan.
The population emerging at this site was drawn from a
single species of the genus Clunio. probably C tsushi-
mensis. Emergence was not synchronized with the day-
night cycle, but with the tidal cycle. Moreover, the pattern
of synchrony changed with season. A bimodal phase ap-
peared in midwinter; but the pattern of synchrony shifted
gradually, during January and February, from morning
low tides to afternoon low tides, and a unimodal phase
appeared in March. This pattern i.e.. synchrony with
afternoon low tides lasted until early October. In mid-
October, the synchrony shifted to the morning low tides.
Only a brief bimodal phase appeared in autumn. The
phase modality was clearly correlated with the height of
tides; i.e., when the low waters in a day were very different
in height, emergence was synchronized only with the lower
one (April to December). During January and February,
the higher low tide, as well as the lower low tide, recedes
considerably. The exposure of the larval habitat at the
higher low tide may stimulate emergence, resulting in bi-
modal phases in midwinter. But the unimodal pattern in
March cannot be accounted for by a simple synchrony
with lower low tide, or with exposure of the larval habitat
to the air; the day-night cycle not only would be one of
the zeitgebers of the tidal rhythm in every season, but also
must participate in the expression of the unimodal phase
in spring. Furthermore, the number of midges that
emerged each day fluctuated with a semilunar cycle with
the season. The phase of this rhythm would be shifted by
water temperature.

Introduction

Tidal and semilunar rhythms of reproductive activity
have been reported in many intertidal and estuarine or-

Received 11 June 1993; accepted 1 February

ganisms (e.g.. see Korringa. 1947; Hauenschild. 1960;
DeCoursey. 1983; Pearse, 1990). Marine midges of the
genus Clunio are among such animals. The imagines
emerge around the full and new moons, and the time of
daily emergence corresponds to that of low tides at the
habitat (Caspers, 1951; Koskinen, 1968; Hashimoto, 1976;
Neumann, 1966; 1976; 1987). But in most of the species
observed, eclosion occurs in a very short period, i.e.. at
most the 4-5 days bracketing the full and new moons. In
these cases, therefore, the rhythm underlying the observed
events in the field i.e., daily or tidal was not clear.

The circadian rhythm of terrestrial animals exhibits at
least two essential and distinctive characteristics: (Da free-
running period approximating the 24-h day-night cycle;
and (2) synchrony with the local light-dark cycle (and
sometimes with 24-h temperature cycles, too). But in ma-
rine organisms, such characteristics are not sufficient evi-
dence of a circadian rhythm. For example, the circatidal
rhythm of larval release in a few semiterrestrial crabs is
surely entrained by 24-h, light-dark cycles (Saigusa, 1986;
1992). We supposed that the emergence rhythm of Clunio
might be similarly timed. As the first step in testing this
hypothesis, the rhythm was examined in the field.

A further problem in tidal rhythm research is the re-
lationship between the modulation of the tidal amplitude
and the modality of the phase. In some of the oceans in
the world, there are two high (or low) tides, of approxi-
mately equal height, per day, at intervals of about 12.4 h.
But in the Pacific Ocean, the two high and low tides on
a given day often show very different heights, and they
recur at asymmetrical intervals (see Saigusa. 1985). En-
right (1963) reported a circatidal rhythm of swimming in
freshly collected specimens of the sand-beach amphipod
Svnchcluliuin sp. A feature of this rhythm was that the
relative amplitudes of activity peaks seemed to reflect
contemporary tidal amplitudes. An isopod. Excirolana

166

THE TIDAL RHYTHM OF AN INTERTIDAI MIDGE

167

33

Figure 1. (A) The site of emergence of the Clitnio population studied (arrow). The Inland Sea is situated
between Honshu and Shikoku (shown by the diagonal lines). As its east and west sides are open to the Pacific
Ocean through the straits, the salinity is not greatly reduced, averaging 28-29 ppt through the year. (B)
Recently emerged male and female imagines shown mating. The pupal eclosion of the female is completed
with the assistance of the male. As soon as he has stripped off the female's pupal skin, the male runs or flies
carrying the female in the mating position shown in this figure. (C) The nylon net used to catch flying
midges. Since many midges fly on the water film, the sampling was carried out with the net touching the
water. (D) Antenna and wing venation of a male Clunio studied: a, the third segment; />. adjoining seven
small segments; c, the ultimate segment; d, length of the wing; e, width of the length. See the text for the
antennal and wing indices.

chi/toni. inhabiting a similar environment exhibited a
similar activity rhythm (Enright, 1972).

Tidal amplitudes vary with the season. Thus, if the ac-
tivity of an organism is correlated with tidal amplitude,
then the pattern of the rhythm must also vary with the
season. We have already reported on the swimming ac-
tivity rhythm of the sublittoral crustacean Dimorphostylis
asiatica, whose pattern was modified seasonally (Akiyama
and Yoshida, 1990). The midge Clunio is also an appro-
priate animal with which to investigate this problem, be-
cause emergence occurs in every season. Oka and Hashi-
moto (1959) reported a seasonal change in daily emer-
gence in Clunio tsushimemis. But their data were only
qualitative (see also Hashimoto, 1976) so it was not clear
whether the daily timing of emergence is correlated with

modulations of tidal amplitude or with other seasonal
factors, such as the ratio of day-night length or the fluc-
tuation of water temperature.

An additional problem is the uncertain classification
of the genus of midges and the distribution of species
along Japanese seacoasts. Hashimoto (1969, 1976) re-
ported a sympatric species, C. aquilonius, whose mor-
phology is quite similar to that of C tsiishimensis. So we
had to identify the species whose imagines were emerging
in our study area along the shoreline of the.Inland Sea.

Materials and Methods

Collections of/lying midges

Field investigations were carried out on the coast of
the Inland Sea of Japan, Okayama Prefecture (Fig. 1A);

168

M. SAIGUSA AND T. AKIYAMA

the exact site was the rocky seashore in front of the Ushi-
mado Marine Laboratory. The Inland Sea is bounded to
the south by Shikoku and is connected to the Pacific
Ocean through straits at both sides of Shikoku. Thus,
heavy waves do not roll ashore except when there are
strong winds. The tidal amplitude varies between - 1 5 cm
(minimum level of low waters in the tide table published
by the Japan Meteorological Agency) and 260 cm (maxi-
mum in high waters) during the year. The tidal pattern
is semidiurnal, but morning low waters recede much fur-
ther than afternoon low waters in winter, and the pattern
is reversed in summer.

Male imagines fly or swarm immediately after eclosion.
They are most abundant within a narrow area a strip
about 3-4 m wide along the water line of the rocky shore.
Outside this narrow strip, the number of flies rapidly de-
creases. The emerged males scurry about on the rocks or
stones while vigorously vibrating their wings or they skim
rapidly over the water. When the male finds a female
pupa, he strips off her pupal cuticle and copulates with
her. The female (Fig. IB) soon lays eggs on the rocky
substrate. The females are always much less numerous
than the males less than 10% of the males emerging at
the same time. In any event, when the tide starts to rise,
the emerged adult midges all die.

We collected the adult males by sweeping the surface
of the water with a nylon net (Fig. 1C). At the beginning
of the emergence, the midges were seen only on the surface
of the water, but around the time of low tide, large num-
bers of males were seen on the rocks as well as on the
water surface. But it was hard to collect them on the rocks
without some type of suction device, so the surface water
was scooped up to capture the midges skimming over the
water.

The sweeping was carried out on the rocks for 5 min
every 30 min: the sampling range was 1-1.5 m along the
water's edge (220-240 strokes per 5-min sampling period).
Most emergence occurred within the 4-5 h surrounding
the time of low water, and the water receded, at most,
50 cm during the period, i.e.. the time between the start
and end of emergence. Therefore, we were not required
to move the collection site frequently as the tide declined;
we always swept only two rocks that differed by about
50 cm in height. The samples were quickly brought to the
laboratory and transferred to a pail containing warm water
(20-30C). The midges rose to the surface of this water
and were picked up with a forceps and counted.

We needed to determine whether the midges emerge
and fly at the time of high tides as well as low. In the
daytime, flying midges can be seen from the shore, but if
only a few specimens were flying on the water surface, we
might overlook them, especially at night. To eliminate
this possibility, we used a light for some collections. Like
other insects, marine midges are attracted to light at night.

The light ( 1 80 W) was placed at the edge of a floating pier
(5-6 m long), and the midges that swarmed around this
spot were collected by sweeping only two strokes with the
net. This method collection under the light was used
only for the initial and preliminary observations, and the
resulting data about emergence at high tide are only in
Figure 4A. All other data were obtained by sweeping
without lighting.

Investigations of the larval habitat

The habitat of the larvae with respect to the level of
tides was examined. While the tide was ebbing, the algae
growing on the rock were taken along with their associated
sand or mud. These substrata (36 cm 2 ) were removed from
various heights within the intertidal zone and transferred,
in the laboratory, to a vessel containing seawater. When
the vessel was shaken by hand, most larvae escaped from
the nest tubes buried in the sand or mud. Each larva was
picked up by forceps and counted. These samples were
taken on 21 and 31 March 1991.

Identification ot the species emerging

The taxonomic classification of the genus Clunio is not
easy because definitive morphological differences dre
lacking. Hashimoto ( 1969) reported a sympatric species,
C. aquilonius. that is extremely similar to C. tsushimensis
and also occurs along the coasts of Japan. The two species
can be distinguished on the basis of three morphological
properties: antennal index, wing index, and body length.
The antennal index is determined on the 1 1 -segmented
antennae of the male: i.e.. the ratio of the length of the
ultimate segment (c) to the length of the 3rd to 10th seg-
ments (a + b) (Fig. ID). The wing index is the ratio of
the width (e) to the length (d) of the wing (Fig. ID, and
Hashimoto, 1968, 1969).

It was critical for us to determine whether both species
are sympatric along the coasts of the Inland Sea, because
different species might emerge during the day and night.
Therefore, specimens were compared under a stereomi-
croscope, not only for the characters described by Ha-
shimoto ( 1 968, 1 969), but also for their emergence during
morning and afternoon low tides.

Results

Morphology of the Inland Sea population
and larval habitat

To determine whether a single species of Clunio emerges
during both the morning and evening low tides, we com-
pared the antennal and wing indexes and the body length
of specimens collected at those times. As shown in Figure
2A, the distribution of the antennal index ( AI) was almost
the same whether the specimens were collected at morning

THE TIDAL RHYTHM OF AN INTERTIDAL MIDGE

169

30

0)

O) 2

03

*-

0)

u

I ,

A

15 2.0 2S 30

0.6 07 08 0.9 10 1.1 12

Al

Figure 2. Comparison of the distribution of antennal index (AI), wing index (WI). and body length (BL)
among the male Clunio population. Date of collection: 30 March 1991. Solid lines: specimens collected at
the afternoon low water. Broken lines: those collected at the morning low water. Total number of the
specimens measured: about 250 individuals at each tide.

low tides (mean 0.83) or afternoon low tides (mean 0.85).
The wing index ( WI) was also much the same (mean 0.49
and 0.48, respectively) among the two groups (Fig. 2B).
Furthermore, as shown in Figure 2C, the mean body
length (BL) was also much the same for specimens emerg-
ing in morning and afternoon (2.43 mm and 2.37 mm,
respectively). The lack of a clear difference between the
specimens collected at the morning and afternoon low
waters suggests that the population of Clunio emerging
in this location is a single species.

Figure 3 summarizes the distribution of the larvae with
respect to the tidal height. The larvae ofC/utiio preferred
the thin, feltlike substratum found in shallow hollows on
the rocks; this substratum consisted of filiform green algae
and sandy mud. The highest larval densities were recorded
at 70-1 10 cm, and the fewest larvae were at the sites lower
than 50 cm.

On this rocky shore (Fig. 1A), Spaniotoma nemalione
(Subfamily Orthocladinae) was also abundant in March.
Imagines of this species were easily distinguished from
those of Clunio: their bodies were much slimmer, they
had a different swarming site (i.e., above oysters exposed
to the air), and their way of flying was different. Although
we could not specifically identify the larvae of this species,
the difference in the swarming site would suggest that
Spaniotoma larvae were not mixed in the substrata
samples.

Tidal rhythm of emergence and its pattern in winter

To determine the relationship between the timing of
emergence and the tidal cycles, collections were made

through the night. As shown in Figure 4A, emergence
did not occur at the time of high tide. Emergence and
swarming started a few hours before the time of low
tide and had ceased by 3-5 hours after. The pattern of
emergence was clearly that of a tidal rhythm, and not
that of a daily rhythm. Another feature of this emer-
gence rhythm is the semilunar timing; i.e., the fluctu-
ation in the number of midges emerging every day is
correlated with the lunar cycle. But the peaks of the
semilunar rhythm did not accurately coincide with the
days of full and new moons; in the data of Figure 4A,
they occurred a few days before the full and new moons,
respectively (see also Fig. 7b).

Figure 4B indicates the time of emergence from January
to February. Emergence is clearly synchronized with the
low tides. Moreover, the phase of the tidal rhythm is bi-
modal; i.e., the timing is clearly synchronized with both
of the low tides that occur each day. A further aspect of
this rhythm is a modulation of the tidal synchrony. As
noticed from the data of the first 17 days (i.e., 9-25 Jan-
uary), the midges emerging during the afternoon low tides
are outnumbered by those emerging during the morning
low tides. During the next 14 days (26 January-8 Feb-
ruary), the number of midges emerging in synchrony with
the morning low tides is about equivalent to the number
emerging during the evening low tides. During the fol-
lowing 20 days in February (Figs. 4B and 5A). the midges
emerging at the morning low tides are outnumbered by
those emerging at the afternoon low tides.

The pattern of emergence in spring and summer

In March, the synchrony with the afternoon low waters
is remarkable. As shown in Figure 5A, very few midges

170

M. SAIGUSA AND T. AKIVAMA

250

200

E 150

100

50

HW1
HW2

50
Number of larvae

100

Figure 3. Relationship between the tidal height and the density of
the larvae. Thick horizontal bars indicate the mean of the number of
the larvae at each height; the error bars show the standard deviations;
74 samples were collected. //'/. HU'2 and Ml'/. Ml? show the two
high and low tides about the time of sampling, respectively. The height
is based on the data in the tide table published by the Japan Meterological

emerged in synchrony with the morning low tides (see
the morning on 13. 14. 16. 17. and 27-30 March): a great
majority of the animals were collected during the after-
noon low tides. Another feature is that the number of
midges emerging in March and April increased drastically
with respect to the population observed in January and
February (compare the data of Fig. 7b and 7c).

After April, the timing in synchrony with the early
morning low tides completely disappeared. Therefore, the
tidal rhythm clearly shows a unimodal phase (Fig. 5 A and
5B). Although field observations were not made frequently
during the early morning low tides from May to Septem-
ber, it is clear that no midges emerged at these times;
rather all midges emerged during the midday and after-
noon low tides (Fig. 5B, 5C, and unpub. data). Compar-
ison of the rhythmic patterns shown in Figure 5. A-D
demonstrates that the time of emergence advances from
spring to summer, with respect to the 24-h day-night cycle.
In Figure 5A, emergence was observed around sunset;
i.e., 1600-2100 in early March, 1400-2000 around the
new moon on 16 March, and 1300-1800 around the full
moon on 30 March. In April and May (Fig. 5B), emer-

gence occurs between noon and sunset. It advances a few
hours further in June and July, when most midges ap-
peared between 1000 and 1600 (Fig. 5C). In August and
September, emergence occurs at midday (Fig. 5D). In ev-
ery case, the daily timing of emergence is clearly syn-
chronized with the times of low tides. If we were to assume
a circadian rhythm underlying the Cliinio emergence
rhythm, then we would have to consider a drastic phase-
advance of the rhythm from March to September. Such
an assumption is not reasonable; these results must be
interpreted in terms of an expression of the tidal rhythm
corresponding to a phase-advance of the peak of the semi-
lunar rhythm (see Fig. 7c-7f).

Alteration of synchrony in autumn

The unimodal tidal pattern was synchronized with the
daytime low tides until September (Fig. 5, A-D). But in
the latter half of October (Fig. 6A), the synchrony of timing
was altered from daytime low tides to nighttime low tides.
Most midges emerge in synchrony with the low tides at
night in November (Fig. 6A) and December (Fig. 6B).
Unlike the winter population (Fig. 4B), the autumnal
emergence shows no clear bimodal phase (see also Fig.
9). A feature of the autumnal tidal rhythm is that the
daytime emergence occurs only for the first several days
of the fortnightly peak of emergence (i.e., 1 7-22 October,
30 October- 2 November, and 16-17 November in Fig.
6 A; and 30 November- 1 December, and 14-16 December
in Fig. 6B).

Seasonal fluctuations in the number of niiil.w*
emerging, senulunar timing, and a seasonal shift
of the peak

Figure 7 summarizes the fluctuations in the number of
flies emerging every day in relation to the lunar cycle. The
number of collected midges is extremely small in January
and February (Fig. 7b); but it suddenly increases there-
after. A large number of the midges were collected in
March and early April (Fig. 7c). In late April and May
(Fig. 7d), the number of emerging midges decreases. Not
many midges emerge in June and July (Fig. 7e), and the
number of midges is very small in August. September
(Fig. 7f ), and early October (Fig. 7g), reaching a maximum
in November (Fig. 7h). The number of midges emerging
every day thus fluctuates seasonally with two peaks: one
in March-April, and another in November. Moreover,
very few midges emerge in January-February and August-
September.

Another feature of seasonal fluctuation is the semilunar
periodicity. But the peak of this rhythmicity shifts several
days in relation to the lunar cycle. In February and March
(Fig. 7b, 7c), it occurs 2-4 days after the full and new
moon; it just coincides with the days of full moon in April

THE TIDAL RHYTHM OF AN INTERTIDAL MIDGE

Time of day

171

21,
Time of day

18

Tide height (m)
05 10 15

Figure 4. Daily timing of emergence of Clunio tsushimensis males in relation to day-night, tidal, and
lunar cycles. (Left) The record from 28 October to 25 November 1990. Midges were collected using a light
trap method. The number of midges is plotted vertically on the horizontal lines, which also show the 5-min
period every 30 min (dot) during which sampling was done. Absence of the horizontal bars indicates no
sampling. 55 and SR represent the times of sunset and sunrise, respectively. HW and L\V connect the
respective times of high tides and low tides at the habitat. Open circle: full moon; black circle: new moon;
semicircles: the first and last quarters of the moon. The vertical scale indicates 300 midges. (Right) Wide
left panel: the pattern of emergence in midwinter (the record from 9 January to 22 February 1991 ). Midges
were collected by sweeping for 5 min every 30 min. The number of midges is plotted vertically on the
horizontal line during which the sampling was made. The vertical scale shows 10 midges. /. 117 and L\V2
connect the times of low tides at the habitat. Other symbols are as in Figure 4A. Narrow right panel:
fluctuations in the height of the two low tides, LI17 and L\V2.

(Fig. 7c, 7d). The peak then advances from the days of
full and new moon: in June and July (Fig. 7e), it appears
4 days before the syzygy, and from August to October
(Fig. 7f, 7g), it advances near the half moon. The peak is
again close to the full and new moon from November to
December (Fig. 7h).

To examine the relationship between the shift of the
semilunar peak and the temperature of the habitat, each
peak of the semilunar rhythm was plotted in relation to
the seasonal fluctuation of water temperature. As shown
in Figure 8, the peak (i.e., the mean of the distribution of
emerging midges in each semilunar cycle) is represented
by the days shifted from each full and new moon. The

water temperature is expressed by the average values of
15 days at each month. The shift of the peak from the
days of full and new moon clearly coincides with the fluc-
tuations of water temperature in the habitat, suggesting
that the phase of the semilunar rhythm is influenced by
the temperature.

Discussion

The emergence of Clunio tsushimensis imagines has a
clear tidal rhythm. Moreover, the pattern of synchrony
changed with season. A bimodal phase appeared in mid-
winter; but the synchrony shifted gradually, during Jan-

A q

1991

Time of day
6 12

Tide height (m)
0,5 10 15

Figure 5. Daily timing of emergence oiClunio males from late Feb-
ruary to mid-September 1991. Vertical scale: 200 midges. Symbols are
the same as in Figure 4. (A) From late February to early April. (B) From
21 April to 15 May. Vertical scale: 50 midges. (C) From 17 June to 12
July. Vertical scale: 100 midges. (D) From 16 August to 10 September.
Vertical scale: 100 midges.

LW2

172

THE TIDAL RHYTHM OF AN INTERTIDAL MIDGE

173

Time of day
6 12 18

I ' ' ' ' ' I

24
> 1

Time of day
6 12 18

1991
Nov27

Dec

24 Tide height (m)
1 I 05 1.0 1.5

-LWl

25-

Figure 6. Daily timing of emergence of Clunio males from mid-October to the end of December 1991.
Symbols are the same as in Figure 4. (Left) In autumn ( 1 7 October-27 November). The number of emerged
midges drastically increased from October to November (compare Fig. 7g and 7h), so the figure was separated
into two panels (horizontal dashed lines) with different vertical scales. The data on emergence in this period
are shown in duplicate in Figure 9. with the tluctuations of the two low tides (L II 7 and L\V2). (Right) From
27 November to 26 December. Vertical scale: 200 midges.

uary and February, from morning low tides to afternoon
low tides, and a unimodal tidal rhythm in synchrony with
afternoon low tides appeared in March. This pattern lasted
until early October. In mid-October, the synchrony shifted
to the morning low tides. As a result, emergence was ob-
served during the day from spring to autumn, at night in
early winter, and in both daytime and nighttime in mid-
winter. Therefore, we must ask a question about the
modulation of the expression of that rhythm: What are
the factors that induce the expression of the bimodal pat-
tern and that cause one of the two low tides to synchronize
the timing?

Relations between the tidal patterns ami the phase
expression of the tidal rhythm

On most Japanese seacoasts fronting the Pacific Ocean,
there are two high and two low tides per day, at mean
intervals of 12.4 h; the amplitude of these tides changes

with a fortnightly periodicity, resulting in spring and neap
tides. But the amplitude of these tides on a given day
varies with season. For example, on 16 January (new
moon; see Fig. 4B), a low low tide (+0.09 m) occurs at
0500. A high high tide (+2.3 m) is followed after 7 h by
this low tide, which is then followed after 6 h by a high
low tide (+0.9 m), and further followed after 5 h by a low
high tide (+1.9 m). The height of the two low tides be-
comes similar 6 days after the new moon. Thus, from late
autumn to winter, a low low tide always occurs in the
morning, and a high low tide always occurs in the after-
noon (Figs. 4B, 6B, and 9). In contrast, as shown in Figure
5, B-D, a low low tide always occurs in the afternoon,
and a high low tide occurs in the morning during spring
and summer.

A similar complex tidal regime is seen on the Pacific
coast of North America. Enright (1963. 1972) recorded
the swimming activity of several kinds of crustaceans
that were freshly collected from the sandy beach of Cali-

174

M. SAIGUSA AND T. AKIVAMA

2000 |-

4

O

20 25

October 1991

5 10

December 1991

Figure 7. Semilunar rhythm of emergence of Cluntu tsushimensii males, and the seasonal fluctuation
of the number emerging every day. The vertical black bars indicate the number collected per day in relation
to lunar cycles. Open circles: full moon; dark circles: new moon: semicircles: the first and last quarters of
the moon. Question marks on the horizontal axis indicate days when the sampling was not made.

fornia. The activity rhythm of the amphipod Syncheli-
diuin sp. was progressively damped as the animal's time
in the laboratory was prolonged, but it was well denned
for the first few days after collection. Another feature of
the activity pattern is that the relative amplitude of its
peaks reflects the amplitude of the tides occurring at the

nearby seacoast (Enright, 1963). Similar activity rhythms
were also recorded from the isopod Excirolana chiltoni
(Enright, 1972). This animal showed a persistent tidal
rhythm for 2 months under the constant conditions in
the laboratory. The temporal variation in activity seems
to parallel predicted changes in the height of high tide.

THE TIDAL RHYTHM OF AN INTERT1DAL MIDGE

175

Phase shift (days)
-5

10

15 20 25

Water temperature

J I I 1 . 1 ,_. L.

30 (C)

3
C

-5

O

3

Figure 8. The relationship between the phase of the semilunar rhythm
and the seasonal change of the mean water temperature at the habitat
(solid curve). Each peak (black circles) of the semilunar rhythm is in-
dicated as the days of shift from full or new moon (i.e.. 'zero' on the
horizontal axis). The error bars indicate a standard deviation within each
peak of the semilunar rhythm. The peaks on the right of 'zero' show a
phase-advance of the semilunar rhythm with respect to the syzygy, and
those on the left of 'zero' show a phase-delay from the syzygy.

To examine whether the tidal synchrony of the rhythm
of emergence of C. tsushimensis could be altered by ir-
regularities in the amplitude of the semidiurnal tides in
the habitat, the water level of low tides was plotted against
the time of emergence. Through the autumn, as shown
in Figure 9, the relative heights of the two low tides i.e..
the low low tide and high low tide vary continuously,
but cyclically, with an interval of 2 weeks. When the two
tides were different in amplitude, emergence clearly co-
incided with low low tides alone, which produced the uni-
modal phase of the tidal rhythm. A bimodal phase ap-
peared for only a few days every fortnight when the heights
of the low tides were close to equal.

Similar relations are also seen in December, when
emergence occurred in synchrony with the low low tides

(i.e.. the nighttime low tides), causing a unimodal phase
except a few days around the half moon (Fig. 6B). The
tidal pattern in January (Fig. 4B) somewhat changes from
that of October-December (Figs. 6B and 9): while the
height of the high low tide exceeds 1 m on many days in
December (Fig. 6B), it is less than 1 m on most days in
January (Fig. 4B). Therefore, in January, the main part
of the larval habitat (see Fig. 3) is exposed to the air two
times per day, except a few days after the half moon. The
decrease in height of the high low tide might influence
the larval habitat, which might have evoked an emergence,
resulting in bimodal phases during a relatively long period
in January and February.

The correlation between the relative difference in the
tidal heights and phase modality was noticed no later than
the first 10 days of March; thereafter it was lost. In March,
for example, the height of both low tides is close to equal
around the full and new moons, yet far more midges
emerged in the afternoon than in the morning (Fig. 5A).
This result cannot be accounted for by a simple synchrony
with low low tide, or by the exposure of the larval habitat
to the air at low tides. Day-night cycles must participate
in the selection of the afternoon low tides. Thus, the syn-
chrony of emergence with the two low tides per day is
correlated, not only with the relative difference in the
heights of the tides, but possibly also with the day-night
cycle.

In May, the timing of emergence is again synchronized
with the lowest of two different low tides, and a unimodal
phase appears in the daytime (Fig. 5B). During June and
September (Fig. 5C and 5D), neither of the two low tides
ebbs less than those of spring. In each case the timing of
emergence is synchronized with the low low tides, and a
unimodal tidal rhythm appears. Since the low low tides
occur in the daytime from the end of April to September,
the unimodal phase also appears in the daytime.

The relationship between the low low tides and the 24-
h day-night cycle is reversed in autumn. As shown in Fig-
ure 9, the low low tides appear in the morning. Emergence
is synchronized with the low low water, and shows a uni-
modal tidal rhythm. As a result, the phase of this tidal
rhythm appears in the morning. Moreover, the high low
tides occur in the afternoon, and do not ebb lower than
0.8-0.9 m. If the larval habitat is not moved in height
from that of March (Fig. 3), there are only a few days
every 2 weeks when the habitat is exposed to the air at
each low tide (i.e.. twice a day). This might have resulted
in a brief bimodal phase in autumn and early winter (Fig.
6A and 6B).

Zeitgeber o/Clunio tidal and semilunar rhythm

Few cyclical environmental factors are known to be
zeitgebers of tidal rhythms. One such is the cycle of water

176

M. SAIGUSA AND T. AKIYAMA

Time of day Tide height (m)

6 12 18 0.5 1.0 1.5

Oct. 20

Figure 9. The relationship between the daily timing of emergence
and the heights, on the same day, of two low tides (L II 7 and! II 2). The
data on emergence are the same as in Figure 6A. To make that relationship
much clearer than in Figure 6A. the numbers of emerged midges are
shown as a percentage of the total number collected per day. The vertical
bar shows the scale of 20%. The right panel shows the fluctuation of the
heights of the two low tides (/JI7 and MI'. 1 ).

turbulence (Enright, 1965). Cyclical or noncyclical
changes in hydrostatic pressure also cause behavioral re-
sponses in amphipods (Enright, 1962; Morgan, 1965).
Hydrostatic pressure fluctuations do not entrain an 'en-
dogenous' rhythm, but since they coincide with the tidal
cycles in the field, they could be the zeitgeber of the tidal
rhythm. But, at least for the swimming activity of intertidal
isopods, the 24-h day-night cycle does not seem to be a
zeitgeber of tidal rhythms (Enright, 1963).

In Chmio emergence rhythm, Neumann (1966, 1976)
showed that the semilunar timing is entrained by the ar-
tificial moonlight given in the laboratory for 3-4 nights
every 30 days. According to Neumann's hypothesis (Neu-
mann, 1976, 1985, 1987), daily timing of emergence is

controlled by a circadian clock that is entrained by a 24-
h day-night cycle only. But the phase of the tidal cycle
differs according to the habitat of animals (e.g.. see Fig.
9 in Saigusa, 1988). Neumann's hypothesis cannot explain
why the phase of the daily emergence rhythm coincides
with the time of low tide in each larval habitat. Moreover,
as indicated by this study, the phase of the daily emergence
cycle was largely shifted through the year, with respect to
the 24-h day-night cycle. Such a large phase-shift could
be explained in terms of the tidal rhythm.

In crab larval release activity (Saigusa, 1986), the 24-h
day-night cycle causes the phase shift of the tidal rhythm
with a unimodal phase. Similarly, as shown in other spe-
cies (Saigusa, 1 992), this factor also causes the phase shift
of the bimodal tidal rhythm. Thus, it is clear that the 24-
h day-night cycle can be one of the leitgebers of circatidal
rhythms.

But it would be impossible that the tidal rhythms are
entrained by the 24-h day-night cycle alone; other factors
correlated with the tidal cycle, such as water turbulence,
must be considered. Moreover, moonlight cycles in par-
allel with the tidal cycle could be the candidate, although
the phase angles of these two environmental cycles differ
according to geographical conditions. Cycles of artificial
moonlight do entrain the tidal rhythm of larval release in
an estuarine terrestrial crab; and the phase relations be-
tween the evoked tidal rhythm and the artificial moonlight
cycle correspond to the phase relations between the time
of high tide and the moonlight cycle in the habitat of a
local population (Saigusa, 1988).

As this study indicates, the timing of emergence is syn-
chronized with the time of low tides and shows a tidal
rhythm through the year. So we can speculate that among
the possible zeitgebers of the Chmio tidal rhythm is the
ebb-flow cycle of the tides, the moonlight cycle, or both.
In addition, as suggested from the experiments with Chmio
(Neumann, 1966, 1976) and crabs (Saigusa, 1986. 1992),
the 24-h day-night cycle could also be the :eitgeber of the
Chmio tsushimensis tidal rhythm. Furthermore, while the
day-night cycle entrains the tidal rhythm in every season,
it would be involved in the expression of a unimodal phase
(see Fig. 5A).

A problem in understanding tidal rhythms having
a semilunar component

Enright ( 1972) reported endogenous swimming activity
rhythm of Excirohma chiltoni inhabiting the sandy
beaches of the Pacific coast of North America. The tidal
regime in this habitat includes semidiurnal inequality in
amplitude. The tidal scheme recurs at double-tidal inter-
vals (i.e.. 24.8 h), which further changes at intervals of 2
weeks. E. chiltoni shows a persistent circatidal rhythm
with a lunar component under constant conditions in the

THE TIDAL RHYTHM OF AN INTERTIDAL MIDGE

177

laboratory; even under such conditions, activity reflects
different heights of the tides (see Fig. 1 in Enright, 1972).
Nevertheless, if the activity were plotted on a 24-h time
scale, the rhythm would appear to be circadian.

Similar phenomena occur in Clunio tsushimensis, as
we have reported here. The tidal rhythm of this animal
showed a unimodal phase except in January and February.
Furthermore, this tidal rhythm features a semilunar tim-
ing, with emergence occurring for several days near the
times of the full and new moon (Fig. 7). From March to
September, emergence is synchronized with the afternoon
low tides (Fig. 5A-5D). If the data for these periods are
plotted against the time of emergence, a daily rhythm
with a peak at the afternoon appears (e.g.. Fig. 10A). On
the other hand, from November to December, emergence
occurs at the time of morning low tides (Fig. 6 A and 6B).
Now if the timing is plotted, a daily rhythm appears with
a peak in the morning (Fig. 10B).

The emergence of Clunio is concentrated around the
times of the full and new moon. So, if the data were ar-
ranged as shown in Figure 10, we could not readily de-
termine whether the rhythm that underlies the daily and
semilunar cycle of emergence is tidally correlated or day-
night correlated. Yet, as stated repeatedly, the daily timing
of emergence is not explicable in terms of a circadian
rhythm or a modified circadian rhythm. It would be a
tidally correlated rhythm that underlies the expression of
a semilunar rhythm. Moreover, this rhythm is surely en-
trained by a 24-h, day-night cycle. Nevertheless, there is
no evidence that the two internal rhythmic systems cir-
cadian and circatidal rhythms couple or interact to-
gether, resulting in a semilunar rhythm (Saigusa, 1986,
1988, 1992). These problems bear on the timing mech-
anism of tidal and semilunar rhythms and require further
consideration.

