THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board DAVID W. BISHOP, Carnegie Institution of JOHN H. LOCHHEAD, University of Vermont Washington y L LooSANOFF) n- s> Fish and wi i d iif e FRANK A. BROWN, JR., Northwestern University Service JAMES CASE, State University of Iowa C. L. PROSSER, University of Illinois JOHN W. GOWEN, Iowa State College BERTA SCHARRER, Albert Einstein College of LIBBIE H. HYMAN, American Museum of Medicine Natural History FRANZ SCHRADER, Duke University J. LOGAN IRVIN, University of North Carolina WM. RANDOLPH TAYLOR, University of Michigan DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 121 JULY TO DECEMBER, 1961 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. 11 THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers $2.50. Subscription per volume (three issues), $6.00. Communications relative to manuscripts should be sent to the Managing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 1 and September 1, and to Dr. Donald P. Costello, P.O. Box 429, Chapel Hill, North Carolina, during the remainder of the year. Second-class postage paid at Lancaster, Pa. LANCASTER PRESS, INC., LANCASTER, PA. CONTENTS ^ MASS. /$/ ^K ^X- No. 1. AUGUST, 1961 PAGE Annual Report of the Marine Biological Laboratory 1 BANG, FREDERIK B. Reaction to injury in the oyster (Crassostrea virginica) 57 BEERS, C. DALE The obligate commensal ciliates of Strongylocentrotus drobachiensis : Occurrence and division in urchins of diverse ages ; survival in sea water in relation to infectivity 69 DALES, R. PHILLIPS Observations on the respiration of the sabellid polychaete Schizobranchia insignis 82 DAVID, CHARLES N., AND ROBERT J. CONOVER Preliminary investigation on the physiology and ecology of luminescence in the copepod, Metridia lucens 92 DETHIER, V. G., AND D. R. EVANS The physiological control of water ingestion in the blowfly 108 DINGLE, HUGH Flight and swimming reflexes in giant water bugs 117 DUNHAM, PHILIP B., AND F. M. CHILD Ion regulation in Tetrahymena 129 GIESE, ARTHUR C. Further studies on Allocentrotus fragilis, a deep-sea echinoid 141 GOLDSMITH, MARY HELEN M., AND HOWARD A. SCHNEIDERMAN A dual effect of carbon dioxide on insects poisoned by oxygen 151 Goss, RICHARD J. Metabolic antagonists and prolonged survival of scale homografts in Fundulus heteroclitus 162 IWASAKI, HIDEO The life-cycle of Porphyra tenera in vitro \73 JENKINS, MARIE M. Respiration rates in planarians. III. The effect of thyroid compounds on oxygen consumption 188 SCHARRER, BERTA, AND MARIANNE VON HARNACK Histophysiological studies on the corpus allatum of Leucophaea maderae. III. The effect of castration 193 No. 2. OCTOBER, 1961 BARLOW, GEORGE W. Intra- and interspecific differences in rate of oxygen consumption in gobiid fishes of the genus Gillichthys 209 79674 iv CONTENTS BLINKS, L. R., AND CURTIS V. GIVAN The absence of daily photosynthetic rhythm in some littoral marine algae. . 230 BUCK, JOHN, AND JAMES F. CASE Control of flashing in fireflies. I. The lantern as a neuroeffector organ. . . 234 BURBANCK, W. D., AND MADELINE P. BURBANCK Variations in the dorsal pattern of Cyathura polita ( Stimpson ) from estu- aries along the coasts of eastern United States and the Gulf of Mexico .... 257 CARLSON, ALBERT D. / Effects of neural activity on the firefly pseudoflash 265 COOK, JAMES R. Euglena gracilis in synchronous division. II. Biosynthetic rates over the life cycle 277 GROSS, WARREN J. Osmotic tolerance and regulation in crabs from a hypersaline lagoon 290 GUNTER, GORDON, L. L. SULYA AND B. E. Box Some evolutionary patterns in fishes' blood 302 HAYES, DORA K., AND W. D. ARMSTRONG The distribution of mineral material in the calcified carapace and claw shell of the American lobster, Homarus americanus, evaluated by means of microroentgenograms 307 MAYNARD, DONALD M. Thoracic neurosecretory structures in Brachyura. I. Gross anatomy 316 RITTER, HOPE, JR. Glutathione-controlled anaerobiosis in Cryptocercus, and its detection by polarography 330 RULON, OLIN Cobalt and glutathione in the preservation of fertility and life of sand dollar eggs 347 SCHONE, HERMANN Learning in the spiny lobster Panulirus argus 354 Abstracts of papers presented at the Marine Biological Laboratory 366 No. 3. DECEMBER, 1961 ALLEN, KENNETH The effect of salinity on the amino acid concentration in Rangia cuneata (Pelecypoda) 419 BAUER, G. ERIC, AND ARNOLD LAZAROW Studies on the isolated islet tissue of fish. IV. In vitro incorporation of C 14 - and HMabeled amino acids into goosefish islet tissue proteins 425 BERNARD, FRANCIS J., AND CHARLES E. LANE Absorption and excretion of copper ion during settlement and metamor- phosis of the barnacle, Balanus amphitrite niveus 438 CASE, JAMES, AND G. F. GWILLIAM Amino acid sensitivity of the dactyl chemoreceptors of Carcinides maenas 449 DETHIER, V. G. Behavioral aspects of protein ingestion by the blowfly Phormia regina Meigen 456 CONTENTS v GEORGE, J. C, AND A. K. SUSHEELA A histophysiological study of the rat diaphragm 471 GROSSO, LEONARD L. The effect of thiourea, administered by immersion of the maternal organ- ism, on the embryos of Lebistes reticulatus, with notes on the adult gonadal changes 481 DE LUQUE, ORLANDO, ALICE S. HUNTER AND F. R. HUNTER Osmotic studies of amphibian eggs. III. Ovulated eggs 497 MARTIGNONI, MAURO E., AND ROBERT J. SCALLION Preparation and uses of insect hemocyte monolayers in vitro 507 PIPA, RUDOLPH L. Studies on the hexapod nervous system. IV. A cytological and cyto- chemical study of neurons and their inclusions in the brain of a cockroach, Periplaneta americana (L. ) 521 SIMMONS, JOHN E., JR. Urease activity in trypanorhynch cestodes 535 SPIEGEL, MELVIN Tryptophan pyrrolase activity in the liver of adult Rana pipiens 547 STEVENSON, J. Ross Polyphenol oxidase in the tegumental glands in relation to the molting cycle of the isopod crustacean Armadillidiurn vulgare 554 WEBB, H. MARGUERITE, AND FRANK A. BROWN, JR. Seasonal variations in Oo-consumption of Uca pugnax 561 WILLIAMS, CARROLL M. The juvenile hormone. II. Its role in the endocrine control of molting, pupation, and adult development in the Cecropia silkworm 572 Vol. 121, No. 1 August, 1961 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE MARINE BIOLOGICAL LABORATORY SIXTY-THIRD REPORT, FOR THE YEAR 1960 SEVENTY-THIRD YEAR I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 15, 1960) ... 1 STANDING COMMITTEES II. ACT OF INCORPORATION 4 III. BY-LAWS OF THE CORPORATION 4 IV. REPORT OF THE DIRECTOR 6 Memorials 8 Addenda : 1. The Staff 11 2. Investigators, Lalor and Lillie Fellows, and Students 14 3. Fellowships and Scholarships 25 4. Tabular View of Attendance, 1956-1960 25 5. Institutions Represented 26 6. Evening Lectures 27 7. Shorter Scientific Papers (Seminars) 28 8. Members of the Corporation 29 V. REPORT OF THE LIBRARIAN 49 VI. REPORT OF THE TREASURER 50 I. TRUSTEES GERARD SWOPE, JR., President of the Corporation, 570 Lexington Ave., New York City A. K. PARPART, Vice President of the Corporation, Princeton University PHILIP B. ARMSTRONG, Director, State University of New York, Medical Center at Syracuse C. LLOYD CLAFF, Clerk of the Corporation, Randolph, Mass. JAMES H. WICKERSHAM, Treasurer, 530 Fifth Ave., New York City EMERITI W. C. CURTIS, University of Missouri PAUL S. GALTSOFF, Woods Hole, Mass. E. B. HARVEY, Woods Hole, Mass. 2 MARINE BIOLOGICAL LABORATORY M. H. JACOBS, University of Pennsylvania School of Medicine F. P. KNOWLTON, Syracuse University CHARLES W. METZ, Woods Hole, Massachusetts W. J. V. OSTERHOUT, Rockefeller Institute CHARLES PACKARD, Woods Hole, Mass. A. C. REDFIELD, Woods Hole Oceanographic Institution LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y. TO SERVE UNTIL 1964 C. LALOR BURDICK, The Lalor Foundation E. G. BUTLER, Princeton University K. S. COLE, National Institutes of Health S. KUFFLER, Harvard Medical School C. B. METZ, Oceanographic Institute, Florida State University ROBERTS RUGH, College of Physicians and Surgeons, Columbia University G. T. SCOTT, Oberlin College E. Z WILLING, Brandeis University TO SERVE UNTIL 1963 L. G. BARTH, Columbia University JOHN B. BUCK, National Institutes of Health AURIN M. CHASE, Princeton University SEYMOUR S. COHEN, University of Pennsylvania School of Medicine DONALD P. COSTELLO, University of North Carolina TERU HAYASHI, Columbia University DOUGLAS A. MARSLAND, New York University, Washington Square College H. BURR STEINBACH, University of Chicago TO SERVE UNTIL 1962 FRANK A. BROWN, JR., Northwestern University SEARS CROWELL, Indiana University ALBERT I. LANSING, University of Pittsburgh Medical School WILLIAM D. MCLROY, Johns Hopkins University C. LADD PROSSER, University of Illinois S. MERYL ROSE, University of Illinois MARY SEARS, Woods Hole Oceanographic Institution ALBERT TYLER, California Institute of Technology TO SERVE UNTIL 1961 ERIC BALL, Harvard University Medical School D. W. BRONK, Rockefeller Institute G. FAILLA, Columbia University, College of Physicians & Surgeons R. T. KEMPTON, Vassar College L. H. KLEINHOLZ, Reed College IRVING M. KLOTZ, Northwestern University ALBERT SZENT-GYORGYI, Marine Biological Laboratory WM. RANDOLPH TAYLOR, University of Michigan TRUSTEES EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES GERARD SWOPE, JR., ex officio, Chairman W. D. MCELROY, 1961 JAMES H. WICKERSHAM, ex officio F. A. BROWN, JR., 1961 ARTHUR K. PARPART, ex officio JOHN BUCK, 1962 P. B. ARMSTRONG, ex officio ALBERT I. LANSING, 1962 KENNETH S. COLE, 1963 STEPHEN KUFFLER, 1963 THE LIBRARY COMMITTEE MARY SEARS, Chairman ANTHONY C. CLEMENT SEYMOUR S. COHEN C. LADD PROSSER THE APPARATUS COMMITTEE ALBERT I. LANSING, Chairman RALPH H. CHENEY HARRY GRUNDFEST FREDERIK BANG HOWARD K. SCHACHMAN THE SUPPLY DEPARTMENT COMMITTEE RUDOLF T. KEMPTON, Chairman GROVER C. STEPHENS SEARS CROWELL DAVID BISHOP THE EVENING LECTURE COMMITTEE PHILIP B. ARMSTRONG, Chairman DONALD P. COSTELLO H. BURR STEINBACH S. MERYL ROSE FRANK A. BROWN, JR. THE INSTRUCTION COMMITTEE JOHN B. BUCK, Chairman BOSTWICK KETCHUM ARNOLD LAZAROW JAMES \V. GREEN TERU HAYASHI THE BUILDINGS AND GROUNDS COMMITTEE EDGAR ZWILLING, Chairman JAMES