A local population of Clunio tsushimensis

The systematics of the genus Clunio is not yet settled
because distinctive morphological features characteristic
of the various geographical strains are lacking. Within C.
marinus, which is widely distributed on the European
coast of the Atlantic Ocean, five races were distinguished
on the basis of the larval habitat and the timing of emer-
gence (Neumann, 1976). Among these local races, the
Baltic Sea population was somewhat different from other
Atlantic populations; it inhabits the sublittoral zone and
tolerates salinities as low as 4-6 ppt in some areas (Palmen
and Lindeberg, 1959; Olander and Palmen, 1968). The
differences in larval habitat and time of emergence tend
to isolate these populations, even at the site where both
are simultaneously distributed, so they might be divisible
into two species (e.g.. see Heimbach, 1978).

Distributions of the antennal index, wing index, and
body length (Fig. 2) all showed a single peak, and there

20

SR

12
Time of day

E 10

SR B

Time of day

Figure 10. A pseudo-daily rhythm. (A) The time ot emergence re-
corded from 17 June to2 July 1991. (B) The time of emergence recorded
from 28 November to 26 December 1991. The vertical axis shows the
percentage of the midges collected. SR and SS indicate the times of
sunrise and sunset, respectively. These times shift only a few minutes in
June, and at most 15 min in December. In this figure, SR and 55 show
the times of sunrise and sunset on 17 June (A) and 28 November (B),
respectively. See text for details.

was no difference between the population collected at the
morning low tides and that collected at the afternoon low
tides. This suggests that the midge Clunio inhabiting the
seacoast of the Inland Sea is a single species. But which
species? To determine whether the collected specimens
are C. tsushimensis or C. aqui/onius, the data in Figure
2 were compared with those obtained by Hashimoto
(1968, 1969) in the Izu Peninsula, Shizuoka Prefecture.
The antennal index (AI) of the individuals collected in
March (Fig. 2A) was between 0.60 and 1.12. and the mean

178

M. SAIGUSA AND T. AKIYAMA

was 0.84, intermediate between the Izu populations of C.
tsitshimensis (mean 1.12) and C. aquilonius (mean 0.62)
collected in the same month. Moreover, the wing index
(WI) of the Inland Sea species (Fig. 2B) ranged from 0.40
to 0.56, about the same as the mean for both C. tsusht-
mensis and C. aquilonius at the Izu Peninsula. The body
length (Fig. 2C) was between 2.00 and 3.10 mm (mean
2.40 mm), much larger than for C. tsushimensis from Izu
collected during the same month (1.4-2. 4 mm; mean
about 1.8 mm). In summary, none of these indices could
provide a positive identification of either C. tsushimensis
or C. aquilonius at our observation site.

In the marine midges studied by Hashimoto (1969,
1976), the AI fluctuates with the season, with a minimum
in early spring, a maximum in late summer, and no over-
lap between C. tsushimensis and C. aquilonius in any
season; that is, whereas the AI (mean) of C. aquilonius
varied from 0.6 in winter to 0.75 in summer, that (mean)
of C tsushimensis varied from 1.05 in winter to 1.4 in
summer (see Fig. 7 in Hashimoto, 1969). The body length
was also closely correlated with the annual change of water
temperature (Hashimoto, 1976). Now, the atmospheric
and water temperatures on the coast of the Inland Sea are
5-6C lower in winter and spring than those on the Izu
Peninsula. We therefore speculate that the decreased AI
and increased body length are due to the temperature
differential between the two larval habitats. Hence, we
conclude that the species reported here is a local popu-
lation of C. tsushimensis. and not a new species.

Acknowledgments

We thank Prof. Masamichi Yamamoto. Director of
Ushimado Marine Laboratory, who gave us every facility
for the present work.

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Reference: Biol. Bull. 188: 179-185. (April. 1995)

The Role of the Cardioregulatory Nerves in Mediating
Heart Rate Responses to Locomotion, Reduced Stroke
Volume, and Neurohormones in Homarus americanus

M. S. GUIRGUIS AND J. L. WILKENS*
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4

Abstract. Control of decapod crustacean heart activity
is believed to be dependent on the regulation of the cardiac
ganglion by external input from the central nervous system
as well as by circulating neurohormones. This study in-
vestigated the roles of these inputs on the heart rates of
lobsters exercising on a treadmill. Heart rate increased
rapidly at the onset of walking in control animals. This
rapid phase was lost after the regulatory nerves were cut,
but small increases still occurred. When stroke volume
was reduced by cutting alary ligaments, the animals com-
pensated by increasing heart rate: this compensation was
lost when the regulatory nerves were cut. In resting ani-
mals, injection of serotonin, octopamine, and dopamine
induced increases in heart rate. After the regulatory nerves
were cut, only dopamine and serotonin injections caused
increases in heart rate, suggesting that these amines act
on the cardiac ganglion as independent effectors.

Introduction

The neurogenic decapod crustacean heart consists of a
single ventricle suspended in the pericardial sinus by an
array of alary ligaments. The energy stored as these liga-
ments are stretched during systole is recovered to re-ex-
pand the heart during diastole. The heart fills by means
of valved ostia and supplies hemolymph to seven arteries.
There is no direct venous supply to the heart.

The basic contraction rhythm of the heart arises from
the bursting discharges of the nine-cell cardiac ganglion
located on the inner dorsal wall of the heart (Alexan-
drowicz, 1932). The cardiac ganglion receives extrinsic

Received 2 September 1994; accepted 8 December 1994.
* To whom correspondence should be addressed.
Abbreviations: f H , heart rate; DA, dopamine; OA, octopamine; 5-HT,
5-hydroxytryptamine.

nerve fibers via the paired dorsal nerves that arise from
the central nervous system (Alexandrowicz, 1932). Each
dorsal nerve contains two accelerator axons and one in-
hibitory axon. In isolated hearts, stimulation of the ac-
celerator nerves speeds the contraction rate, and stimu-
lation of the inhibitory nerves slows or stops the heart
(Maynard, 1953; Florey, 1960: Wilkens and Walker,
1992). En passant recordings from the dorsal nerves in
semi-intact animals reveal periodic increases in inhibitory
nerve firing rates: these increases cause bradycardia (Field
and Larimer, 1975; Young, 1978). The role of this au-
tonomic-like control system in regulating heart rate re-
sponses in intact animals has not been studied.

Heart rate (/ H ) is also modified by all of the neurohor-
mones that have been identified in the pericardial organs
(see reviews by Wilkens, 1987; Wilkens and McMahon.
1992). Each of the aminergic neurohormones, dopamine
(DA), octopamine (OA) and serotonin (5-HT), trigger
tachycardia in isolated hearts (Cooke, 1966; Florey and
Rathmayer, 1978; Grega and Sherman. 1975) and in in-
tact animals (Wilkens el a/., 1985). In intact animals the
possibility cannot be discounted that the neurohormones
act indirectly via the nervous system as well as directly
(Berlind et ill. 1970). The experiments reported here were
designed to test the hypothesis that / H in intact lobsters is
under continuous modulatory control by cardioregulatory
nerves and pericardial aminergic neurohormones.

Materials and Methods

Source ami holding condition of lobsters

Homarus americanus of 490-670 g mass were obtained
from a commercial supplier and maintained in flowing
artificial seawater at 1 2C. No differences in performance
were observed between males and females.

179

180

M. S. GUIRGUIS AND J. L. WILRENS

f H measurement and surgical procedures

Heart beat was measured by electrodes implanted next
to the heart in the pericardia! sinus. Wires were inserted
through holes drilled in the carapace and cemented in
place. Signals were amplified (Grass PI 5 amplifier) and
digitized by a Mac Lab/8 and visualized on the Macintosh
SE/30 computer running CHART/8 software. In some
cases data were also displayed on a Gould RS200 chart
recorder.

For surgical interventions, animals were first packed in
crushed ice for about 30 min to effect cold "anesthesia."
Next, the carapace and hypodermis over the heart were
removed. A dam molded from dental impression wax was
placed around the opening to prevent hemolymph from
spilling over into the bath. The wax was secured to the
carapace with cyanoacrylate adhesive. With the heart so
exposed, the two dorsal anterior alary ligaments, the dorsal
nerves, or both could be cut. The dorsal nerves approach
the heart along the dorsal surface of the posterior dorsal
alary ligaments and traverse the posterior half of the ven-
tricle in the connective tissue covering the heart before
penetrating to the cardiac ganglion. Superficial cuts
through the connective tissue over the heart severed these
nerves without impairing the alary ligaments. After sur-
gery, the square of carapace was replaced and sealed with
melted dental wax and cyanoacrylate adhesive. Sham op-
erations consisted of removing the square of carapace and
opening the hypodermis only. Following the return of
animals to seawater, / H usually returned to preoperation
rest levels in 2 h; however, tests were not run until the
next day.

Induced locomotion

To measure cardiac responses to exercise, lobsters were
trained to walk on an underwater treadmill. The treadmill
consisted of a cloth-backed sanding belt passed around a
motor driven and a tensioning drum. The treadmill
chamber was submerged in aerated seawater siphoned
from the holding tank and was maintained at 13-14C.
Some lobsters walked readily from the outset, but others
required several training runs before they would exhibit
continuous walking. Touching the telson and uropods
with a test tube brush was usually an adequate stimulus
to induce walking. The treadmill belt traveled at
1.7m- min" 1 . All observations in this study were carried
out in the treadmill chamber. The walls of the chamber
were covered with black plastic to isolate the animal from
visual stimuli.

Administration of neurohormones

Saline or neurohormones dopamine (DA), octopa-
mine (OA), and serotonin (5-HT) (Sigma Chemical Co.)

dissolved in lobster saline (Cole, 1941) were injected into
the lateral pericardial sinus of resting animals. A syringe
pump was used to deliver the injection over a period of
1 min. An Intermedic polyethylene tube (P.E. 160) ran
from the syringe to a needle that was inserted into the
sinus through a hole that had been drilled into the carapace
and covered with dental dam. The injection volume was
adjusted to the weight of the animal so that no more than
240 //I would produce a circulating concentration of 1 nAI.
assuming a 30% hemolymph volume (Gleeson and Zub-
koff, 1977). Animals were not handled during the injection
and did not appear to be disturbed by the saline injections.
They often became agitated, however, during hormone
injections. When more than one test was conducted during
a day, an average of 4-5 h was allowed for recovery be-
tween injections.

Statistical tests

Significance was evaluated with a two-tailed paired
Student's / test. A P value < 0.05 was considered signifi-
cant. Paired / tests were used to compare the heart rates
during the last 6 min of the rest period to the first 6 min
after the onset of walking or following injections. Ex-
amination of the significance of denervation was carried
out with an analysis of covariance. In this case the slope
of heart rate change, during exercise, in denervated ani-
mals was compared to the change seen in intact animals.

Results

Role of dorsal nerves in heart response to startle
and exercise

In settled animals, novel stimuli such as shadows,
movements in the visual field, and touches to the carapace
always triggered periods of bradycardia known as startle
responses. These responses diminished when the stimuli
were repeated (Fig. 1). Successful cutting of the dorsal
nerves was confirmed by the loss of the startle response.

Settled lobsters resting in the treadmill chamber exhib-
ited regular heart rates of 54 13 min" 1 . At the onset of
walking, / H rapidly increased to 87 5 min ' over the
first 2-3 min and then more slowly to 90 5 min ' by
the end of the 30-min walk (Fig. 2). While an animal was
walking, the swimmerets beat and the abdomen and che-
lipeds were held slightly elevated and extended. During
the 30-min exercise period, animals often stopped and
then, when they had drifted back to the bottle brush, ac-
celerated and then resumed a steady pace. The frequency
and length of pauses tended to increase with time during
the exercise period. At the point of exhaustion, animals
would not respond to tactile stimulation. Heart rate and
walking behavior were not different between sham-op-
erated and unoperated control animals.

REGULATION OF LOBSTER HEART RATE

181

B

10 sec

Figure 1. The startle response, a period of bradycardia, seen after passing a shadow (A) across the visual
field of a settled lobster. The startle response is usually diminished if the same or a different stimulus is
repeated within a few minutes of a first stimulus, in this case tapping the carapace (B). Parts A and B are
continuous records.

Following denervation surgery, heart rate returned to
previous resting rates in 2-3 h, but it gradually increased
over several days thereafter. The data shown in Figure 2
were collected over several days following surgery, and
the slightly elevated settled rates reflect this gradual in-
crease. Walking behavior was unchanged from that of
controls; however, the rapid phase of tachycardia did not
occur. Heart rates did increase slowly from 62 min~' at
rest to a maximum of 72 min" 1 , an increase significantly
below that observed in controls. Rates during the last

6 min of walking did not differ significantly from those
recorded during the 6-min period prior to walking.

Role of dorsal nerves in response to cardiac impairment

In Carcinus maenas. cutting the two dorsal anterior
alary ligaments reduced cardiac output of semi-isolated
hearts by 25% (Wilkens and McMahon, 1992). One day
after these same two ligaments were cut in lobsters, the
resting /H was elevated by 20.6% above controls (Fig. 3).

120 r

100

I

1 80

60

D

(D

40

20

* Control
o Denerved

-10

10

20

Time (min)

Figure 2. The effects of exercise on the heart rate of intact lobsters
(n = 11) and following sectioning of both dorsal cardioregulatory nerves
(n = 3). The dotted line is drawn at the mean heart rate of denervated
animals for the 6-min period before the start of walking. The treadmill
was started at time zero and traveled at 1 .7 m min" 1 . Mean SD.

0)

-*-J

D

D

0)

X

120 r

100

80

60

40

20

JJJJJJJJJ556505555555 00000000

sari

* Control

o Ligament Loss

-10

10

20

30

Time (min)

Figure 3. The effect ot severing the two dorsal-anterior alary ligaments
on heart rate in resting and exercising lobsters (a = 11 for control, n
= 5 for operated). The control data are reproduced from Figure 2. The
five operated lobsters were taken from that group. The treadmill was
started at time zero. Mean SD.

182

M. S. GUIRGUIS AND J. L. WILKENS

During walking, / H increased on average 11.7% above
controls ( 102 2 min '). These animals showed no im-
pairment in walking behavior.

On the second or third day after the ligaments were
cut, the heart chamber was reopened and the dorsal nerves
were sectioned. After this procedure the compensatory
increases seen in resting / H after ligament loss were re-
versed (Fig. 4). During locomotion, / H increased slowly
and steadily. The final rates after 25 min were significantly
lower than those of control or ligament-loss animals. The
rates were not significantly different from those observed
during the same period in animals with only dorsal nerve
loss (Fig. 2). These animals were reluctant to walk and
continued to do so only if continuously prodded. Three
ot the animals refused to walk longer than 21 min. The
chelae often dragged along the belt, and after about 10 min
of walking the animals seemed to be trying to recruit the
chelae to aid walking. The abdomen also dragged.

Responses of heart to uniinergic neurohormones

When settled lobsters were injected with enough of each
of the aminergic neurohormones to produce a circulating
concentration of 1 n\f (assuming complete mixing), the
result was significant and prolonged tachycardia (Fig. 5),
whereas injection of the same volume of saline had no
effect. DA caused the largest increase (83 2 min" 1 ), fol-
lowed by 5-HT(79 2 min" 1 ) and OA (71 3 min ').
Heart rate remained elevated for more than an hour in
almost all cases.

The maximum /H values following injection of DA and
5-HT in denervated settled animals were similar to those

120 r

100

80

O

60

o

^ 40
20

Control

Ligament loss and

Denervation

-10

10

20

Time (min)

Figure 4. The compounded effects of alary ligament loss and de-
nervation on the heart rate of exercising lobsters (n = 11 for control,
same animals illustrated in Figs. 2 and 3; n = 5 for operated, the same
denervated animals illustrated in Fig. 3). The treadmill was started at
time zero. Mean SD.

o

I

120

100

80

60

40

20

100

80
60
40
20

-10

DA injection
Saline injection

10

20

30

***********

OA injection
Saline injection

10

20

30

5-HT injection
Saline injection

10

20

30

Time (min)

Figure 5. The effects of saline, OA, DA, and 5-HT injections on
heart rate of settled lobsters (/; = 5). The responses to saline injections
are copied in each panel as a reference trace. The amines were presented
in random order from animal to animal. Mean SEM.

in the control animals (Fig. 6), but they were not main-
tained at the maximum levels as they were in the controls.
All of these animals showed elevated rates at rest com-
pared to controls. OA injections had no effect on / H ; how-
ever, the preinjection /H values were at the same level as
in control animals following OA.

Injection of the same amounts of 5-HT into control
lobsters just before the treadmill was turned on caused
similar increases in / H , but prevented walking for as long
as 15 min. The animals appeared to be stiff" and did not

REGULATION OF LOBSTER HEART RATE

183

I

(D

D

^_

-4 '
i_

O
<U

X

120 r
100

80

60

40

. 100 -

80

60

100 -

80 -

60

40

20

-10

T J

t

DA injection
Saline injection

20
120

i

1 1

i

10 20

30

40

OA injection

Saline inject

on

i i

i

20
-10 10 20

30

120 r

5-HT injection
Saline injection

10

20

30

Time (min)

Figure 6. The effects of saline, OA, DA, and 5-HT injections on
heart rate in denervated lobsters (n = 5). The responses to saline injections
are copied in each panel as a reference trace. The amines were presented
in random order from animal to animal. Mean SEM.

walk even when prodded. Over the next 30 min the will-
ingness to walk gradually increased. DA and OA did not
impair walking.

Discussion

Role of dorsal nerves in heart response to exercise

The loss of startle-induced bradycardia after dorsal
nerve cutting indicates that the cardioinhibitor nerves are
the causative inputs for this behavior. The heart rate re-

sponse to exercise involves two phases. Phase I is rapid-
onset tachycardia, which occurs within the first 2 to 3 min
of exercise (Fig. 2). The control system is rather like an
on-off switch and does not seem to be graded with duration
of exercise. Phase II is a sustained and gradually increasing
/H throughout the period of exercise. The role of the car-
dioregulatory innervation in these two phases was eval-
uated following nerve sectioning. In the absence of the
dorsal excitatory and inhibitory nerves, the regulation of
the heart activity can occur only via hemolymph-borne
factors. Such factors could include neurohormonal secre-
tions from the pericardial organs lining the pericardial
cavity and metabolite end products produced during
walking. The absence of Phase I in denervated animals
demonstrates the ineffectiveness of these secondary pro-
cesses in compensating for cardiovascular stress. There-
fore, the cardioaccelerator nerves must be responsible for
this response, whereas the pericardial neurohormone se-
cretions are not. This finding is consistent with prior in
vitro investigations, which demonstrated that stimulation
of accelerator nerves causes a quick increase in / H (within
30 s of stimulation; Maynard, 1953; Florey, 1960; Hagi-
wara, 1961). The small increases in / H during Phase II of
exercising in control, denervated, and dual-operated (lig-
ament loss and denervation) animals may arise from neu-
rohormonal inputs even though the rates never reached
control levels. Apparently pericardial secretions alone do
not contribute to Phase I, but they may play a role in
Phase II. Therefore, Phase II is most likely due to dual
action of nervous input and pericardial secretions.

Role of dorsal nerves in response to cardiac impairment

The effects of reduced stroke volume, resulting from
ligament loss, were compensated for by an increase in / H
both at rest and while walking. This supports the finding
of McGaw ct at. ( 1 994) that the primary mechanism used
to offset the effects of cardiovascular stress is an increase
in / H . The subsequent denervation of these hearts elimi-
nated the elevated resting rates, although the very slow
increases in / H still occurred during exercise. Following
the dual insults on cardiac output of ligament loss and
denervation. lobsters were reluctant to walk for more than
short periods and appeared to fatigue rapidly. These results
demonstrate that cardioregulatory innervation plays an
important role in primary compensatory cardiovascular
responses to exercise.

An examination by McGaw et al. (1994) of hemolymph
flow patterns during short periods of spontaneous walking
showed preferential hemolymph flow to the sternal artery
that supplies the periopods and scaphognathites (venti-
latory pumps). This redistribution of hemolymph flow is
thus another compensatory measure used by animals in
the face of cardiovascular stress. Those authors also found

184

M. S. GUIRGU1S AND J. L. WILKENS

that short-term cardiovascular stress was accompanied by
an increase in / H and stroke volume.

Role of extrinsic nerves in mediating
neurohormone action

Injection of any one of the three aminergic neurohor-
mones (OA, DA, and 5-HT) into resting animals brought
about a significant increase in the / H that was maintained
for more than 30 min. The circulating half life of these
three animals in crayfish and crabs varies from 2 to 1 3 min
(Hoeger and Florey, 1989; Hoeger, 1990; Bininda et a!.,
1992). These clearance studies were performed at tem-
peratures similar to those used during the present study.
Clearance rates in lobsters have not been determined, but
if we can assume that they are similar to those in crayfish
and crabs, then the duration of the elevation in / H is greater
than the expected residence time of the hormones. In de-
nervated animals, only DA and 5-HT produced short-
term increases in resting / H ; OA injection produced no
change. The initial increase in / M brought about by DA
and 5-HT injection lasted for only about 10 min and
gradually declined thereafter. The initial responses may
have arisen from the period when circulating amines were
highest, while the decline thereafter may indicate that the
dorsal nerves play a role in maintaining neurohormone
action beyond the expected amine residence time. In other
words, the prolonged elevation of / H in intact animals
may arise from long-term increases in cardioaccelerator
or decreases in cardioinhibitor tonic firing rates. The ex-
ogenously applied DA may be mimicking the action of
the cardioaccelerator nerves, because recent evidence
suggests that DA may be the neurotransmitter released
by the cardioaccelerator nerves ( Yazawa and Kuwasawa,
1992, 1994).

Effect of neurohormones on posture and walking

The injection of 5-HT and octopamine into lobsters
induces stereotypical postures thought to resemble those
seen in dominant (5-HT) and subordinate (OA) animals
(Livingstone et a/.. 1980; Kravitz, 1990). 5-HT caused
animals to stand tall on the tips of their walking legs with
their chelae extended and open and their abdomens flexed.
OA injections caused the animals to stand low to the sub-
strate with their claws and walking legs extended and
pointing forward and with their abdomens hyperextended
upward. In our experiments, lobsters injected with similar
doses of 5-HT also assumed the expected stance, but were
quite stiff and unable to walk for up to 15 min. They
slowly regained ability to walk over the following 30 min.
In Carcintis, 5-HT triggers a rigid posture and reduces an
animal's ability to right itself when placed on its back
(McPhee and Wilkens, 1989). Perhaps it is inappropriate
to suggest that the 5-HT-induced posture mimics the

dominant stance displayed by untreated lobsters in natural
settings, because these animals are unable to move. On
the other hand, the ability of lobsters to walk was unaf-
fected by OA injections.

Conclusions

The focus of this investigation was to determine the
role of cardioregulatory nerves in regulating the / H of//.
americanits. Our data support the conclusions that the
cardioinhibitor nerves are responsible for startle-induced
bradycardia and that the cardioaccelerator nerves cause
the rapid-onset tachycardia associated with walking. The
continued gradually increasing heart rate during long
walks may be partially accounted for by hemolymph-
borne factors including neurohormones. The cardioac-
celerator nerves are also responsible for the compensatory
increases in / H in animals whose stroke volume was ar-
tificially reduced.

Acknowledgments

This study was supported by a grant from the Natural
Science and Engineering Research Council of Canada
to JLW.

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\\ilkens, J. L. 1987. Cardiac and circulatory control in decapod Crus-
tacea with comparisons to molluscs. Experientia 43: 990-994.

\\ilkens, J. L., and B. R. McMahon. 1992. Intrinsic properties and
extrinsic neurohormonal control of crab cardiac hemodynamics. Ex-
pcncntia 48: 827-834.

Wilkens, J. L., and R. L. Walker. 1992. Nervous control of crayfish
cardiac hemodynamics. Co/up Physiol. 11: 115-122.

\\ ilkens, J. L., A. J. Mercier, and .J. Evans. 1985. Cardiac and ventilatory
responses to stress and to neurohormonal modulations by shore crab
Carcimtx maenas Camp. Bittclmn. Pliyxiol. 82C: 337-343.

Yazawa, T., and K. kuvvasavva. 1992. Intrinsic and extrinsic neural and
neurohormonal control of the decapod heart. Experientiu 48: 834-
840.

Yazawa, T., and K. Kuwasawa. 1994. Dopaminergic acceleration and
GABAergic inhibition in extrinsic neural control of the hermit crab
heart. / Own/'- Physiol. A 174: 65-75.

Young, R. E. 1978. Correlated activities in the cardioregulator nerves
and ventilatory system in the Norwegian lobster, Nephrops norvegicus
(L.). Cm/> Biocliem. Physiol. 61A: 387-390.

Reference: Biol. Bull 188: 1S6-I%. (April.

The FMRFamide-Related Peptides Fl and F2 Alter
Hemolymph Distribution and Cardiac Output in the

Crab Cancer magister

I. J. McGAW* AND B. R. Me MAHON

Department of Biological Science, University of Calgary, Calgary. Alhcrta. T2N 1N4. and Bamfield
Marine Station. Bamfield. British Columbia, I 'OR 1 BO Canada

Abstract. The FMRFamide-related peptides Fl and F2,
originally isolated from lobster pericardia! organs, have
been shown to exert cardioexcitatory effects on isolated
or semi-isolated crustacean hearts. The present study
sought to determine the in vivo effects of Fl and F2 on
cardiac and circulatory performance of Cunccr magixter
using a pulsed-Doppler technique. In general the effects
of Fl and F2 were similar; however, Fl was more potent
and its effects were of longer duration than those exerted
by F2. Infusion of either Fl or F2 caused a decrease in
heart rate and subsequent periods of acardia. These de-
creases in rate occurred concurrently with a short-term
increase in stroke volume of the heart, followed by a
longer-term decrease in stroke volume. Hemolymph flow
rates through the anterior aorta, anterolateral arteries,
sternal artery, and posterior aorta also showed the same
trend, with an initial short-term increase in flow rate fol-
lowed by a longer-term decrease with periods of ischemia.
Hemolymph flow through the paired hepatic arteries sim-
ply decreased, but recovery to pretreatment levels was
faster than in the other arterial systems. Threshold for
these responses occurred at circulating concentrations be-
tween 1CT 9 mol- 1~' and 10~ 8 mol- 1~' for Fl and some-
what higher, between 10 s mol- 1 ' and 10 7 mol- 1 ',
for F2.

The neuropej
was first isoL

Introduction

FMRFamide (Phe-Met-Arg-Phe-
d and sequenced from the clam

Received 13 June 1444, am-pted 25 January 1995.

* Present address: Department of Biology. College of Charleston,
Charleston, SC 29424.

Abbreviation-, FaRPs, FMRFamide-related peptides; Fl, TNRN-
FLRFamide: F2, SDRNFLFRamide: CNS. central nervous system.

Macrocallista nimhosa (Price and Greenberg, 1977).
FMRFamide is now known to belong a large family of
neuropeptides, collectively called the FMRFamide-related
peptides (FaRPs), that share the sequence Arg-Phe-NH :
and exist throughout the invertebrate and vertebrate
kingdom (for reviews see Greenberg and Price, 1983;
RafTa, 1988; Price and Greenberg, 1989). FaRPs generally
function as neurohormones or neurotransmitters; their
action on the cardiovascular system is predominantly ex-
citatory, but inhibitory or biphasic effects occur in some
species (e.g.. Painter and Greenberg, 1982; Cuthbert and
Evans. 1989; Price el al.. 1990: Duve et a!.. 1993; Lesser
and Greenberg. 1993).

In crustaceans, FMRFamide-like immunoreactivity has
been found throughout the nervous system, the highest
amounts being concentrated in the pericardia! organs
(Kobierski el al.. 1987; Marder et al.. 1987; Trimmer et
al.. 1987; Krajniak, 1991; Mercier et al.. 1991). Two of
these peptides have been isolated and sequenced from the
pericardia! organs of the lobster Homarus americanus
(Trimmer et al.. 1987) and have been identified as having
the sequences TNRNFLFRamide (Fl) and SDRN-
FLFRamide (F2). Both have since been reported in the
stomatogastric system of the rock crab Cunccr horealis
(Weimann et al.. 1993). The high concentration of these
peptides in the pericardial organs suggests that they may
be released into the circulatory system to directly affect
the heart or cardioarterial valves. Both Fl and F2 excite
isolated lobster (// americanus; Kravitz et al.. 1987) and
blue crab (Callincclcs sapidus; Krajniak, 1 99 1 ) hearts, and
increase amplitude and frequency of hearts isolated from
crayfish (Procambarus clarkii: Mercier and Russenes,
1992) and shore crab (Carcinus maenas; Wilkens and
McMahon, 1992). In the lobster and crayfish, these pep-

186

MODULATION OF CIRCULATION BY FARPS

187

tides restrict hemolymph flow by their actions on the car-
dioarterial valves (J. L. Wilkens, pers. comm.).

In addition to their action on the heart, Fl and F2 also
activate the pyloric and gastric rhythms in Cancer boreal is
(Weimann et al.. 1993), and Fl has powerful effects on
the phasic extensor muscle of the lobster (Pasztor and
Golas, 1993). There are very few reports of the effects of
neurohormones on the cardiovascular dynamics of whole-
animal preparations. We recently developed a pulsed-
Doppler technique for measuring blood flow and cardiac
output in Cancer magister in vivo (Airriess el al., 1994).
This technique is minimally invasive and allows simul-
taneous measurement of blood flow in all arteries leaving
the heart. This permits calculation of cardiac output
which, when divided by heart rate, yields stroke volume.

A number of amine and peptide neurohormones have
been shown to have distinctive effects on cardiac and cir-
culatory function in crustaceans //; vivo compared with
isolated or semi-isolated preparations (McMahon and
Reiber, 1991; Airriess and McMahon, 1992: McGaw et
al., 1994a). Thus the aim of the present study was to de-
termine the effects of the FMRFamide-related peptides
Fl and F2 on hemolymph flow and cardiac output in vivo
in the Dungeness crab C. magister.

Materials and Methods

Adult male intermolt Cancer magister weighing 600-
850 g were purchased from local fishermen and held at
Bamfield Marine Station, British Columbia, Canada, in
filtered running seawater at a temperature of 12 1C
and a salinity of 33 1% for at least 1 week prior to
experimentation. Crabs were usually fed chopped fish
twice a week but were isolated from food supplies for
2 days prior to experimentation.

A 545C-4 directional pulsed-Doppler flowmeter
(Bioengineering, University of Iowa) was used to measure
hemolymph velocity in each of the major arteries. This
technique of minimally invasive flow measurement and
probe calibration is described in detail in Airriess and
McMahon (1994) and Airriess et al. (1994). Briefly, pi-
ezoelectric crystal probes (Iowa Doppler Products, Crystal
Biotech) were implanted in grooves abraded to the dermis
of the carapace directly above each artery except the ster-
nal and hepatic arteries, which were monitored by means
of internal catheter mounted probes. Hemolymph loss
during the latter procedure was minimal. Probe perfor-
mance was first optimized manually, then fine focused
electronically to obtain maximum signal amplitude and
fixed in place using cyanoacrylate glue and dental wax.
Output from the flowmeter was recorded on a Gould 6-
channel oscillograph.

Heart rate was determined by counting the peaks on
the arterial flow traces. This method, evaluated by Airriess

and McMahon (1994), gives values analogous to those
produced by the more familiar impedance conversion
technique as long as there is measurable hemolymph flow
through at least one arterial system. Cardiac output was
calculated by summation of the mean flow through each
artery (values for paired arteries were doubled), and this
value was divided by mean heart rate to obtain a mean
value for cardiac stroke volume. Scaphognathite beat fre-
quency was recorded with a hydrostatic pressure trans-
ducer (Statham/Gould) connected via a saline-filled cath-
eter to the right branchial chamber.

During experiments, crabs were held in a covered
acrylic plastic box, dimensions 28 X 20 X 10cm, that
was supplied with a constant flow of aerated seawater.
The size of the box allowed the crabs minimum movement
and thus restricted damage to the probe implants. After
electrodes were implanted, animals were allowed to settle
for at least 24 h before experimentation. Experiments were
carried out in constant darkness at a temperature of
12 1C.

Fl was obtained from Bachem Bioscience, Inc., and
F2 was a gift from Dr. Joffre Mercier. The peptides were
dissolved in Cancer saline (Morris and McMahon, 1989)
and diluted to achieve final calculated circulating con-
centrations of 10"* 1 to 10~ 12 mol- 1~'. Test solutions were
infused directly into the lateral pericardia! sinus by means
of a chronically implanted polyethylene catheter (PE20).
A syringe pump (Sage Instruments) was used to infuse
350 n\ of the test solution followed by 1 50 j/1 of saline for
catheter washout over a 3-min period. This interval was
long enough to ensure that the hormone did not reach
the pericardia! sinus as a concentrated bolus of injectate
but was distributed slowly and homogeneously (Airriess
and McMahon. 1992). Control infusions were carried out
with Cancer saline. Each experimental animal received
the entire concentration range of either Fl or F2.

Heart rate, hemolymph flow rates, and scaphognathite
beat frequency were determined for 1 1 animals tested with
Fl and for 10 animals tested with F2. Recordings were
carried out at 10-min intervals during a 30-min control
period, during and immediately after infusion of either
saline or peptide solution, and at regular intervals after
infusion up to a total time of 120 min for each hormone
concentration.

One-way analysis of variance with repeated measures
design (Potvin et al.. 1990) was carried out on the data
to test for significant differences between pre- and post-
treatment levels; any missing values were statistically es-
timated (Zar, 1984).

Results

The heart rate, scaphognathite rate, and hemolymph
flow through each of the arterial systems leaving the heart

188

I. J. McGAW AND B. R. MrMAHON

were determined for 1 1 crabs tested with Fl and 10 crabs
tested with F2 (Figs. 1-3 and 8). The figures show mean
responses (with SE) to a control infusion of Cancer sa-
line and a circulating concentration of Fl and F2
( 1CT 7 mol I ' ) that had distinct effects.

Saline infusion of 350 n\ caused no significant change
( ANOVA P > 0.05) in any of the measured cardiovascular
parameters (Figs. 1-3 and 8, dashed line). In general, the
effects of Fl and F2 were similar, but Fl was more potent
and its mediated effects tended to be of longer duration
than those elicited by F2.

Infusion of either Fl or F2 caused a decrease in heart
rate (Fig. la and c) (F = 6.18 and 4.47, P < 0.01) for up
to 60 min after peptide treatment, often with periods of
acardia. Both the magnitude and duration of these effects
were greater with Fl . This response was observed between
circulating concentrations of 10 y and 10~ 8 mol- 1~' for
Fl, while the threshold for F2 was somewhat higher, be-
tween 10~ s and 10 7 mol 1 '. This long-term decrease
in heart rate was initially accompanied by a short-term
increase (5 min) in the stroke volume of the heart. Mean

stroke volume increased from about 0.15 ml/beat to
0.28 ml/beat with Fl (Fig. Ib) and from about 0.22 ml/
beat to 0.3 1 ml/beat upon administration of 10~ 7 mol 1 ~ '
F2 (Fig. Id). Thereafter, stroke volume decreased in both
cases and pretreatment values were not regained until after
the end of the 2-h measurement period.

Fl and F2 also elicited similar responses in patterns of
hemolymph flow through each of the five major arterial
systems. Hemolymph flow through the anterior aorta (Fig.
2a, d), the left anterolateral artery (Fig. 2b, e), the posterior
aorta (Fig. 3a, c), and the sternal artery (Fig. 3b, d) showed
a short-term increase (<5 min). These changes proved to
be statistically significant in all cases (ANOVA, P < 0.05)
except for the anterior aorta treated with F2 (/" = 1.40, P
> 0.05). Thereafter hemolymph flow in each arterial sys-
tem decreased significantly and often could not be detected
for extended periods. In most animals pretreatment flow
levels were not regained within the experimental time pe-
riod (120 min), and often took 3-4 h to recover fully. This
long-term decline in hemolymph flow was most apparent
in the anterolateral arteries, which also showed evidence

90 -
80 -
70 -
60
50 -
40
30
20 -
10 -
-

-30

30 60

Time (min)

90

120

90 -

80 -

I 70-

^ 60 -
19

. 50 -
40 -

10

~ 30 "

S 20 -

X

10 -

-

-30

30 60

Time (min)

120

0.35 -
0.30 -
0.25 -
0.20 -
0.15 -
0.10 -
0.05 -

J

-30

30 60

Time (mm)

90

120

J

30 60

Time (mm)

90

120

Figure 1. Changes in (a, c) mean heart rate (SE) and (h, d) mean stroke volume of the heart of Cancer
nuiai-'ilcr after infusion of 350 ^1 of saline (dashed line) and 350 jil of 10 7 mol 1 ' Fl (a, h) and F2 (c, d)
(solid line) at time min (arrow).

0.028
0.024

_ 0.020

c

E

( 0.012
)

0.008

0.004

6-

5-

!= 4-
1 3-

MODULATION OF CIRCULATION BY FARPS
0.4 -

0.3 -

"H
1

Jo.2-

189

o

-30

-30

0.1

30 60

Time (mini

90

120

-30

6 -I

1 3-1

I M

J r

30 60

Time (mini

90

120

-30

8 -

c i

7-

6-

c

1

5-

1

4 -

o

3 -

2-

, , , ;

1 -

1 ] Vrft~n i ^

-

-30 30 60 90 120

Time (mini

6 -

1 *

X
_o
LL

2 -

f I

30 60

Time (mini

90

120

30 60

Time (min)

90

120

o J i . . i i i i ' ' i i "-

-30 30 60

Time (min)

Figure 2. Changes in hemolymph flow (mean SE) through the anteriorly directed arteries of Cancer
magisterin response to a 350 ^1 saline injection (dashed line) or a 350 ^1 treatment of 10~ 7 mol- I" 1 Fl or
F2 (solid line) at min (arrow), (a, d) Anterior aorta, (b, e) left anterolateral arteries, and (c, f) right hepatic
arteries after Fl and F2 infusion respectively.

90

120

of a slight reduction in flow at the lower concentrations
ofFl andF2 tested (10"' : mol- P 1 to 10"'mol- r 1 ).

Hemolymph flow through the right hepatic artery (Fig.
2c, f) did not show the initial short-term increase in re-
sponse to Fl or F2. Flow through this artery decreased
after treatment with Fl , although owing to the large vari-
ance there was no significant decrease for F2 (F = 1 .49,
P > 0.05). The effect of Fl and F2 on hemolymph flow
through the hepatic arteries was of shorter duration than
in the other arterial systems, and pretreatment levels were

usually regained within 30-45 min after hormone infu-
sion.

There was large inter-individual variability in both the
duration and the magnitude of hormone-induced changes
in hemolymph flow through each arterial system (duration
varied from 20 s to 10 min). Because such changes tended
to be obscured when shown only as mean responses (Figs.
1-3), representative examples are given in Figures 4 and
5. Increases in flow through each arterial system were not
simultaneous. In the majority of animals tested there was

190

1. J. McGAW AND B. R. McMAHON

0.4 -

0.3 -

1

1 0.2-

LJ_

0.1 -

0-

-30

30 60

Time (min)

90

120

0.8 -i

0.7 -

0.6 -

I 0.5 -

. 0.4

| 0.3-
ti_

0.2 -

0.1

-30

30 60

Time (min)

90

120

16 -i

14 -

12-

I 10-

*
o

-30

30 60

Time (min)

90

120

16 -
14 -
12 -

2 -

-

-30

30 60

Time (min)

90

120

Figure 3. Changes in hemolymph flow (mean SE) through the ventrally directed arteries of Cancer
mogi.er after infusion at min (arrow) of 350 fd of saline (dashed line) or l() ' mol 1 ' Fl and F2 respectively
(solid linel. (a.c| Posterior aorta, and (b. d) sternal artery.

a tendency for the anteriorly directed arteries to he affected
first and for the ventrally and posteriorly directed systems
to respond slightly later (Fig. 4). After the initial short-
term increase in hemolymph flows there was a decrease
followed by periods of ischemia (Figs. 4 and 5), and evi-
dence suggested that this interval of both decreased flow
and ischemia was dose dependent.

Dose response curves (heart rate, /; = 11) for Fl and
F2 are presented in Figure 6. Heart rate decreased in a
simple dose-dependent manner. Fl significantly decreased
heart rate at concentrations between 10 Q mol- 1~' and
10~ 8 mol 1 "', whereas the threshold for F2 was somewhat
higher, between 10" 8 mol- r 1 and 10~ 7 mol-l"'.

Total mean cardiac output was calculated by sum-
mation of mean flows in all arteries (values for paired
arteries were doubled) and expressed as a percentage of
flow through each artery (Figs. 7a, b, and c). Saline in-
fusion did not alter the percentage of hemolymph deliv-
ered through each system (Fig. 7a). The sternal artery re-
ceived about 45% of the total cardiac output, about 25%

was channeled into each of the hepatic and anterolateral
artery systems, and less than 5%. was delivered to the
smaller-diameter posterior and anterior aortae.

Fl not only produced an overall long-term reduction
in cardiac output infusion of 350 /jl of 10 7 mol -1 ', it
also radically altered the distribution of cardiac output
(Fig. 7b). The anteriorly directed arteries were affected
first: within a minute of hormone infusion a short-term
increase in percentage output delivered to the anterolateral
arteries and a decrease to the hepatic arteries occurred,
while flow through the anterior aorta was routinely low.
After about 3 min the percentage of output delivered to
the sternal artery and posterior aorta increased for up to
10 min. The longer-term decrease in all arteries except
for the hepatic arteries (which recovered to pretreatment
flows before the rest of the systems) meant that after
15 min most of the cardiac output was delivered through
the hepatic arteries. Similar changes in the distribution of
cardiac output resulted from infusion of F2 (Fig. 7c), but
the longer duration of flow inhibition in most arteries

MODULATION OF CIRCULATION BY FARPS

191

Fl

-to

-5

Time (mini

10

15

100 -,

80 -

<o
o>

.Q
CD

cr.

60 -

40 -

20 -

-

-10

Time (min)

- 0.4

Figure 4. Changes in hemolymph How (ml/min) and cardiac function in a single specimen of Cancer
magister after administration of 350 n\ of ICT 7 mol 1~' Fl at min. (a) Anterior aorta, (b) left anterolateral
artery, (c) right hepatic artery, (d) posterior aorta, and (e) sternal artery, (f ) Represents changes in heart rate
(solid), cardiac stroke volume (dotted), and scaphognathite beat frequency (dashed)

192

I. J. McGAW AND B R. McMAHON

0.26

a

F2

i

E 11 - 34 1

^

_

X

o o -I

20.46 -i

-4

I I 1 >~

2345

-10

r~
-5

10

15

Time (mini

100 n

J r

-10

Time (min)

- 0.8

Figure 5. Changes in hemolymph flow (ml/min) and cardiac function in a single specimen of Cancer
magisler after administration of 350 iA of 10~ 7 mol 1"' F2 at min. (a) Anterior aorta, (b) left anterolateral
artery, (c) right hepatic artery, (d) posterior aorta, and (e) sternal artery, (f) Represents changes in heart rate
(solid), cardiac stroke volume (dotted), and scaphognathite beat frequency (dashed).

MODULATION OF CIRCULATION BY FARPS

193

100 -

o -
I -100-

- -200 -

o>

c
ia

.c
u

-300 -
-400 -

-500 -

-600 -

Saline ID' 12 10'" lO" 10 10' 9 10' 6

Concentration (moM~ 1 )

10"

10'

Figure 6. Dose response curves for Fl and F2. depicting cumulative
percentage decrease in heart rate (mean SE) over a 60-min period
after hormone infusion.

extended the period during which most of the flow passed
through the hepatic arteries. Note that the hepatic artery
flow is not potentiated concurrently. Diversion of a greater
percentage of flow into the hepatic arteries at this time is
a function of an overall reduction in cardiac output, cou-
pled with a much faster restoration of flow in this arterial
system.

The fastest and most dramatic effects of Fl and F2
infusion were on the scaphognathite beat frequency (F
= 1 1.91 and 4.52, P < 0.01), and they occurred at lower
concentrations (10~ 9 mol- r 1 and 10~ 8 mol- 1 ' respec-
tively) than observed for the cardiac or circulatory indi-
cators. Within 1 min of the start of infusion, beating was
disrupted, and subsequently ceased (Fig. 8a, b). As with
changes in heart rate, reductions by Fl were of greater
magnitude and longer duration than those of F2.

Discussion

Investigation of neuropeptidergic modulation of car-
diovascular dynamics in decapod crustaceans has focused
largely on the effects on isolated or semi-isolated prepa-
rations. The present study provides evidence of the actions
of the FaRPs Fl and F2 on heart and circulatory function
in whole-animal preparations.

In isolated heart preparations of Callinectes sapiclus
(Krajniak, 199 1 ), Procambarus clarkii (Mercier and Rus-
senes, 1992; J. L. Wilkens, pers. comm.), and Cardnus
maenas (Wilkens and McMahon, 1992), Fl and F2 are
positively chronotropic and the thresholds for responses
tend to be lower than those reported in the present study.
Interestingly, Fl and F2 also excite the isolated heart of
Cancer magister (McGaw, Wilkens, McMahon, and Air-
riess, in prep.), elevating frequency for up to 30 min

Sternal artery
Hepatics
Anterolaterals
Posterior aorta
Anterior aorta

2 3 4 5 10 15 20 25 30 45 GO 90 120

Time (min)

Sternal artery
\y Hepatics
Anterolaterals

Anterior aorta

45 10 15 20 25 30 45 60 90 120

Time (min)

S' Sternal artery
Hepatics
Anterolaterals
Posterior aorta
Anterior aorta
4 5 10 15 20 25 30 45 60 90 120

Time (min)

Figure 7. (a) Percentage of total cardiac output delivered through
each arterial system after infusion of 350 \A of saline, (h) infusion of
350 ^1 of 10~ ; mol- r' Fl. and (c) infusion of 350/tl of 10 7 mol- r'
F2. Where values for percentage cardiac output delivered through the
sternal artery are obscured, they are shown as horizontal lines on the
bars for the hepatic artery.

194

I. J McGAW AND B. R McMAHON

30 60

Time (min)

30 60

Time (min)

120

Figure 8. Changes in mean scaphognalhite beat frequency after administration of 350 fj\ of saline (dashed
line) or 10~ 7 mol 1~' Fl (a) and 2 (b) (solid line) at time min (arrow).

after hormone application. The threshold for response
of the isolated heart is between 10 10 mol-l ' and
1(T Q mol -I" 1 , which is higher than for intact C. magister
hearts (Fig. 6). Despite their published reputation as car-
dioexcitatory peptides, both Fl and F2 induced a rapid
bradycardia in intact C. nnigiMcr, followed by periods of
acardia (Figs, la and c, 4f, and 5f). Similar responses to
Fl occur in the lobster Homurus umerieanns (McMahon
and Reiber. 1991), although the threshold for inhibition
is some 100,000-fold lower, occurring at 10 n mol 1 '
instead of at 10~ 8 mol- 1 ' as in C. magister.

Fl and F2 also increase the amplitude of contraction
of the isolated heart of P. clarkii ( Mercier and Russenes.
1992; J. L. Wilkens, pers. comm.). However, this increase
in contractility is persistent and, in contrast to the present
study, F2 was more potent; similar increases in cardiac
stroke volume occur in isolated C. magister preparations
(McGaw et a/., in prep.). In contrast. Fl and F2 have
negative inotropic effects on isolated C. maenas hearts
(Wilkens and McMahon, 1992), whereas no clear pattern
is observed in C. sapidus (Krajniak, 199 1 ). The action of
these hormones on cardiac stroke volume of intact C.
magister was more complex. Both Fl and F2 caused an
initial increase in stroke volume that lasted between 30 s
and 10 min. This increase was followed by a longer-term
depression; in many cases pretreatment levels of mean
stroke volume were not regained within the 120-min test
period (Fig. Ib and d).

The discrepancy in cardiac function between isolated
and whole-animal preparations suggests that in intact C.
magister Fl and F2 either exert their effects on the heart
by means of inhibitory innervation from the central ner-
vous system (CNS) or cause secondary release of other
cardioregulatory hormones (Groome and Watson, 1989).
The rapid action of these peptides in disruption and sub-

sequent cessation of scaphognathite beating (Fig. 8) also
suggests inhibitory nervous input directly from the CNS.

Modulation of hemolymph flow by Fl and F2 in each
of the arterial systems was also complex, and changes were
not as uniform as those reported for the heart (Figs. 2 and
3). Duration and magnitude of responses in arterial flow
varied quite considerably, not only between animals (Figs.
4 and ?) but even within single animals. Such variability
in circulatory function may be associated with the many
(but invisible) changes in physiological state that may oc-
cur in quiescent animals held under similar conditions
(McGaw et ul.. 1994b). Hemolymph flow increased briefly
through all vessels except the paired hepatic arteries and
decreased thereafter (Figs. 2 and 3). The initial increase
in flow in all arteries except the hepatics must have been
brought about by increases in the stroke volume of the
heart, since heart rate fell (Fig. 1 ), and might also involve
differential contraction of the cardioarterial valves at the
base of each arterial tree. The subsequent decreases in
flow resulted from decreases in both heart rate and stroke
volume (Fig. 1 ) and might be associated with simultaneous
contraction of all the cardioarterial valves. F2 has not
been tested on flow in other crustaceans, but Fl causes
similar biphasic effects on hemolymph flow through ar-
teries of the lobster. A short-term increase followed by
longer-term decreases and pauses in hemolymph flow oc-
curs in the anterior aorta and the lateral and sternal ar-
teries. A simple decrease occurs in the posterior aorta at
higher concentrations (10"" mol-l" 1 and above). At
lower concentrations (10 ~ 13 mol- 1~') Fl has purely in-
hibitory effects on lobster hemodynamics (McMahon and
Reiber, 1991).

In the present study, the effects of Fl and F2 on the
anterolateral arteries were not wholly consistent. In about
75% of the animals tested, flow initially increased; in the

MODULATION OF CIRCULATION BY FARPS

195

remainder, it just decreased. Similar responses are also
reported for the lobster (McMahon and Reiher, 1991).
The anterolateral arteries are often subject to periodic cy-
cles of hemolymph flow without any apparent zeitgeber
in C, magister (McGaw el a/.. 1994b) and, therefore, re-
sponses may depend on the underlying cycle and the time
of hormone infusion. Certainly modification of the pyloric
and gastric rhythms of C. horealis by Fl or F2 depends
on the original state of each of these rhythms (Weimann
etal.. 1993). AtFl or F2 concentrations of 1(T 7 mol- 1~'
and above, hemolymph flow through the anterolateral ar-
teries of C. niagixtcr often did not regain pretreatment
capacity until 3-6 h after hormone infusion (unpubl. obs.).
The cardioarterial valves of the anterolateral vessels in P.
clarkii show a persistent increase in resistance to Fl (J.
L. Wilkens, pers. comm.). Thus it is possible that long-
term contraction of the valves in C. magister also de-
creased blood flow through these arteries.

The paired hepatic arteries, which perfuse the hepa-
topancreas (Pearson, 1908; McLaughlin. 1983), were the
only vessels that did not show an initial increase in flow.
However, these arteries recovered pretreatment levels of
flow before the other systems, and therefore most of the
cardiac output was diverted to the digestive gland (Fig.
7a and b). The FaRPs are thought to be implicated in
digestive processes in crustaceans (Mercier el ai, 1991; J.
L. Wilkens, pers. comm.). They initiate pyloric and gastric
rhythms in C. borealis (Weimann et ai. 1993) and act on
the cardioarterial valves of the lobster to increase cardiac
output to vessels supplying the gut: they also increase
hindgut motility in P. clarkii (J. L. Wilkens, pers. comm.).
This explanation does not, however, account for the long-
term decrease in perfusion of the anterolateral arteries
that supply the foregut of C magister (Pearson, 1908;
McLaughlin, 1983). A further anomaly is echoed in work
on the isolated heart of the crayfish P. clarkii. in which
the hepatic artery valves contract in response to Fl ad-
ministration and thus decrease perfusion of the hepato-
pancreas (J. L. Wilkens, pers. comm.).

The overall effect, therefore, of Fl and F2 in C. magister
is to induce a temporary increase in perfusion of the lo-
comotory structures, mouthparts. CNS, brain, and telson
via the sternal, anterolateral, anterior, and posterior ar-
terial systems (Fig. 7a. b, c). These structures are thus
perfused either periodically (Figs. 4 and 5) or at a lower
rate, and the largest part of a reduced cardiac output is
delivered to the hepatopancreas via the hepatic arteries
(Fig. 7b, c). The functional significance of this is unclear:
however, the overall pattern is not dissimilar to that char-
acteristic of burrowing, when hemolymph is diverted to
the locomotory structures and mouthparts. Once sub-
merged in the mud, crabs become quiescent and perfuse
the system only periodically (McGaw and McMahon, un-
publ. obs. ). While an animal is burrowed, channeling car-

diac output to the hepatopancreas may aid digestion,
which implicates FaRPs in a postprandial role in crus-
taceans.

In the present study, Fl was about 10 times more potent
than F2 (Fig. 6), although the activity of these two peptides
differs amongst the decapod crustaceans and appears to
depend upon the species under investigation (Krajniak,
1991; Mercier and Russenes, 1992; Wilkens and Mc-
Mahon, 1992; J. L. Wilkens, pers. comm.).

The function of the FaRPs is still not fully understood
in other invertebrate phyla their action on the cardiovas-
cular system is variable, and excitatory, inhibitory, or bi-
phasic responses vary with the concentration of the hor-
mone or the species of organism (e.g.. Painter and Green-
berg, 1982: Cuthbert and Evans, 1989; Price et ai. 1990;
Duve et ai, 1993; Lesser and Greenberg, 1993). Future
work will focus on the sites of action of these peptides to
ascertain their role in cardioregulation, and measurement
of circulating levels will determine whether they are re-
leased from the pericardia! organs in amounts sufficient
to effect the reported changes.

Acknowledgments

This work was supported by NSERC grant #A5762 to
BRM and a grant from WCUMBS (Bamfield Marine Sta-
tion) to IJM. We are grateful to Dr. Joffre Mercier for
donation of F2 and to Dr. Kevin Krajniak for helpful
discussion.

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The Functional Morphology of Starfish Tube Feet:
The Role of a Crossed-Fiber Helical Array in Movement

R. SKYLER McCURLEY AND WILLIAM M. KIER 1

Department of Biology, CB#3280 Coker Hall, University of North Carolina,
Chapel Hill. North Carolina 27599-3280

Abstract. The morphology and mechanics of the tube
feet, ampullae, and lateral and radial canals of the water
vascular systems of Luidia clathrata and Astropecten ar-
ticulatm (Echinodermata, Asteroidea) were analyzed.
Histological methods, based on embedding in both par-
affin and glycol methacrylate, were used to document the
arrangement of muscle and connective tissue. The tube
foot wall includes longitudinal muscles and connective
tissue fibers, the latter arranged in a crossed-fiber helical
array, with a fiber angle of about 67 in elongated tube
feet. No evidence was found for the circular rings of con-
nective tissue reported in earlier studies; the appearance
of rings is probably an artifact of folding. The ampullae
are bilobed and include circumferentially arranged muscle
fibers and connective tissue fibers aligned 90 to the mus-
cle. The lateral canals are short and equipped with one-
way flap valves similar to those described for other echi-
noderms. The radial canal is thin-walled, nonmuscular,
and enclosed in the connective tissue and ossicles of the
ambulacrum. Frame-by-frame video analysis of both in-
tact animals and animals with "windows" cut in the arm
wall was used to document the movements of the tube
feet and ampullae. No evidence was found for the pre-
viously suggested role of the radial canal in protracting
the tube feet. The ampullae protract the tube feet and
antagonize the tube foot musculature. The fiber angle of
the connective tissue allows protraction and prevents di-
lation of the tube feet, and limits elongation of the am-
pullae.

Introduction

The water vascular system of asteroids serves crucial
roles in locomotion, food handling, respiration and, in

Received 13 May 1994; accepted 10 January 1995.
1 To whom correspondence should be sent.

many species, burrowing. The major components of the
system are the circumoral ring canal, the radial canals
extending from the ring canal down each arm. and the
tube feet with their associated ampullae that are connected
to the radial canal by the lateral canals. Studies of the
tube feet and associated ampullae in asteroids have in-
cluded analyses of general morphology and function
(Mangold, 1908; Hamilton, 1921; Paine, 1926, 1929;
Smith, 1937, 1946, 1947; Kerkut, 1953; Heddle, 1967;
Nichols. 1966, 1969, 1972), of innervation and neuro-
muscular control (Smith. 1945, 1950a, b: Bargmann et
al. 1962; Cobb, 1967, 1970, 1987; Cobb and Laverack,
1967; Cavey and Wood, 1981), of ultrastructure (Souza
Santos and Silva Sasso, 1968, 1970; Dolder, 1972;Engster
and Brown, 1972; Wood and Cavey, 1981), and of per-
meability and maintenance of fluid volume (Binyon,
1962, 1964; Prusch and Whoriskey, 1976; Prusch, 1977:
Ferguson, 1990a, b). Although the tube foot-ampulla
complex of asteroids has been well studied, two crucial
aspects of its function have not been completely resolved.
The first concerns connective tissue fiber reinforcement
of the walls of the complex, and the second concerns the
role of the radial canal in tube foot movements.

The production of force and movement in these ap-
pendages depends on a hydraulic mechanism in which
contraction of muscle displaces water vascular fluid from
one portion of the system to another. In most hydrostatic
skeletal support systems that rely on such a mechanism,
the walls of the hydraulic appendage or body are rein-
forced with connective tissue fibers arranged in a specific
pattern, referred to as a 'crossed-fiber helical array' (for
reviews, see Chapman, 1958; Clark, 1967;Trueman, 1975;
Wainwright et al., 1976; Alexander, 1983). In such a sys-
tem, sheets of connective tissue fibers wrap the structure
in regular arrays of right- and left-hand helices. The re-
inforcement provided by the fibers allows shape change

197

198

R. S. McCURLEY AND W M. K1ER

and bending and prevents torsion. Although crossed-fiber
helical arrays of connective tissue fibers in the walls of
the tube feet of ophiuroids have been described and an-
alyzed (Woodley, 1967, 1980), previous studies of the tube
feet of asteroids do not describe crossed-fiber helical re-
inforcement of the tube foot walls. Instead, the connective
tissue reinforcement has been reported as consisting of
circular rings (Smith, 1946. 1947). The present study was
therefore undertaken to reexamine the connective tissue
and muscle in the tube feet of two asteroid species, Luidea
clathrata and Astwpecten articulatus. Our analysis has
revealed crossed-fiber helical connective tissue reinforce-
ment of the tube foot wall, as in other hydraulic systems.
Many of the previous studies cited above have empha-
sized the interdependence of the tube foot and ampulla
in movement; i.e., extension of the tube foot results from
contraction of the ampulla, and distension of the ampulla
results from contraction of the tube foot. However, recent
reviews (Nichols, 1969, 1972) of tube foot functional
morphology describe the radial canal in asteroids (species
not specified) as being directly involved in extending the
tube feet and being capable of accommodating water vas-
cular fluid from contracted tube feet. Indeed, Lawrence
(1987, citing Nichols, 1972) describes the radial canal as
being responsible for most of the elongation of the tube
feet in asteroids. But in the species analyzed in the present
study, the radial canal is unlikely to serve in these roles.

Materials and Methods

Experimental animals

Specimens of the grey sea star, Luidia clathrata, and
the margined sea star, Astropecten articulatus. were sup-
plied by Gulf Specimen Supply. Inc.. Panacea, Florida.
They were maintained in a recirculating artificial seawater
system in the Department of Biology, University of North
Carolina, Chapel Hill.

Histology

Segments of the arms of L. clathrata and A. articulatus
were removed from specimens that had been anesthetized
with a 1:1 mixture of 7.5% MgCl : -6H 2 O and seawater
(Messenger et a!.. 1985). The tissue was fixed in 10% for-
malin in seawater for 24 h. The tissue was then decalcified
in a solution of 0.7 g/1 ethylenediaminetetraacetic acid,
tetrasodium; 8 mg/1 sodium potassium tartrate; 99.2 ml/1
hydrochloric acid; 0.14 g/1 sodium tartrate (S/P Decalci-
fying Solution, Baxter Scientific Products, McGaw Park,
IL) and then washed in water for 2 h. The fixed and de-
calcified arm tissue was cut into segments that included
three to four pairs of tube feet, and these tissue blocks
were embedded in paraffin (L. clathrata and A. articulatus)
or glycol methacrylate plastic (/.. clathrata).

For paraffin embedding, the fixed, decalcified tissues
were dehydrated in ethanol, cleared in Histo-Clear (Na-
tional Diagnostics. Manville, NJ), and embedded in Par-
aplast Plus (MP 56C) (Monoject Scientific, St. Louis,
MO). The blocks were then serially sectioned on a rotary
microtome at 10 nm in three mutually perpendicular
planes. The sections were stained with picro-ponceau with
Weigert iron hematoxylin (see Kier, 1992). For glycol
methacrylate embedding, the fixed and decalcified tissues
were partially dehydrated in an ethanol series to 95%
ethanol and then infiltrated with unpolymerized glycol
methacrylate plastic (Reichert-Jung Historesin, Leica In-
struments GmbH, Heidelberg, Germany). Following po-
lymerization, the blocks were sectioned at 0.5-3.0 ^m
with glass knives. The sections were stained with Lee's
methylene blue-basic fuchsin stain (Bennett et a/.. 1976)
and were examined with brightfield. phase contrast, and
polarized light microscopy.

In addition to the sectioned material, whole mounts
were also examined with brightfield, phase contrast, and
polarized light microscopy. Tube feet and ampullae were
dissected from fixed and decalcified arm tissue and were
partially macerated in 1.0 M potassium hydroxide and
50%. glycerine for 2-3 days (Woodley, 1967). The epider-
mis was removed from the tube feet, and whole mounts
of the tube feet and ampullae were prepared.

Computer-assisted three-dimensional reconstruction

The morphology of the valve located between the tube
foot-ampulla complex and the radial canal was examined
with the aid of a computer program for three-dimensional
reconstruction (PC3D, Jandel Scientific, Corte Madera.
CA). Parasagittal sections (defined here as vertical planes
parallel to the long axis of the arm) and frontal sections
(defined here as horizontal planes) were used for the re-
constructions of L. clathrata tissues. A microscope
equipped with a camera lucida was used to trace, from
sections, the outline of the internal and external surface
of the tube foot, the profile of the valve tissue, and the
position of the valve muscle fibers. The tracings were
aligned according to the visual best-fit method (Gaunt
and Gaunt, 1978; Young et al.. 1985) and digitized with
a Numonics 2210 digitizing tablet. The PC3D software
stacked the tracings of the internal structures to produce
a three-dimensional image that could be viewed in any
orientation with a Gateway 2000 4DX2-66V microcom-
puter. The reconstructions were plotted with a Hewlett-
Packard HP 7475A plotter.

1 'ideo recordings

Locomotion and feeding movements of L. clathrata in
a glass-bottom aquarium were videotaped from the side
and from below with a Panasonic AG-450 S-VHS camera-

TUBE FOOT FUNCTIONAL MORPHOLOGY

199

recorder. In addition, movements of the tube feet during
burrowing were recorded by placing the animals in the
aquarium, but with a thin layer of sand on the bottom,
and filming from below. The movements were analyzed
frame by frame with a Panasonic AG- 1 960 videocassette
recorder.

Direct observations of ampullae

Under anesthesia, the distal portion of an arm and a
portion of the dorsal body wall of L. c/at/irala were re-
moved. After such an operation, the animals appear to
behave normally. Movements of the ampullae and their
associated tube feet were observed directly, both during
normal movement of the animal and in response to man-
ual mechanical stimulation of individual tube feet with a
dissecting probe.

Results

Morphology of the tube foot-ampulla complex

This study focuses on the components of the water vas-
cular system associated with the radial canal: the tube
feet, ampullae, and lateral canals (see Fig. 1 ). The following
morphological description is based on L. clathrata. Any
observed differences between Luidia and Astropecten are
noted below.

Tube feet. The tube feet project from the ambulacral
groove on the ventral surface of the arm; they are cylin-
drical and have conical ends. Each arm has more than
100 tube feet arranged in pairs along the length of the
arm, thus constituting two parallel rows, one on either
side of the ambulacral groove. Most of the tube foot is
external to the body wall (Fig. 2).

The tube foot epithelium is covered by a thin cuticle
continuous with that covering adjacent areas of the am-
bulacrum (See Engsterand Brown, 1972, for details). The
appearance of the epithelium in sectioned histological
material depends on the state of elongation or contraction
of the tube foot. In retracted tube feet, the epithelium is
thick and the epithelial surface is highly folded into an-
nular rings (Fig. 3). In protracted tube feet, the epithelium
appears thinner and the folding is reduced. The epithelium
of the distal conical end appears to be secretory, it consists
of tall columnar epithelial cells with intensely staining
cell inclusions (Fig. 3).

Underlying the epithelium is a layer of nervous tissue,
similar in disposition to the basiepithelial (ectoneural)
plexus described previously in other echinoderms (see
Smith, 1937; Coleman, 1969; Raymond, 1979). Under-
lying the nervous tissue layer is a dense layer of fibrous
connective tissue. Fibers in this layer show staining re-
actions typical of collagen and are highly birefringent when
viewed with polarized light microscopy. Grazing sections

CT

Figure 1. Schematic cutaway drawing of the arrangement of the tube
feet (T). ampullae (A), and the lateral (LC) and radial canals (R). showing
the trajectories of the connective tissue fibers (CT), longitudinal muscle
(L) of the tube feet, and circular muscle (C) of the ampullae.

through the connective tissue layer show that it is com-
posed of connective tissue fibers arranged as a crossed-
fiber helical array (Fig. 4). Such an array consists of con-
nective tissue fibers that wrap the tube foot in both left-
and right-hand helices. The fiber angle, defined as the
angle that the connective tissue fibers make with the long
axis of the tube foot, was measured in whole mounts and
in grazing sections of protracted tube feet; the angle is
about 67 and is relatively constant along the length of
the tube foot. In retracted tube feet, the crossed-fiber he-
lical connective tissue layer becomes somewhat folded,
and grazing sections may then sometimes give the mis-
leading impression that this layer consists of circumfer-
ential rings of connective tissue. Because the fibers are
highly birefringent, their arrangement as a crossed-fiber
helical array is more easily observed with polarized light
microscopy (Fig. 4). The whole-mount preparations were
also useful in visualizing the disposition of the connective
tissue fibers.

Internal to the crossed-fiber helical connective tissue
layer is a robust layer of muscle fibers (see Dolder, 1972.
for details) (Figs. 2, 3, 4). The fibers are arranged longi-
tudinally, parallel to the long axis of the tube foot. No
striations were observed in the muscle cells. The internal
lumen of the tube foot is lined with epithelium (see Wood
and Cavey, 1981, for details).