CASE MORRIS ROCKSTEIN DANIEL GROSCH THE RADIATION COMMITTEE G. FAILLA, Chairman WALTER L. WILSON ROGER L. GREIF WALTER S. VINCENT CARL C. SPEIDEL THE RESEARCH SPACE COMMITTEE PHILIP B. ARMSTRONG, Chairman MAC V. EDDS, JR. ARTHUR K. PARPART W T ILLIAM D. MCLROY 4 MARINE BIOLOGICAL LABORATORY II. ACT OF INCORPORATION No. 3170 COMMONWEALTH OF MASSACHUSETTS Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves with the intention of forming a Corporation under the name of the Marine Biological Laboratory, for the purpose of establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural his- tory, and have complied with the provisions of the statutes of this Commonwealth in such case made and provided, as appears from the certificate of the President, Treasurer, and Trustees of said Corporation, duly approved by the Commissioner of Corporations, and recorded in this office; Nozv, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi- ner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and successors, are legally organized and established as, and are hereby made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB- ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties, and restrictions, which by law appertain thereto. Witness my official signature hereunto subscribed, and the seal of the Commonwealth of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord One Thousand Eight Hundred and Eighty-Eight. [SEAL] HENRY B. PIERCE, Secretary of the Commonwealth. III. BY-LAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY I. The members of the Corporation shall consist of persons elected by the Board of Trustees. II. The officers of the Corporation shall consist of a President, Vice President, Director, Treasurer, and Clerk. III. The Annual Meeting of the members shall be held on the Friday following the second Tuesday in August in each year at the Laboratory in Woods Hole, Massachusetts, at 9:30 A.M., and at such meeting the members shall choose by ballot a Treasurer and a Clerk to serve one year, and eight Trustees to serve four years, and shall transact such other business as may properly come before the meeting. Special meetings of the mem- bers may be called by the Trustees to be held at such time and place as may be designated. IV. Twenty-five members shall constitute a quorum at any meeting. V. Any member in good standing may vote at any meeting, either in person or by proxy duly executed. VI. Inasmuch as the time and place of the Annual Meeting of members are fixed by these By-laws, no notice of the Annual Meeting need be given. Notice of any special BY-LAWS OF THE CORPORATION meeting of members, however, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting, at least fifteen (15) days before such meeting, to each member at his or her address as shown on the records of the Corporation. VII. The Annual Meeting of the Trustees shall be held promptly after the Annual Meeting of the Corporation at the Laboratory in Woods Hole, Mass. Special meetings of the Trustees shall be called by the President, or by any seven Trustees, to be held at such time and place as may be designated, and the Secretary shall give notice thereof by written or printed notice, mailed to each Trustee at his address as shown on the records of the Corporation, at least one ( 1 ) week before the meeting. At such special meeting only matters stated in the notice shall be considered. Seven Trustees of those eligible to vote shall constitute a quorum for the transaction of business at any meeting. VIII. There shall be three groups of Trustees: (A) Thirty-two Trustees chosen by the Corporation, divided into four classes, each to serve four years. After having served two consecutive terms of four years each. Trustees are ineligible for re-election until a year has elapsed. In addition, there shall be two groups of Trustees as follows : (B) Trustees ex officio, who shall be the President and Vice President of the Cor- poration, the Director of the Laboratory, the Associate Director, the Treasurer, and the Clerk : (C) Trustees Emeriti, who shall be elected from present or former Trustees by the Corporation. Any regular Trustee who has attained the age of seventy years shall con- tinue to serve as Trustee until the next Annual Meeting of the Corporation, whereupon his office as regular Trustee shall become vacant and be filled by election by the Corpora- tion and he shall become eligible for election as Trustee Emeritus for life. The Trustees ex officio and Emeriti shall have all the rights of the Trustees except that Trustees Emeritus shall not have the right to vote. The Trustees and officers shall hold their respective offices until their successors are chosen and have qualified in their stead. IX. The Trustees shall have the control and management of the affairs of the Cor- poration ; they shall elect a President of the Corporation who shall also be Chairman of the Board of Trustees and who shall be elected for a term of five years and shall serve until his successor is selected and qualified ; and shall also elect a Vice President of the Corporation who shall also be the Vice Chairman of the Board of Trustees and who shall be elected for a term of five years and shall serve until his successor is selected and qualified ; they shall appoint a Director of the Laboratory ; and they may choose such other officers and agents as they may think best ; they may fix the compensation and define the duties of all the officers and agents ; and may remove them, or any of them, except those chosen by the members, at any time ; they may fill vacancies occurring in any manner in their own number or in any of the offices. The Board of Trustees shall have the power to choose an Executive Committee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient. They shall from time to time elect members to the Corporation upon such terms and conditions as they may think best. X. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. MARINE BIOLOGICAL LABORATORY XI. The consent of every Trustee shall be necessary to dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees. XII. The account of the Treasurer shall be audited annually by a certified public accountant. XIII. These By-laws may be altered at any meeting of the Trustees, provided that the notice of such meeting shall state that an alteration of the By-laws will be acted upon. IV. REPORT OF THE DIRECTOR To : THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY Gentlemen : I submit herewith the Report of the seventy-third session of the Marine Bio- logical Laboratory. 1. Policy During the past year there were several extended discussions on the advisability of developing year-round programs in Marine Biology at the Laboratory. Several alternatives were suggested with reservations expressed on the advisability of establishing a year-round program staffed with permanent personnel, if these staff members were to be employed by the Marine Biological Laboratory. It was voted that the Laboratory should do everything possible to assist in establishing in the Woods Hole area an independent institute for basic research in the broad field of Marine Biology. 2. Land Acquisitions Four parcels of land were acquired during the year, including the Veeder prop- erty, the Broderick property and the Tinkham property on Albatross Street. In- cluded were four residences. The small cottage on the Tinkham Property is in very bad condition and unfit for housing purposes. The Breakwater Hotel property on Bar Neck Road was also purchased and the hotel razed, leaving a free area of 1.4 acres designed to be used as a site for a combination dormitory-dining hall for which tentative plans have been developed. The total land area acquired by these purchases increases the land holdings of the Laboratory in the immediate vicinity of its campus by 2.2 acres. 3. New Laboratory Building One of the feature events of the year was the completion of the new laboratory building in time for summer occupancy. Included in this building are 61 labora- tories, 10 being used as general radiobiological service laboratories, 5 constant tem- perature rooms, 16 dark rooms, 1 aquarium room, 1 conference room, 1 lecture REPORT OF THE DIRECTOR / room accommodating 140 people, 1 photo laboratory and 4 dry rooms. The sum- mer occupancy demonstrated the adaptability of the new building to a wide variety of research activities. Also, the building stood up very effectively through Hurri- cane Donna and other severe storms, and proved to be a watertight building, con- trary to the Laboratory's experience with buildings of brick construction. 4. Personnel Changes It is the policy of the Laboratory that the heads of the various training programs will serve for a period of five years. Dr. Nelson T. Spratt, Jr., will serve as head of the training program in Experimental Embryology during the summer of 1961 and will be succeeded by Dr. James D. Ebert. Dr. Clark P. Read succeeds Dr. Grover C. Stephens as head of the training program in Invertebrate Zoology. By action of the Executive Committee, three additional training programs at the post- doctoral level will be established starting in the summer of 1962. These will be in Marine Microbiology, Problems of Fertility, and Comparative Physiology, to be headed respectively by Drs. W. D. McElroy, C. B. Metz and C. Ladd Prosser. 5. Laboratory Fees During the past several years, the fees paid by investigators for laboratory space and the included services have covered only one-sixth of the cost of operation of such a laboratory. It was voted by the Executive Committee to gradually increase these fees over a period of two years so as to finally increase the fees to cover one- third of these costs. The Executive Committee also voted to have a weekly inclu- sive dormitory charge to cover both board and room, patterned after the usual operation as seen in the colleges and universities. 6. Plant Changes During the summer of 1960 all of the training programs operated throughout the summer instead of two groups, each group operating for half the summer. In order to accommodate all of the training programs concurrently, the Old Lecture Hall was completely remodeled to accommodate the training program in Experi- mental Embryology. The ground floor provides a general student laboratory, the second floor a series of laboratories for special research procedures. 7. Grants, Contracts and Contributions, in Support of Laboratory Activities, Including Training Grants The total income from these sources of support amounts to $373,000 in 1960. This represents 44% of the total income and is made up of the following accounts : Training grants for the courses from NIH and NSF, support for regular re- search activities from NIH, NSF, AEC and ONR and gifts from the MBL Asso- ciates, Josephine C. Crane Foundation, The Rockefeller Foundation, The George F. Jewett Foundation and the following pharmaceutical companies : The Merck Co. Foundation, Carter Products, Inc., C. I. B. A. Pharm. Products Inc., Abbott Laboratories, Schering Foundation, Inc., Eli Lilly & Company, the Upjohn Com- pany and E. R. Squibb & Sons. 8 MARINE BIOLOGICAL LABORATORY 8. Future Plans With the acquisition of the Breakwater Property the Laboratory now has a proper site for the location of the projected dormitory-dining hall. The Officers of the Corporation are exploring various sources of funds for this construction which is so necessary for the solution of problems of congestion and parking difficul- ties in our campus. In addition funds are being sought for the construction of additional cottages in the Devil's Lane Tract. Respectfully submitted, PHILIP B. ARMSTRONG, Director MEMORIALS Ross GRANVILLE HARRISON :' ' ' ., by ; Chester L. Yntema Ross Granville Harrison died September 30, 1959, after a full life of 89 years. During his lifetime biology became a modern science. His contributions based on critical experi- mentation were a great factor in this maturation and his example has been an inspiration to biologists. Dr. Harrison was born January 13, 1870, in Germantown, Pennsylvania. His under- graduate and graduate work was done at Johns Hopkins ; he received his Doctorate of Philosophy in 1894. The thesis on the embryological origin of the rays of the fins in teleosts was done with Dr. Brooks as his teacher. Five years later, after intermittent study in Germany, he was awarded the degree of Doctor Medicine by the University of Bonn. After receiving his Ph.D., Dr. Harrison taught at Bryn Mawr for a year and then studied for a year in Germany. In 1896 he returned to Johns Hopkins to join the depart- ment of anatomy headed by Dr. Mall. In 1907 he accepted the Bronson professorship of comparative anatomy at Yale and the remainder of his career continued with Yale as its base. Early in his stay at Yale, the Osborn Laboratories were built for the Departments of Botany and Zoology and these buildings continue to serve the departments. In addi- tion, the science departments at Yale became University departments as he demanded. This recognition is so generally given today that it is difficult to realize that the issue once had to be made and pressed. Dr. Woodruff and Dr. Petrunkevitch joined Dr. Coe and Dr. Harrison; these four became central and lasting figures in a zoology department which was outstanding in both its undergraduate and graduate programs. An increasing number of graduate students and foreign fellows came to the Osborn Laboratories and for many years there was a group of students pursuing their thesis work under Dr. Harrison. In his scientific research Dr. Harrison furthered the concept of an experimental approach to embryology initiated by Roux and Driesch and he devised means of analyzing development. In part, his genius consisted of picking a critical experiment bearing on a basic problem and performing the experiment in an uncomplicated way. This approach is illustrated by his cultivation of neuroblasts from the neural tube of the frog embryo in hanging drops of frog lymph, and following the growth of the processes from these cells by repeated microscopic observations. By this one procedure he settled the con- troversy over the origin of nerve cell processes, and in addition devised the technique REPORT OF THE DIRECTOR 9 of tissue culture for animal cells and tissues which has come to be a standard biological procedure. After other pioneer studies with explanation, he developed and refined means of transplantation for amphibian embryos which he and many others have used. The analyses of development he undertook included studies of the lateral line organs, the neural crest, the polarization of the limb and the internal ear, and growth rates in heteroplastic transplants. In each of his many reports, the same standard of perfection is maintained. In a clear and obvious way, a basic problem is resolved by results from simple experiments ingeniously devised and applied. Dr. Harrison was granted an honorary master's degree from Yale University in 1907 and honorary doctor's degrees from Yale and Cincinnati in 1920. These honors were followed by similar recognition from several other institutions in this country and in Europe. He was a member of many learned societies and the recipient of awards given in recognition of his achievements. Dr. Harrison's memberships in societies and academies of other countries indicate the regard held for him. He was a member of the Royal Society of Lund, the Royal Society of Uppsala, and the German Anatomical Society, of which he was a president. He was a corresponding member of the Gottingen Academy of Science, the German Academy of Sciences, the Bavarian Academy of Science, the Academy of Sciences of the Institute of France, and the Society of Biology of Paris. He was an honorary member of the Royal Academy of Turin and the Royal Academy of Belgium. He was a foreign correspondent of the Academy of Science of the Institute of Bologna. He was a foreign associate of the Academy of Medicine of Paris. He was a foreign member of the Royal Netherlands Academy of Science, the Norwegian Academy of Science, the National Academy of Rome, the Royal Swedish Academy of Stockholm, the Zoological Society of London and the Royal Society of London. The professional activities of Dr. Harrison included many administrative responsi- bilities in addition to those that he met at Yale. His interest in marine laboratories is evident from his connections with such institutions. He served as a Trustee of this Laboratory from 1908 to 1940 and then became a Trustee Emeritus. In addition he was a Trustee of the Woods Hole Oceanographic Institution and the Bermuda Biological Station. He served as president of several scientific societies. He was a member of the boards of the Rockefeller Institute of Medical Research and the Jane Coffin Childs Fund for Medical Research. From 1903 to 1946, Dr. Harrison was Managing Editor of the Journal of Experi- mental Zoology; he imprinted upon this publication a standard of excellence which is a challenge to contributors. For many years, Dr. Harrison was a councilor and member of the executive com- mittee of the National Academy of Sciences. After his retirement from Yale he was chairman of the National Research Council from 1938 to 1946. During this period, which included the Second World War, he handled responsibilities for the national gov- ernment with which the National Academy and the National Research Council were faced. During the same period he was a member of the science committee of the National Resources Planning Board. Later he served on the United States National Committee for the United Nations Educational Scientific and Cultural Organization. With all his accomplishments, Dr. Harrison was modest, self-contained and retiring. He had a deep regard for the individualities of others. This was particularly evident to those who were graduate students under him. He himself set an example of application and devotion to his work ; others could determine their own pace and ways without com- ment or persuasion. He had no understanding of incompetence but he overlooked human foolishness and foolhardiness. His practice of sharing his luncheon hour with students was of great value which came to be appreciated more fully with passing years. During 10 MARINE BIOLOGICAL LABORATORY this hour of sandwiches and tea no mention of scientific interests was recognized. Con- sequently a variety of topics was covered under the wise and sympathetic aegis of Dr. Harrison. We came to appreciate and be influenced by his wide range of knowledge and interests, his great understanding, and his whimsical humor. Dr. Harrison is survived by his wife, Mrs. Ida Lange Harrison, whom he met in Germany and married in 1896. Also surviving him are their three daughters and two sons. In honoring the memory of Dr. Harrison we wish to convey to his family a sense of our indebtedness and appreciation for his many years with us. LEWIS VICTOR HEILBRUNN by H. B. Steinbach This is a note in memory of Lewis Victor Heilbrunn who died in an automobile acci- dent early last fall. If a memorial could echo the nature of the man, it would be vig- orous, terse, highly intelligent and very human. Lewis Victor Heilbrunn was one of the most influential figures of modern cellular biology, not only through his books and scientific papers but through his impact on his students and associates. He had the special knack of bringing out and fostering the intellectual best of those who worked with him. In large part this must have been due to the fact that he spent his life as an eager searcher after truth, not as a repository of the truth. Thus, those who talked to him of their problems, scientific and otherwise, found themselves discussing the problems and arriving at conclusions rather than being told answers. Surely this is at the heart of all good teaching, and Heilbrunn was its best exponent. His life was intimately associated with the Marine Biological Laboratory, and he loved the institution with a fierce devotion. The records show him first appearing here as a student investigator in 1912 at the age of twenty. He was elected a Member of the Corporation in 1915. The Director's report for this year records that Heilbrunn, with a few others, was