Figure 2. Photomicrographs of a transverse section of an arm from Luidia cluthrata in the region of a
pair of tube feet (T) and their associated hilobed ampullae (A). A 10-fim-thick paraffin section stained with
picro-ponceau and iron hematoxylin was photographed with brightfield illumination, (a) Low power view
showing relative proportions of the arm and tube foot-ampulla complex. The section includes a portion of
the pylonc cecum (P) on each side of the arm. Scale bar, I mm. (b) Higher power view showing the radial
canal (R) located dorsal to the radial nerve cord (N). The epithelium on the roof of the radial canal has

200

TUBE FOOT FUNCTIONAL MORPHOLOGY

201

Ampullae. Located within the coelomic cavity of the
arm are the bulbous ampullae. In both L. clathralu and
A articulalus the ampullae are bilobed, one lobe extending
laterally from the union with the tube foot and the other
extending medially (Figs. 2, 5). Each lobe is elongate, cy-
lindrical, and curved toward the oral surface. In /.. cknli-
rala. the lateral lobe is longer than the medial lobe,
whereas in A. articuluius the medial lobe is the longer.
The long axes of the two lobes are roughly parallel and
in the same plane, and so form what is essentially a single
cylindrical tube more or less perpendicular to the long
axes of both the tube foot and the arm. The connection
between the tube feet and the ampullae is made by a
slightly narrowed neck that is displaced laterally relative
to the axis of the tube foot (Figs. 2, 5). Two bands of
tissue, which Smith ( 1950b) named 'seams' (see also Cobb.
1967; Cobb and Laverack, 1967), run dorsoventrally in
the ampullar neck: one seam occupies a medial position
on the portion of the neck closest to the central axis of
the arm, and the other is located laterally on the opposite
side of the ampullar neck (Fig. 5). The tube foot and am-
pullar wall are continuous through the neck region.

A layer of epithelium covers the outer surface of the
ampulla (i.e., the surface exposed to the coelomic cavity
of the arm). Beneath the epithelium is the ectoneural ner-
vous tissue layer. Beneath the nervous tissue layer is a
thin layer of dense, fibrous, birefringent connective tissue
that shows staining reactions typical of collagen. The con-
nective tissue fibers of this layer are closely packed and
aligned in parallel. Grazing sections show the fibers to be
oriented parallel to the long axis of the ampulla (Fig. 6).
Underneath the dense connective tissue sheet is a robust
layer of muscle fibers. The muscle fibers are arranged in
circumferential bands around the lumen of the ampullae
in planes perpendicular to the long axis of the ampulla.
Thus, the muscle cells are arranged at right angles to the
connective tissue fibers of the dense connective tissue
layer. No striations were observed in the muscle cells. The
lumen of the ampulla is lined with a simple epithelium.

Lateral canal and valve. Short lateral canals extend from
each side of the radial canal to connect to each tube foot
along the length of the arm (Fig. 7). The internal lumen
of the lateral canals is lined with a simple squamous epi-
thelium that appears to be continuous with that of the
tube foot and the radial canal. The canals are wrapped
by fibrous birefringent connective tissue continuous with
the connective tissue that surrounds the arm ossicles and
forms the structure of the arm.

Figure 3. Photomicrograph of a longitudinal section of an individual
tube foot (T) from I.iiului flullmila. The epithelium (EP) of the tip in-
cludes tall columnar cells. Folding of the epithelium on the sides of the
foot is visible. Underlying the epithelium is a layer of nervous tissue (N).
and underneath this is the crossed-fiber helical connective tissue layer
(CT). Longitudinal muscle ( L) is visible under the connective tissue layer
and is separated from the tube foot lumen (T) by a thin epithelium.
Scale bar. 100 yum. A 10-jum-thick paraffin section stained with picro-
ponceau and iron heivuiUmlin was photographed under brightfield il-
lumination.

The lateral canal joins the tube foot wall at the top of
the tube foot. A pair of flaps are present on either side of
the opening of the lateral canal into the tube foot, forming
a valve (Figs. 7, 8). The flaps are parallel to one another

pulled away from the ambulacral connective tissue; glycol methacrylate sections (see Fig. 9) show the epithelium
to be attached to the ambulacral connective tissue. The longitudinal muscle (L) of the tube foot (T) and the
circumferential muscle (C) of the ampulla (A) are also visible. A portion of a pyloric cecum (P) is visible.
Scale bar. 0.5 mm.

202

R S McCURLEY AND W M. KIER

Figure 4. Photomicrograph, taken under polan/cd light, of a partialK
macerated portion of a tube loot wall from l.iiii/ui cUilhrulii. The long
axis of the tube foot is oriented vertically. T he crossed lines indicate the
approximate trajectories of the crossed-liher helical connective tissue
array. The longitudinal muscles of the luhe foot wall are oriented vertically
in the photomicrograph and can he seen behind the crossed fibers. Scale
bar, 25 pm.

and extend down from the roof of the tube foot and lat-
erally from the medial wall of the tube foot on either side
of the opening of the lateral canal into the tube foot. Mus-
cle fibers originate on the tube foot roof and wall and
insert on the side of the flap opposite to that facing the
opening of the lateral canal (Figs. 7, 8). The trajectory of
these muscle fibers is such that their contraction pulls the
two flaps away from one another, opening the connection
between the lateral canal and the tube foot. The flaps
consist of a thin sheet of connective tissue covered by a
simple squamous epithelium that appears to be contin-
uous with the epithelium of the tube foot and lateral canal.
On either side of the valve, the roof of the tube foot is
slightly domed.

Radial canal. The radial canal is adjacent to the ventral
surface of the arm and extends from the ring canal to the
tips of the arms. It is encased in the connective tissue and
calcite ossicles of the ambulacrum and is dorsoventrally
flattened in cross section (Fig. 9). The radial canal is lined
with a simple squamous epithelium, lacks musculature,
and is surrounded by connective tissue. The connective
tissue is continuous with that surrounding the calcite os-
sicles of the ambulacrum. No valves or sphincter muscles
were observed along its length. The floor of the radial
canal is raised as a transverse ridge midway between each
pair of tube feet along the length of the arm. The ridges
are formed by a bundle of connective tissue and muscle
fibers, called 'transverse ambulacra! muscles' (Hyman,
1955), that connect ambulacra! ossicles on opposite sides
of the arm.

'I'uhc /C('/ and ampullae kinematics

Two general categories of movement were observed in
the tube feet: length change and bending. As for length
change, the tube feet of a large L. clathrata (arm length
= 9 cm) are about 1 2 mm long when fully protracted and
2 mm when retracted. During such a retraction, the di-
ameter of the tube foot, measured midway between the
base and tip, increases from 0.9 to 1 .3 mm. The tube foot
wall is opaque, white, and thrown into a series of closely
spaced annular folds when retracted. When protracted,
the folding is reduced, and the tube foot wall becomes
translucent.

The tube feet may bend either in combination with
change in length or at constant length, and in virtually
any direction relative to the axis of the arm. Both localized
and general bending movements were observed. Localized
bending typically occurs at the base, while the remainder
of the tube foot remains essentially straight and thus pivots
about the base. General bending occurs along the entire
length of a tube foot, sometimes causing the tip of the
tube foot to be oriented at 90 to the base.

During locomotion, the tube feet undergo repeated,
stereotyped stepping cycles. An individual tube foot first
elongates and bends at the base so that the tip of the foot
points in the direction of motion. Once the tube foot is
fully protracted, it bends downward until the tip contacts
and attaches to the substratum. The tube foot then bends
at its base and moves the animal over the point of at-
tachment. In the final phase of the cycle, the tube foot
releases its attachment and retracts. The cycle is then re-
peated. The tube feet do not move synchronously but are
coordinated so that those on different arms create move-
ment in a single direction. L. claihrata may reach speeds
of 2 cm/s when moving across the surface of a crushed
oyster shell substratum.

Tube foot movements during burrowing involve bend-
ing away from the ambulacrum toward the sides of the

TUBE FOOT FUNCTIONAL MORPHOLOGY

203

Figure 5. Photomicrograph of a transverse section of an arm from Litului clalhraui showing the bilobed
ampulla. A thin connective tissue layer (CT) surrounds the circumferential muscles (C) of the ampulla (A).
The ampullar seam (S) is visible. Also included are the radial canal (R). radial nerve (N). and a portion of
a pyloric cecum (P). Scale bar, 0.25 mm. A 10-^m-thick paraffin section stained with picro-ponceau and
iron hematoxylin was photographed under brightlield illumination.

Figure 6. Photomicrograph, taken under polarized light, of a grazing section of an ampulla from I.nu/ui
cUilltruiii The long axis of the ampulla is oriented horizontally. The tenuous fibers in the connective tissue
layer (CT) are also horizontally disposed, i.e.. parallel to the long axis of the ampulla. The robust, vertically
oriented circumferential muscle fibers (C) lie underneath the connective tissue layer. The epithelium (EP)
and ectoneural nervous tissue layer (N) are also visible. Scale bar. 25 nm. A 10-/jm-thick paraffin section
stained with picro-ponceau and iron hematoxylin was photographed under polarized light.

204

R. S. McCl'RLEV AND W. M K.1ER

.! B

Figure 7. (Top) Photomicrograph of a parasagittal section (vertical section parallel to long axis of the
arm) of the arm of l.uu/iu clailuala showing the valve at the entrance to the lateral canal. The valve flaps
(V) project down from the roof of the tube foot into the tube foot lumen (T). Muscle fibers (M) originate
on the roof of the tube foot and insert on the valve flaps. Scale bar. 100 /um. A 10-^m-thick paraffin section
stained with picro-ponceau and iron hematoxylin was photographed under brightfield illumination. (Bottom)
Photomicrograph of a frontal section of the arm of LniJia cttithrulu showing details of the valve structure.
The lateral canal (LC) extends from the radial canal (R) which is oriented horizontally in the figure. The
valve flaps (V) project into the tube foot lumen (T) on either side of the entrance of the lateral canal. Scale
bar, 100 ^m. A 10-^m-thick paraffin section stained with picro-ponceau and iron hematoxylin was photo-
graphed under brightfield illumination.

arm. As in locomotion, the burrowing movements of the
tube feet are stereotyped and cyclical. An individual tube
foot is first protracted downward from the ambulacrum
into the sediment. Next, the tube foot bends laterally,
sweeping sediment from under the arm. The bending
during this phase occurs both at the base of the tube foot

and along its entire length. The tube foot then retracts
and bends back toward the ambulacrum to repeat the
cycle.

The tube feet are also involved in conveying food down
the length of the ambulacral groove to the mouth. As a
particle of food approaches a given tube foot, it bends

TUBE FOOT FUNCTIONAL MORPHOLOGY

205

8a

Figure 8. Computer-assisted three-dimensional reconstructions
showing the morphology of the valve and valve muscles at the entrance
to the lateral canal. Scale bar, 100 pm. (a) Reconstruction showing valve
flaps (V) projecting down into the tube foot lumen from the tube foot
roof. The (laps are longer adjacent to the wall of the tube foot (not
shown, right side of figure) and become shorter as they extend away from
the wall along the roof. The opening to the lateral canal is located between
the flaps and is thus hidden from view, (b) Reconstruction showing lo-
cations of valve muscles (M). Muscles originate on the tube foot roof
and insert on the sides of the valve flaps. Additional muscles originate
on the tube foot wall and insert near the free edges of the valve flaps.

toward the particle and protracts until the tip adheres to
the food. Once attached, the tube foot retracts, pulling
the food toward the mouth. At the same time, neighboring
tube feet bend toward the food, adhere, and contract. The
particle may move beyond the location of a given tube
foot while it is still attached, and thus the tube foot elon-
gates further. Whether this elongation involves active
protraction by the individual tube foot or passive elon-
gation due to the contraction of neighboring tube feet
attached to the same particle remains unclear. The final
steps in the cycle are release of the attachment of the tube
foot to the particle, and retraction.

The experiments in which the distal portion of the arm
and part of the dorsal body wall of L. clathrata were re-
moved show that contraction of an ampulla occurs during
protraction of its associated tube foot and vice versa.
Elongation of the tube foot appears to occur only when

its associated ampulla decreases in volume, and expansion
of an ampulla occurs only when its associated tube foot
contracts. There was no evidence of elongation of the tube
feet without contraction of their associated ampullae.

Discussion

Basic tube foot mechanics

The functioning of the tube foot-ampulla complex of
L. clathrata and A. articitlatus relies on a hydraulic mech-
anism similar to that proposed originally by Smith ( 1 945,
1946, 1950a). As described by Kier (1988), in hydraulic
systems, force transmission for movement and muscular
antagonism results from localized muscle contraction that
displaces fluid from one portion of the system to another.
The functioning of this system relies on the incompres-
sibility of the hydraulic fluid in this case the water vas-
cular fluid. Because the fluid is an aqueous liquid and no
gas-filled spaces are present, a decrease in volume in one
portion of the system results in an increase in volume in
another rather than in compression of the fluid.

Tube foot elongation. Elongation of a tube foot is caused
by a decrease in volume of its associated ampulla. The
musculature of the bilobed ampulla is arranged circum-
ferentially in the ampulla wall. Upon contraction, the
muscle decreases the volume of the lumen, displacing wa-
ter vascular fluid from the lumen into the tube foot. The
connective tissue of the ampullar wall plays a crucial role
in controlling the shape change of the ampulla (see below).
Contraction of the ampullar musculature creates a pres-
sure difference between the lumen of the tube foot and
the lumen of the lateral canal. Because the flaps of the
valve at the entrance of the lateral canal protrude into the
tube foot, the difference in pressure closes the valve, pre-
venting water vascular fluid in the tube foot from backing
into the lateral and radial canals. The tube foot is therefore
inflated by the fluid displaced from the ampulla, and this
increase in volume causes elongation of the tube foot. As
in the ampulla, the connective tissue fibers of the tube
foot wall play a crucial role in controlling shape change
during tube foot elongation (see below). When the tube
foot elongates, its longitudinal muscle is extended.

Tube foot shortening. This is caused by contraction of
the longitudinal muscle of the tube foot wall. When the
tube foot shortens, its volume decreases, and water vas-
cular fluid moves into the ampulla, which expands. Again,
the valve at the entrance to the lateral canal closes, pre-
venting fluid from leaking into the water vascular canals,
as described above. The increase in fluid volume that ex-
pands the ampulla also reextends the circumferential
muscle of the ampulla. Thus the circumferential muscle
of the ampulla and the longitudinal muscle of the tube
foot function as antagonists during length change in the
tube foot.

206

R. S. McCURLEY AND W. M. K1ER

\

Figure 9. Photomicrograph of a transverse section of the radial canal (R) of Luiclia clathrata. The
connective tissue (CT) of the ambulacrum surrounds the radial canal. No muscle fibers encircle the radial
canal. The section is slightly oblique and includes a portion of the valve (V) on the left side of the figure.
The radial nerve (N) is located in the center, between the two tube feet (T). Scale bar, 100 ^m. A \-nm-
thick glycol methacrylate section stained with Lee's methylene blue-basic fuchsin and photographed under
bnghtlield illumination.

Tube foot bending. Bending of the tube foot involves
the same musculature as that used for elongation and
shortening. During bending, however, the longitudinal
muscles of the tube feet and the circumferential muscles
of the ampullae operate synergistically rather than
antagonistically. Bending of a hydrostatic structure re-
quires unilateral longitudinal muscle contraction on the
inside radius of the bend. The force of this contraction
not only bends the structure, but also tends to decrease
its length. For bending without shortening, the longitu-
dinal compressional force must be opposed by a resistance
to expansion of the ampulla, which prevents displacement
of the fluid from the tube foot. Expansion of the ampulla
is resisted by contractile activity of the circumferential
muscles. Thus, bending requires simultaneous unilateral
longitudinal muscle contraction of the tube foot and cir-
cumferential muscle contraction of the ampulla. Gener-
ation of a localized rather than a more generalized bend
is dependent on the degree to which the pattern of activity
of the longitudinal musculature of the tube foot is local-
ized. No morphological subdivision of the longitudinal
musculature at the base of the tube foot was observed in
the species examined in this study.

The role of the radial canal. There is no evidence in
the species studied here that the radial canal plays a role
in protraction, as has been suggested previously (e.g.. Ni-
chols. 1969, 1972). Nichols (1972) described the radial

canal as being highly muscular in modern starfish (species
not identified) and capable of receiving fluid from re-
tracted tube feet, and then contracting to distribute the
fluid. In the species analyzed in the present study, the
radial canal completely lacks musculature. There is no
evidence for sphincter muscles or other structures that
might allow the radial canal to be partitioned along its
length. Further, it is wrapped by the connective tissue and
calcite ossicles of the ambulacrum and is thus prevented
from expanding. Our results are in agreement with those
of Smith ( 1946) who suggested, on the basis of both direct
observation of the tube feet and ampullae and calculations
of volume accommodated by the ampullae during tube
foot retraction (Astropeeten irregularis. Axterias rubens),
that "little, if any fluid enters or leaves the tube foot am-
pulla system during these movements" (p. 280). The spe-
cies examined by Nichols (1972) were not identified, so
we can say only that participation by the radial canal in
tube foot elongation is not a universal feature of the water
vascular system of asteroids.

The role of the connective tissue. The connective tissue
fibers observed in both the tube foot and the ampullar
wall play a crucial role in the mechanics of the tube foot
and the ampulla. Consider first the requirement that an
increase in pressure (due to contraction of the ampulla)
causes elongation of a tube foot. The stress distribution
in a pressurized cylinder is such that the hoop stress (which

TUBE FOOT FUNCTIONAL MORPHOLOGY

207

tends to increase diameter) is twice as large as the longi-
tudinal stress (which tends to increase length) (see Wain-
wright ct nl.. 1976; Wainwright, 1982; for details). Thus,
in a pressurized cylinder lacking fiber reinforcement, the
result of increasing pressure is swelling of the diameter of
the cylinder, rather than elongation. The crossed-fiber he-
lical array of connective tissue fibers described here rein-
forces the wall so that an increase in pressure causes an
increase in length, rather than diameter. Previous reports
(Smith, 1946, 1947) of circular "rings" of connective tissue
fibers in the tube foot wall (Astropecten irregularis, As-
terias rubens) are probably the result of misinterpretations
of folding of the connective tissue in the wall of retracted
tube feet. No circular rings of connective tissue were seen
in any of the material from the species examined in this
study.

To understand how fiber reinforcement can control
shape change, consider a simple geometrical model de-
rived from those described by Chapman (1958), Clark
and Cowey (1958), Cowey (1952), and Woodley (1980).
The tube foot can be modeled as a right circular cylinder
wrapped by a single turn of an inextensible helical fiber.
Because the tube foot is essentially cylindrical and the
connective tissue fibers are likely to be collagenous and
thus quite stiff in tension, the assumptions of this simple
model are reasonable. For a helical fiber of given length,
the volume of such a cylinder is a function of the length
of the cylinder and the fiber angle (the angle that the fiber
makes with the long axis of the cylinder) (Fig. 10). The
fiber angle of the helical fiber ranges from 90 (when the
cylinder length is at a minimum) to (when the cylinder
length is at a maximum). The maximum volume occurs
at an intermediate length, when the fiber angle is 5444'.
If the fiber angle of the cylinder is greater than 5444', an
increase in its volume causes an elongation and a decrease
in the fiber angle (see Fig. 10). If the fiber angle is initially
less than 5444', an increase in volume causes the cylinder
to shorten. At an angle of 5444', the stiffness in the hoop
direction is double the stiffness in the longitudinal direc-
tion, and the fibers therefore resist both an increase in
length and an increase in diameter. The fiber angle of the
crossed-fiber helical array thus determines the shape
change that results from an increase in volume of the tube
foot.

This model predicts that the fiber angle of the connec-
tive tissue fibers in the wall of the tube foot must be greater
than 5444' if the increase in volume due to contraction
of the ampulla is to cause elongation. Indeed, a fiber angle
of about 67 was measured in the connective tissue fibers
of elongated tube feet of L. clathmta. Although the fiber
angle in retracted tube feet could not be measured or cal-
culated due to the folding of the wall, shortening of the
tube foot from the elongated state must increase the fiber
angle to greater than 67.

10

bJ
13

60

CYLINDER LENGTH

HIGH 6

LOW 9

Figure 10. Plot of the length versus volume of a cylinder wrapped
by a single inextensihle helical fiber. The fiber angle 6, defined as the
angle that the helical fiber makes with the long axis of the cylinder, is
diagrammed above the plot and is included parametrically on the plot.
The volume is maximal when the fiber angle is 5444'. Note that in-
creasing the volume of such a cylinder causes an increase in length only
when the initial fiber angle is greater than 5444' (high in diagram
below the plot). If the initial fiber angle is less than 5444', increasing
the volume decreases the cylinder length (low 6 in diagram below the
plot).

The connective tissue fiber reinforcement of the am-
pullar wall is also functionally important. Contraction of
the circumferential musculature of an ampulla increases
the pressure of the fluid in the ampulla, generating a stress
that tends to elongate the ampulla. Lacking longitudinal
muscle fibers (i.e., fibers aligned parallel to the long axis
of the ampulla), some other component of the structure
must resist this longitudinal stress. The connective tissue
fibers of the ampulla are ideally oriented i.e., parallel to
the long axis and they therefore will resist the longitu-

208

R. S. McCURLEY AND W. M. K.IER

dinal stress directly. This is a novel example of fiber re-
inforcement of hydrostatic skeletons; in all other cases,
the fibers are in a crossed-fiber helical array (Wainwright
et a/.. 1976; Wainwright, 1982), which allows length
change, smooth bending without kinking, and resistance
to torsion about the long axis. These are important char-
acteristics of tube feet, but not ampullae, where the most
important role of the connective tissue is to resist elon-
gation.

The role of the valve. The valves located at the opening
of the lateral canals into the tube feet also have an im-
portant function in the water vascular system of asteroids.
As described above, the valve flaps are arranged so that
an increase in pressure in the tube foot-ampulla complex
tends to close the valve, preventing loss of fluid from the
tube foot (Markel and Roser, 1992). Since forceful move-
ment of the tube feet requires significant pressure, fluid
is probably lost from the system due to ultrafiltration
through the tube foot and ampulla wall (Binyon, 1964,
1984; Prusch and Whorisky, 1976). As a result of active
pumping of K + , the fluid in the water vascular system is
hyperosmotic to the surrounding seawater and to the per-
ivisceral (coelomic) fluid (Binyon, 1962, 1964; Prusch and
Whorisky, 1976; Prusch, 1977; Ferguson. 1990a). Because
the hydrostatic pressure in temporarily inactive tube feet
is low, uptake of water probably occurs across the tube
foot wall and ampullar wall. Perhaps uptake in one portion
of the water vascular system can replenish the fluid lost
in others; the valves could open, allowing fluid to flow
via the radial canals. Active opening of the valves is likely
because, as described above, the flaps are equipped with
muscle fibers with appropriate trajectories. In addition,
the madreporite may contribute water to the water vas-
cular system and peri visceral coelom (Ferguson, 1989,
1990b). This mechanism would also require that the valves
open to allow fluid to flow from the radial canals into the
tube foot-ampulla complex. Active opening of the valves
by contraction of the valve musculature, allowing water
vascular fluid to flow from contracting tube feet into the
radial canal, has been described for ophiuroids (Buchanan
andWoodley, 1963; Woodley. 1980), asteroids (Nichols,
1 972), and for echinoids under extreme conditions (Mar-
kel and Roser, 1992). For the species studied here, our
observations (described earlier) suggest that the tube foot-
ampulla complex functions as an autonomous unit during
normal activity. Significant flow of water vascular fluid
in and out of the radial canal during normal movement
appears unlikely.

Implications for neural control. The anatomy, function,
and coordination of the nervous system of the tube foot-
ampulla complex of asteroids has received considerable
attention previously (Smith, 1945. 1946, 1950a. 1950b;
Cobb, 1967, 1970, 1987; Cavey and Wood, 1 98 1 ). Several
points arising from the analysis of the present study have

not been previously considered in discussions of the neural
control of the tube foot-ampulla complex. Although
bending of the tube foot in any direction was known to
require localized control of the musculature of the tube
foot, the role of synergistic ampulla muscle contraction
in providing the support required for bending was not
recognized. These two muscle groups operate sequentially
during elongation and retraction, and simultaneously
during bending movements, so their neural control is
complex.

In addition, complexity of nervous control is also im-
plied by the ability of the tube feet either to bend locally
and pivot around the base or to form a more generalized
bend along the length of the tube foot. In a study of the
innervation of the tube feet and ampullae of Astropecten
irregularis, Smith ( 1950b) observed a separate innervation
restricted to the muscle at the base of the tube foot (which
he referred to as postural muscles), as well as a distinct
innervation of the musculature of the tube foot wall (which
he called retractor muscles). This pattern of innervation
is to be expected if the musculature at the base of the tube
foot acts alone to create a localized bending while that of
the tube foot wall is responsible for more generalized
bending.

Acknowledgments

The authors are grateful to S. Johnsen and K. K. Smith
for comments on the manuscript. This research was sup-
ported by a National Science Foundation Presidential
Young Investigator Award (DCB-8658069) and NSF Re-
search Experiences for Undergraduates Supplement.

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Reference: Bail. Hull 188: 210-21X. (April. 1445)

Ammonium Metabolism in the Green Hydra Symbiosis

P. J. McAULEY

School of Biological ami Medical Sciences. Sir Harold Mitchell Building. University of St. Andrews.

St. Andrews. Fife. KYI 6 9TH. L'nited Kingdom

Abstract. Inhibitors of enzymes of ammonium assimi-
lation were used to test if assimilation of ammonium in
the green hydra-Chlorella symbiosis was due to host or
symbionts. Both methionine sulphoximine (MSX, an in-
hibitor of glutamine synthetase, found in both host and
symbionts) and azaserine (AZS, an inhibitor of 2-oxoglu-
tarate amido transferase, not found in the host) inhibited
ammonium uptake by the intact symbiosis. MSX was
taken up and caused predictable changes in pools of glu-
tamate and glutamine in both freshly isolated symbionts
and cultured ex-symbiotic C/ilorel/u. However, after
treatment of the intact symbiosis with MSX. no MSX
was found in the symbiotic Chlorella. and glutamine and
glutamate pools of both host and symbionts were unaf-
fected. Although both MSX and AZS inhibited ammo-
nium uptake by Chlorella. MSX caused seven times as
much ammonium release from the intact symbiosis as
did AZS. AZS treatment of the intact symbiosis caused
an increase in glutamine pools in both host and symbionts,
and AZS also competitively inhibited glutamine uptake
by Chlorella. Further, ammonium treatment of intact hy-
dra did not affect the nitrogen status of the algal symbionts,
although it did cause a small increase in the number of
algae in each digestive cell of the host. It is suggested that
primary ammonium assimilation in the green hydra sym-
biosis occurs by means of animal glutamine synthetase,
and that the resulting glutamine may be taken up and
further processed by the symbiotic algae. Freshly isolated
symbionts were able to process glutamine into glutamate
even when incubated at low pH, which causes them to
release a substantial proportion of fixed carbon as maltose.

Introduction

Invertebrate-microalgal symbioses are able not only to
reassimilate catabolically produced ammonium but also

Received 10 March 1 444; accepted 12 January 1495.

to take up and assimilate ammonium from the environ-
ment (Kawaguti, 1953: Gates and McLaughlin, 1976;
Szmant-Froelich and Pilson, 1977; Muscatine and D'Elia,
1978; Muscatine el at.. 1979; Wilkerson and Muscatine,
1984; Wilkerson and Trench. 1986; Rees, 1986; McAuley,
1990). However, there is some controversy over whether
ammonium assimilation is due to the symbiotic algae, or
to the animal host, or to the combined activities of both
(Miller and Vellowlees, 1989). The possibility that animal
rather than algal enzymes may be responsible, in part or
in whole, for assimilation of ammonium has important
implications for the mechanisms by which animal hosts
regulate cell division in populations of symbiotic algae,
since a number of workers have suggested that the growth
of populations of symbiotic algae may be nitrogen-limited
(Rees. 1986; Cook and D'Elia. 1987; McAuley. 1987a).

Miller and Yellowlees (1989) pointed out that in corals
symbiotic with dnioflagellates (zooxanthellae), levels of
the ammonium assimilatory enzyme NADPH-glutamate
dehydrogenase (NADPH-GDH) are higher in host tissue
than in symbionts, although the catabolic production of
ammonium probably exceeds the capacity of host
NADPH-GDH (Rahav etui.. 1989; Falkowski el al.. 1993;
Spencer-Davies, 1992), which has a low specificity for
ammonium (Catmull el al.. 1987). More recently, glu-
tamine synthetase (GS) activity was reported in giant clam
( Tridacna gigas) and coral (Pocillopora damicornis) host
tissues (Fitt et a/.. 1993; Yellowlees et al.. 1994). Because
there are relatively high levels of ammonium-assimilating
enzyme in host tissues, it is possible that much of the
ammonium present in natural seawater concentrations is
assimilated before it reaches the symbiotic zooxanthellae
( Yellowlees et al., 1994).

However, several lines of evidence suggest that some if
not all ammonium assimilation is due to the zooxan-
thellae. Assimilation is light dependent and does not occur
in aposymbiotic animals (for review, see Rees, 1986).

210

AMMONIUM METABOLISM IN GREEN HYDRA

Rates of uptake by intact associations are similar to those
of freshly isolated symbionts (D'Elia et al.. 1983: Wil-
kerson and Trench, 1986), and intact associations exhibit
uptake kinetics which suggest that symbionts assimilate
ammonium from seawater by depleting ammonium in
host tissue and causing a diffusion gradient to form (D'Elia
and Cook, 1988). Finally, addition of ammonium to sea-
water causes increases in zooxanthellar mitosis, biomass,
and amino acid levels (Cook ct a/., 1988; Muscatine el
a/.. 1989; Hoegh-Guldberg and Smith, 1989; Dubinsky
el al.. 1990; Fitt and Cook, 1990; Stambler el ai. 1991;
McAuley, 1994; Muller- Parker el al.. 1994; McAuley and
Cook, 1994), and reduces ammonium uptake rates (Yel-
lowleest 1 / al.. 1994). Ammonium assimilation is assumed
to involve the coupled glutamine synthetase/2-oxoglutar-
ate amido transferase (GS/GOGAT) pathway. At present.
GS but not GOGAT has been detected in zooxathellae
(Summons and Osmond, 1981; Wilkerson and Muscatine,
1984), although azaserine, a potent inhibitor of GOGAT,
prevents ammonium uptake in intact corals (Rahav et
al.. 1989), and the presence of GS/GOGAT activity is
supported by the data of Summons et al. ( 1986).

In contrast to marine symbioscs. in the symbiosis be-
tween the freshwater cnidarian Hydra viridissima (green
hydra) and Chlorella algae, the host rather than the algal
symbionts may be responsible for assimilation of am-
monium. Ammonium assimilation by maltose-releasing
Chlorella is inhibited at the low pH that stimulates maltose
release (Rees, 1989). Recent evidence suggests that in
symbiosis the algae are carbon- rather than nitrogen-lim-
ited, in that in vivo release of maltose consumes consid-
erable amounts of photosynthetically fixed carbon that
would otherwise be used to assimilate ammonium into
amino acids (McAuley. 1992). Treatment of the intact
symbiosis with the photosynthetic inhibitor DCMU does
not cause ammonium excretion into the medium, and
rates of ammonium uptake by freshly isolated symbionts
are only 40% of those of the intact symbiosis (Rees, 1986;
McAuley, 1990). Finally, the hosts possess GS, and levels
of GS activity are higher in symbiotic than aposymbiotic
animals (Rees, 1986).

Although green hydra do not normally excrete am-
monium into the medium, release can be induced by
treatment with methionine sulphoximine (MSX), which
inhibits GS in intact hydra (Rees, 1986). However, the
effect of MSX on algal GS has not been tested, because
of the difficulty in isolating sufficient numbers of sym-
bionts uncontaminated by host material. This paper de-
scribes the effects of inhibitors of enzymes of ammonium
assimilation on perturbation of animal and algal internal
pools of glutamate and glutamine, amino acids closely
associated with ammonium assimilation (Miflin and Lea,
1976). Recovery of symbiotic algae after SDS washing,
which is necessary to remove host contamination, is low

(McAuley. 1986a), but unlike assays for enzyme activity,
measurement of free amino acid pools by HPLC requires
only small numbers of algae (the equivalent of I0 4 cells
or fewer per sample injection). Uptake of MSX by algae
;/; vitro and in the intact symbiosis was also measured
using HPLC.

Materials and Methods

Maintenance of organisms

Green hydra of the European strain (EE hydra) were
grown in unbuffered 'M' solution (Muscatine and Lenhofi,
1965) at 15Cin constant light (60 jumol photons m 2 s '
PAR). Cultures were fed each Monday, Wednesday, and
Friday with freshly hatched brine shrimp. All hydra used
in experiments had not been fed for the previous 3 days
to allow complete digestion of food. Cultures of the
3N813A strain of maltose-releasing Chlorella, grown in
Kessler's medium (Kessler et ai. 1963), pH 6.3, were
maintained in a shaking, illuminated incubator in growth
conditions similar to those of hydra cultures.

Isolation of symbiotic algae from hydra

The SDS-washing technique (McAuley, 1986a) was
used to isolate symbiotic Chlorella algae from hydra.

Determination of numbers of algae per digestive cell

Five gastric regions of hydra were isolated in a drop of
macerating fluid (David. 1973). After 10 min, pieces of
hydra were teased apart into individual cells and examined
using phase contrast microscopy. Numbers of algae were
determined in 100 randomly selected digestive cells in
each preparation.

Measurement of ammonium uptake

Ammonium uptake was measured as depletion from
the medium of either hydra or algae. Duplicate samples
(200 ^1) were taken at the beginning and end of experi-
ments, and amounts of ammonium were determined with
a scaled-down hypochlorite-nitroprusside colometric assay
(Rees, 1986). After reagents were added, samples were
incubated for 20 min in darkness, then absorbence was
read at 630 nm and compared to that of ammonium sul-
phate standards (reagents and standards from Sigma
Chemical Company).

Inhibitors

Methionine sulphoximine (MSX), an inhibitor of GS
(Ranziorfa/.. 1969; Meister, 1974), and azaserine (AZS),
an inhibitor of 2-oxoglutarate amido transferase (GO-
GAT) (Wallsgrove et al.. 1977; Elrifi and Turpin, 1986),
were purchased from the Sigma Chemical Company.

212

P. J. McAULEY

Stock solutions were stored at 4C and routinely tested
by measuring their effectiveness in inhibiting ammonium
uptake by cultured 3N8 1 3 A cells. In all experiments, MSX
was used at a final concentration of 200 fiM and AZS at
1 mM.

Extraction ami measurement of free cimino acid pools

Samples of algae, or of animal homogenates centrifuged
at 1000 X g for 5 min to remove algae, were added to
absolute ethanol to give 80% ethanol (v:v) and extracted
for 24 h at 4C. Protein contents of homogenates were
determined using the Bradford method ( Bradford, 1976).
In some cases, algal samples were filtered at low vacuum
through Whatman GF/C filters, which were then extracted
twice in 80% ethanol. Extracts were dried //; vacno and
resuspended in an appropriate volume of 12.5/uA/ a-
amino-butyric acid (A ABA), which acted as an internal
standard. Amino acid contents of aliquots of extracts were
determined by ophthaldialdehyde pretreatment and re-
verse-phase HPLC as previously described (McAuley,
1992). MSX but not AZS was detectable using this system;
the MSX peak occurred 0.8 min after that of serine.

Glutamine and methylamine uptake

Glutamine uptake was determined from cultured
3N813A cells resuspended in 10 mM MES buffer pH 7
at a density of 5 X 10 7 cells ml '. After 30 min of prein-
cubation, the assay was started by adding 2-20 pM glu-
tamine containing 3.7 ^/Bq [U- M C]-glutamine ( Amersham
International; specific activity 9.25 GBq mmol ') with or
without additional 20 \iM AZS. At intervals of 1. 2, and
3 min after addition of radioactivity, 200-^1 samples were
filtered onto Whatman GF/C filters at low vacuum and
washed with 20 ml distilled water. Filters were dried in
scintillation vials, 10 ml of scintillation fluid was added
(McAuley, 1988), and samples were counted on an LKB
1214 Rackbeta scintillation counter. V max and K M were
determined from Lineweaver Burke double reciprocal
plots of uptake rates against substrate concentration.

Uptake of [ l4 C]-methylamine was determined by the
same method as glutamine uptake, except that [ M C]-me-
thylamine (Amersham International; specific activity
2.22 GBq mmor ') was added to give a final concentration
of 5 juA/. At intervals. 100-jul samples were filtered onto
Whatman GF/C niters and radioactivity was determined
as described above.

Enzyme assay

About 1 00 hydra, previously starved for 3 days, were
homogenized in extraction buffer as described by Rees
(1986). The homogenate was centrifuged at 13000 X g
for 5 min at 4C, and the supernatant was assayed for

GOG AT activity by determining the rate of oxidation of
NAD(P)H at 340 nm by the method of Bhandari and Ni-
cholas (1981). Blanks were run without addition of n-
ketoglutarate.

Results

Effect oj ammonium on symhionts in intact hydra

Incubation of EE hydra for 7 days in M solution sup-
plemented with 50 nAf ammonium chloride had no effect
on the size of the glutamate and glutamine pools of their
algal symbionts (Table I). However, significantly more
algae (one-way ANOVA. P < 0.05, n = 6 independent
experiments) were present in digestive cells of hydra
maintained in ammonium-supplemented medium (24.94
1.71 SD) than in controls (22.58 1 .48).

Effect of MSX and A/.S on ammonium uptake

As previously observed (Rees, 1986; McAuley, 1990).
MSX not only inhibited ammonium assimilation but also
caused release of ammonium from both intact hydra and
cultured 3N813A algae (Table II). AZS inhibited am-
monium assimilation but did not cause release in 3N8 1 3A
algae; ammonium release by green hydra treated with AZS
was seven times lower than that observed with MSX
treatment.

Effect of MSX ami AZS on internal pools of glutamate
and K/iitaminc in 3NS13A

3N8 1 3A algae were incubated with either MSX or AZS
in the absence or presence of a nitrogen source (either
glutamine or ammonium), and amino acid pools were
compared to those of untreated controls (Table III). MSX
and AZS had distinct effects on internal glutamine and
glutamate pools irrespective of the nitrogen supply, and

Table I

Effect <>t inciihalion / inlucl hydra in ammonium on glittamaic anil
i>o<il\ ol algal vrw/i;uv

Ammonium

Controls

treated

Glu

574.1

38.0

578.9

56.7

Gin

150.3

18.5

146.7

29.3

Gln:Glu

0.26

0.03

0.26

0.06

EE Hydra were starved for 7 days in M solution 50 n,\l ammonium
chloride; incubation solutions were changed every day. After 7 days,
hydra were washed five times in large volumes of M solution and ho-
mogenized, and amino acid pools in SDS-washed freshly isolated sym-
bionts were determined as described in Materials and Methods. Values
are the means SD of amino acid content (amol cell' 1 ) of glutamate
and glutamine pools determined from three independent experiments.

AMMONIUM METABOLISM IN GREEN HYDRA

213

Table II

Effect of MSX and AZS on uptake (+) or release (-) of ammonium hy
intact green hydra and 3NSI3.4 algae

Hydra
(nmol hydra"

h ')

3N813A
(fmol ceir 1 h*')

Control

MSX

AZS

+0.192 + 0.045
-0.220 0.036
-0.031 +0.006

+ 1.51 0.35
-0.86 0.25
+0.05 + 0.03

Hydra were preincubated in MSX or AZS for 24 h and washed twice
in large volumes of growth medium before being placed in new medium;
3N813A algae (5 X 10 7 cells ml" 1 ) were preincubated for 30 min in 10
nM MES pH 7. Uptake of ammonium was measured by the difference
in ammonium content of aliquots taken immediately after addition of
ammonium chloride to give a concentration of 50 nM (hydra) or 100
li M (algae) and of aliquots taken after 1 h. Figures are the means + SD
of three (hydra) or four (algae) independent experiments. All treatment
values are significantly different from controls (one-way ANOVA, P
< 0.001).

these effects were consistent with inhibition of GS or GO-
GAT respectively. MSX significantly decreased glutamine
and AZS significantly decreased glutamate in all treat-
ments. In algae without a nitrogen source or supplied with
glutamine, MSX also significantly decreased glutamate
pools; in algae without a nitrogen source or supplied with
ammonium, AZS significantly increased glutamine pools.
Supply of nitrogen also affected glutamine and gluta-
mate pools. Glutamine increased in algae supplied with
ammonium and glutamine, and glutamate increased in
algae supplied with glutamine (one-way ANOVA. P <
0.05). Tukey post-hoc tests showed that the size of the
glutamine pools was similar in all MSX and all AZS treat-
ments (P > 0.05). The size of the glutamate pools was
also similar in all AZS treatments (P > 0.05), but gluta-
mate levels were higher in algae treated with MSX in the
presence of glutamine than in the presence of ammonium
or the absence of a nitrogen source.

Effect of MSX and AZS on internal pools of glutamate
and glutamine in freshly isolated symbionts and in the
intact symbiosis

The effect of maltose release on the changes the two
inhibitors produce in glutamate and glutamine pools was
tested on freshly isolated symbionts treated at pH 5 and
at pH 7. Maltose release is pH dependent (Cernichiari et
a!.. 1969) and occurs at the former but not the latter pH.

When freshly isolated symbionts were treated with
MSX and AZS at pH 7, the effects were similar to those
seen when 3N8 1 3A algae were treated without a nitrogen
source: MSX caused a significant decrease in glutamine
and glutamate; AZS caused a significant decrease in glu-
tamate and an increase in glutamine (Table IV). At pH

Table III

Effect of MSX anil .1 ZS on tree glulamalc and glutamine pools
ol 3X81 3A algae

Control

MSX

AZS

-N

Glu

379.9 24.2

295.9 40.3*

68.4 + 20.2*

Gin

77.3 + 10.3

11.4+ 2.3'

437.7 + 72.8*

+NH4

Glu

335.0 14.2

300.4 22. 1

50.5 13.1*

Gin

166.1 47.2

11.5+ 5.5*

438.4 51.0*

+Gln

Glu

625.4 99.0

407.1 62.7*

56.8 6.3*

Gin

330.1 62.3

49.7 16.4*

495.9 +91.6

Algae were incubated with MSX or AZS or in 10 mM MES buffer
pH 7 for 30 min before addition of 100 ^M ammonium chloride, glu-
tamine, or an equivalent volume of distilled water (-N). After I h. aliquots
were filtered at low vacuum onto Whatman GF/C filters, washed, and
extracted for amino acid analysis. Figures are the means SD of amino
acid content (amol cell" 1 ) of glutamate and glutamine pools determined
from three independent experiments.

* Significantly different from control, one-way ANOVA, P < 0.05.

5, AZS treatment also caused a significant decrease in
glutamate and an increase in glutamine, but whereas MSX
treatment decreased glutamate, glutamine levels tended
to increase, although this trend was not significant. Uptake
of MSX at pH 5 was considerable (over 3000 amol ceir 1 ),
and the large MSX peak, adjacent to that of glutamine,
may have interfered with detection of glutamine. When
an amount of MSX equivalent to that found in the algae
was added to a sample containing 1 pmol glutamine, the
apparent amount of glutamine detected by HPLC in-
creased 30%. The smaller MSX peak at pH 7 did not
interfere with glutamine detection.

Table IV

Effect of MSX or A ZS on free glutamate and glutamine pools of freshly
isolated symbiotic algae

Control

MSX

AZS

pH 7.0

Glu

334. 1

+ 62.3

182.2

29.1*

63.6 +

13.1*

Gin

120.1

27.2

50.9 +

12.7*

297.1 +

102.3*

pH 5.0

Glu

1 56.0

+ 4.5

84.0 +

22.1*

62.1 +

14.7*

Gin

60.6

+ 6.5

95.4

23.1

89.2

11.4*

Algae were incubated with MSX or AZS or in 10 mM MES buffer
only (controls) for 90 min. and then aliquots were filtered at low vacuum
onto Whatman GF/C filters, washed, and extracted for amino acid
analysis. Figures are the means SD of amino acid content (amol cell' ')
of glutamate and glutamine pools determined from three independent
experiments.

* Significantly different from control value, one-way ANOVA, P
< 0.05.

214

P. J. McAULEY

Treatment of freshly isolated symbionts at pH 5 caused
a significant reduction in both glutamate and glutamine
pools compared to those at pH 7 (one-way ANOVA, P
<0.05).

Incubation of intact green hydra in MSX caused no
significant change in the glutamate or glutamine pools of
either the symbiotic algae or the host (Table V). Only 16.2
30.2 SD amol cell ' MSX was detected in algal pools
after 24-h treatment of intact hydra, compared to high
levels in freshly isolated symbionts after only 90 min of
treatment (3126.0 227.6 amol cell ' in freshly isolated
symbionts treated at pH 5; 495.3 175.7 amol cell ' in
freshly isolated symbionts treated at pH 7). Treatment of
intact hydra with AZS increased glutamine pools in both
hydra and algae, although the increase was significant only
in hydra. AZS treatment did not cause a reduction in
either host or algal glutamate pools, in contrast to the
consistent effect of AZS on glutamate pools measured in
both freshly isolated symbionts and 3N813A irrespective
of pH or nitrogen supply.

Effect of MSX and AZS on glutamine ami methylamine
uptake by 3N813A algae

[ u C]-glutamine uptake by 3N8 1 3A algae was competi-
tively inhibited by a low concentration of AZS (Fig. 1).
K M was 17.3 ^M in the absence of AZS and 28.6 /uA/ in
the presence of AZS. In both cases, V max was 250 amol
glutamine cell ' h '.

There was linear uptake of the ammonium analog [ I4 C]-
methylamine in the presence of both MSX and AZS, al-
though uptake rates were reduced by 25.1% and 33.4%
respectively, in comparison to controls (Fig. 2). In con-
trast, ammonium chloride inhibited methylamine uptake

Table V

Effect ofinciihatmx hyilra in MSX or A7S on free s-lulamate and
glutamine pooh <>t hvclra (pmol ng protein' 1 ) and symbiotic algae
(amol cell' 1 )

0.06 -

Control

MSX

AZS

Hydra

Glu

24.3

10.4

24.3

12.8

25.1

12.0

Gin

1.3

0.4

1.8

0.9

5.9

3.0*

Algae

Glu

551.3

82.7

571.5

143.6

554.8

94.6

Gin

146.2

54.3

156.6

65.8

224.1

128.1

One hundred and twenty hydra were incubated in MSX or AZS for
24 h, washed five times in large volumes of M solution and homogenized,
and amino acid pools in homogenized hydra and in SDS-vvashed freshly
isolated symbionts were determined as described in Materials and Meth-
ods. Figures are the means SD of six independent experiments.

* Significantly different from control value, one-way ANOVA. P
< 0.05.

0)
O

0>

c

E
ro

*

"5

E

CO

0.04 -

0.02 -

0.1

Figure I . Effect of the absence

0.2

0.3

0.4

- 1 (urn)

and presence (O-

0.5

-O)

of AZS on the apparent K M value of the glutamine transport system of
3N8 I 3A algae. Algae were preincuhated in 10 m/U MES buffer pH 7 for
30 min before addition of 2-20 nM [ H C]-glutamine with or without
20 11 M AZS. Uptake of glutamine and K M and V ma , was determined as
described in Materials and Methods.

by 98%- for the first 4 min, although the uptake increased
gradually thereafter. Calculations based on ammonium
uptake rates in Table I indicated that the algae would
have greatly depleted ammonium concentration at the
time that uptake of methylamine began to increase.

L 'pi iikc ofglutamiiic by freshly isolated symbionts

Short-term experiments showed that incubation of
freshly isolated symbionts in 100 n,\l glutamine caused
an increase in internal glutamine and glutamate pools at
bothpH Sand pH 7 (Fig. 3).

GOGAT activity

Neither NADH- or NADPH-specific GOGAT activity
was detectable in hydra tissue.

Discussion

Several lines of evidence suggest that assimilation of
ammonium in the green hydra-C/ilorella symbiosis is due
to GS in the animal host rather than in the algal symbiont.
Incubation of green hydra in medium supplemented with
ammonium chloride caused a small, significant increase
in numbers of algae per host digestive cell, but there was

<u
o

<u

w

s

"o
E

lOO-i

80-

60-

AMMONIUM METABOLISM IN GREEN HYDRA

1200-]

1000-

800-

600-

215

a)

Time (minutes)

Figure 2. Effect of MSX, AZS, and ammonium on uptake of [ 14 C]-
methylamine by 3N813A algae. Algae were preincubated in 10 mM MES
buffer pH 7 for 30 min before addition of 5 pM [' 4 C]-methylamine to-
gether with either 10 pM MSX ( ). 10 nM AZS (O O),

10 nM ammonium chloride (A A), or an equivalent volume of

buffer only as control (D D) to give a final density of 5 > 10 7 cells

ml' 1 . At intervals, 100-^1 samples were filtered and radioactivity was
determined.

no effect on glutamine and glutamate pools or on the Gin:
Glu ratio of the algae (Table I), the latter being an indicator
of nitrogen sufficiency in microalgae (Flynn ci til.. 1989).
In marine symbioses. in which it is believed that symbionts
are able to assimilate ammonium from seawater, am-
monium supplementation not only causes large increases
in the population density of symbionts (Muscatine et ai,
1989; Hoegh-Guldberg and Smith. 1989; Dubinsky et a!..
1 990; Fitt and Cook, 1990; Stambler et /.. 1991; Muller-
Parker et ai. 1994). but. in a coral and a hydroid, also
increases the size of internal glutamine pools of symbionts
and hence their Gln:Glu ratio (McAuley, 1994; McAuley
and Cook, 1994).

Further, although MSX inhibits ammonium uptake by
intact hydra (Rees, 1986; McAuley. 1990; Table II. this
paper), almost no MSX was detected in symbiotic algae
isolated from hydra that had been incubated in MSX for
24 h, and there was no change in algal internal pools of
glutamate and glutamine. In contrast, freshly isolated
symbionts treated with MSX rapidly accumulated large
amounts of the inhibitor, and MSX treatment caused
predictable changes in glutamate and glutamine pools of
freshly isolated symbionts and cultured 3N8 1 3 A algae in
a variety of conditions (Tables III and IV).

o

T3
'0

O
03

"o

Time (minutes)

Figure 3. Effect of glutamine uptake on internal pools of glutamine

(O O) and glutamate (D D) in freshly isolated symbionts.

Freshly isolated symbionts ( 10 7 cells ml"') were preincubated for 30 min
in 1 mM Mcllvaine's buffer at either pH 5 (a) or pH 7 (b). Ten minutes
after the start of the experiment, glutamine was added to give a concen-
tration of 100/i.U. Samples (200 nM) taken at each time point were
filtered onto GF/C disks under low vacuum, washed with 20 ml ice-cold
distilled water, and extracted for amino acid analysis.

216

P. J. McAULEY

In addition to inhibiting GS, MSX has been shown to
be a noncompetitive inhibitor of active ammonium
transport in Anahaena jlos-aquae (Turpin et a/.. 1984).
In 3N813A, both MSX and AZS caused a degree of in-
hibition of uptake of methylamine (Fig. 2). a structural
analog of ammonium that is taken up by the same trans-
port system (Hackette et a/.. 1970; Pelley and Bannister,
1979; Smith, 1982; Wright and Syrett, 1983). However,
if both MSX and AZS caused noncompetitive inhibition
of entry of ammonium into symbionts, release of am-
monium from the intact symbiosis would presumably be
the same in both treatments. Instead, release was seven
times higher in hydra treated with MSX than in hydra
treated with AZS (Table II). As shown in 3N813A cells,
MSX blocks assimilation of ammonium into glutamine
via GS (Ranzio et al., 1969; Meister, 1974), leading to
low glutamine pools and release of ammonium. AZS
blocks subsequent metabolism of glutamine to glutamate
via GOGAT (Wallsgrove et al.. 1977; Elrifi and Turpin,
1986), leading to accumulation of glutamine, low gluta-
mate pools, and no ammonium release. That hydra release
more ammonium when treated with MSX than with AZS
is consistent with inhibition of GS activity pathways rather
than with noncompetitive inhibition of ammonium
transport into the algae. Because very little MSX was de-
tected in symbionts after treatment of intact hydra, the
ammonium release observed during MSX treatment of
the intact symbiosis must have been due to inhibition of
host GS.

No GOGAT was detected in crude, alga-free extracts
of homogenized green hydra. However, treatment of intact
hydra with AZS, an inhibitor of GOGAT, prevented up-
take of ammonium from the medium by the intact sym-
biosis and caused a significant increase in host glutamine
pools (Tables II and V). This may be explained if gluta-
mine resulting from activity of host GS was taken up by
the algae and converted into glutamate via GOGAT. Up-
take of glutamine by algae would also explain why MSX
treatment did not cause an increase in host glutamine
pools.

AZS treatment may have affected this coupling in two
ways. First, AZS may have specifically inhibited algal
GOGAT, although AZS treatment of the intact symbiosis
did not cause the reduction in glutamate and increase in
glutamine consistently observed in 3N813A or in freshly
isolated symbionts. Second, AZS, which competitively
inhibited uptake of glutamine in 3N813A algae (Fig. 1),
prevented entry of glutamine into either the perialgal vac-
uole or the symbiotic algae themselves. In either case, the
glutamine transport system must be specific for glutamine
and AZS but not MSX. Given that symbionts accumu-
lated MSX in vitro but not /// hox/iice. it is possible that
transport systems in the host perialgal vacuolar membrane

differentially control entry of substances into the perialgal
vacuole.

Supply of nitrogen to algal symbionts as glutamine or
other amino acids rather than as ammonium may be im-
portant because a supply of carbon is required for assimi-
lation of ammonium into amino acids (Turpin, 1991),
and symbionts release a high proportion of fixed carbon
to their host in the form of maltose (Mews, 1980). In
cultured 3N813A, maltose synthesis and release inhibits
ammonium uptake because it diverts photosynthetically
fixed carbon from assimilation of ammonium into amino
acids (McAuley, 1992). Maltose release is stimulated by
low pH, and ammonium uptake by freshly isolated sym-
bionts and cultured maltose-releasing algae falls as pH is
reduced and maltose release increases (Rees, 1989). Below
a critical pH value, the algae actually begin to release am-
monium, not only because fixed carbon is diverted from
the TCA cycle to maltose release, but also because amino
acids may be deaminated to provide carbon skeletons for
maltose synthesis (Rees, 1990).

Given that high rates of maltose release and ammonium
assimilation are incompatible, amino acids may provide
an alternative source of nitrogen for algae in symbiosis
with green hydra. Freshly isolated symbionts have active
amino acid transport systems (McAuley, 1 986b), and algae
in the intact symbiosis can assimilate a variety of amino
acids supplied by host feeding (McAuley, 1987b, 1988,
1991). Further, the rate of glutamine uptake by freshly
isolated symbionts peaks between pH 4 and 5, the same
range over which release of maltose is maximum (Mc-
Auley, 1991). Maltose-releasing symbionts using gluta-
mine as a nitrogen source would require carbon skeletons
in the form of 2-oxoglutarate to produce two molecules
of glutamate from each molecule of glutamine. But even
if one molecule of glutamate was deaminated, the carbon
recycled to 2-oxoglutarate, and the ammonium released
to be reassimilated by the host, the algae would still gain
a new molecule of glutamate for each molecule of glu-
tamine taken up. Short-term experiments showed that
uptake of glutamine by freshly isolated symbionts at both
pH 5 (with maltose release) and pH 7 (without maltose
release) caused an increase in the size of internal glutamate
pools (Fig. 3). Although the increase in glutamate pools
was similar, it should be noted that the rate of glutamine
uptake by freshly isolated symbionts is more than 5 times
higher at pH 5 than at pH 7 (McAuley. 1991 ).

This scheme is qualitatively but not quantitatively dif-
ferent from that in marine algal-invertebrate symbioses.
in which symbiotic zooxanthellae appear to assimilate
ammonium via coupled GS/GOGAT (for review, see
Spencer-Davies, 1992). In green hydra, although both GS
and GOGAT are involved in ammonium assimilation,
they may be located in different compartments, and the
necessity for glutamine to traverse the perialgal vacuole

AMMONIUM METABOLISM IN GREEN HYDRA

217

would produce a point at which nitrogen supply to the
algae could be regulated.

Acknowledgments

This work was supported by grants from the Royal So-
ciety and the Natural Environmental Research Council.
My thanks to Mr. Harry Hodge for his help in HPLC
analysis of amino acids, and to an anonymous referee for
helpful comments on a draft of this paper.

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On the Giant Octopus (Octopus giganteus) and
the Bermuda Blob: Homage to A. E. Verrill

SIDNEY K. PIERCE 1 , GERALD N. SMITH, JR. 2 , TIMOTHY K. MAUGEL 1 ,

AND EUGENIE CLARK 1

^Department of Zoology, University of Maryland, College Park. Maryland 20742: and

2 Department of Medicine, Division of Rheumatology, Indiana University

School of Medicine. Indianapolis. Indiana 46202

"The substance looks like blubber, and smells like blubber
and it is blubber, nothing more or less."

F. A. Lucas. 1897

Abstract. We have obtained samples of two large car-
casses. One washed up on a beach in St. Augustine, Flor-
ida, in 1896 and has been occasionally attributed to a
species of gigantic octopus (Octopus giganteus). The other
carcass washed up on Bermuda in 1 988 and has remained
unidentified, although its gross morphology, except for a
much smaller total mass, was remarkably similar to the
Florida carcass. We have subjected both samples to elec-
tron microscopic and biochemical analyses. Our results
show that both carcasses are masses of virtually pure col-
lagen. Furthermore, neither sample has the biochemical
characteristics of invertebrate collagen, nor the collagen
fiber arrangement of octopus mantle. Instead, they are
large pieces of vertebrate skin, the Bermuda sample from
a poikilotherm and the Florida sample from a huge ho-
miotherm. We conclude that there is no evidence to sup-
port the existence of Octopus giganteus.

Introduction

The first evidence that seemed to document the exis-
tence of a species of gigantic octopus washed ashore on
an oceanic beach at St. Augustine, Florida, late in the
year of 1896. The so-called carcass was taken in charge
by a local physician. Dr. DeWitt Webb, who was also
president of the St. Augustine Scientific Society (Wood,
1971). Webb set about to photograph the body, dig it out

Received 31 October 1994; accepted 25 January 1995.

of the sand, haul it up above the high tides (a task that
required the efforts of several horses and men), and to
inform the scientific community of its existence. Because
Webb believed the carcass to be the remains of a huge
octopus, one of the scientists he contacted was the pre-
eminent invertebrate naturalist. Professor A. E. Verrill at
Yale (Fig. 1 ). Based at first only upon a letter from Webb,
Verrill reported the finding, speculating that it might be
a specimen ofArchiteuthis(Veml\, 1897a). Shortly there-
after, having received additional correspondence and
photographs from Webb, Verrill concluded that it was a
"true Octopus, of colossal size . . . one of those upon
which the sperm whale feeds regularly." On the basis of
the descriptive and photographic evidence, Verrill pro-
posed to name the species Octopus giganteus (Verrill,
1897b). However, almost immediately, Verrill changed
his mind. Based upon more photographs, measurements,
and descriptions of the carcass after it had been entirely
unearthed from the beach sand, along with several for-
malin-preserved pieces of the tissue, Verrill retracted his
rapidly drawn, initial conclusions, writing that "the crea-
ture could not have been an Octopus" (Verrill, 1897c, d).
Instead, Verrill, together with some other biologists of the
time, came to the conclusion that the carcass was from a
large vertebrate, most likely a whale (Lucas. 1 897; Verrill,
1 897d, e). However, other biologists, notably Dr. William
H. Dall, then the Curator of Mollusks at the National
Museum of Natural History, still favored Verrill's original
species diagnosis according to correspondence between
him, Webb, and Verrill (archived at the Smithsonian In-
stitution, Washington. DC).

Although Verrill disavowed his species description of
Octopus giganteus several times, the carcass was never
properly identified. The matter rested quietly for 70 years.

219

220

S K. PIERCE ET AL

Figure 1. A. E. Verrill. Courtesy of the Marine Biological Laboratory
Archives.

and then a report appeared stating "it can be safely said
that the gigantic mass of tissue that washed up on the
beach at St. Augustine in 1896 was the remains of an
octopus. . . 200 feet . . ." between tentacle tips (Wood,
1971). That report continued to detail the chronology of
the events summarized above and also indicated that siz-
able pieces of the carcass had been preserved and were
held at the Smithsonian Institution. A cursory histological
comparison of the tissue from the Smithsonian with that
of "contemporary" squid and octopus was carried out.
Although the specific squid and octopus tissues that were
compared to the Florida carcass were not reported, no
cellular structure was found in any of the tissues. Instead,
a connective tissue fiber network in all three tissues was
revealed with polarized light. The conclusions reached
were that none of the samples looked mammalian, that
the St. Augustine tissue looked much more like the oc-
topus fiber network than that of the squid, and therefore,
"the St. Augustine sea monster was in fact an octopus"
(Gennaro, 1971). Because the report of these histological
studies was written for a general, rather than scientific
audience, it lacked a rigorous description of protocol and
observations.

The matter rested again, this time for another decade
and a half, until the appearance of a report about the
amino acid composition of an acid hydrolysate of the St.
Augustine tissue, by now almost 100 years old. Although
neither hydroxylysine nor hydroxyproline concentrations
were determined, the amino acid composition suggested
that the St. Augustine tissue was likely to have been a
huge mass of collagen (Mackal, 1986), explaining the lack

of cellular structure found in it by Gennaro (1971) (but
not the lack of such structure in the "contemporary" tis-
sues) and the persistence of the carcass as it lay on the
beach. (According to the correspondence between Webb
and Dall, the carcass was still on the beach on March 17,
1897, almost 4 months after the initial discovery.) Mackal
( 1986) concluded that the amino acid data together with
some inconclusive Cu and Fe measurements "support the
original identification of the tissue and carcass by A. E.
Verrill as an exceptionally large cephalopod, probably oc-
topus, not referable to any known species," in spite of
both VerriU's change of mind and the complete lack of a
suitable test of taxonomic relationships in Mackal's data.
In the end, while the existence of the St. Augustine carcass
is well documented and the discovery often cited [most
recently in the popular press (Ellis, 1994) and in a bio-
logical science text book (Milne, 1995)], there is no un-
equivocal evidence at all that it belonged to a giant octopus
or, indeed, to any particular species.

During the summer of 1988 another carcass washed
into a lagoon on the island of Bermuda. This unrecog-
nizable carcass ( 2.50 X 1.25 X 0.30 m), immediately
labeled the "Bermuda Blob" by the popular press, was
photographed and sampled by Teddy Tucker, a renowned
local diver and fisherman who often works with scientists
on Bermuda. While considerably smaller than the St. Au-
gustine discovery, the Bermuda carcass fit Verrill's de-
scription exactly, on a gross level. "No bones or hard parts.
. . . Instead of being muscular . . . [the tissues] are firm,
tough and elastic, and composed mainly of much inter-
laced fibers and large bundles of tough fibrous, white con-
nective tissue. [The tissue is] difficult to cut or tear apart
.... Some large irregular canals permeate the [tissue].
These may have contained blood vessels originally. From
the inner surface of some of the pieces large cords of elastic
fibers proceeded inward" (Verrill, 1897c). Gennaro's ad-
ditional description of the St. Augustine tissue also fit the
Bermuda carcass exactly. "White as soap . . . the con-
nective tissue was so tough that it dulled four blades . . .
the same homogenous, tough, white, fibrous texture
[throughout]" (Gennaro, 1971).

We have been able to obtain small pieces of both the
St. Augustine and Bermuda carcasses. We have subjected
both to electron microscopic examination as well as bio-
chemical analyses to test the similarity between the two
tissue masses and to determine their taxonomic origin.
In addition, we have carried out light and electron mi-
croscopic examinations of octopus (Bathypolypus arcticus)
mantle tissue and humpback whale (Megaptera novaean-
gclue) blubber.

Materials and Methods

Elect mn microscopy

Bermuda and St. Augustine Carcasses. Specimens from
both the Bermuda and St. Augustine tissue masses were

THE GIANT OCTOPUS AND THE BERMUDA BLOB

221

SillSK'.fcAi'S "^jSfa&jSfeVj-kSW&SS',.:

Figure 2. Low magnification transmission electron micrographs of sections of the Bermuda (A) and St.
Augustine (B) carcasses. The collagen fibers of both tissues run in layers that are perpendicular to each other.
Within each layer the fibers appear to be organized in bundles (see the upper half of A). This type of fiber
organization is typical of skin collagen (see Discussion). Other than the fibers, no other cellular elements
were found. Bacteria and bacterial spores (arrows) were scattered throughout the fiber layers in both samples.

prepared for electron microscopic examination. Although
the exact composition of the original preservation medium
was unknown for either tissue, the distinct odor of for-
malin was obvious in both. Indeed, Webb, in his corre-
spondence with Dall, indicated that he had put several
pieces of the body in formalin before sending them off to
both Dall and Verrill (cited in Wood, 197 1 ). We cut sev-
eral pieces ( 1 mm 1 ) from each of the original tissue sam-
ples and placed them directly into 2.5% glutaraldehyde/
2% paraformaldehyde in 0.1 M cacodylate buffer con-
tainingO.3 M sucrose (~ 1 100 mosm) (Bermuda sample)
or 2% glutaraldehyde in 0.15 M cacodylate buffer con-
taining 0.58 M sucrose (~ 1 100 mosm) (Florida sample).
The tissue samples remained in the fixative for several
days. After fixation, the tissue pieces were post-fixed in
2% OsO 4 in the cacodylate-sucrose buffer, treated en bloc
with 2% aqueous uranyl acetate, dehydrated in an ethanol
series (35-100%), and embedded in epoxy resin (Spurr's
medium). Thin sections showing a silver interference color
were cut with a diamond knife on an ultramicrotome
(Reichert, Ultracut E). The sections were mounted on
copper grids and stained with 2% uranyl acetate for 5 min.

followed by 0.2% lead citrate for 1.5 min (Venable and
Coggeshall, 1965). The stained sections were examined
with a transmission electron microscope (Zeiss EM 10
CA) at 80 kV.

Octopus nuinlle. The specimen of B. arcticus (USNM
catalogue #884 1 84) that provided a mantle tissue sample
had been collected by trawl off the New Jersey coast during
a 1 98 1 cruise of the R/ V Delaware II and had been placed
immediately into formalin upon its capture. At some
point, the octopus was transferred into isopropyl alcohol
and had been stored in that solution until we were given
access to it. We cut a section of the mantle offthe octopus,
rehydrated it and placed it into 2% glutaraldehyde. The
tissue was then prepared for electron microscopy, as de-
scribed above for the Florida sample. For light microscopy,
thick sections were cut from the same specimen, stained
with Richardson's stain (a mixture of methylene blue and
Azure II). and viewed with bright-field optics (Zeiss. Pho-
tomicroscope II).

Whale blubber. We cut a small sample of blubber from
a much larger piece that came from a male humpback
whale that had died at sea and washed onto a beach in

222

S. K. PIERCE /:'/ I/

Figure 3. Higher magnification transmission electron micrographs showing the periodicity along the
fibers n!" the St. Augustine collagen (A), rat tail tendon collagen (B). and the Bermuda collagen (C). all at
the same magnification. The fibers of the St. Augustine and Bermuda samples are thinner than those in the
rat tail tendon a characteristic of skin collagen. The somewhat indistinct banding pattern of the Bermuda
fibers is hkcl\ due to the poor original fixation. The dense deposits in this sample also derive from the
original fi\ati\e solution.