responsible for raising the sum of twelve dollars to enable the library to subscribe to the British Journal of Physiology. He was elected a Trustee in 1931 and to the Executive Committee in 1934, and over the years continued to serve the MBL in a variety of capacities. While his services to the Laboratory may lose their sharpness with the death of the man, they do not cease. At least eighteen active members of the Corporation received their doctorate degrees under his direction, as did four who have served or are serving on the Executive Com- mittee. An equal number of workers active in the interests of the Laboratory gladly would acknowledge their direct debt to his training. In 1917 and 1918 his name does not appear on the attendance records of the Labora- tory. During these years he served as a pilot in the then new air force of his country. In a parenthetical way it could be noted that it is quite consistent with the essential daring of the man that he should be an accomplished pilot of an aircraft fifteen years before he learned to drive a car. In 1919 the record shows him in attendance as an Independent Investigator from Brooklyn, New York, his home town. The record does not spell out the circumstances but one can be sure that he paused but briefly at home upon demobilization and then took off at once for his beloved Woods Hole. Heilbrunn's professional career is recorded in other places and need not be repeated here. He was a great man, not to be illustrated by a recital of data. Time may dim the memory but his influence will be great for years to come. In the absence of ade- REPORT OF THE DIRECTOR 11 quate words the true nature of the man will be found residing in the memories of those who had the pleasures and the jolts of working with him. He was a catalyst, an arouser. Some awoke to anger, to difference but this was productive; some he awoke to curiosity, to the equable search; some he awoke to fire, to the necessity of looking and thinking and doing day and night, in dreams as in waking, for the truth that man must seek in the laboratory, in the university, in life. To work with Heilbrunn was to be a part of his family. The interests of the world were the subjects of his cosmic classroom, housed alike in the laboratory, ice cream socials and the soft-ball field. We extend to his widow, Ellen Donovan Heilbrunn and to his daughter Constance our understanding sympathy and our gratitude for sharing him with us. ZOOLOGY I. CONSULTANTS F. A. BROWN, JR., Professor of Zoology, Northwestern University LIBBIE H. HYMAN, American Museum of Natural History A. C. REDFIELD, Woods Hole Oceanographic Institution II. INSTRUCTORS GROVER C. STEPHENS, Associate Professor of Zoology, University of Minnesota, in charge of the course MILTON FINGERMAN, Assistant Professor of Zoology, Newcomb College, Tulane Uni- versity BERNARD L. STREHLER, Chief, Cellular and Comparative Physiology, Division of Geron- tology, National Institutes of Health PAUL P. WEINSTEIN, Laboratory of Tropical Disease, National Institutes of Health RICHARD C. SANBORN, Professor of Zoology, Department of Biological Sciences, Purdue University JAMES CASE, Associate Professor of Zoology, State University of Iowa A. FARMANFARMAIAN, Research Associate, University of California G. F. GWILLIAM, Assistant Professor of Biology, Reed College III. ASSISTANTS ROBERT ASHMAN, Wabash College JONATHAN P. GREEN, University of Minnesota EMBRYOLOGY I. INSTRUCTORS MAC V. EDDS, JR., Professor of Biology, Brown University, in charge of the course PHILIP GRANT, Assistant Professor of Pathobiology, The Johns Hopkins University LIONEL I. REBHUN, Assistant Professor of Biology, Princeton University JOHN W. SAUNDERS, JR., Professor of Zoology, Marquette University NELSON T. SPRATT, JR., Professor of Zoology, University of Minnesota MAURICE SUSSMAN, Associate Professor of Biology, Brandeis University II. LABORATORY ASSISTANTS DAVID SONNEBORN, Brandeis University RICHARD WHITTAKER, Yale University 12 MARINE BIOLOGICAL LABORATORY PHYSIOLOGY I. CONSULTANTS MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania OTTO LOEWI, Professor of Pharmacology, New York University School of Medicine ARTHUR K. PARPART, Professor of Biology, Princeton University ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Marine Biological Laboratory II. INSTRUCTORS W. D. MCELROY, Director, McCollum-Pratt Institute, The Johns Hopkins University; in charge of the course FRANCIS D. CARLSON, Associate Professor of Biophysics, The Johns Hopkins University BERNARD D. DAVIS, Professor of Pharmacology, Harvard Medical School DONALD R. GRIFFIN, Professor of Zoology, Harvard University TIMOTHY H. GOLDSMITH, Society of Fellows, Harvard University ROBERT B. LOFTFIELD, Massachusetts General Hospital BOTANY I. CONSULTANT WILLIAM RANDOLPH TAYLOR, Professor of Botany, University of Michigan II. INSTRUCTORS RICHARD C. STARR, Associate Professor of Botany, Indiana University, in charge <>! Ihr course WALTER R. HERNDON, Associate Professor of Botany, University of Alabama JOHN M. KINGSBURY, Assistant Professor of Botany, Cornell University III. COLLECTOR JOYCE FLETCHER, New York Botanical Garden IV. LABORATORY ASSISTANTS MELVIN GOLDSTEIN, Indiana University PHILIP COOK, Botany Department, Indiana University ECOLOGY I. CONSULTANTS PAUL GALTSOFF, U. S. Fish and Wildlife Service, W'oods Hole ALFRED S. REDFIELD, Woods Hole Oceanographic Institution BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution EDWIN T. MOUL, Rutgers University CHARLES E. JENNER, University of North Carolina HOWARD T. ODUM, University of Texas REPORT OF THE DIRECTOR 13 II. INSTRUCTORS EUGENE P. ODUM, Alumni Foundation Professor of Zoology, University of Georgia, in charge of the course JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution HOWARD L. SANDERS, Woods Hole Oceanographic Institution WALTER R. TAYLOR, Chesapeake Bay Institute, The Johns Hopkins University III. LABORATORY ASSISTANTS RICHARD B. WILLIAMS, Harvard University and Marine Institute, University of Georgia ELIJAH V. SWIFT, Swarthmore College 1. THE LABORATORY STAFF, 1960 HOMER P. SMITH, General Manager MRS. DEBORAH LAWRENCE HARLOW, Librarian ROBERT KAHLER, Superintendent, CARL O. SCHWEIDENBACK, Manager of the Buildings and Grounds Supply Department ROBERT B. MILLS, Manager, DC- IRVINE L. BROADBENT, Office Manager velopment of Research Service GENERAL OFFICE MRS. LILA S. MYERS MRS. MARION C. CHASE MRS. VIVIEN R. BROWN MRS. VIVIAN I. MANSON MRS. VIRGINIA M. MOREHOUSE MRS. RUTH MAYO LIBRARY MRS. GWENDOLYN S. BLOMBERG JANICE PARENT ALBERT K. NEAL MAINTENANCE OF BUILDINGS AND GROUNDS ROBERT ADAMS RALPH H. LEWIS ELDON P. ALLEN RUSSELL F. LEWIS GARDNER GAYTON ALAN G. LUNX ROBERT GUNNING ALTON J. PIERCE ROBERT W. HAMPTON ROBERT H. WALKER, JR. WALTER J. JASKUN JAMES S. THAYER DONALD B. LEHY DEPARTMENT OF RESEARCH SERVICE GAIL M. CAVANAUGH CAROLINE MCDAXIEL SEAVER R. HARLOW SUPPLY DEPARTMENT DONALD P. BURNHAM BRUNO F. TRAPASSO MILTON B. GRAY MRS. PATRICIA TRAVARES GEOFFREY J. LEHY JOHN J. VALOIS ROBERT O. LEHY JARED L. VINCENT ROBERT M. PERRY HALLETT S. \\~AGSTAFF 14 MARINE BIOLOGICAL LABORATORY 2. INVESTIGATORS; LALOR AND LILLIE FELLOWS; AND STUDENTS Independent Investigators, 1960 ADAMS, RALPH G., National Institutes of Health ADELMAN, WILLIAM J., JR., Physiologist, National Institutes of Health ALLEN, M. JEAN, Professor of Biology and Chairman, Wilson College AMATNIEK, ERNEST, Biophysicist, College of Physicians and Surgeons AMBERSON, WILLIAM R., Marine Biological Laboratory ANDERSON, JOHN MAXWELL, Professor of Zoology, Cornell University ARMSTRONG, PHILIP B., Professor and Chairman, Department of Anatomy, Upstate Medical Center ATWOOD, KIMBALL C, Associate Professor of Medical Genetics, University of Chicago BALTUS, ELYANE, Research Associate, Free University of Brussels, Belgium BANG, FREDERIK B., Chairman, Department of Pathobiology, Johns Hopkins School of Hygiene BARTH, L. G., Professor of Zoology, Columbia University BAYLOR, MARTHA B., Marine Biological Laboratory BEEVERS, HARRY, Professor of Biology, Purdue University BELTON, PETER, College of Physicians and Surgeons, Columbia University BENNETT, MICHAEL V. L., Assistant Professor of Neurology, College of Physicians and Surgeons BERENDSEN, HERMAN J. C., Staff Member, Massachusetts Institute of Technology BERGER, CHARLES A., Chairman, Biology Department, Fordham University BERNSTEIN, MAURICE H., Assistant Professor, Wayne State University BORGESE, THOMAS A., Research Fellow in Medicine, Harvard Medical School BRADLEY, DAN F., Scientist, Commissioned Corps of U. S. Public Health Service BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University BUCK, JOHN B., Physiologist, National Institutes of Health BUDINGER, THOMAS F., Woods Hole Oceanographic Institution BURBANCK, W. D., Professor of Biology, Emory University BURKE, JOSEPH A., Assistant Professor of Biology, Loyola College CABRERA, GUILLERMO, Assistant Professor of Biochemistry, New York University College of Dentistry CARLSON, FRANCIS D., Associate Professor, The Johns Hopkins University CASE, JAMES, Assistant Professor of Zoology, State University of Iowa CHAET, ALFRED B., Associate Professor of Biology, The American University CHALAZONITIS, N., Charge de Recherches, Faculte des Sciences, Lyon, France CHANDLER, WILLIAM K., Medical Officer, Public Health Service CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College CHILD, FRANK M., Instructor in Zoology, University of Chicago CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School CLARK, ARNOLD M., Professor of Biology, University of Delaware CLEMENT, ANTHONY C., Professor of Biology, Emory University COHEN, BERNARD, Visiting Scholar, Columbia University COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health COLLIER, JACK R., Marine Biological Laboratory COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve School of Medi- cine COPELAND, EUGENE, Chairman, Zoology Department, Tulane University COSTELLO, DONALD P., Kenan Professor of Zoology, University of North Carolina CRANE, ROBERT K., Associate Professor of Biological Chemistry, Washington University Medi- cal School CROWELL, SEARS, Associate Professor in Zoology, Indiana University DAVIS, BERNARD D., Head, Department of Bacteriology, Harvard Medical School DAVISON, JOHN A., Assistant Professor of Zoology, Florida State University DAVSON, HUGH, Medical Research Council and University College, London DETTBARN, WOLF D., Research Associate, College of Physicians & Surgeons DOWBEN, ROBERT M., Assistant Professor of Medicine, Northwestern University REPORT OF THE DIRECTOR 15 DuBois, ARTHUR B., Associate Professor of Physiology, University of Pennsylvania, Graduate School of Medicine EDDS, MAC V., JR., Professor of Biology, Brown University EDWARDS, CHARLES, Associate Professor of Physiology, University of Minnesota EHRENPREIS, SEYMOUR, Assistant Professor, College of Physicians & Surgeons EICHEL, HERBERT J., Research Associate, Hahnemann Medical College FAILLA, G., Professor, Columbia University FAILLA, PATRICIA, Columbia University FARMANFARMAIAN, ALLAHVERDI, Research Associate, University of California FELDHERR, CARL, Physiologist, College of Physicians & Surgeons FIELD, JAMES B., Senior Investigator, National Institutes of Health FINGERMAN, MILTON, Assistant Professor of Zoology, Newcomb College of Tulane University FISCHER, SIEGMUND, Research Associate, Albert Einstein College of Medicine FLAKS, JOEL G., Associate, Department of Biochemistry, University of Pennsylvania School of Medicine FULTON, CHANDLER M., Assistant Professor of Biology, The Rockefeller Institute FUORTES, M. G. F., Chief, Neurophysiology Section, National Institutes of Health FURSHPAN, EDWIN J., Instructor in Neurophysiology, Harvard Medical School GLADE, RICHARD W., Assistant Professor of Zoology, University of Vermont GOLDSMITH, TIMOTHY H., Junior Fellow, Harvard University GRANT, PHILIP, Assistant Professor of Pathobiology, The Johns Hopkins University School of Hygiene GREEN, JAMES W., Associate Professor of Physiology, Rutgers University GREGG, JAMES H., Associate Professor of Biology, University of Florida GRIFFIN, DONALD R., Professor of Zoology, Harvard University GROSCH, DANIEL S., Professor of Genetics, North Carolina State College GROSS, PAUL R., Associate Professor of Biology, New York University GRUNDFEST, HARRY, Associate Professor of Neurology, College of Physicians & Surgeons GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College GWILLIAM, GILBERT F., Assistant Professor of Biology, Reed College HAGERMAN, DWAIN D., Associate in Biological Chemistry, Harvard Medical School HAGINS, WILLIAM A., Physiologist, National Institutes of Health HARDING, CLIFFORD V., Assistant Professor of Physiology, College of Physicians & Surgeons HARVEY, ETHEL BROWNE, Marine Biological Laboratory HAYASHI, TERU, Professor of Zoology, Columbia University HEGYELI, ANDREW F., Biochemist, Institute for Muscle Research, Marine Biological Laboratory HEINMETS, FERDINAND, Head, Physiology Laboratory, Research and Engineering Center, Natick HENLEY, CATHERINE, Research Associate, University of North Carolina HERNDON, WALTER R., Associate Professor of Botany, University of Alabama HERVEY, JOHN P., Senior Electronic Engineer, Marine Biological Laboratory HIRSHFIELD, HENRY I., Associate Professor of Biology, New York University HOLTZER, HOWARD, Associate Professor of Anatomy, University of Pennsylvania, School of Medicine HOLZ, GEORGE G., JR., Associate Professor of Zoology, Syracuse University HOSKIN, FRANCIS C. G., Assistant Professor, College of Physicians & Surgeons HURWITZ, JERARD, Assistant Professor of Microbiology, New York University ISENBERG, IRVIN, Institute for Muscle Research, Marine Biological Laboratory JOHNSON, SISTER MARIA BENIGNA, Professor of Biology, Saint Joseph College KAAN, HELEN W., Marine Biological Laboratory KAMINER, BENJAMIN, Senior Lecturer in Physiology, University of Witwatersrand, Johannes- burg, S. A. KANE, ROBERT E., Assistant Professor of Biochemistry, Brandeis University KEMPTON, RUDOLF T., Professor of Zoology, Vassar College KEOSIAN, JOHN, Professor of Biology, Rutgers University KESSEL, RICHARD G., Instructor in Anatomy, Bowman Gray School of Medicine of Wake Forest KINGSBURY, JOHN M., Assistant Professor of Botany, Cornell University KISHIMOTO, UICHIRO, Rockefeller Fellow, National Institutes of Health KITAI, STEPHEN T., Graduate Student, Wayne State University and Lafayette Clinic 16 MARINE BIOLOGICAL LABORATORY KRANE, STEPHEN M., Assistant in Medicine, Massachusetts General Hospital KUFFLER, STEPHEN W., Professor of Neurophysiology, Harvard Medical School KURY, LIVIA REV., Institute for Muscle Research, Marine Biological Laboratory LAMBERT, FRANCIS L., Jacques Loeb Associate, Rockefeller Institute LANSING, ALBERT I., Professor of Anatomy, University of Pittsburgh School of Medicine LAURIE, JOHN S., Assistant Professor of Experimental Biology, University of Utah LAZAROW, ARNOLD, Professor and Head, Department of Anatomy, University of Minnesota LEVY, MILTON, Professor and Chairman, Department of Biochemistry, New York University College of Dentistry LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont LOEWENSTEIN, WERNER R., Associate Professor of Physiology, Columbia University LOFTFIELD, ROBERT B., Associate Biochemist, Massachusetts General Hospital LONDON, IRVING M., Professor of Medicine, Albert Einstein College of Medicine LOOMIS, WILLIAM F., Professor of Biochemistry, Brandeis University DELoRENZO, A. J., Director Anatomical Research Laboratories, Johns Hopkins Medical School LOVE, WARNER E., Assistant Professor of Biophysics, The Johns Hopkins University Luco, J. V., Professor of Neurophysiology, Catholic University Medical School, Santiago, Chile LYNCH, REV. WILLIAM F., St. Ambrose College MAHLER, HENRY R., Professor of Chemistry, Indiana University MANN, DAVID E., Professor of Pharmacology, Temple University MANN, THADDEUS R. R., Director, Unit of Reproductive Physiology and Biochemistry, Uni- versity of Cambridge, England MARGALEF, RAMON, Institute Investigaciones Pesqueras, Paseo Nacional, Barcelona, Spain MARSLAND, DOUGLAS, Professor of Biology, New York University, Washington Square College MATEYKO, G. M., Assistant Professor of Biology, New York University, Washington Square College MATHEWS, MARTIN B., Associate Professor, University of Chicago MCELROY, W. D., Chairman, Department of Biology and Director, McCollum-Pratt Institute METZ, CHARLES B., Professor of Zoology, Florida State University METZ, CHARLES W., Marine Biological Laboratory MIDDLEBROOK, W. ROBERT, Institute for Muscle Research, Marine Biological Laboratory MILLER, JAMES A., Professor of Anatomy, Emory University MITCHISON, J. M., Reader, University of Edinburgh, Scotland MOORE, JOHN W., Associate Chief, Laboratory of Biophysics, National Institutes of Health MORI, SYUITI, Professor, Zoological Institute, Kyoto University, Kyoto, Japan MORRILL, JOHN B., Assistant Professor of Biology, Wesleyan University MOSCONA, A. A., Associate Professor, University of Chicago MOYED, HARRIS S., Associate in Bacteriology, Harvard Medical School MULLINS, L. J., Associate Professor of Biophysics, Purdue University MUSACCHIA, X. J., Associate Professor in Biology, Saint Louis University NACE, PAUL FOLEY, Associate Professor of Zoology, McMaster University NELSON, LEONARD, Associate Professor of Physiology, Emory University ODUM, EUGENE P., Alumni Foundation Professor of Zoology, University of Georgia OIKAWA, TOSHIHIKO, Assistant Professor, Department of Physiology, Tohoku University School of Medicine, Sendai, Japan OSTERHOUT, W. J. V., Rockefeller Institute PALINCSAR, EDWARD E., Instructor in Biology, Loyola University PAPENFUSS, GEORGE F., Professor of Botany, University of California PARKER, JOHNSON, Assistant Professor in Plant Physiology, Yale University PARPART, ARTHUR K., Chairman and Professor of Biology, Princeton University PATERSON, MABEL C., Assistant Professor of Zoology, Vassar College PERSON, PHILIP, Veterans Administration Hospital, Brooklyn PO-CHEDLEY, DONALD S., Professor and Chairman of Biology Department, D'Youville College POLGAR, GEORGE, Research Fellow, University of Pennsylvania, Graduate School of Medicine POTTER, DAVID D., Instructor in Neurophysiology, Harvard Medical School RAPPORT, MAURICE M., Professor of Biochemistry, Albert Einstein College of Medicine REBHUN, LIONEL I., Assistant Professor of Biology, Princeton University REUBEN, JOHN P., Research Associate, Columbia University REPORT OF THE DIRECTOR 17 REYNOLDS, LESLIE B., Assistant in Physiology, Medical College of South Carolina ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of Medi- cine ROSE, S. MERYL, Professor of Zoology, University of Illinois ROSENBERG, EVELYN K., Associate Professor, New York University-Bellevue Medical Center ROSENBERG, PHILIP, Special Postdoctoral Trainee Fellow, College of Physicians & Surgeons ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College RUGH, ROBERTS, Associate Professor of Radiology, Columbia University RUSHTON, W. A. H., Chief, Neurophysiology Section, National Institutes of Health RUSTAD, RONALD C, Assistant Professor of Physiology, Florida State University RYTHER, JOHN H., Marine Biologist, Woods Hole Oceanographic Institution SANBORN, RICHARD C., Professor of Zoology, Purdue University SANDERS, HOWARD L., Research Associate, Woods Hole Oceanographic Institution SATO, HIDEMI, Dartmouth Medical School SAUNDERS, JOHN W., Professor and Chairman, Department of Biology, Marquette University SCHACHMAN, HOWARD K., Professor of Biochemistry, University of California SCHUH, JOSEPH E., Associate Professor of Biology, Saint Peter's College SCOTT, ALLAN C., Professor of Biology, Colby College SCOTT, SISTER FLORENCE MARIE, Professor and Chairman, Department of Biology, Seton Hill College SCOTT, GEORGE T., Professor and Chairman, Department of Zoology, Oberlin College SEGAL, JOHN R., Physiologist, Veterans Administration Hospital, Boston SELIGER, HOWARD H., Research Associate, The Johns Hopkins University SMELSER, GEORGE K., Professor of Anatomy, Columbia University SMITH, LESLIE FRANK, Department of Biochemistry, Cambridge, England SONNENBLICK, B. P., Professor of Biology, Rutgers University SPECTOR, ABRAHAM, Instructor, Howe