THE GIANT OCTOPUS AND THE BERMUDA BLOB

223

Figure 4. Light micrographs of cross sections taken through the width of the mantle of Bathypolypus
arcliais. The epidermis, which consists of an epithelium underlain with a thin layer of dispersed collagen
fibers, has been removed. (Micrograph A) The mantle consists of two primary layers of muscle bundles of
about equal width. These layers are separated by a space that contains blood vessels (oval structure in the
center) (magnification = 62X). (Micrograph B) The outer surface of the mantle consists of small bundles of
longitudinal muscles (L) covered by a very thin layer of collagen (arrowhead C). Underlying the longitudinal
muscles are muscle bundles that also run longitudinally, but containing individual fibers within each bundle
that are not parallel to each other (M). Interspersed between these deeper bundles run thin radial muscles
(R) which span the width of the mantle (see micrograph A. also) (magnification = 390X). (Micrograph C)
Below the blood vessel layer run additional muscle bundles containing fibers with a wide array of orientations
(M). Small bundles of collagen (arrowheads C) containing fibers that run parallel to each other occur oc-
casionally between the muscle bundles. The hollow, tubular structure of the muscle cells is evident in this
section (magnification = 390> ). (Micrograph D) The inner surface of the mantle is also covered by a thin
layer of collagen (arrowhead C) that extends upward between (arrow C) adjacent bundles of the longitudinal
muscles (L). Although not shown in this section, the radial muscles insert between the longitudinal muscle
bundles (see micrograph A). The longitudinal muscle bundles are much larger on this aspect of the mantle
than those on the outer surface (compare with micrograph B). Immediately above the longitudinal muscles
runs a thin bundle of circularly oriented muscle fibers (magnification = 390X).

Virginia Beach County, Virginia, in October 1992. Ac-
cording to the Virginia Museum of Science collection re-
cord, this carcass was 906 cm long (about 200 cm longer
than the St. Augustine carcass) and only moderately de-

cayed. The original piece of the blubber, still attached to
the epidermis, had been preserved in formalin and de-
posited at the Smithsonian (catalogue #VMSM 921025).
Our sample was transferred into 1% glutaraldehyde, small

ff ' : , .... .-. . ..

'" .-^^^'f*' ":>...:''.. ''y^-'A

I .;;;,

..

Figure 5. Electron micrographs of Bathypolypus arcticus mantle. (Micrograph A) This cross section
shows the arrangement of the contractile proteins in bundles that radiate from the hollow center of each
tubular muscle cell (M). Adjacent muscle fibers are separated by occasional bundles of collagen fibers (C).
(Micrograph B) The fibers within the collagen bundles (C) always run parallel to each other. Adjacent layers
of perpendicularly running collagen fibers were never seen. (Micrograph C) The contractile proteins within

224

THE GIANT OCTOPUS AND THE BERMUDA BLOB

225

pieces were cut from immediately under the epidermis,
the middle, and the inner regions of the blubber, and all
were prepared for electron microscopy as described above
for the Florida sample.

Rat tail tendon and skin. Because our initial micro-
scopic examination of sections from the carcasses found
fibers that resembled collagen, we proceeded to measure
the periodicity of the banding pattern of the fibers to con-
firm that identification. We used collagen fibers from rat
tail tendon as an internal standard for these measure-
ments. A piece of tail was obtained from a white rat that
had been decapitated and immediately frozen for other
experimental purposes. The tail was thawed, skinned, and
the tail tendon removed. Pieces (1 mm 3 ) were cut from
both the skin and tendon and fixed in 2% glutaraldehyde
in 0.12 M phosphate buffer (pH 7.4). Following initial
fixation, the tissue pieces were washed in the above buffer
and then postfixed in 2% OsO 4 in the phosphate buffer.
Subsequent preparative steps were the same as described
above for the Florida sample.

Amino acid analysis

In the case of both samples, the small piece of tissue
that remained following the microscopy (256 mg, Ber-
muda, 216mg, St. Augustine) was soaked in several
changes of artificial seawater (940 mosm) overnight at 4C
to wash out the preservative solution. The tissue was then
cut into pieces (2 mm 3 ) and hydrolyzed for 24 h in 6 N
HC1 at 100C. Both samples dissolved within 15 min of
being placed into the acid. The hydrolysate was neutral-
ized with NaOH and the amino acids extracted with an
equal volume of 95% ethanol. The extract was centrifuged
at 20,000 X g(4C), and the supernatant was freeze-dried
overnight. The residue was dissolved in 0.2 N lithium
citrate buffer (pH 2.2), and the amino acid composition
of this last solution determined with an automatic amino
acid analyzer with ninhydrin detection (Beckman. System
Gold). Amino acid concentrations were calculated by the
System Gold software with norleucine as an internal stan-
dard, and then converted to residues/ 1 000 residues for
each individual amino acid. These data were graphically
compared to the amino acid compositions of the collagens
of 97 species from diverse phyla, according to the protocol
described by Matsumura (1972).

Briefly, the Matsumura protocol consists of calculating
the sum of each of the hydrophobic (valine, methionine,
leucine, isoleucine, tyrosine, and phenylalanine), hy-

droxylated (hydroxyproline, threonine, serine, tyrosine.
and hydroxylysine), and polar (aspartate, glutamate, hy-
droxylysine, lysine, histidine, ornithine, and arginine)
amino acid residues per 1000 in the collagen hydrolysate.
Each sum was divided by the grand sum of the three amino
acid groups and then multiplied by 1000 to yield a set of
three of Matsumura's R values. A vector R is then plot-
ted on a triangular coordinate graph using the R values
(Rhydrophobicj ^hydroxyiated- and R po \- dr ) as coordinates (Mat-
sumura, 1972).

Results

The electron microscopy and the amino acid analysis
indicate that both the Bermuda and St. Augustine car-
casses are made up almost exclusively of collagen fibers.

The pieces of tissue from the Bermuda and St. Augus-
tine carcasses contain layers of fibers showing banding
patterns that are characteristic of collagen, a few scattered
bacteria and bacterial spores, and no other cellular struc-
tures (Fig. 2). In both specimens, adjacent layers of the
collagen fibers run perpendicular to each other. Although
the fixation of the Bermuda sample is not very good, lon-
gitudinal sections of the two specimens appear very similar
at low, and even at high, magnifications. Certainly, the
banding periodicity along the fibers is similar both to each
other and to rat tail tendon collagen (Fig. 3). We measured
the distance between the major periods along several fibers
in several sections at both low and high magnification.
The averages were 54.3 nm (2.72 SD, n = 150 mea-
surements) for the St. Augustine collagen and 57.9 nm
(5.37 SD, ;; = 154 measurements) for the Bermuda fi-
bers. Although these values are slightly less than the usu-
ally published periodicity for collagen banding (60 nm),
control samples of both rat tail tendon (Fig. 3B) and rat
skin collagen yielded a banding periodicity of 55.7 nm
(5.6 SD) with our protocol. The diameter of the indi-
vidual fibers was also determined from micrographs of
both cross sections and longitudinal sections. The average
for the St. Augustine collagen was 109 nm (25.2 SD, n
= 68), 156 nm (34.7 SD, ;; = 106) for the Bermuda col-
lagen, and 1 73 nm (82.0 SD, n = 3 1 ) for rat tail tendon
collagen.

Microscopic examination of Bathypolypm arcticus
mantle revealed a structure that is dramatically different
from that of the two carcasses. In particular, the massive,
perpendicular collagen fiber arrangement characteristic
of the two carcasses is completely absent in the octopus

the sarcomeres of the mantle muscle cells are not in register. (Micrograph D). The radial muscles (R) that
span the width of the mantle between the rest of the mantle musculature (M) are attached to the inner and
outer layer of collagen (C) by junctional complexes (J). The dorsal surface of the mantle lies to the left of
this micrograph.

V <

*j

W V 'V; :,: .
5=5=3, ^^ ;',''; ';> ., '*;'

5-^W^.^IS^S

ir. -V'" .v : &i''?'iv jL-ii.i^ "s?f -., ''4 : 'V~

Figure 6. Electron micrographs of hlubber from McKtiplcru novaeangelae. (Micrograph A taken near
the epidermis) Large bundles of perpendicularly oriented collagen libers are interspersed with cells (most of
which appear to be h'hroblasts), lipid deposits (L), and occasional elastin fibers (E). (Micrograph B) Collagen

226

THE GIANT OCTOPUS AND THE BERMUDA BLOB

227

mantle. Instead, the bulk of the mantle is composed of
muscle. Small amounts of collagen are located in thin
sheets, one between the epithelial cell layer of the epider-
mis and the outer surface of the mantle, the other covering
the inner surface of the mantle. In addition, collagen also
occurs in an internal network of small bundles of parallel
fibers running between the muscle bundles (Fig. 4). The
banding periodicity along the octopus collagen fibers was
46.6 nm (8.6 SD, n = 90), 15% smaller than the peri-
odicity of the St. Augustine collagen.

The muscle fibers that make up the bulk of the mantle
are arranged in several layers (Fig. 4). Immediately under
a thin external collagen layer is a layer of parallel muscle
fibers that runs longitudinally from the mantle edge. These
fibers are separated laterally from each other by thin
downward extensions of the covering collagen sheet (Fig.
4B). A similar muscle layer, although consisting of larger
diameter fibers, is located immediately inside the collagen
layer that forms the inner surface of the mantle (Fig. 4D).
The center of the mantle is occupied by a space that con-
tains blood vessels (Fig. 4A). Ventrally between the
blood vessel space and the inner, longitudinal muscle
layer lie several layers of muscle fibers that run at a va-
riety of angles oblique to the cross-section (Fig. 4C). Dor-
sally, between the blood vessel space and the outer lon-
gitudinal muscle layer, the muscle fibers generally run in
the same direction as the fibers of the outer muscle bundles
(Fig. 4B). Lastly, the outer and inner collagen sheets are
occasionally connected to each other by thin, radial mus-
cle bundles that traverse the width of the mantle, perpen-
dicular to the rest of the musculature and attached to the
collagen sheets by a junctional complex (Figs. 4A, B, C;
5D). All of this structure is supported by occasional thin
bundles of parallel-running collagen fibers (Fig. 5A, B).
Altogether, there is nothing in the octopus mantle mor-
phology that resembles anything in the two relics.

The mantle muscle cells are basically hollow tubes, ta-
pered at each end. The contractile proteins are arranged
in bundles that are rectangular in cross-section and radiate
out from the hollow center of the cell like tightly packed
spokes (Fig. 5 A). The nucleus and mitochondria are lo-
cated in the center of the cell. The arrangement of sar-
comeres in the octopus mantle muscle gives the appear-
ance of oblique striations in occasional sections, but the

contractile filaments within each sarcomere are not
striated (Fig. 5C). Thick filaments, reminiscent of para-
myosin, surrounded by thin filaments are evident in cross
sections of the muscle cells. Squid mantle muscle cells
have a similar fine structure (Ward and Wainwright,
1972).

Microscopic examination of the blubber from the
humpback whale revealed a massive matrix of collagen
fibers present throughout the entire thickness of the tissue
(Fig. 6). Fat deposits and poorly fixed cellular structure
were evident between layers of collagen fibers in the sec-
tions of the blubber taken both close to the epidermis and
from the medial region (Fig. 6A, C). Neither fat nor any
remnant of cellular structure was found in either the Flor-
ida or Bermuda carcass. The sections taken from the inner
aspect of the blubber layer contained only collagen fibers
in very large bundles, interspersed with occasional larger
fibers that appeared to be elastin (Fig. 6D). At all levels
of section, the collagen fiber arrangement of the blubber
was exactly that of the Florida and Bermuda carcasses,
namely, bundles arranged in layers running perpendicular
to adjacent layers (Fig. 6A, B, C). The banding periodicity
along the whale collagen fibers was 54.6 nm (5.1 SD, /;
= 83), essentially identical to the banding of the St. Au-
gustine collagen.

The amino acid analyses of the tissues from the two
carcasses were also suggestive of collagen. Glycine ac-
counts for about one third of the amino acid residues in
both tissues, and both hydroxylysine and hydroxyproline
were present as well; these features are virtually diagnostic
of collagen (Table I). However, the amino acid compo-
sitions of the hydrolysates of the two carcasses are quite
different from each other. In particular, the St. Augustine
carcass is very rich in proline (169 residues/ 1000) and
quite low in lysine (0.4 residues/ 1000), in comparison to
both the Bermuda collagen (88 residues/ 1000 and 10 res-
idues/1000, respectively) and skin collagens from several
other species (Table I). Of course, the unusually low lysine
values in the St. Augustine sample may be an artifact of
a century in formalin. Only whale skin collagen (species
not reported in Eastoe, 1955) has proline residues/ 1000
that are anywhere near those from the St. Augustine col-
lagen (Table I). In addition, the amino acid composition
of the St. Augustine collagen is very different from that

fibers arranged in bundles running perpendicular to each other are everywhere throughout the entire thickness
of the blubber. The size of the bundles varies, depending upon location within the width of the blubber.
(Micrograph C from the middle of the blubber layer). The lipid deposits (L) in this region of the blubber
were larger and more frequent than the other areas examined. The perpendicular arrangement of the collagen
bundles surrounding the fat deposits was still evident (lower right hand corner and upper left hand corner).
(Micrograph D taken from the inner aspect of the blubber layer) The collagen bundles were very large in
this region. The expanse of collagen fibers shown here are all in cross section, and were bounded by equally
large expanses of perpendicularly running collagen fibers. Very few cells or lipid deposits were encountered
in this region of the blubber, although elastin fibers (E) were quite common.

228

S. K. PIERCE ET AL.
Table I

Comparative amino acid compositions of skin collagens of several species and the Bermuda ami Si. Augustine carcasses
(values are amino acid residues/ 1 000 residues)

Ammo
acid

Bermuda
carcass

St. Augustine
carcass

Octopus

mantle'

Squid
mantle 2

Carp-'

Whale
skin"

Shark

skin 5

Asp

52

50

53

58

48

46

43

Thr

27

28

28

26

25

24

23

Ser

47

45

52

47

43

41

61

OH-Pro

79

54

95

89

82

89

60

Pro

88

164

101

96

117

128

106

Glu

83

82

64

86

69

70

68

Gly

339

330

324

308

326

326

338

Ala

113

106

100

89

119

11 1

106

Val

25

18

19

21

18

21

25

Cys

8

4

Met

6

8

14

5

18

lieu

14

11

22

21

11

11

15

Leu

32

28

30

32

22

25

25

Tyr

5

s

3

4

3

Phe

16

14

8

12

14

13

13

OH-Lys

13.1

15.3

15.7

16.1

7.1

6

5.5

Lys

10

0.4

11

15

25

26

27

His

6

4

3

7

5

6

13

Arg

55

48

58

59

52

50

51

1 Pepsin-extracted collagen from Octopus viilgaris body wall (Kimura el <//.. 1969).
: Pepsin-extracted collagen from Todarodes pacificus body wall (Kimura et at . 1969).

3 Gelatin from skin (Piez and Gross, 1960).

4 Whale skin gelatin, species not reported (Eastoe. 1955).

5 0.5 A/ acetic-acid-extracted skin collagen from Squalus acanthus (Piez et nl , 1963).

of both squid ( Todarodes pacificus) and octopus (Octopus
vulgaris) mantle (Kimura et al. 1969), especially with
respect to the proline, hydroxyproline and lysine residues
(Table I).

Finally, the ratios of hydrophobic, hydroxylated, and
polar amino acids, calculated according to the procedures
described by Matsumura (1972), were 183, 353. and 464
for the Bermuda sample and 172, 345, and 483 for the
St. Augustine sample. These values mapped both collagens
into Matsumura's "S range" along with most striated,
structural collagens from both invertebrate and vertebrate
species. Interestingly, within the S range, the values for
the St. Augustine sample were quite close to those for a
frog (Rana temporaria) skin, while the Bermuda sample
mapped near a number of bovine collagens, as well as
chick bone, carp skin, and wallaby tail tendon collagens.
The results of this comparison must be viewed with some
caution, because the lengthy formaldehyde fixation of the
Bermuda and St. Augustine fibers may have caused some
changes in the amino acid composition of the protein. In
addition, the small amount of tissue we had to work with
limited the number of analyses we could do for statistical
purposes. Nevertheless, neither of the relic specimens
mapped close to invertebrate collagens within the S range,
including those of octopus and squid (Matsumura, 1972).

Discussion

The microscopy and amino acid analyses clearly dem-
onstrate that both the St. Augustine and Bermuda car-
casses were large pieces (extremely large, in the case of
the former) of almost pure collagen. Apparently, both
pieces of tissue had been in the ocean long enough, post
mortem, that bacterial action had recycled all but the most
resistant of the proteins. Neither carcass is from a giant
octopus nor any other invertebrate, but they are also not
from the same species.

The electron micrographs of both the St. Augustine
and Bermuda samples show fibers with the characteristic
banding pattern of collagen. The width of the repeating
unit along the fibers is essentially identical to that of rat
tail tendon collagen, and the intraperiod banding, al-
though somewhat indistinct, is typical of collagen. In ad-
dition, the whale blubber collagen banding periodicity was
the same as that of the St. Augustine collagen, while that
of the octopus mantle collagen was much less than any
of the other samples. This comparison must be viewed
cautiously, however, since the differences in the original
fixations may have produced artifacts in the banding pat-
terns.

The organization of the collagen fiber bundles in the
two relic samples is typical of dermis from a number of

THE GIANT OCTOPUS AND THE BERMUDA BLOB

229

vertebrate groups, including fish, amphibians, and reptiles,
where the bundles are distinctly layered (Moss. 1972). A
similar layering pattern of the collagen fibers was nowhere
to be found in the octopus mantle tissue we examined
here. Instead, the octopus mantle is composed mainly of
a complex network of muscle fibers containing only small
amounts of widely dispersed collagen fibers, as might be
expected of an animal so capable of shape-changing. We
found absolutely nothing in the octopus mantle mor-
phology that was comparable to the collagen fiber ar-
rangement in the two carcasses, nor has anything similar
been reported in squid or cuttlefish mantle (reviewed by
Packard, 1988). In contrast, the similarity between the
layering pattern of the collagen fiber support matrix of
the humpback whale blubber and the fiber pattern in the
carcasses is quite obvious. In addition, unlike the octopus
mantle, but very much like the Florida and Bermuda tis-
sues, collagen fibers are the main component of the blub-
ber. The whale tissue we examined here also contained
fat deposits and cellular structures that were not present
in either the Florida or the Bermuda carcass. However,
the humpback whale that provided our blubber sample
had only recently expired and did not approach the ad-
vanced state of decay of the tissues of the two relics. Thus,
the fine structure indicates that both carcasses were ac-
tually only the collagenous remains of skin, rather than
an entire animal, and the organization of the skins is rem-
iniscent of both lower vertebrates and whale blubber. In
addition, the thickness of the St. Augustine carcass
[3.5 inches (Webb's letter to Verrill dated Jan. 14, 1897)
to 10.5 inches (Webb's letter to Dall dated Feb. 12, 1897)]
is consistent with whale blubber.

Collagen fiber diameters within an organism range
widely from the very thin fibers typical of cartilage and
cornea to the comparatively thick fibers of dermis and
tendon. The thickness of the fibers within both our sam-
ples is also consistent with the diagnosis of their origin in
skin. Furthermore, while the diameter of skin collagen
fibers can vary both with the age of the organism and the
distance from the epithelium (Flint el a/., 1984), the uni-
modal distribution of fiber diameter and the tight pack-
aging of the fibers within both our specimens are typical
of a "non-active skin" characteristic of mammals (in-
cluding pygmy sperm whale blubber, Craig et al.. 1987)
and birds. In contrast, collagen fiber diameters from the
"active" skins of fish, sharks, and reptiles usually have
either a bimodal distribution or a distribution skewed to-
wards larger diameters (Craig et a/.. 1987).

The amino acid compositions of the hydrolysates of
the St. Augustine and Bermuda specimens confirm the
collagen identification indicated by our microscopy. In
addition, they provide some indication of the phyletic
source of the two carcasses. One third of the amino acid
residues in both samples are glycine. That large an amount

of glycine, taken together with the presence of hydroxy-
proline and hydroxylysine, is diagnostic of collagen. The
absence of methionine from both samples and the ex-
tremely low lysine values in the St. Augustine sample are
unusual and are, perhaps, the result of exposure to what-
ever preservation chemicals were actually used. The
number of proline residues in the St. Augustine collagen
is surprisingly high, and the level of that imino acid may
be the most important clue to its specific origin. The de-
naturation temperature of collagen is directly proportional
to its total imino acid content. In particular, the imino
acids provide the collagen molecule with a degree of ther-
mal stability required at the elevated body temperatures
of the homiothermic species (Rigby. 1968; Hochachka
and Somero, 1984). Thus, the collagens from invertebrates
and poikilothermic vertebrates are relatively low in total
imino acid residues (generally less than 200 residues/
1000). while homiothermic vertebrate collagens have
a combined imino acid total that is usually higher
(210 residues/ 1000 or greater; for examples see the data
tabulated in Kimura el al., 1969; Eastoe, 1955). Thus,
the total imino acid content of the Bermuda sample
(167 residues/ 1000), together with the rest of the amino
acid composition and morphological data presented
above, suggests that the source of that collagen was the
skin of a poikilothermic vertebrate. Indeed, the relatively
small mass of the carcass is easily within the size range of
either a large teleost or an elasmobranch. On the other
hand, the elevated imino acid content of the St. Augustine
collagen (223 residues/ 1000) together with its amino acid
composition, fine structure, and size of the carcass all in-
dicate that it was the remains of the skin of an enormous
warm-blooded vertebrate.

Altogether, and with profound sadness at ruining a fa-
vorite legend, we find no basis for the existence of Octopus
giganteus. We concur with Verrill's (1897e) and Lucas'
(1897) final words on the matter, that the St. Augustine
sea monster was "the remains of a whale, likely the entire
skin [blubber layer] . . . nothing more or less."

Acknowledgments

Samples of the St. Augustine and Bermuda carcasses
were kindly provided by Professor Joseph Gennaro of the
University of Florida and Teddy Tucker of Bermuda, re-
spectively. Samples of Bathypolypus arciicus mantle were
kindly provided by both Dr. Clyde Roper of the Smith-
sonian Museum of Natural History and Dr. Don Flecher
of the NOAA Laboratory in Woods Hole, Massachusetts.
The sample of humpback whale blubber was kindly pro-
vided by Dr. Charles Potter, also of the Smithsonian Mu-
seum of Natural History. The sample of rat tail was kindly
provided by Dr. Tom Castonguay of the Department of
Nutrition and Food Science at the University of Maryland.

230

S. K. PIERCE ET AL.

This work was supported with funds provided by the Uni-
versity of Maryland Agricultural Experiment Station and
the National Science Foundation (IBN-91 17248). It is
contribution #7 1 from the Laboratory for Biological Ul-
trastructure at the University of Maryland and contri-
bution #313 from the Tallahassee, Sopchoppy & Gulf
Coast Marine Biological Association.

Literature Cited

Craig, A. S., E. F. Eikenberry and D. A. D. Parry. 1987. Ultrastructural
organization of skin: classification on the basis of mechanical role.
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Eastoe, J. E. 1955. The amino acid composition of mammalian col-
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Ellis, R. 1994. Monster* of the Sea A. A. Knopf. New York. Pp 301-
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CONTENTS

RESEARCH NOTES

Zimmer-Faust, Richard K., Christopher M. Finelli,

N. Dean Pentcheff, and David S. Wethey

Odor plumes and animal navigation in turbulent
water flow: a field study

Toro, J. E., and A. M. Vergara

Evidence for selection against heterozygotes: post-
settlement excess of allozyme homozygosity in a
cohort of the Chilean oyster, Ostrea chilensis Philippi,
1845 . 117

Scholtz, Gerhard

Expression of the engrailed gene reveals nine pu-
tative segment-anlagen in the embryonic pleon of
the freshwater crayfish Cherax destructor (Crustacea,
Malacostraca, Decapoda) 157

ECOLOGY

Saigusa, Masayuki, and Tadashi Akiyama

The tidal rhythm of emergence, and the seasonal
variation of this synchrony, in an intertidal midge

166

REVIEWS

NEUROBIOLOGY AND BEHAVIOR

del Castillo, Jose, David S. Smith, Ada M. Vidal,
and Cesar Sierra

Catch in the primary spines of the sea urchin Eu-
cidaris tribuloides: a brief review and a new inter-

pretation

120

CELL BIOLOGY

Weidner, Earl, S. B. Manale, S. K. Halonen, and
J. W. Lynn

Protein-membrane interaction is essential to normal
assembly of the microsporidian spore invasion tube 1 28

DEVELOPMENT AND REPRODUCTION

Guirguis, M. S., and J. L. Wilkens

The role of the cardioregulatory nerves in me-
diating heart rate responses to locomotion, reduced
stroke volume, and neurohormones in Homarm

amencanus

179

PHYSIOLOGY

McGaw, I. J., and B. R. McMahon

The FMRFamide-related peptides Fl and F2 alter
hemolymph distribution and cardiac output in the
crab Cancer magister 186

McCurley, R. Skyler, and William M. Kier

The functional morphology of starfish tube feet:
the role of a crossed-fiber helical array in movement

McAuley, P. J.

Ammonium metabolism in the green hydra sym-
biosis . 210

197

Abraham, V. C., A. L. Miller, and R. A. Fluck

Microtubule arrays during ooplasmic segregation

in the medaka fish egg (Oryzias latipes) 136

Webb, Tamika A., Wendy J. Kowalski, and Richard

A. Fluck

Microtubule-based movements during ooplasmic
segregation in the medaka fish egg (Oryzias latipes) 146

SEA MONSTERS

Pierce, Sidney K., Gerald N. Smith, Jr., Timothy
K. Maugel, and Eugenie Clark

On the giant octopus (Octopus giganteus) and the
Bermuda Blob: homage to A. E. Verrill 219

Volume 188

THE

Number 3

BIOLOGICAL
BULLETIN

JUNE, 1995

Published by the Marine Biological Laboratory

THE

BIOLOGICAL BULLETIN

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CONTENTS

No. 1, FEBRUARY 1995

RESEARCH NOTES

ECOLOGY AND EVOLUTION

Fluck, Richard A.

Responses of the niedaka fish egg (Onzias latipes)
to the photolysis of microinjected nitrophenyl-

EGTA, a photolabile calcium chelator

Wittenberg, Jonathan B., and Jeffrey L. Stein
Hemoglobin in the symbiont-harboring gill of the
marine gastropod Alvinichoncha hr.ulcri

BIOMINERALIZATION

Giles, R., S. Manne, S. Mann, D. E. Morse, G. D.
Stucky, and P. K. Hansma

Inorganic overgrowth of aragonite on molluscan
nacre examined by atomic force microscopy . . .

DEVELOPMENT AND REPRODUCTION

Bates, William R.

Direct development in the ascidian Mtilgulu retor-
tijimnis (Verrill, 1871) 16

Chang, Wen-Teh, and Robert J. Lauzon

Isolation of biologically functional RNA during
programmed death of a colonial ascidian 23

Hamel, Jean-Francois, and Annie Mercier

Prespawning behavior, spawning, and development

of the brooding starfish Leplfi.<,lcri(i.\ pohins 32

Mead, Krishna S., and Mark W. Denny

The effects of hydrodynamic shear stress on fer-
tilization and early development of the purple sea
urchin Strongylocentrotus purpurcitus 4li

Thorn, Kurt, Robert M. Cerrato, and Mark L. Rivers
Elemental distributions in marine bivalve shells as
measured by synchrotron x-ray fluorescence ... 57

Gherardi, Francesca, and Paul M. Cassidy

Life history patterns of Discorsopagurus .vhniilti, a
hermit crab inhabiting polychaete tubes 68

NEUROBIOLOGY AND BEHAVIOR

Carlberg, Mats, Karin Alfredsson, Sven-Olle Nielsen,
and Peter A. V. Anderson

lain ine-like immunoreactivity in the motor nerve
net of the jellyfish C\<n>fa capillata 78

Miklosi, Adam, Jozsef Haller, and Vilmos Csanyi
The influence of opponent-related and outcome-
related memory on repeated aggressive encounters
in the paradise fish (Macropodiu operations) .... 83

Young, Craig M., and Roland H. Emson

Rapid arm movements in stalked crinoids 89

PHYSIOLOGY

Ferguson, John C.

The structure and mode of function of the water
vascular system of a brittlestar, Ophioderma upmMiiii 98

No. 2, APRIL 1995

RESEARCH NOTES

Zimmer-Faust, Richard K., Christopher M. Finelli,
N. Dean Pentcheff. and David S. Wethey

Odor plumes and animal navigation in turbulent
water flow: a field study

Toro, J. E., and A. M. Vergara

Evidence for selection against heterozygotes: post-
settlement excess of allozyme homozygosity in a
cohort of the Chilean oyster, Os/mi fhilenxis Philippi,
1845 .

11

117

REVIEWS

del Castillo, Jose, David S. Smith, Ada M. Vidal,
and Cesar Sierra

Catch in the primary spines of the sea urchin F.n-
fi<lari.\ trihulni<lr\: a brief review and a new inter-

pretation

CELL BIOLOGY

Weidner, Earl, S. B. Manale, S. K. Halonen, and
J. W. Lynn

Protein-membrane interaction is essential to normal
assembly of the microsporidian spore invasion tube

1 20

128

CONTENTS

DEVELOPMENT AND REPRODUCTION

Abraham, V. C., A. L. Miller, and R. A. Fluck

Microlubule arrays during ooplasmic segregation

in the niedaka fish egg (Or\zias latipft) 136

Webb, Tamika A., Wendy J. Kowalski, and Richard

A. Fluck

Microtubule-based movements during ooplasmic
segregation in the medaka fish egg (On'zias latipes) 146
Scholtz, Gerhard

Expression of the engrailed gene reveals nine pu-
tative segment-anlagen in the embryonic pleon of
the freshwater crayfish Cherax destructor (Crustacea,
Malacostraca, Decapoda) 137

ECOLOGY

Saigusa, Masayuki, and Tadashi Akiyama

The tidal rhythm of emergence, and the seasonal
variation of this synchrony, in an intertidal midge

NEUROBIOLOGY AND BEHAVIOR

Guirguis, M. S., and J. L. Wilkens

The role of the cardioregulatory nerves in me-
diating heart rate responses to locomotion, reduced
stroke volume, and neurohormones in Homaru*

166

PHYSIOLOGY

McGaw, I. J., and B. R. McMahon

The FMRFamide-related peptides Fl and F2 alter
hemolymph distribution and cardiac output in the
crab Cancer magister 186

McCurley, R. Skyler, and William M. Kier

The functional morphology of starfish tube feet:

the role of a crossed-fiber helical array in movement 1 97

McAuley, P. J.

Ammonium metabolism in the green hydra sym-
biosis . .... 210

179

SEA MONSTERS

Pierce, Sidney K., Gerald N. Smith, Jr., Timothy
K. Maugel, and Eugenie Clark

On the giant octopus (Octopus giganteus) and the
Bermuda Blob: homage to A. E. Verrill 219

No. 3, JUNE 1995

RESEARCH NOTE

ECOLOGY AND EVOLUTION

Stokes, M. D., and N. D. Holland

Ciliary hovering in larval lancelets (=amphioxus) 231

PHYSIOLOGY

Black, Nancy A., Richard Voellmy, and Alina M.
Szmant

Heat shock protein induction in Montastraea favro-
luta and Aijitiisui fxilliila exposed to elevated tem-
peratures 234

CELL BIOLOGY

Leys, Sally P.

Cytoskeletal architecture and organelle transport
in giant syncytia formed by fusion of hexactinellid
sponge tissues

Chen, Jiun-Hong, and Christopher J. Bayne

Bivalve mollusc hemocyte behaviors: characteriza-
tion of hemocyte aggregation and adhesion and
their inhibition in the California mussel (Mytilus rnl-
ifornianus)

Nicosia, Santo V., and Janice M. Sowinski

Cytological analysis of the urn cell complex of Si-
/jitnrulus niuliit before and after serum-induced se-
cretion . 267

241

Weedon, Michael J., and Paul D. Taylor

Calcitic nacreous ultrastructures in bryozoans: im-
plications for comparative biomineralization of lo-
phophorates and molluscs 281

Byrne, M.

Changes in larval morphology in the evolution of
benthic development by Patiriella rxigua (Asteroidea:
Asterinidae), a comparison with the larvae of Pati-
riella species with planktonic development 293

I Ian. Micha

Reproductive biology, taxonomy, and aspects of
chemical ecology of Latrunculiidae (Porifera) . . . 306

Wikfors, Gary H., and Roxanna M. Smolowitz
Experimental and histological studies of four life-
history stages of the eastern oyster, Crassostrea vir-
ginica, exposed to a cultured strain of the dinofla-
gellate Prorocentrum minimum 313

Carlon, David B., and John P. Ebersole

Life-history variation among three temperate her-
mit crabs: the importance of size in reproductive
strategies 329

Index to Volume 188

338

Reference: Bio/. Bull. 188: 231-233. (June, 1995)

Ciliary Hovering in Larval Lancelets (=Amphioxus)

M. D. STOKES AND N. D. HOLLAND

Marine Biology Research Division, Scripps Institution of Oceanography,
La Jo/la, California 92093-0202

Larvae oflancelets (=amphioxus) are of special interest
because they figure prominently in debates about vertebrate
origins (1), can sometimes grow into a giant "amphiox-
ides" form (2. 3), have a puzzling right-left asymmetry
(4), and constitute a major zooplankton resource in parts
oj the Atlantic (5). By using improved methods (6, 7) to
culture and observe healthy pre-metamorphic larvae in
relatively deep containers, we demonstrated a prominent
hovering behavior. The larvae spend most of their time
suspended in midwater by metachronal beating of epider-
mal cilia. The body is usually tilted at an angle such that
the anterior end and ventral side are oriented towards the
water surface. This posture is maintained in the dark and
in the light, although there is directional photosensitivity.
Hovering may help account for the giant "amphioxides"
and may be related to the curious asymmetry of the larval
body.