Laboratory SPEIDEL, CARL C., Professor of Anatomy, University of Virginia, School of Medicine SPIEGEL, MELVIN, Assistant Professor of Zoology, Dartmouth College SPINDELL, WILLIAM, Associate Professor of Chemistry, Rutgers University SPRATT, NELSON T., JR., Chairman and Professor of Zoology, University of Minnesota SPYROPOULOS, CONSTANTINE S., Neurophysiologist, National Institutes of Health STARR, RICHARD C., Associate Professor of Botany, Indiana University STEIN, MYRON, Instructor in Medicine, Harvard Medical School STEINBACH, H. BURR, Professor and Chairman, Department of Zoology, University of Chicago STEPHENS, GROVER C., Associate Professor of Zoology, University of Minnesota STONE, WILLIAM, JR., Director, Ophthalmic Plastics Laboratory, Massachusetts Eye and Ear Infirmary STREHLER, BERNARD L., Chief, Cellular and Comparative Physiology Section, National Insti- tutes of Health STRITTMATTER, CORNELIUS F., Assistant Professor of Biological Chemistry, Harvard Medical School STRITTMATTER, PHILIPP, Assistant Professor of Biochemistry, Washington University Medical School STRUMWASSER, FELIX, Neurophysiologist, National Institutes of Health STUNKARD, HORACE W., Research Associate, The American Museum of Natural History STURTEVANT, A. H., Professor of Genetics, California Institute of Technology SUDAK, FREDERICK N., Assistant Professor of Physiology, Albert Einstein College of Medicine SUSSMAN, MAURICE, Associate Professor, Brandeis University SZENT-GYORGYI, ALBERT, Director, Institute for Muscle Research, Marine Biological Laboratory SZENT-GYORGYI, ANDREW, Institute for Muscle Research, Marine Biological Laboratory TAKEUCHI, AKIRA, Fellow of Rockefeller Foundation, University of Utah TASAKI, ICHIJI, Chief, Special Senses Section, National Institutes of Health TAYLOR, ROBERT E., Physiologist, National Institutes of Health TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan TAYLOR, W. ROWLAND, Assistant Professor of Oceanography, The Johns Hopkins University TEWINKEL, Lois E., Professor of Zoology, Smith College DETERRA, NOEL, Post-doctoral Fellow, Rockefeller Institute 18 MARINE BIOLOGICAL LABORATORY TORCH, REUBEN, Assistant Professor of Zoology, University of Vermont TRACER, WILLIAM, Associate Professor, Rockefeller Institute TRAVIS, DAVID M., Assistant Professor of Pharmacology, University of Florida TROLL, WALTER, Associate Professor, New York University TWEEDELL, KENYON S., Assistant Professor of Biology, University of Notre Dame VAN NORMAN, EARL, SR., Princeton University DEVILLAFRANCA, GEORGE W., Associate Professor of Zoology, Smith College VILLEE, CLAUDE A., Associate Professor of Biological Chemistry, Harvard University VINCENT, WALTER S., Assistant Professor of Anatomy, Upstate Medical Center WARREN, LEONARD, Visiting Scientist, National Institutes of Health WATANABE, AKIRA, Research Associate, College of Physicians & Surgeons WEBB, H. MARGUERITE, Associate Professor, Goucher College WEINSTEIN, PAUL P., Senior Scientist, National Institutes of Health WEISS, LEON P., Associate Professor of Anatomy, Johns Hopkins Medical School WERMAN, ROBERT, Visiting Scholar, College of Physicians & Surgeons WHITING, ANNA R., Guest Investigator, University of Pennsylvania WICHTERMAN, RALPH, Professor of Biology, Temple University WIERCINSKI, FLOYD J., Associate Professor of Biological Science, Drexel Institute of Technology WILBER, CHARLES G., Professorial Lecturer in Biology, Loyola College WILLEY, CHARLES H., Professor and Chairman, Department of Biology, New York University WILSON, THOMAS HASTINGS, Assistant Professor of Physiology, Harvard Medical School WILSON, WALTER L., Assistant Professor of Physiology, University of Vermont College of Medicine WITTENBERG, JONATHAN B., Associate Professor of Physiology, Albert Einstein College of Medicine WRIGHT, PAUL A., Associate Professor of Zoology, University of New Hampshire YEANDLE, STEPHEN, Assistant Professor, George Washington University ZIGMAN, SEYMOUR, Research Associate, Massachusetts Eye and Ear Infirmary ZIMMERMAN, ARTHUR M., Instructor in Pharmacology, Downstate Medical Center ZWILLING, EDGAR, Professor of Biology, Brandeis University Lalor Fellows, 1960 MANN, THADDEUS R. R., Molteno Institute, Cambridge, England DOWBEN, ROBERT M., Northwestern University FLAKS, JOEL G., University of Pennsylvania School of Medicine HURWITZ, JERARD, New York University College of Medicine STRITTMATTER, CORNELIUS F., Harvard Medical School WARREN, LEONARD, National Institutes of Health Lillie Fellow, 1960 MOSCONA, A. A., University of Chicago Grass Fellows, 1960 KITAI, STEPHEN T., Massachusetts General Hospital Luco, J. V., Catholic University Medical School, Santiago, Chile REYNOLDS, LESLIE B., Medical College of South Carolina Beginning Investigators, 1960 ALSUP, PEGGY ANN, Harvard Medical School BEEBE, CURT, University of Vermont, College of Medicine BINSTOCK, LEONARD, National Institutes of Health BOLEYN, BRENDA J., University of Rhode Island BROWN, GEORGE W., North Carolina State College REPORT OF THE DIRECTOR 19 CAMPBELL, JAMES W., Rice Institute CIUCHTA, HENRY, Temple University, School of Pharmacy COOKE, IAN M., Harvard University CORRIDEN, FRANKLIN E., University of Delaware COSTELLO, ROBERT CHARLES, University of North Carolina CURTIS, DAVID R., Australian National University DOOLITTLE, RUSSELL F., Harvard University DUBNAU, DAVID, Columbia University DUDEL, JOSEF, Harvard Medical School DUNHAM, PHILIP B., University of Chicago EISENBERG, ROBERT S., Harvard College FAUST, ROBERT G., Princeton University FILOSA, MICHAEL F., Johns Hopkins University FRUMENTO, A. S., University of Buenos Aires GASSELING, MARY T., Marquette University HANLORE, MARY S., University of California HEX SHAW, EDGAR C, Harvard Medical School HUVER, CHARLES W., Yale University HWANG, JOSEPH CHI-CHIU, University of Oregon ISSELBACHER, KURT J., Massachusetts General Hospital JACKSON, JAMES A., Western Reserve University KATZ, GEORGE M., College of Physicians & Surgeons KROPF, ALLEN, Amherst College MORAN, JOSEPH F., JR., Russell Sage College ORKAND, RICHARD K., University of Utah PETERSON, R. PRICE, University of Pennsylvania, School of Medicine RUDOMIN, PEDRO N., Rockefeller Institute SCHUEL, HERBERT, New York University SHEPARD, DAVID, University of Chicago SIMMONS, JOHN E., Rice Institute SJODIN, R. A., Purdue University SUDDUTH, SOLON S., Johns Hopkins School of Medicine WHITELEY, GEORGE C., JR., The Hill School WHITFIELD, SYLVIA G., Tulane University WILF, RUTH T., University of Illinois WOOD, ROBERT W., Sloan-Kettering Division, Cornell University WORMSER, EVA H., Johns Hopkins University Research Assistants, 1960 ABBOTT, JOAN, University of Pennsylvania Medical School ANTLEY, RAY MILLS, Emory University AREND, WILLIAM P., Columbia Medical School ASHMAN, ROBERT F., Wabash College BAIRD, SPENCER, Institute for Muscle Research, Marine Biological Laboratory BARNWELL, FRANKLIN H., Northwestern University BAUER, ADELIA C., Marine Biological Laboratory BAUER, G. ERIC, University of Minnesota BERMAN, LAWRENCE JOSEPH, Harvard Medical School BERMAN, PAUL ELIOT, Upstate Medical Center BIANCHI, CARLA, Northwestern University BITO, LASZLO Z., Columbia University BLEYMAN, LEA K., Columbia University BLUMSTEIN, JOYCE R., Albert Einstein College of Medicine BOSLER, ROBERT, Harvard Medical School BRANHAM, JOSEPH M., Florida State University BURDICK, CAROLYN, Harvard Medical School BYRNE, PAUL M., National Institutes of Health 20 MARINE BIOLOGICAL LABORATORY CANBY, DIANE MARIE, Smith College CECCARINI, COSTANTE, St. Peter's College CICAK, ANNA, Albert Einstein College of Medicine CLARK, ELOISE E., Columbia University COOK, PHILIP WILLIAM, Indiana University CORDES, EUGENE, Brandeis University COUSINEAU, GILLES H., New York University CROWE, PRISCILLA, Seton Hill College DEWEL, WILLIAM C, Wesleyan University DIETRICH, THOMAS S., Wayne State University College of Medicine DINGLE, AL. D., University of Illinois DOWNS, PATRICIA, Colby College DUBIN, DONALD, Harvard Medical School DUBNAU, EUGENIE J., Columbia University EDWARDS, JOAN F., Wilson College EIGNER, ELIZABETH ANN, Massachusetts General Hospital ELEFANT, HELENE, Bellevue Medical Center ELEK, MARIA E., Johns Hopkins School of Hygiene ERSKINE, LOUISE, Institute for Muscle Research, Marine Biological Laboratory EVAN, GERALD L., University of Vermont EWING, RICHARD D., Reed College FEHRENBAKER, LAWRENCE G., Wayne State University, College of Medicine FELDSHUH, DANA, Massachusetts Eye and Ear Infirmary FINKEL, ARNOLD, New York University College of Medicine FISHER, SYLVIA S., Saint Louis University FLATHERS, ANN R., University of New Hampshire FLETCHER, JOYCE, New York City FONG, BETTY ANN, New York University FORAN, ELIZABETH H., Smith College GIBBON, CHARLOTTE A., Indiana University GOLDSTEIN, MELVIN E., Indiana University GRABSKE, ROBERT, Kansas University GRANT, DAVID C., Yale University GREEN, JONATHAN, University of Minnesota GREEN, SAMUEL A., JR., Claymont, Delaware HALEY, BARBARA, Brandeis University HALL, ZACH W., Emory University HALPERN, EVELYN, Western Reserve University Medical School HAMMOND, CONSTANCE, Radcliffe College HANSON, FRANK E., JR., State University of Iowa HATHAWAY, RALPH R., Florida State University HAYWARD, GEORGE, National Institutes of Health HENRY, ELEANOR, Hahnemann Medical College HESSLER, ANITA Y., Woods Hole, Massachusetts HIRSCH, CARL A., Harvard Medical School HOLSTEIN, IRMA, University of Pennsylvania, Graduate School of Medicine HOLSTEN, GEORGE H., Ill, Yale University HUFNAGEL, LINDA, University of Vermont HUMPHREYS, TOM D., University of Chicago HUTTRER, ANNICK, Mount Holyoke College JACKSON, THOMAS J., Lehigh University JAFFREY, IRA S., New York State University KELLOCK, MARGERY, College of Physicians & Surgeons KENNEN, DANE E. M., American University KIMBALL, SALLY P., Columbia University KREWSON, CARRIE R., Vassar College LAUFENBERG, HENRY J., Saint Peter's College LEHV, JANE WENDY, Vassar College REPORT OF THE DIRECTOR 21 LEINING, JUDITH M., Massachusetts Eye and Ear Infirmary LEMMA, AKLILU, Johns Hopkins University School of Hygiene LENOX, MARILYN, Philippi, West Virginia LIBBIN, DICK, University of Cincinnati LOOMIS, WILLIAM F., JR., Loomis Laboratory LORING, JANET M., Harvard Medical School MCKENZIE, SHARON G., American University MAcNicHOL, EDWARD F., JR., Johns Hopkins University MAKINEN, PAULA M., University of Minnesota MILLER, HEDWIG B., Wellesley College MILLS, NANCY L., College of Physicians & Surgeons MINGIOLI, ELIZABETH S., Harvard University MUSICK, ROY, American University NAGABHUSHANAM, R., Tulane University NAUMANN, DOROTHY C., Smith College NORRIS, ELAINE, Wesleyan University GETTING, BONNALIE J., Northwestern University OTERO-VILARDEBO, Luis R., University of Puerto Rico OWENS, DEAN PAUL, Johns Hopkins University PALMER, JOHN D., Northwestern University PHILPOTT, CHARLES W., Tulane University PHILPOTT, LORALEE, Tulane University POLLACK, MATTHEW, National Institutes of Health RANLETT, MARY, Dartmouth College RAY, FRANCES L., Bellevue Medical Center ROBERTS, MARY Lou, Washington University Medical School RODGERS, PATRICIA E., New York University ROSENBLUTH, RAJA, Columbia University ROSSMAN, RONALD E., Princeton University SCOTT, NANCY F., University of Vermont SCRICCO, ELAINE ANN, Howe Laboratory SEIDMAN, AARON, Brandeis University SIMON, BARBARA, Rutgers University SMALLER, BERNARD, Argonne National Laboratory SMITH, ISSAR, Columbia University SONNEBORN, DAVID R., Brandeis University SPENCER, JOYCE, Harvard Medical School SPRITZER, RUTH C., New York University School of Medicine SRINIVASAN, DOBLI, College of Physicians & Surgeons STEINBERG, SONIA N., Brandeis University STERN, EDWARD L., University of Chicago SUTHERLAND, KERSTIN E., Institute for Muscle Research, Marine Biological Laboratory SWIFT, ELIJAH, Swarthmore College SZENT-GYORGYI, EVE, Institute for Muscle Research, Marine Biological Laboratory SZENT-GYORGYI, MARTA, Institute for Muscle Research, Marine Biological Laboratory THOMAS, CYNTHIA, Massachusetts Eye and Ear Infirmary TULCHIN, NATALIE, New York University WATKINS, DUDLEY T., Oberlin College WATTERS, CHRISTOPHER, Notre Dame University WEINTRAUB, ARTHUR H., New York University WEIS, PEDDRICK, New York University College of Dentistry WELLINGTON, FREDERICA M., Harvard Medical School WHITTAKER, J. RICHARD, Yale University WILKENS, JERREL L., Tulane University WILLIAMS, RICHARD B., University of Georgia WILSON, JOAN, Rice Institute ZAMBERNARDI, JOSEPH, Tulane University ZIMINSKY, ALVIN C., National Institutes of Health 22 MARINE BIOLOGICAL LABORATORY Library Readers, 1960 ARVANITAKI, ANGELIQUE, Director, Faculte de Sciences, Lyon, France BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School BECK, LYLE V., University of Pittsburgh School of Medicine BLUM, HAROLD F., Physiologist, National Cancer Institute and Princeton University BODANSKY, OSCAR, Chief, Division of Enzymology and Metabolism, Sloan-Kettering Institute BOVEE, EUGENE C, Associate Professor, University of Florida BRIDGMAN, ANNA JOSEPHINE, Professor of Biology, Agnes Scott College BUTLER, ELMER G., Professor of Zoology, Princeton University CHANUTIN, ALFRED, Professor of Biochemistry, University of Virginia School of Medicine CHASE, AURIN M., Associate Professor of Biology, Princeton University CLARK, ELIOT R., University of Pennsylvania COHEN, SEYMOUR S., Professor of Biochemistry, University of Pennsylvania EDER, HOWARD, Professor of Medicine, Albert Einstein College of Medicine EISEN, HERMAN N., Professor of Medicine, Washington University FLAVIN, MARTIN, National Heart Institute, National Institutes of Health FLESCH, PETER, Associate Professor of Research Dermatology, University of Pennsylvania FRIES, E. F. B., Associate Professor, The City College of New York GINSBERG, HAROLD S., Associate Professor, Western Reserve University GOLDTHWAIT, DAVID A., Assistant Professor of Biochemistry, Western Reserve University GREEN, MAURICE, Assistant Professor, Saint Louis University School of Medicine HERRMANN, ROBERT L., Assistant Professor of Biochemistry, Boston University School of Medicine HOBERMAN, HENRY D., Professor of Biochemistry, Albert Einstein College of Medicine HURWITZ, CHARLES, Chief, General Medical Research Laboratory, Veterans Administration Hospital JACOBS, M. H., Professor Emeritus, University of Pennsylvania JENNISON, MARSHALL W., Chairman, Department of Bacteriology and Botany, Syracuse Uni- versity KARUSH, FRED, Professor of Immunochemistry, University of Pennsylvania School of Medicine KLEIN, MORTON, Professor of Microbiology, Temple University School of Medicine KLOTZ, IRVING M., Professor of Chemistry and Biology, Northwestern University LENHOFF, HOWARD M., Howard Hughes Memorial Institute LEVINE, RACHMIEL, Chairman, Department of Medicine, Michael Reese Hospital LIONETTI, FABIAN J., Associate Professor of Biochemistry, Boston University School of Medicine LUBIN, MARTIN, Assistant Professor of Pharmacology, Harvard Medical School MCDONALD, SISTER ELIZABETH SETON, Chairman, Department of Biology, College of Mt. St. Joseph MOUL, EDWIN T., Associate Professor of Botany, Rutgers University NELSON, THOMAS C., Senior Microbiologist, Eli Lilly and Company NOVIKOFF, ALEX B., Research Professor, Albert Einstein College of Medicine PEABODY, RICHARD A., Assistant Professor of Biochemistry, Albany Medical College PICK, JOSEPH, Professor of Anatomy, New York University Medical Center PULLMAN, BERNARD, Professor, University of Paris ROOT, WALTER S., Professor of Physiology, College of Physicians & Surgeons ROTH, REV. OWEN H., Associate Professor of Zoology, St. Vincent College SCHLAMOWITZ, MAX, Associate Cancer Research Scientist, Roswell Park Memorial Institute SCHWARZ, KLAUS, Chief, Section on Experimental Liver Diseases, National Institutes of Health SPIRTES, M. A., Associate Professor of Pharmacology, Hahnemann Medical College STETTEN, DEWiTT, Associate Director in Charge of Research, National Institutes of Health STETTEN, MARJORIE R., Biochemist, National Institutes of Health SULKIN, S. EDWARD, Professor and Chairman, Department of Microbiology, University of Texas Southwestern Medical School TOLKSDORF, SIBYLLE, Senior Biochemist, Schering Corporation TRURNIT, HANS J., Principal Scientist, Research Institute for Advanced Study VILLANI, FRANK J., Senior Research Chemist, Schering Corporation WAINIO, WALTER W., Professor of Biochemistry, Rutgers University REPORT OF THE DIRECTOR 23 WARNER, ROBERT C., Associate Professor of Biochemistry, New York University College of Medicine WEIGLE, WILLIAM O., Assistant Professor of Immunochemistry in Pathology, University of Pittsburgh School of Medicine WEXLER, HARRY, Director of Research, U. S. Weather Bureau WHEELER, GEORGE E., Assistant Professor of Biology, Brooklyn College YNTEMA, CHESTER L., Professor of Anatomy, Upstate Medical Center at Syracuse Students, 1960 All students listed completed formal course program, June 21-July 30. Asterisk indicates students completed Post Course Research Program, August 1-September 3. BOTANY AUYANG, SHIH-CHEN, Clark University *BONAMO, PATRICIA M., Cornell University BROOKS, AUSTIN E., Wabash College *CYRUS, RODNEY V., University of Michigan DEUTSCH, ELIZABETH J., Radcliffe College ERICKSON, PAUL A., Clark University FALCON, GISELA, Ave. Galipan No. 16, San Bernardino, Caracas, Venezuela FRANKLIN, SANDRA E., Acadia University *HALL, NANCY V., Vassar College KOETZNER, KENNETH L., Lycoming College KREMER, PETER R., Cornell University LANG, NORMA J., Indiana University MITCHELL, ROBERT A., Cornell University MULLIN, MICHAEL M., Harvard University *NICHOLS, HERBERT W., University of Alabama NOODEN, LARRY D., Harvard University *WATERS, ANNETTE, Indiana University *WESTERDALE, THOMAS H., University of Michigan EMBRYOLOGY *ALLISON, WILLIAM S., Brandeis University ARNOLD, JOHN M., University of Minnesota *CLARKE, RICHARD B., University of Illinois *COHEN, NICHOLAS, University of Rochester EISENSTADT, JEROME, Brandeis University *ESPER, HILDEGARD, Columbia University *GREEN, SANDRA J., University of Minnesota HOVINGH, PETER, Johns Hopkins University *JAFFEE, ROBERT L., University of Rochester *KIMMEL, DONALD L., JR., Temple University LICHTENBERG, INGEBORG, University of Chicago MARSHALL, LEE ANN, University of Michigan ORLOFF, SERVE, Brussels, Belgium *PLATT, JOHN R., University of Chicago RACE, JAMES, JR., State University of Iowa *RITTENHOUSE, ELIZABETH W., University of Michigan SLATER, DONALD W., Indiana University SWEENY, PHILLIP R., Brown University WILLE, JOHN J., JR., Indiana University *WINESDORFER, JOHN E., Johns Hopkins University 24 MARINE BIOLOGICAL LABORATORY ECOLOGY *ALEXANDER, DOUGLAS G., University of North Carolina BEARDOW, JANE M., Drew University *BROUGHTON, WILLIAM S., University of Georgia *DE LA CRUZ, ARMANDO, University of the Philippines, Pasay City, Philippines GOLD, KENNETH, New York University *GUSTAFSON, ALTON H., Bowdoin College *GUTKNECHT, JOHN W., University of North Carolina *KRAMER, DANA D., City College of New York *PLATZMAN, SARA J., Yale University *STERNS, CAROL W., Peekskill, New York VANDENACK, SISTER JULIA MARIE, Catholic University of America *WILKENS, JERREL L., Tulane University *ZiEG, ROGER G., University of Nebraska PHYSIOLOGY ALBERTS, BRUCE M., Harvard College *BOASS, AGNA, Radcliffe College *BRODY, STUART, Stanford University *COLLIER, ROBERT J., Harvard University *DOLAN, MICHAEL F., Johns Hopkins University *FORREST, HELEN F., Rutgers University *FREEMAN, ALAN R., Hahnemann Medical College FRIDOVICH, IRWIN, Duke University *HALL, ZACH W., Emory University *HEMPFLING, WALTER P., Yale University HOLTZMAN, ERIC, Columbia University *MADDUX, WILLIAM S., Princeton University McEwEN, BRUCE S., Rockefeller Institute NADING, Louis K., Oberlin College *NATHENSON, STANLEY G., Washington University *NORRIS, JOHN L., Vanderbilt University ORR, CHARLES W. M., Johns Hopkins University *PATRICK, NOEL V., Columbia University RICHARDSON, G. S., Harvard Medical School ROSENFIELD, CAROL, New York University College of Medicine *RozE, ULDIS, Washington University Medical School SCHINDLER, FREDERICK J., University of Pennsylvania *SCHWARTZ, NORMAN M., Syracuse University SNIPES, CHARLES A., Duke University *STONE, HENRY O., Duke University TANG, JIEN-NAN JORDAN, Oklahoma Medical Research