We raised developing lancelets (Branchiostoma flori-
dae) in the laboratory (6, 7) in 8-cm diameter containers
filled to a depth of 6 cm with seawater, and we used a
television camera fitted with a macro lens (8) to record
behavior at room temperature (23C). Locomotion of the
embryos (hatching to 2 days after fertilization) was by the
spiral, ciliary swimming that has been described previously
(9). In contrast, during the month-long stage of the pre-
metamorphic larva, the behavior differs markedly from
most previously published accounts, which were often
based on gradually starving animals maintained in very
shallow dishes. We found that the larvae cultured in deeper
water spent most of their time hovering almost motion-
lessly in midwater at various depths in the culture vessel.
The body of each hovering larva was oriented at an angle
of about 60 from horizontal, with the anterior end and
ventral side oriented toward the water surface (Fig. 1 ).

Received 9 December 1994; accepted 28 March 1995.

The motive force for larval hovering was the beating
of epidermal cilia in metachronal waves that pass down
the body from anterior to posterior at about 0.3 mm/s
(Fig. 2). Brief exposure to 0.1% glutaraldehyde in seawater
arrested ciliary beating, and the larvae sank, usually an-
terior first, at about 0.25 mm/s, close to values previously
found for larvae of another lancelet species (10). This
sinking indicates that the larvae do not use gas bubbles
or a high lipid content to remain suspended. Drag was
calculated by the formula for the low Reynolds number
drag of a cylinder moving parallel to its long axis ( 1 1 ). By
equating this drag to the forces (buoyant and gravitational)
acting on the larvae, we estimated that approximately
1.4 X 10~ 9 Newtons were required for ciliary hovering.

The hovering larvae maintain their characteristic
slanted posture in the dark and in moderate light. They
do, however, show some directional photosensitivity: as
seen from above, they will slowly orient themselves with
their heads away from an eccentrically placed light source
(Fig. 3). Because complete orientation requires about
20 min, it is difficult to understand what, if any, function
this could have in the natural environment. Significantly,
however, the phenomenon indicates that lancelet larvae
have directional photoreceptors. As recently proposed
(12), such receptors could well be the neurons associated
with the pigment spot at the anterior end of the cerebral
vesicle. When the head of a hovering larva is oriented
directly away from an eccentric light source, the pigment
spot would provide maximal shading to the putative pho-
toreceptive cilia of the anterior neurons.

Our results vindicate Willey (13), who clearly described
larval hovering in Branchiostoma lanceolatum, but whose
observations were never repeated and were strongly
doubted by some (10, 14). In our cultures, the few larvae
that were not hovering swam for a few seconds at infre-
quent intervals by muscular undulation or occasionally
crawled on the bottom of the container by means of their

231

232

M. D. STOKES AND N. D. HOLLAND

Figure 1 . Lateral view (from video) of a hovering. 4-day-old lancelel
larva, illuminated by white light. The body axis is oriented 60 from the
horizontal and the anterior end and ventral surface is uppermost, directed
towards the surface. The long axis of the body is at an angle (a) of about
60 from horizontal; for a random sample of 4-day-old larvae (n = 115)
the mean angle was 61 (1 SD = 6.7). Scale bar, 0.5 mm.

ft it

:*(

ii

f

\",

I

fi!

?'

I

l

r r

\ r-

V

I ?
fl
ft

3

r

if

K 1 * 1

4

i?i*.

!

(

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j

' 1

{]

i

1
Vi

cj

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u

!/

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Seconds

Figure 2. Lateral views of a hovering 4-day-old larva from video
(30 frames/s) taken at 5-frame intervals. Animals were observed under
darkfield laser Schlieren illumination (25) to visualize the beating of the
epidermal cilia. The arrowheads follow the movement of one of a series
of metachronal waves moving down the body from the anterior (top) to
the posterior (bottom) at approximately 0.3 mm/s. Scale bar, 0.25 mm.

270

180

Figure 3. Orientation of 4-day-old hovering larvae, as viewed from
above, in the presence and absence of an incandescent light source (in-
tensity approximately 2.6 log Lum/m 2 , positioned 50cm from the ob-
servation vessel). The anterior ends of the larvae point in the direction
of the arrowheads, while the length of the arrow shaft shows the number
of larvae in each 10 sector. The number of larvae in some sectors is
indicated. Orientations of a group of larvae (n - 125): (a) after 3 h of
darkness; (b) after 20-min exposure to a light source originating from
one side; (c) 20 min after moving the light to the opposite side of the
observation vessel.

epidermal cilia. Most previous studies of larval lancelets
were under conditions (very shallow culture containers
and insufficient food) that did not permit hovering, but
mainly favored ciliary crawling interspersed with brief ep-
isodes of undulatory swimming. Some studies (14, 15)
even used such unsanitary culture conditions that the lar-
vae became heavily infected with bacteria, moribund, and
attached artifactually to the substratum.

Pre-metamorphic larval lancelets are not infrequently
captured in the plankton (5, 10, 16), sometimes even far
above the ocean bottom, although it has never been de-
termined how they remain in midwater. Plankton ecol-
ogists usually imply that such larvae use muscular un-
dulation continuously or sporadically to remain in the
water column ( 10, 14, 17); however, we prefer the minority
view (9, 13) that lancelet larvae under field conditions
remain suspended chiefly by the beating of their epidermal
cilia. In comparison to undulatory swimming, ciliary
hovering probably conserves energy, favors continuous
filter feeding, is less attractive to predators, and permits
the larvae to attain a relatively large planktonic size.

Lancelet larvae lose most of their cilia at metamorphosis
(7). However, the "amphioxides" larvae (2, 3, 18, 19) delay

HOVERING LANCELET LARVAE

233

metamorphosis and evidently the loss of cilia. Such larvae
could use energy-efficient ciliary hovering to remain con-
tinuously in the plankton where they can then attain
lengths up to 1 3.8 mm. In contrast, the post-metamorphic
lancelets that are occasionally (and temporarily) present
in the plankton (20) must remain in the water column
entirely by muscular undulation, which is probably less
efficient than ciliary hovering.

The ciliary hovering of lancelet larvae could well be
related to their curious asymmetry, which has been ex-
plained by some as inherited from an ancestor (21, 22),
but by others as a larval adaptation (23, 24). We favor
the latter view and suggest that larval hovering co-evolved
with a laterally compressed body that, by optimizing the
surface-to-volume ratio, increased the effectiveness of the
ciliary propulsion. Such lateral compression may in turn
be related to the asymmetric displacement of the larval
mouth to one of the broad body surfaces [as previously
suggested by Bone (2)] simply to ensure an opening large
enough to permit effective filter feeding.

Acknowledgments

We thank J. Lawrence and D. Mahoney for providing
facilities and assistance in Tampa, Florida, and P. K.
Dayton and L. Z. Holland for providing critical comments
on this manuscript.

Literature Cited

1. Cans, C. 1989. Stages in the origin of vertebrates: analysis by means
of scenarios. Biol. Rev 64: 221-268.

2. Bone, Q. 1957. The problem of the 'amphioxides' larva. Nature
180: 1462-1464.

3. Wickstead, J. H. J. 1964. The status of the 'amphioxides' larvae.
J. Linn. Soc. (Zool.) 45: 201-207.

4. Jefferies, R. P. S. 1986. The Ancestry of the I'erlehrali-s. British
Museum of Natural History, London.

5. Flood, P. R., F. Gosselk, and J. G. Braunn. 1982. Larvas de
Branchiostoma" en el area de "upwelling" del NW de Africa. Boll,
lust. Kxpan. Oceanogr. 1: 73-86.

6. Holland, N. D., and L. Z. Holland. 1994. Embryos and larvae of
invertebrate deuterostomes. Pp. 21-32 in Essential Developmental
Biology: a Practical Approach. C. Stern and P. W. H. Holland, eds.
IRL Press, Oxford.

7. Stokes, M. D., and N. 1). Holland. 1995. Embryos and larvae of
a lancelet. Branchiostoma tloriilue. from hatching to metamorphosis:
growth in the laboratory and external morphology. Acta Zool. Stockh
76: 105-120.

8. Holland, N. D., J. R. Strickler, and A. B. Leonard. 1986. Particle
interception, transport and rejection by the feather star O/igometra
serripinnu (Echinodermata: Crinoidea), studied by frame analysis
of videotapes. Mar. Biol. 93: 1 1 1-126.

9. Bone, Q. 1958. Observations upon the living larva of amphioxus.
Puhhl. Sta:. Zool. Napoli 30: 458-471.

10. Webb, J. E. 1969. On the feeding and hehaviour of the larva of
Branchiostoma lanceolatum. Mar. Biol. 3: 58-72.

1 1. Cox, R. G. 1970. The motion of long, slender bodies in viscous
tluids. Part I. General theory. J. Fluid Mech. 44: 791-810.

12. Lacalli, T. C., N. D. Holland, and J. E. West. 1994. Landmarks
in the anterior central nervous system of amphioxus larvae. Phil.
Trans Roy. Soc. B 344: 165-185.

13. VV'illey, A. 1891. The later larval development of amphioxus. Q.
J. Microsc. Sa. 32: 183-234.

14. van Wijhe, J. W. 1927. Observations on the adhesive apparatus
and the function of the ilio-colon nng in the living larva of amphioxus
in the growth period. Proc. Konin. Akad. Iff/. 30: 991-1003.

15. Orton, J. H. 1914. On a hermaphrodite specimen of amphioxus
with notes on experiments in rearing amphioxus. Mar. Biol. Assoc.
U. K. 10: 506-512.

16. Wickstead, J. H., and Q. Bone. 1959. Ecology of acraniate larvae.
Nature 184: 1849-1851.

17. Wickstead, J. H. 1975. Chordata: Acrania (Cephalochordata). Pp.
308-314 in Reproduction of Marine Invertebrates. A. C. Giese and
J. S. Pearse, eds. Academic Press, New York.

18. Goldschmidt, R. 1906. Amphioxides und Amphioxus. Zool. An:.
30: 443-448.

19. Gibson, H. O. S. 1910. The Cephalochorda: "Amphioxides."
Trans. Linn. Soc. Loud. Zool. 13: 213-256.

20. Boschung, H. T., and R. F. Shaw. 1988. Occurrence of planktonic
lancelets from Louisiana's continental shelf, with a review of pelagic
Branchiostoma (order Amphioxi). Bull. Mar. Sci. 43: 229-240.

21 Klaatsch, H. 1898. Uber den Bau und die Entwickelung desTen-
takelapparates des Amphioxus. Verh. Anal. Ges. Jena 12: 184-195.

22. MacBride, E. W. 1909. The formation of the layers in amphioxus
and its bearing on the interpretation of the early ontogenetic processes
in other vertebrates. Q J Microsc. Sci. 54: 279-345.

23. van Wijhe, J. W. 1913. On the metamorphosis of amphioxus lan-
ceolatus. Proc Konin Akiul. \Velcn. Sci. Sect. 16: 574-583.

24. Garstang, W. 1928. The morphology of the Tunicata, and its
bearings on the phylogeny of the Chordata. Q J. Microsc. Sci. 72:
51-187.

25. Strickler, J. R. 1985. Feeding currents in calanoid copepods: two
new hypotheses. Pp. 459-485 in Physiological Adaptations for Ma-
rine Animals M. S. Laverack, ed. Soc. Exp. Biol.. New York.

Reference: Bio/. Bull. 188: 234-240. (June, 1995)

Heat Shock Protein Induction in Montastraea

faveolata and Aiptasia pallida Exposed

to Elevated Temperatures

NANCY A. BLACK 1 , RICHARD VOELLMY 2 , AND ALINA M. SZMANT 1 *

] Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science,

University of Miami, Miami, Florida 33149; and 2 Department of Biochemistry and Molecular

Biology. School of Medicine. University of Miami, Miami, Florida 33136

Abstract. Frequent widespread episodes of coral bleach-
ing have made researchers aware of the sensitivity of reef
corals to moderately elevated temperatures and led us to
investigate mechanisms of temperature stress tolerance in
this group. One such mechanism may be the induced syn-
thesis of heat shock proteins (hsps), which have been
shown to play a role in thermotolerance in other organ-
isms. However, induced synthesis of hsps in scleractinian
corals was not reported until recently.

Experiments were conducted in which Montastraea
faveolata was exposed to high temperatures (up to 35C)
for short periods (2 h). Under the conditions tested, the
corals produced seven different hsps with approximate
molecular weights of 95, 90, 78. 74, 33, 28, and 27 kDa.
Another zooxanthellate species, the sea anemone Aiptasia
pallida, also synthesized hsps during temperature stress,
but fewer and with different molecular weights (82, 72,
68, and 48 kDa) than those produced by Montastraea. It
now remains to be determined whether hsps are involved
in differences in thermotolerance and susceptibility to
bleaching within and between the various species of
Montastraea. and between species of reef cnidarians.

Introduction

Heat shock proteins (hsps) are also referred to as "stress
proteins" because their expression can be induced by a
number of stressful conditions, only one of which is heat
shock (reviewed in Craig, 1985). These proteins are highly
conserved evolutionarily: they have been found in all

Received 8 June 1994; accepted 17 March 1995.
* Author to whom correspondence and reprint requests should be
sent.

phyla examined thus far, from bacteria and yeast to hu-
mans.

Several functions have been proposed for hsps, includ-
ing the transport of newly transcribed proteins to the en-
doplasmic reticulum and mitochondria and involvement
in the insertion of these proteins into these organelles (re-
viewed in Gething and Sambrook, 1992; Parsell and
Lindquist, 1993). Hsps may also play a role in translation
and, during stress, may interact with unfolding (denatur-
ing) proteins to either keep them in a refoldable confor-
mation or target them for degradation (Welch, 1992). The
production of hsps has also been correlated with the ac-
quisition of enhanced thermotolerance (Parsell and Lind-
quist. 1993). Because of their important functions in nor-
mal metabolic and repair processes, moderate amounts
of many hsps are present in unstressed cells at all times,
but synthesis increases after a stress. In some cases, this
increase in hsp synthesis is accompanied by a decrease in
the production of other non-hsp proteins (Parsell and
Lindquist, 1993).

Interest in the effects of elevated temperature on scler-
actinian reef-building corals and their ability to produce
heat shock proteins has increased with recent frequent,
widespread episodes of coral bleaching (Glynn. 1991;
Williams and Bunkley- Williams, 1990). Corals have a
complex symbiotic relationship with photosynthesizing
dinoflagellates, called zooxanthellae, that are contained
within the gastrodermal cells of corals. Bleaching results
from the loss of the zooxanthellae, the degradation of
chlorophyll pigments within zooxanthellae, or a combi-
nation of both. Many conditions have been shown to in-
duce bleaching or are implicated in its production (re-
viewed in Williams and Bunkley- Williams, 1 990), but the

234

HEAT SHOCK PROTEINS IN CORALS

235

condition most often correlated with recent bleaching
events is prolonged exposure to moderately elevated tem-
peratures.

Hsp induction has been reported in a number of cni-
darians. Two thermotolerant species of the hydrozoan
Hydra were found to increase the synthesis of hsp60 when
exposed to elevated temperature or to the chemical in-
ducers sodium azide and cadmium chloride; six more
thermosensitive species failed to produce this hsp (Bosch
el a/., 1988). In the scyphozoan jellyfish Aurelia aurita,
six hsps were identified when various developmental stages
were exposed to elevated temperatures for a few hours
(Black and Bloom, 1984). An anemone, Anemonia viridis,
produced six different hsps in response to elevated tem-
peratures or copper chloride (Miller el a/., 1992). Hsp70
was found in the coral Goniopora djiboutiensis (Sharp el
ai, 1994), and other reports of hsp production in zoo-
xanthellate cnidarians have been presented at recent sci-
entific meetings.

The objective of this research was to determine whether
the Caribbean reef coral Montastraeafaveolata (Ellis and
Solander) formerly Montastmea annularis (Knowlton
el ai, 1992; Weil and Knowlton, 1994) produces hsps;
and if so, which ones. Individual colonies and sibling spe-
cies of this coral have various degrees of susceptibility to
temperature-induced bleaching (Gates, 1990; Szmant and
Gassman. 1990; Black, 1993). Samples of this coral were
subjected to acute exposure to higher temperature fol-
lowed by variable recovery periods at control tempera-
ture and then tested for elevation of hsp synthesis. Sea
anemones, Aiptasia pallida (Verrill), were also used to
work out techniques and to illustrate hsp production by
another zooxanthellate cnidarian species. The exposure
conditions tested for each species are summarized in
Table I.

Methods and Materials

Collection and maintenance of the animals

Multiple cores from large colonies of Montastraea
faveolata were collected from a depth of 5-7 m on a reef

near Joulters Cay (25 19' N, 78 05' W), north of Andros
Island, Bahamas, in 1988 and 1989. A pneumatic drill
fitted with a hole-saw and powered by compressed air
from a scuba tank was used to obtain short cores, 5 cm
in diameter, with live tissue only on the top. The corals
were used in nondestructive and nonstressful laboratory
experiments involving temperature and light exposure and
then returned to uncontrolled ambient laboratory con-
ditions for several months before the start of these exper-
iments.

Sea anemones of the species Aiptasia pallida were ob-
tained from holding tanks at the Florida Keys Marine
Laboratory on Long Key in February 1993. They were
allowed one week to acclimate to laboratory conditions
before experiments were started.

Specimens were maintained in flowing, sand-filtered
seawater at ambient seawater temperature (ca. 28C for
the corals used in experiment 1, 20-24C for the cor-
als and anemones used in experiments 2-4, Table I)
under cool-white fluorescent or metal halide lights (ca.
200/iEin m~-s"' of light) on a 14:10 hour light:dark
schedule. Thawed frozen brine shrimp were provided as
food once a week.

Incubation procedures

For all experiments, the organisms were incubated in
filtered (Whatman GF/A) seawater with 5 /iCi/ml of either
3 H-leucine ( 156.0 or 179.6 Ci/mmol specific activity) or
"S-methionine/cysteine (1 141.0 Ci/mmol specific activ-
ity) purchased from New England Nuclear.

Incubation was either in glass dishes with gentle aeration
in a dark incubator or in covered beakers in a shallow
water bath. The specimens were placed in the containers
with the radiolabeled amino acid at the beginning of the
temperature exposure, left there for the duration of the
recovery periods of each experiment, then rinsed three
times with filtered seawater (to remove excess radiolabel
before processing). Each treatment group contained two
or three corals or four anemones, but anemones were
pooled for processing.

Table I

Summary of the animals, conditions, and isotopes used in the heat shock experiments

Exp. no.

Species

Control temp. (C)

Treatment conditions

Recovery conditions

N per treatment*

Isotope

1

Montastraea faveolata

28

31C: 1 week. 34C: 2 h

none

T

3 H

2

M. faveolata

22

27,29, 31, 33. 35C: 2 h

22C: 2 h

3

35 S

3

M. faveolata

22

33C: 2 h

22C: 4 h

2 corals

"s

Aiptasia pallida

4 anemones

4

A. pallida

22

27, 29, 31, 33. 35C: 2h

22C: 2 h

4

35 S

5

it

33C 2 h

22C: 0. 1,2, 4, 24 48 h

4

35 S

* Because of their small size, tissues of sea anemones were pooled for analysis.

236

N. A. BLACK ET AL

Tissue preparation

Coral tissues were removed from the skeletons with a
jet of 0.1 M Tris (pH 6.8) sprayed through an airbrush;
resulting tissue slurry volumes ranged from 3 to 13 ml.
Preparations were kept on ice throughout processing. The
tissue slurry was homogenized and centrifuged (850 X g,
15 min. 4C) to separate the coral animal fraction (su-
pernatant) from the zooxanthellae (pellet). Anemones
were ground in 2 ml of cold Tris buffer (0.1 M, pH 6.8)
in a glass tissue grinder with a Teflon-tipped pestle. Zoo-
xanthellae and unbroken cells were removed by centrif-
ugation (6600 X g. 20 min, 4C).

In the first experiment, the supernatants were centri-
fuged again at 1 2,000 X g ( 1 5 min, 4C) to remove cellular
debris and larger organelles (Ford and Graham, 1991).
To 100 n\ of the latter supernatants was added 50 n\ of
2X protein loading buffer [PLB; 0. 1 25 M Tris, 20% glyc-
erol, 2% 2-mercaptoethanol, and 4% SDS (Gallagher and
Smith, 1 99 1 )]. The amount of radioactivity in a subsample
of each supernatant was measured with a Beckman model
LSI 80 scintillation counter. The remainder of each sample
was boiled for 1 min and frozen at -80C.

In the later experiments, proteins were precipitated
from the supernatants with 8% trichloroacetic acid (TCA).
Proteins were pelleted by centrifugation (12,000 X g,
20 min. 4C). The pellets were rinsed three times with
8% TCA and three times with acetone to remove TCA-
soluble and acetone-soluble contaminants, and then re-
dissolved in 1 ml of PLB. A subsample was removed for
scintillation counting and the rest was boiled and frozen
at -80C.

Gel electrophoresis and autoradiography

Proteins were separated by one-dimensional SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) (10%
running gel; 3% stacking gel). Equal amounts of radio-
activity per sample (approx. 400,000 dpm) were loaded
in each lane, and protein molecular weight standards were
run in parallel. The electrophoresis buffer consisted of
0.025 M Tris, 0. 1 92 M glycine, and 0. 1 % SDS (Laemmli,
1970). After electrophoresis, gels were fixed (50% meth-
anol, 5%- glacial acetic acid, 30 min), rinsed in water (three
times, 10 min each), incubated in a solution of 10% sal-
icylic acid and 3%> glycerol (20 min), and vacuum dried
at 80C (2 h.). Autoradiograms were produced by expos-
ing Kodak X-Omat AR film to the dried gels for several
days at -80C. The approximate molecular weights (M r )
of protein bands on the autoradiograms were estimated
by comparison with the positions of molecular standards
on the gels.

Because the gels were loaded with equal amounts of
radiolabel rather than equal amounts of protein, the results
provide us with a relative comparison of how radiolabel

was incorporated within each treatment, but do not allow
us to compare rates of synthesis of the various hsps be-
tween treatments.

Densitometry

The autoradiogram from experiment 4 (Fig. 2A). in
which specimens of Aiptasia pallida were incubated in a
temperature series from 22 to 35C (Table I), was ana-
lyzed with densitometry to compare quantitatively the
densities of the hsp68 and hsp72 bands from animals ex-
posed to the various temperatures. The densitometry im-
age analysis system consisted of a NEC single-chip color
video camera to produce the image, a Targa + 64 frame-
grabber board to capture the image, and the MOCHA
software package (Jandel Scientific) to do the density scan
of the image. Equal areas of each temperature lane were
scanned, and the density values associated with each band
summed. Figure 3 includes the density scans for each lane
(A), and histograms that present the density and the per-
centage of total density of each hsp in each lane (B, C).

Results

Exposure to elevated temperature increased the syn-
thesis of several discrete proteins (hsps) in both corals and
sea anemones. Each species produced its own character-
istic set of hsps. The results of each experiment are sum-
marized separately for each species.

Montastraea faveolata (Fig. 1)

31 & 34C vs. 2SC. Corals exposed to 34C expressed
several hsps with M r of 33. 74, 78, 90, and 95 kDa. In
this experiment, but not subsequent ones, there was re-
duced synthesis of other proteins such as a 40 kDa protein
presumed to be actin and an unknown 99 kDa protein
(Fig. la). The patterns of proteins synthesized at 28 and
31C were similar and did not include any discernible
hsp bands.

27-35C vs. 22C. Hsp synthesis was tested at five
temperatures from 27 to 35C. No hsps were synthesized
at 27, 29, or 3 1C (not shown). The only hsps that were
distinguishable in this experiment were hsp74 in the 33C
(faint) and 35C (darker) treatments as well as two bands
of ca. 27 and 28 kDa at 35C (Fig. Ib). A repeat of the
33 C exposure experiment produced a pattern of hsps at
90. 78, and 74 kDa (Fig. Ic) similar to that seen before
at 34C.

Aiptasia pallida (Figs. 2. 3)

27-35C vs. 22C. In this experiment, anemones were
exposed to five different 2-h heat treatments (Fig. 2a). At
22 (control), 27, and 29C, the anemones did not appear
stressed. At 3 1 C, however, the anemones were partially

HEAT SHOCK PROTEINS IN CORALS

237

B

28

31 34

22

i

,95
-90
-78
74

-74

-

*

*

-33

-28
27

f^rilMh ^^fe ^^ife

Figure 1. Autoradiograms showing proteins synthesized by Montastraea faveolata after exposure to
various temperatures. (A) Synthesis after 1 week at 31 C or 2 h at 34C compared to controls (28C). The
positions of hsps produced at approximately 95, 90, 78, 74. and 33 kDa are indicated. (B & C) Synthesis
after 2 h at 33 or 35C compared to controls (22C). The positions of hsps produced at approximately 74,
28. and 27 kDa (B) and 90. 78. and 74 kDa (C) are indicated. Each lane represents the proteins of an
individual coral, with two or three replicate corals per temperature treatment, a = protein presumed to be
actin.

contracted during heat exposure, but recovered to normal
appearance during the 2-h recovery period at 22C. At
33 and 35C, the anemones were severely stressed: all
were totally contracted and some were lying on their sides.
The 33C anemones returned to normal appearance dur-
ing the recovery period, but the 35 C anemones did not.
Only two hsps (68 and 72 kDa) could be distinguished
visually in this experiment (Fig. 2A). The densitometry
was used to quantify the amounts of radiolabel incorpo-
rated into each band with respect to the total amount of
radiolabel in protein in each sample (Fig. 3). The density
scans for each lane are presented in Figure 3A and the
relative densities within the hsp68 and hsp72 bands in
Figures 3B and 3C, respectively. The 72 kDa hsp was
present as a faint band in the control (22C) and 27C
anemones (2.8% of total density for each), with up to a
32% increase in the 29, 31, and 33C animals (3.7%,
3.2%, and 3.3% of total, respectively), and then declined
at 35C (to 2.3% of total). Hsp68 was present at very low
levels (1.9% of total) at the lowest two temperatures, in-

creased by 30% to 50% at the intermediate temperatures,
(29, 31, and 33C: 2.6%, 2.4%, and 3.0% of total, re-
spectively), and then increased dramatically by 280% at
the highest temperature (35C: 7.25% of total). In some
experiments additional hsps were detected at 82 and
48 kDa (Fig. 2B), that were apparently induced to a lower
extent than the hsps 68 and 72.

33C vs. 22 C. variable recovery period. This experi-
ment examined the turnover of hsps synthesized during
the temperature stress and post-stress recovery periods.
Increased amounts of hsps 68 and 72 (and 48 and 82, to
a minor extent) were synthesized after a 2-h heat treatment
at 33C (comparison of lanes C and in Fig. 2C; especially
notice the increased density of the hsp68 and hsp82 bands
in lane 0). Both of these hsps remained elevated for up
to 24 h at 22C (Fig. 2C), but were less evident in the 4-
h and 48-h samples. Because of the experimental design
(specimens were incubated with the radiolabel during both
the stress and recovery periods), we cannot distinguish
between radiolabeled hsps produced during the stress itself

238

A

N. A. BLACK ET AL.

B C

22 27 29 31 33 35 22 33 C 1 2 4 24 48 C

ill*

72
-68

Figure 2. Autoradiogram showing proteins synthesized by Aiptasia palliila after exposure to various
temperatures. (A) Synthesis after 2 h at 27. 29. 31. or 35C compared to controls (22C). (B) Synthesis
after 2 h at 33C compared to controls (22C). (C) Synthesis after 2 h at 33C with variable recovery times
(for 0. 1.2, 4, 24. or 48 h) at 22C compared to controls (22C). Each lane contains a sample consisting of
pooled tissues from four sea anemones, a = protein presumed to be actin. The positions of hsps produced
at approximately 72 and 68 kDa (A) and 82. 72. 68. and 48 kDa (B, C) are indicated.

and subsequently during recovery. However, in other
studies in which we have incubated reef corals with ra-
diolabeled amino acids, the medium was depleted of more
than 80% of the radiolabel within the first 3 h (FitzGerald
and Szmant. 1988, in prep.). Thus, it is likely that most
of the radiolabeled hsps (and other proteins) in the samples
were synthesized during the stress period and the early
hours of the recovery period.

Discussion

Several hsps were found in both Montastraeafaveolata
and Aiptasia pallida after brief exposures to elevated tem-
perature. Overall, hsp74 for Montastraea, and hsps 68
and 72 for Aiptasia were the dominant hsps produced in
response to heat stress. In the latter species, hsp72 appears

to be constitutive, with moderately increased synthesis
after moderate temperature shock, and down-regulation
at the highest temperature tested (35C). In Aiptasia,
hsp68 appears to be the major one produced with more
extreme temperature stress. Given the near-background
levels of this hsp in the control and 27C samples. hsp68
may be inducible rather than constitutive.

Comparison of our results with those reported for other
cnidarian species reveals a few common hsps and hsp
families (Table II). According to Craig (1985). nearly all
species produce hsps in the same three size classes: hsp90
(80 to 90 kDa), hsp70 (68 to 74 kDa), and small hsps (18
to 30 kDa). Variability regarding the number and size of
hsps is generally greater for small hsps. Five of the cni-
darian species studied produced one hsp in the 80 to 90

HEAT SHOCK PROTEINS IN CORALS

239

200

100

200 300

DISTANCE

400

500

22 oC 27 oC 23 oC 31 oC 33 oC 36 oC

2000

8

6 5

u.
_0

27 29 31 33

TEMPERATURE

35

IfflBand Density % Total Density

2000

27 29 31 33

TEMPERATURE

35

Figure 3. (A) Density profile along the central portion of each lane
shown in Figure 2A, extending lengthwise about the mid-third of each
lane. The scan width was about two-thirds the width of each lane. See
text tor further details. (B) The total density assigned to the hsp68 band
of each sample, and as a percentage of total lane density. (C) Same as
(B) but for the hsp72 band.

be applied to determine whether the hsps detected are
members of the known hsp families. This approach has
been used recently by Sharp cl al. ( 1 994) to identify mem-
bers of the hsp70 family in a coral and a sea anemone
(Table II).

Hsps in Aiptasia appear to have a moderate turnover
rate. Radiolabeled hsps synthesized during and after the
2-h heat shock were still present 24 h after temperature
exposure, but had apparently been degraded by 48 h after
treatment. Experiments should be conducted to determine
the duration of heat stress during which these animals can
continue to synthesize hsps.

An attempt to determine whether zooxanthellae in
Montastraea produce hsps failed because tissue homog-
enates of the zooxanthellae preparations contained too
little radiolabel (4% or less) to allow detection of hsps.
Although hsps have not yet been reported in zooxan-
thellae, the degradation and loss of pigments from the
chloroplasts of zooxanthellae in bleached corals (Glad-
felter, 1988; Kleppel et al.. 1989; Porter et al.. 1989;Glynn
and D'Croz, 1990; Szmant and Gassman, 1990; Black,
1993) would indicate that the zooxanthellae are heat
stressed and could also benefit from hsps.

In this study, we have shown that Montastraea faveolata
can produce hsps in response to short exposures (several
hours) to relatively highly elevated temperatures (>33C).
Our experimental conditions differ from those associated
with natural bleaching, which is thought to be caused by
exposure to more moderately elevated temperatures (29-
30C) for longer periods (several weeks to months) (Glynn
and D'Croz, 1990; Black, 1993; Black and Szmant. in
prep.). No hsp production was observed in corals exposed
to moderately elevated temperatures (27-31C) for
2 hours to one week. In addition, the duration of our ex-
periments was too short to expect any visible bleaching.
Susceptibility of corals to bleaching varies between dif-
ferent species (Williams and Bunkley- Williams, 1990).
The present results suggest that further work is warranted
to determine whether hsps are involved in differences in
thermotolerance and susceptibility to bleaching within
and between reef cnidarian species.

range, and all but Hydra produced one or more hsps in
the 68 to 74 range.

As has been observed for other species, the number and
relative quantities of hsps synthesized in our experiments
increased with increasing severity of heat treatment. There
was, however, variability between experiments in the
amount and type of hsps produced, especially in Mon-
tastraea. Some of this variation may be due to individual
differences in thermotolerance or ability to turn on hsp
synthesis. This possibility should be addressed by repeating
these experiments with greater replication. In addition,
immunochemical techniques (i.e., Western blots) should

Conclusions

( 1 ) Both Montastraea faveolata and Aiptasia pallida
produce heat shock proteins in response to short periods
of extreme but sublethal thermal stress. Most of the hsps
produced appear to fall into a molecular weight range
similar to those found in other cnidarian species. Western
blots are needed to learn whether the hsps produced by
Montastraea and Aiptasia belong to the same families as
the hsps found in other groups. (2) The involvement of
heat shock proteins in coral bleaching remains to be de-
termined.

240 N. A. BLACK ET AL

Table II

Comparison of the hem v/jor/,- proteins ohwn'ed in six different cnidarian species

Species

Class Molecular weight (kilodaltonsl

Hvdra attenuatu*

Hvdrozoa 28

60

80

Amelia aurelia*

Scyphozoa 39 45

68 70

83

93

Anemonia n//i//\'

Anthozoa 30 31 35 39

69

82

Anemonia vindis' 1

Anthozoa 28 29

(joniopora djiboutiensis*

Anthozoa

70

Aiptasia pallida'

Anthozoa 48

68 72

82

Mi 'iiiaslraea faveolata*

Anthozoa 27 28 33

74

78 90

95

"Bosch et al. (1988).
"Black and Bloom (1984).
c Miller el al. (1992).
d Sharp el ul (1994).
' This studv.

Acknowledgments

We thank Ruben Baler for his advice and assistance
and the Florida Keys Marine Laboratory for providing
the anemones. The manuscript was greatly improved by
helpful comments from two anonymous reviewers. This
research was supported by NSF grant OCE-91 14424 to
A. M.S.

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1988. Thermotolerance and synthesis of heat shock proteins: These

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FitzGerald, L. M., and A. M. Szmanl. 1988. Amino acid metabolism.

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Gates, R. D. 1990. Seawater temperature and sublethal coral bleaching

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Gelhing, M. J.. and ,1. Sambrook. 1992. Piotein folding in the cell.

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Glynn, P. \V. 1991. Coral reef bleaching in the 1980's and possible
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Glynn, P. \\ ., and L. D'Croz. 1990. Experimental evidence for high
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Kmmlton, N., E. Weil, L. A. Weigt, and H. M. Guzman. 1992. Sibling
species in Montastraea annularis, coral bleaching, and the coral cli-
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Miller, D., B. E. Brown, V. A. Sharp, and N. Nganro. 1992. Changes
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Parsell, D. A., and S. Lindquist. 1993. The function of heat-shock
proteins in stress tolerance: degradation and reactivation of damaged
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Porter, J. \\ ., W. K. Fitt. H. J. Spero, C. S. Rogers, and M. W. White.
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sponses. Proc. Null. Acad. Sci. USA 86: 9342-9346.

Sharp, V. A., D. Miller, J. C. Bythell, and B. E. Brown. 1994.
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sponse to heat shock. / Exp. Mar Biol. Ecol. 179: 179-193.

Szmant, A. M., and N. J. Gassman. 1990. The effects of prolonged
bleaching of the reef coral Montaslrea annularis on the tissue biomass
and reproduction. Coral Reefs 8: 217-224.

Weil, E., and N. Knowlton. 1994. A multi-character analysis of the
Caribbean coral Montastraea annularis (Ellis and Solander, 1786)
and its two sibling species, M faveolata (Ellis and Solander. 1786)
and M tninksi (Gregory. 1895). Bull Mar Sci 55: 151-175.

Welch, W. J. 1992. Mammalian stress response: cell physiology, struc-
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Williams, E. H.. and I,. Bunkley-Williams. 1990. The world-wide coral
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Reference: Biol. Bull 188: 241-254. (June, 1995)

Cytoskeletal Architecture and Organelle Transport

in Giant Syncytia Formed by Fusion

of Hexactinellid Sponge Tissues

SALLY P. LEYS

Department of Biology. University of Victoria. Victoria. British Columbia V81V2Y2. Canada

Abstract. Dissociated tissue from the hexactinellid
sponge Rhabdocalyptus dawsoni adheres to coated sub-
strates and aggregates by the fusion of tissue pieces to
form a giant syncytium. Video microscopy shows that the
pieces contact each other by way of lamellipodia or filo-
podia. Fusion, corroborated by evidence of dye spread,
occurs about 1 hour after plating and is characterized by
two-way transport of individual organelles. including nu-
clei, at an average rate of 2. 1 5 yum s~', and bulk streaming
of cytoplasm at an average velocity of 1.72 //m-s~'. In
the cellular sponge Haliclona, by contrast, dye does not
spread through aggregates and no streaming can be seen.
That transport in Rhabdocalyptus is microtubule-based
is indicated by the reversible inhibition of streaming
caused by colcemid and nocodazole. Immunofluorescence
and electron microscopy reveal an extensive network of
microtubule bundles within the aggregates. The cyto-
skeleton also includes microfilament bundles that traverse
aggregates and run around the periphery and giant, actin-
dense rods that extend from the edges. Cytochalasin B
reversibly disrupts the microfilamentous framework
without blocking streaming.

In contrast to demosponges where the cytoskeleton is
organized on the basis of individual cells, in hexactinellids
it provides a supporting framework and transport path-
ways within vast, multinucleate tissue masses. If we take
this preparation as a model for tissue organization in the
intact sponge, these findings support the view that hex-
actinellids are syncytial organisms, probably the largest
in the animal kingdom, and suggest that food products
may be distributed through the sponge intracellularly

Received 14 December 1994; accepted 28 March 1995.
A video of cytoplasmic streaming and fusion in Rhabdocalyptus daw-
son: is available from the author at cost.

rather than by wandering amoebocytes. The findings
strengthen the case for establishing the Hexactinellida as
a subphylum within the Porifera.

Introduction

The syncytial organization of hexactinellid sponge tis-
sue has been in question since the histology of dredged
specimens was first examined. These animals, commonly
known as glass sponges because of their siliceous skeleton,
inhabit deep waters throughout the world's oceans, mak-
ing their retrieval in good condition difficult. Early sponge
researchers reported that there were no discernible mem-
brane boundaries between nuclei (Schulze, 1887; Ijima,
1901). Although two cell types, archaeocytes and theso-
cytes, could be distinguished, most of the sponge was
thought to be syncytial. At the time, however, many ani-
mal tissues were considered to be syncytial, including the
pinacoderm of demosponges (Hyman, 1940), but phase
contrast and electron microscopy have since revealed their
cellular nature. Thus, the idea that hexactinellids were
syncytial animals received little serious attention from
modern sponge workers until recently.

Reiswig ( 1979) investigated hexactinellid histology us-
ing several populations accessible by scuba on the coast
of British Columbia, Canada. Although his first attempts
at electron microscopy with Aphrocallistes vastus and
Chonelasnui caly.\ were thwarted by difficulties with ul-
trastructural preservation, Reiswig nonetheless found no
evidence to contradict Schulze's and Ijima's conclusions.
Perseverance with electron microscopy with Rhabdoca-
lyptus dawsoni (Fig. la) resulted in an improved fixation
technique that provided ultrastructural evidence for two
kinds of reticular, multinucleate tissues, the trabecular
syncytium and the choanosyncytium, and for several types
of cells (Mackie and Singla, 1983) (Fig. Ib). Most inter-

241

242

S. P LEYS

Figure 1. (A) Drawing of the hexactinellid sponge Rhabdocalyptus dawmmi with its osculum partially
cut open. (B) Diagram illustrating the tissues of whole sponges. Water flows into choanoflagellate chambers
(FC) as shown by the curved arrows, and is pumped out directly up from the center of the chamber.
Trabecular syncytium (TS), collar body (CB), choanoblast (Chb), plugged junction (PJ). mesohyl (M). dermal
membrane (DM), nucleus in the syncytium (N s ), nucleus of an archaeocyte (N af ), spherulous cell (SC),
archaeocyte (AR).

estingly, the syncytial cytoplasm was found to be con-
nected to cellular components either by open cytoplasmic
bridges or by a unique osmiophilic perforated plugged
junction (Mackie, 1981). Sclerocytes were the only cell
type not connected by plugs to the syncytium.

Concurrent electrophysiological studies with Rhabdo-
calyptus showed that following mechanical or electrical
stimulation this sponge was capable of propagating signals
at a rate of 0.26 cm s ' that stopped the flow of feeding
currents throughout the whole animal, presumably by
causing flagellar arrest (Lawn cl til., 1981: Mackie et al.,
1983). Because no nerves could be found, the syncytial
tissues were proposed as the pathways for conduction,
though no recordings of propagated electrical signals could
be obtained.

Plugged junctions have since been reported in five of
the six hexactinellids examined by electron microscopy,
the exception being Dactylocalyx pnmicciis (Reiswig,
1 99 1 ). In this animal, no such junctions were found, but
their presence could not be completely ruled out. Because
of the remarkable differences in cellular organization be-
tween hexactinellids and other sponges and the physio-
logical evidence of signal conduction, it was proposed that
this group be separated from cellular sponges at the sub-
phylum level (Reiswig and Mackie, 1983). Nonetheless,
conclusive proof of syncytial organization from dye ex-
change experiments or from observations of cytoplasmic
movements in living tissues has not been obtained, and
recent investigations have raised doubts about the syn-
cytial nature of hexactinellid larvae (Boury-Esnault and
Vacelet, 1994).

When demosponges are dissociated by squeezing them
through a fine mesh, the cells have the ability to reaggre-

gate, forming a new individual (Wilson, 1907). The
mechanisms underlying reaggregation have been exten-
sively studied. The process is homeotypic and therefore
of interest in relation to self and non-self discrimination
in the earliest metazoa (Curtis, 1962; Moscona, 1968;
McClay, 1971; Miiller, 1982). Studies of newly dissociated
demosponge cells shows that they exhibit rapid nondi-
rectional crawling along the substrate (Noble and Peter-
son, 1972; Gaino el nl.. 1985). Initial contacts between
cells may be made by membrane bridges (Evans and
Bergquist, 1974), whereas the formation of secondary ag-
gregates is by species-specific cell adhesion molecules
(Miiller, 1982). Whether aggregation is brought about by
cell migration or artificially, for example, by the rotary
technique of Humphreys ( 1963), aggregates rapidly grow
in size, becoming opaque under the light microscope.

In earlier work (Pavans de Cecatty, 1982), aggregates
in hexactinellids, like those in demosponges, were found
to form large spherical masses, and their contents could
not be observed with light microscopy. In the present
study, however, substrates containing sponge tissue extract
or concanavalin A were used (Leys, 1995). The tissues
adhere and spread out on these substrates, permitting ob-
servation of the cytoskeleton in vitro. The findings re-
ported here show that, in such preparations of R/iabdo-
calyptus. tissue aggregation leads to the formation of giant
syncytia in which multidirectional streaming occurs over
great distances in a manner unique among the Porifera.

Materials and Methods

Specimens of Rhabdocalyptus dawsoni were collected
by scuba from a depth of 30 m in Barkley Sound and

CYTOSK.ELETON IN A SYNCYTIAL SPONGE

243

Saanich Inlet. British Columbia, and kept in flow-through
seawater tanks at the Bamfield Marine Station and at the
University of Victoria. Specimens of Haliclona sp. were
collected intertidally at Clover Point, Victoria, British
Columbia, for use in dye exchange experiments only.

Preparation of substrate

The preparation of tissue extract dried onto coverslips
or plastic petri dishes, to which dissociated tissue of R.
dawsoni adheres, is described elsewhere (Leys, 1995). Al-
ternatively, 50 ^1(100 yug- ml ')Concanavalin A (Con A)
was pipetted onto coverslips and allowed to air dry before
being used as an adhesion substrate for dissociated tissue.

Preparat ion of aggregates

Pieces ( 1 cm 3 ) of cleaned whole sponge tissue were
squeezed through lOO^m Nitex mesh into a beaker to
make 3.0-5.0 ml of dissociated tissue, then diluted to
200 ml with seawater. About 2 ml of the suspension was
poured into 1.8 cm diameter plastic petri dishes contain-
ing coated coverslips and held at 1 1C either by floating
dishes on seawater or by placing them in an incubator.
Alternatively, for video microscopy, the dissociated tissue
was briefly pelleted at 1000 X g for 1 5 s to remove spicule
debris, and the top of the pellet was pipetted onto a coated
coverslip in a dish of seawater.

Computer-assisted light microscopy

Preparations were viewed with a compound microscope
equipped with phase contrast and differential interference
contrast (DIC) optics and with a cooling stage. Images
were captured with a Panasonic digital color CCT V video
camera and OMNEX digital image processor (Imagen
Inc.). Photographs were taken from the screen of a
Technitron television monitor.

Immunolabeling and vital staining

To prevent depolymerization of the cytoskeleton due
to excess calcium, aggregates were transferred to calcium-
free seawater (CFSW) for 30 min prior to fixation. For
micronlament labeling, preparations were lysed at 2, 6,
12, and 24 h after plating the tissue, in a PEM buffer con-
sisting of 50 mM piperazine-A r ,jV'-bis[2-ethane sulfonic
acid], 1 mM ethyleneglycol-bis-(/3-aminoethyl eiher)N,N'-
tetraacetic acid, 0.5 mM MgCl : at pH 6.9 with 10% di-
methylsulfoxide and 0.1% Triton X-100 for 2 min and
fixed in 2% paraformaldehyde in CFSW with 10 nM
EGTA and 0.04% tannic acid for 10 min. For microtubule
labeling, preparations were fixed without lysing, in 2%
paraformaldehyde in PEM buffer, at 30 min, 1 , 6, and
12 h after plating. After one 30-min wash in 0.05 M Tris
buffer pH 7.0 with 0.1% TX-100, coverslips were incu-

bated overnight in tubulin antibodies or rhodamine-phal-
loidin (Molecular Probes, Inc.) to visualize actin micro-
filaments. The tubulin antibodies used included a poly-
clonal anti-tubulin antibody (Chemicon); a monoclonal
antibody against flagellar axonemes or isolated basal ap-
paratus of Polytomella (Protista, Chlorophyceae), desig-
nated 5A6 (kindly provided by Dr. David Brown, Uni-
versity of Ottawa); a monoclonal antibody against yeast
tubulin clone YOL1/34 (Sera Labs); a monoclonal anti-
body against native chick brain alpha tubulin (Amer-
sham); and a monoclonal antibody against Drosophila
(insect) beta tubulin, designated E7, which was developed
by M. Klymkowski and obtained from the Developmental
Studies Hybridoma Bank maintained by the Department
of Pharmacology and Molecular Science, Johns Hopkins
University School of Medicine, Baltimore, Maryland, and
the Department of Biological Sciences, University of Iowa,
Iowa City, Iowa. After a 30-min rinse, preparations were
incubated in their respective secondary antibody for 5 h,
rinsed thoroughly in phosphate buffered saline (PBS) pH
7.2, and mounted in PBS-glycerol with w-propyl gal-
late. To stain the nuclei, coverslips were incubated in
lOOyug-mr' Hoechst #33342 (Sigma) for 5 min at 1 PC,
and immediately observed with a 40X water immersion
lens. To examine the microtubule network in severed
streams, a stream was cut with a scalpel while the prep-
aration was still in a dish of seawater on an inverted mi-
croscope. After the material downstream of the wound
had completely drained away, the preparation was fixed
and labeled for anti-tubulin immunofluorescence as
above.

Electron microscopy

For scanning electron microscopy (SEM), dissociated
tissue from Rhabdocalyptus adhered to coverslips was
transferred to CFSW for 30 min prior to fixation. Prep-
arations were briefly lysed for 5-15 s, or fixed directly, in
a fixative containing 2% glutaraldehyde, 1% OsO 4 , 0.45 M
sodium acetate buffer at pH 6. 4 (Harris and Shaw, 1984),
10% sucrose and 5 nM EGTA final concentration, for 2 h
on ice. Coverslips were dehydrated through a graded
ethanol series, critical-point dried in CO : , mounted on
stubs with silver conducting paint, coated with gold in an
Edwards S150B sputter coaler, and examined in a JEOL
JSM-35 scanning electron microscope.

For transmission electron microscopy (TEM), whole
mounts were prepared on extract-coated, Formvar-coated
gold grids, lysed for 2 min, and fixed as above. Whole
preparations, adhered to 5 cm diameter plastic petri
dishes, were acclimated to CFSW for 30 min and fixed as
above. Grids were critical-point dried and viewed in a
Hitachi H-7000 electron microscope. Whole preparations,
fixed in plastic petri dishes, were treated with 4% hydro-

244

S. P. LEYS

fluoric acid overnight to remove silica, dehydrated in
ethanol, and embedded in Epon. For cross sections, the
embedded material was taken out of the petri dish and
reembedded in Epon. Thin sections were cut on a Reichert
UM2 ultramicrotome and stained with uranyl acetate and
lead citrate.

Pharmacological manipulations

To demonstrate the effect of various cytoskeletal dis-
ruptors on streaming, drugs (cytochalasin B, colcemid,
nocodazole, Taxol, EGTA) were added to 2 ml seawater
in a Falcon petri dish containing one preparation. To
demonstrate reversibility of the effects, preparations were
incubated in these drugs for 5 min and 2 min respectively,
and transferred immediately to clean seawater every
15 min thereafter for 4.5 h. Preparations were kept at
1 1C for the duration of the experiment.

Dye exchange experiments

To confirm that fusion of tissues and exchange of cy-
toplasm occurred during aggregation in syncytial sponges
but not in cellular sponges, dissociated tissues of both
sponges were loaded with fluorescent, membrane-im-
permeable dyes and allowed to aggregate. Dissociated tis-
sue from both Rhalnlocalyptus (R) and Haliclona (H) was
briefly centrifuged at 1000 x g for 15-3()s to remove
spicule and other debris. Calcein acytoxymethyl ester
(CAM) and Calcein blue acytoxymethyl ester (CBAM)
(Molecular Probes, Inc., Eugene, OR) were made up in
DMSO to stock concentrations of 1 and 2 mAf respec-
tively. The dyes were added to the sponge tissue at a final
concentration of 10 nM in microfuge tubes that were left
at 10C for 1 5 min. The tissue was then rinsed three times
by pelleting the tissue, drawing off the remaining seawater
by pipette and resuspending the pellet in fresh seawater.
After the final rinse, equal volumes were plated in a 1.8-
cm, extract-coated plastic petri dish as follows: (R)-CAM
with (R)-CBAM; (R)-CAM with (H)-CBAM; (H)-CAM
with (H)-CBAM.

Results

Fusion

Immediately after being plated, the dissociated tissues
consisted of unattached rounded masses of various sizes,
some of which could be identified by their collars and
flagella as parts of choanoflagellate chambers. All pieces
adhered to the coverslip within seconds of plating and
flattened and spread out between 5 s and 10 min after
plating (Fig. 2). The larger (10pm diameter) pieces de-
veloped a broad, skirt-like lamellipodium or extended long
filopodia into which the cytoplasm streamed. These ad-
hered tissue pieces incorporated other smaller, rounded

Figure 2. Fusion in adhered aggregates of Rhabdocalyptus taken from
a video recording of tissue aggregation. Upon plating, pieces ot tissue
adhere and spread a skirt-like lamellipodium or long filopodia. After
about I h of spreading, such pieces encounter each other, as shown by
the overlapping lamellipodia (B. arrow). Tissues fuse and exchange cy-
toplasm between 5 and 30 min after first contact (C). Fused tissue pieces
continue to grow in diameter, incorporating some tissue pieces (* in A
and Bland fusing with others (D. arrow). Minutes after plating dissociated
tissue: A. 33 min; B. 54 min;C, 102 min; D, 134 min. Bar: 10 ^m. (Video
available: see footnote on title page.)

CYTOSKELETON IN A SYNCYTIAL SPONGE

245

Figure 3. Twenty-four hours after plating sponge tissue, a single tissue mass covers an entire petri dish.
(A) Streams (arrowheads) are abundant and run in opposite directions, winding throughout the entire coverslip.
A 5-s shutter exposure allowed moving objects to leave trails. Bar: 20 p.m. (B) Hoechst-labeled nuclei are
visible in both streaming (SIR) and stationary cytoplasm. Nuclei move in streams at just over 2 ^ms"'. A
30-s shutter exposure causes all moving nuclei to leave a trail of light marking their path. Bar: 20 ^m. (C)
Images from a video monitor showing bulk transport of material in streams (*) beside organelles that are
being transported individually (arrow and arrowhead). Streams move continuously but at a slower rate than
individual organelles. The organelle marked by an open arrow moves steadily, leaving the field of view in
the third frame, while the organelle marked by an arrowhead moves haltingly and eventually stops. Frames
shown are at 8-s intervals. Bar: 10 /^m. (Video available; see footnote on title page.)

pieces of tissue by drawing them in to a central location
within the aggregate. Independent adhered pieces en-
countered each other by way of the lamellipodia or filo-
podia. Fusion did not always occur immediately upon
contact of lamellipodia. Lamellipodia even overlapped
one another for 10-30 min before the exchange of organ-
elles could be clearly detected (Fig. 2b, 54 min). A clear
sign of fusion, however, was a shared lamellipodium at
the point of touching, followed by exchange of organelles.
Within minutes effusion there was no evidence that the
single piece of tissue had been otherwise. Lamellipodial
extension continued in all directions, and streams of cy-
toplasm started to become clearly visible circumnavigating

the aggregate in both directions. Fusion continued with
other tissue pieces that were encountered.

Cyloplasmic streaming

As aggregates increased in size by incorporating neigh-
boring tissue masses, organelle movement became orga-
nized into wider and straighter streams, until eventually
the entire coverslip was covered in tissue that consisted
of dramatic rivers of flowing cytoplasm traversing the
coverslip, sometimes running parallel in opposite direc-
tions (Fig. 3a) and even crossing each other without ap-
parent interruption in volume or velocity of flow. Streams

246

S. P. LEYS

Figure 4. The effect of severing streams of cytoplasm. (A) When cut,
the cytoplasm downstream (DS) of the wound (arrowhead) continues to
drain in the direction of the arrow, while that upstream (US), builds up.

of cytoplasm flowed uninterrupted for distances up to
several centimeters, limited only by the area of coated
substrate available. Large streams continually changed
direction and both gained and lost volume. Under DIC
microscopy, bulk cytoplasm in streams could be seen to
contain spicule debris and vesicles of various sizes. With
electron microscopy, cytoplasm fixed while it was stream-
ing showed similar objects along with numerous mito-
chondria, nuclei, Golgi bodies, and some archaeocytes.
Fluorescent staining with Hoechst #33342 showed nuclei
in both the flowing and stationary cytoplasm (Fig. 3b).
Individual nuclei could be followed in streams for dis-
tances up to several millimeters.

Organelles resolvable by video-enhanced contrast mi-
croscopy in thin areas of tissue moved at an average rate
of 2. 15 0.33 ^m-s" 1 (// = 100), while bulk streams
moved at an average rate of 1.72 0.30 /^m s~" (n = 100)
(Fig. 3c). However, some organelles moved haltingly,
sometimes reversing direction or apparently bumping into
each other; these organelles seemed to buckle or bend as
they reversed. Organelles moving in broad lamellipodia
followed no one direction, and occasionally paused for
some seconds. In some streams the bulk cytoplasm that
had been flowing diminished in volume to isolated or-
ganelles, to be followed once again by bulk cytoplasm.

Cutting a stream with a scalpel caused cytoplasm to
build up on the upstream side of the wound. Downstream
of the wound, cytoplasm continued to flow away until no
movements of organelles could be detected, but birefrin-
gent tracks could still be seen by phase contrast and DIC
microscopy (Fig. 4a). Immunofluorescence of tubulin on
the depleted side showed that microtubule bundles re-
mained, even though all streaming along them had ceased
(Fig. 4b). Eventually the cytoplasm upstream of the wound
began to turn back upon itself, initiating a new stream
parallel to the original one but in the reverse direction.
At the same time, lamellipodial and filopodial processes
were extended toward the downstream side until, after
several minutes, contact was once again made, followed
by fusion; a few organelles, soon followed by the full
stream, then began to flow along the original track. These
stages are summed up diagrammatically in Figure 4c.

With time, the tissue amassed in central locations and
gradually withdrew from the substrate until an opaque

Bar: 30 ^m. (B) Anti-tuhulin labeling of microtubules in the wounded
stream. Microtubules remain, even though all visible organelle movement
has ceased downstream of the wound. Bar: 30 ^m. (C) Diagram illus-
trating the sequence of events after wounding a stream. The cut cytoplasm,
represented by the dashed line in ( I ). builds up on the upstream side
(2). eventually forging a new path in the reverse direction (3). as indicated
by arrowheads. Filopodial and lamellipodial projections extend across
the wound, making contact with the original tracks (4). Single organelles,
rapidly followed by the bulk cytoplasm, begin to stream along the former
path (4). Arrows indicate direction of flow.

CYTOSKELETON IN A SYNCYTIAL SPONGE

247

Figure 5. The actin cytoskeleton in adhered aggregates after lysing. (A) Rhodamine-phalloidin labeling
of aggregates 2 h after plating reveals tissue masses 30-100 ^m in diameter that possess well-defined rods
projecting from their periphery (arrowheads). Bar: 20 ^m. (B) Six hours after plating dissociated tissue,
aggregates are already 0.5 mm in length and can be much larger. A portion of a fused aggregate shows giant

248

S. P. LEYS

sphere was formed. Such spheres could temporarily adhere
once again if new substrate was offered.

The act in cytoskeleton

Only by briefly lysing preparations before fixation was
it possible to obtain a clear picture of actin distribution
in adhered tissue preparations. Attempts to label micro-
filaments with a polyclonal antibody to actin and with
rhodamine-phalloidin in whole preparations gave, at best,
weak labeling of microfilament bundles whose precise lo-
cation could not be determined. However, lysing the tissue
for 5 s-2 min resulted in detachment of the superficial
part of the tissue mass, exposing the layer adjacent to the
substrate in which actin labeling produced clearer results.

Two hours after plating, aggregates were 30-100 f*m in
diameter and possessed blunt rods containing thick actin
bundles that projected from the periphery (Fig. 5a). The
edges of large lamellipodia labeled strongly with rhoda-
mine-phalloidin and stress fibers some 20 nm in length
transected the tissue. After 6 h, these masses had joined,
and no membranes demarcating cell boundaries could be
seen within the tissue mass by phase-contrast microscopy.
Rhodamine-phalloidin-labeled microfilament bundles up
to several hundred micrometers long were observed to
run around the edges of adhered aggregates (Fig. 5b). A
network of fine filaments was apparent beneath the stress
fibers throughout the syncytium. One-day-old adhered
aggregates showed no change in general morphology or
in actin distribution, although the distances over which
bundles of microfilaments could be followed now ex-
ceeded 500 ^m. After 24 h. much of the tissue had been
drawn into central, opaque areas. The edges of such dense
tissue masses possessed blunt rod-like extensions reaching
some 18.0 4.5 jum (n = 20) out from the edge of the
lamellipodium, forming a "hairbrush effect" (Fig. 5c).
These extensions labeled very strongly for actin (Fig. 5d).
The organization of actin in aggregates older than 48 h
did not change significantly, because most tissue was cen-
tralized at that time, anchored firmly by massive projec-
tions from the edges. Scanning electron microscopy of the
giant rods in intact and lysed preparations revealed thick
actin bundles forming their core (Fig. 5e. 0.

The microtubule cytoskeleton

Preservation of microtubules required the use of a spe-
cial fixation method (see Immunolabeling and vital stain-

ing in the Materials and Methods), after which it was pos-
sible to visualize microtubule bundles by phase contrast
and immunofiuorescence microscopy. These bundles were
still visible after cytochalasin B treatment, but not after
treatment with nocodazole or colcemid. Of five anti-tu-
bulin antibodies, only two monoclonal antibodies one
prepared against beta tubulin in Drosophila and the other
against chick brain alpha tubulin gave good immuno-
fiuorescence in whole mounts. Control experiments
showed that all antibodies labeled microtubules in neurons
and cilia of tunicate branchial basket and veliger larvae.

Immunofluorescence microscopy revealed a remark-
able change in the microtubule network over the course
of aggregation. At 30 min, microtubules formed a fine
meshwork in tissue pieces up to 80 /um in diameter; mi-
crotubules appeared delicate, some directly crossing and
others winding around the tissue mass. Nuclei appeared
randomly scattered among the microtubules. After 6 h,
the microtubules were already well organized into striking
bundles, many of which traversed an entire coverslip (Fig.
6a). Where many bundles of microtubules converged into
one path they became rigidly straight (Fig. 6b). At the
edge of preparations, the entire bundle gave way to a re-
ticular network of lines (Fig. 6c). These often wound
through giant lamellipodia before joining the main stream
and traversing the coverslip again.

Double labeling of actin and tubulin proved unsatis-
factory because the lysing procedure required for clear
visualization of the microfilament network usually de-
stroyed the continuity of microtubules. However, double
labeling of nuclei and microtubules clearly demonstrated
that nuclei were randomly distributed among microtu-
bules (Fig. 80.

The microtubules were difficult to fix for electron mi-
croscopy. The fixative used by Mackie and Singla ( 1983)
does not stabilize microtubules except in flagella. The fix-
ative of Harris and Shaw ( 1984) gave excellent results for
both general ultrastructure and microtubule preservation.
In cross sections of the adhered tissue, microtubules were
seen both in bundles and lying individually (Fig. 7a and
inset). In thicker areas of the tissue, large bundles were
seen at the surface of the preparation as well as through
the depth of the tissue. Rarely in these areas did they
occur singly, and rarely were they on the bottom of thick
tissue masses. In horizontal section, microtubule bundles
could be traced for many hundreds of micrometers (Fig.

bundles of microfilaments delineating the border (filled arrows). A network of microfilament bundles traverses
the basal layer adjacent to the substrate (arrowheads) and small, actin-dense rods lie within lamellipodia
(open arrow). Bar: 20 fim. (C, D) Adhered aggregates older than 12 h possess giant rods projecting from the
periphery as a "hairbrush" (C: phase contrast; D: rhodamine-phalloidin labeling of unlysed tissue.) Bar:
10 ^m. (E) SEM shows several giant rods extending from the lamellipodium (LM). (F) High magnification
SEM of a lysed actin-dense rod. E, F. bar: 0.5

CYTOSKELETON IN A SYNCYTIAL SPONGE

249

Figure 6. Immunofluorescence of the microtubule cytoskeleton in
adhered aggregates. (A) Six hours after plating, microtubule bundles al-
ready stretch up to a centimeter across an entire coverslip. A-C, bar:
20 pm. (B) Microtubule bundles are rigidly straight (STR) in streams,
while those leaving or converging on a stream (arrowheads) are often
curved. (C) At the edges of preparations, bundles give way to a fine
meshwork of microtubules that reach into lamellipodia (arrowheads).

7b). Nuclei, mitochondria, coated vesicles, and tubulo-
vesicular organelles were all found adjacent to microtubule
bundles as well as lying free in the cytosol. Tracts of mi-
crotubules ran beside, but not through, clusters of ar-
chaeocytes and spherulous cells. In whole mounts of ag-
gregates adhered to tissue extract on Formvar-coated gold
EM grids, organelles of various sizes were also seen as-
sociated with linear structures with diameters of approx-
imately 22 nm, presumably microtubules (Fig. 7c).

Inhibition experiments

Organelle movement was reversibly inhibited by both
nocodazole and colcemid but was unaffected by cyto-
chalasin B (Table I), although the latter caused the tissue
to detach from the substrate. Neither Taxol nor \Q nM
EGTA had any effect on rate of organelle transport.

Dye exchange

Attempts to inject the fluorescent dyes carboxyfluores-
cein or lucifer yellow through glass capillary microelec-
trodes were not successful because of difficulty in obtaining
stable penetrations. The surface membrane of adhered
aggregates either blocked the electrode or did not reseal
around the electrode after penetration. However, fluores-
cent dyes coupled to acytoxymethyl esters such as Calcein
AM (CAM) and Calcein blue AM (CBAM) could be
readily loaded into dissociated tissue. Tissue loaded with
CAM plated in the same culture dish with tissue loaded
with CBAM produced fused aggregates in which streaming
occurred. After 6-12 hours, these aggregates were uni-
formly blue-green (Fig. 8a, b, c). In control experiments
in which dissociated tissue from the cellular sponge Hali-
clona penno/is loaded with CAM was plated with tissue
from Rhabdocalyptus loaded with CBAM, Haliclona cells
rapidly formed large round aggregates, did not mix with
Rhabdocalyptus tissue, and did not take up the blue dye
(Fig. 8d). Haliclona tissue, even if adhered, showed no
sign of cytoplasmic streaming. When two samples of
Haliclona tissue were loaded with CAM and CBAM re-
spectively and plated together, there was no exchange of
dye, although after 1 2 h aggregates were mosaics of both
colors (Fig. 8e).

Discussion

This remarkable preparation provides the first evidence
effusion during aggregation of dissociated hexactinellid
sponge tissues and reveals an entirely novel form of cy-
toplasmic streaming. It further reveals a cytoskeletal
framework that is unique in the histology of sponges and,
perhaps, the entire metazoa, for sheer size. Given the ev-
idence of syncytialization in other hexactinellids, stream-
ing may be widespread within the group. The syncytial

S. P. LEYS

Figure 7. Electron microscopy of adhered aggregates. (A) In cross section, cellular components (such as
archaeocytes) lie within the multinudeated cytoplasm, which is surrounded above and below by a continuous
membrane. Microtubule bundles lie at the surface of streams and throughout the tissue (arrows). Bar: 1 nm.
Inset shows an enlargement of microtubules from within the box. Bar: 0. 1 jim. Nuclei, N; archaeocyte, AR;
Golgi, G; mitochondria, M. (B) Horizontal section through a stream showing a bundle of microtubules
(MT) with associated organelles. Bar: 0.5 ^m. (C) A whole-mount preparation showing an organelle associated
with linear structures. Bar: 0.2 ^m.

condition of hexactinellid sponges appears to be significant
in two ways. First, syncytialization would allow the trans-
port of nutrients throughout the animal in the absence of
mobile archaeocytes (Mackie and Singla, 1983). The
present findings suggest that cytoplasmic streams may be
the transport routes. Second, the lack of membrane bar-
riers within the syncytium presumably makes possible the
propagation of impulses coordinating flagellar arrests

(Lawn ctal.. 1981: Mackie et a/.. 1983). Cellular sponges,
lacking nerves and gap junctions, have no such capability.

Formation oj a syncytium

Membrane fusion is the means by which Rhabdoca-
lyptux forms a syncytium during aggr