Foundation *TURNEY, TULLY, JR., University of North Carolina WEBB, GEORGE D., University of Colorado Medical School INVERTEBRATE ZOOLOGY ALEXANDER, KATHLEEN, University of North Carolina *BALLARD, JULIET L., Drew University BOTTOMLY, GAIL, University of Massachusetts BRIGGS, RICHARD G., Cornell University *BROCH, EDMUND S., Cornell University *BRUNO, MERLE S., Syracuse University CHAICHARN, AIMORN, University of New Hampshire CLARRIDGE, JILL E., University of Michigan REPORT OF THE DIRECTOR 25 *CLELAND, CHARLES F., Wabash College COSTELLO, ROBERT C., University of North Carolina *D'AcosTiNO, ANTHONY S., New York University DRUMMOND, SISTER THERESE, Catholic University of America EAGLESON, LOUISE J., Spellman College EDLIN, GORDON J., University of Oregon EMLEN, JOHN M., University of Wisconsin FARRELL, CAROLYN ROSE, Marquette University *FENNER, BARBARA, Vassar College FOURCADE, MIGUEL, S. J., Fordham University GAUTHIER, GERALDINE F., Harvard Medical School *HADDAD, LAMIA, Brown University HARMAN, MARY, Radcliffe College HOLLAND, NICHOLAS D., Carleton College HOLT, PORTIA, Colorado College *HOPPER, FRED A., JR., University of Oklahoma JARVIS, SISTER JULIE, Catholic University of America KANESHIRO, EDNA S., Syracuse University KECK, CARL W., Lafayette College KEE, JAMES W., JR., Massachusetts Institute of Technology *KIRCHENBERG, RALPH J., DePaul University KNOWLTON, ROBERT E., Bowdoin College KREWSON, CARRIE R., Vassar College LUCKENBILL, LOUISE M., Washington University MAHOWALD, ANTHONY P., S. J., Johns Hopkins University MORRISON, ROBERTA A., Smith College PORCARO, CAROL A., Marymount College *SCHOPF, THOMAS J. M., Oberlin College SMITH, STEPHEN D., Wesleyan University SQUADRONI, JOSE, S. J., Fordham University THEROUS, ROGER B., Bureau of Commercial Fisheries VOGEL, STEVEN, Tufts University WAUGH, MARY, Wilson College WESTHOFF, DAVID D., St. Louis University ZWEIG, CHARLES H., Brandeis University 3. FELLOWSHIPS AND SCHOLARSHIPS, 1960 Calkins Scholarship : RICHARD BRIGGS, Invertebrate Zoology Course Bio Club Scholarship : DANA KRAMER, Ecology Course Lucretia Crocker Fellowships : KENNETH GOLD, Ecology Course GISELA FALCON, Botany Course 4. TABULAR VIEW OF ATTENDANCE, 1956-1960 1956 1957 1958 1959 1960 INVESTIGATORS TOTAL 304 326 410 427 458 Independent 184 186 203 215 231 Under Instruction 20 23 39 45 42 Library Readers 50 42 54 51 50 Research Assistants 50 75 114 116 135 26 MARINE BIOLOGICAL LABORATORY STUDENTS TOTAL 140 139 138 134 122 Invertebrate Zoology 55 55 55 49 43 Embryology 28 27 22 23 20 Physiology 30 30 27 27 28 Botany 18 18 18 20 18 Ecology 9 9 16 15 13 TOTAL ATTENDANCE 444 465 548 561 580 Less persons represented as both investigators and students 2 3 5 4 2 442 462 543 557 578 INSTITUTIONS REPRESENTED TOTAL 130 129 142 143 144 By Investigators 97 94 110 98 83 By Students 33 35 74 73 61 SCHOOLS AND ACADEMIES REPRESENTED By Investigators 1 1 2 8 5 By Students 3 5 12 2 FOREIGN INSTITUTIONS REPRESENTED By Investigators 9 11 20 29 11 By Students 6 5 6 9 3 5. INSTITUTIONS REPRESENTED, 1960 Acadia University American Museum of Natural History American University Amherst College Bowdoin College Brandeis University Brown University Carleton College Catholic University of America City College of New York Clark University Colby College Colorado College Columbia University Columbia University College of Physicians and Surgeons Cornell University Dartmouth College DePaul University Drew University Duke University D'Youville College Drexel Institute of Technology Emory University Florida State University Fordham University George Washington University Goucher College Hahnemann Medical School Harvard University Harvard University Medical School Indiana University Institute for Muscle Research Johns Hopkins University Lafayette College Loyola College Marquette University Marymount College Massachusetts Institute of Technology McMaster University Medical College of South Carolina National Institutes of Health New York University Heights New York University, College of Dentistry New York University, College of Medicine New York University, Washington Square College North Carolina State College Notre Dame University Oberlin College Oklahoma Medical Research Foundation Princeton University Purdue University Queens College Radcliffe College Reed College Rice Institute Rockefeller Institute Rockefeller Foundation Russell Sage College Rutgers University Saint Joseph College Saint Louis University Seton Hill College Single Cell Research Foundation Smith College Spellman College State University of Iowa REPORT OF THE DIRECTOR 27 State University of New York, Upstate Medi- cal College State University of New York, Downstate Medical College Syracuse University Temple University Tufts College Tulane University University of Alabama University of California University of Chicago University of Colorado Medical School University of Delaware University of Florida University of Georgia University of Illinois University of Massachusetts University of Michigan University of Minnesota University of Nebraska University of New Hampshire University of North Carolina University of Oregon University of Pennsylvania University of Pennsylvania School of Medicine University of Pittsburgh School of Medicine University of Utah University of Vermont U. S. Bureau of Commercial Fisheries Vassar College V. A. Administration Hospital at Brooklyn Wabash College Washington University Washington University Medical School Wayne State University Wesleyan University Western Reserve University School of Medi- cine Wilson College Woods Hole Oceanographic Institution Yale University FOREIGN INSTITUTIONS REPRESENTED, 1960 Free University of Brussels, Belgium Faculte Des Sciences, Lyon, France University College, London University of Witwatersrand, Johannesburg, South Africa Catholic University Medical School, Santiago, Chile University of Cambridge, England University of Edinburgh, Scotland Kyoto University, Kyoto, Japan McMaster University, Canada Tohoku University School of Medicine, Sen- dai, Japan University of Buenos Aires University of the Philippines, Pasay City, Philippines Institute Investigaciones, Barcelona 3, Spain SUPPORTING INSTITUTIONS AND AGENCIES, 1960 Associates of the Marine Biological Labora- tory Atomic Energy Commission Josephine B. Crane Foundation The Grass Foundation The Lalor Foundation The Merck Company Foundation National Institutes of Health National Science Foundation Office of Naval Research The Rockefeller Foundation Schering Foundation, Inc. CORPORATE ASSOCIATES Abbott Laboratories CIBA Pharmaceutical Products, Inc. Carter Products, Inc. Eli Lilly and Company E. R. Squibb & Sons The Upjohn Company 6. EVENING LECTURES, 1960 June 24 G. ADRIAN HORRIDGE St. Andrews University, Scotland July 1 A. A. MOSCONA University of Chicago, Frank R. Lille Fellow at MBL "Electrophysiological and anatomical anal- ysis of primitive ganglia" "Experimental studies on tissue synthe- sis : problems and prospects" 28 MARINE BIOLOGICAL LABORATORY July 8 THADDEUS R. R. MANN Molteno Institute, University of Cambridge, Senior Lalor Fellow at MBL July 15 CLIFFORD V. HARDING Columbia University, College of Physicians & Surgeons July 22 J. V. Luco Catholic University of Chile, Alex- ander Forbes Lecturer at MBL July 25 J. V. Luco July 29 DEWITT STETTEN, JR. National Institutes of Health August 5 LASZLO LORAND Northwestern University August 12 SEVERO OCHOA New York University School of Medicine August 19 ERNST A. SCHARRER Albert Einstein College of Medicine August 26 GEORGE L. CLARKE Harvard University "Comparative aspects of sperm physiol- ogy" "The control of cell division" "Physiological studies during Wallerian degeneration" "The trophic effect of neuron activity" "The metabolism of gout" "The chemical basis of the clotting of blood" "Metabolism of propionic acid in animal tissues" "Neurosecretion" "Ecological aspects of daylight and bio- luminescence in the sea" 7. TUESDAY EVENING SEMINARS, 1960 July 5 MARTIN B. MATHEWS HAROLD F. BLUM July 12 RONALD C. RUSTAD DONALD P. COSTELLO C. C. SPEIDEL R. H. CHENEY July 19 ALBERT SZENT-GYORGYI IRVIN ISENBERG BENJAMIN KAMINER ANDREW HEGYELI July 26 W. ROBERT MIDDLEBROOK HERMAN J. C. BERENDSEN ALEX B. NOVIKOFF August 2 PHILIP PERSON ALBERT FINE KLAUS SCHWARZ S. EHRENPREIS "Some comparative biochemistry of con- nective tissue ground substance" "Complexity and organization" "X-ray induced dissociation of the mi- totic and micromere 'clocks' " "The giant cleavage spindle of the egg of Polychocnis cannelcnsis" "Motion pictures of radiation-induced modifications of fertilization and early development of the sea urchin Arbacia" "Energy and charge transfer" "Spin resonance studies of riboflavin semiquinones and riboflavin complexes" "Contractile responses in the presence of charge transfer complexes" "Detection of electron donors" "The action of trypsin on acetylated myo- sin" "The structure of water in tissue, as stud- ied by nuclear magnetic resonance" "Phagocytosis, pinocytosis and lysosomes : Cytochemical and electron microscopic studies" "The role of free radical formation during indophenol blue synthesis by respira- tory enzymes" "A role of trivalent chromium in glucose utilization" "A receptor protein : Isolation and drug binding properties" REPORT OF THE DIRECTOR 29 August 9 JAMES CASE "Excitation of firefly light organ" JOHN BUCK R. A. SJODIN "Cation permeability in muscle" P. BELTON "Effects of ions on potential in lepidop- teran muscle fibers" August 16 F. D. CARLSON "A scheme for the mechanochemistry of muscle" A. G. SZENT-GYORGYI "Studies on actin. I. Reversibility of actin depolymerization in presence of KI" T. HAYASHI "Studies on actin. II. Polymerization and RAJA ROSENBLUTH the bound nucleotide" 8. MEMBERS OF THE CORPORATION, 1960 1. LIFE MEMBERS BRODIE, MR. DONALD M., 522 Fifth Avenue, New York 18, New York CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania CARVER, DR. GAIL L., Mercer University, Macon, Georgia COLE, DR. ELBERT C., 2 Chipman Park, Middlebury, Vermont COWDRY, DR. E. V., Washington University, St. Louis, Missouri CRANE, MRS. W. MURRAY, Woods Hole, Massachusetts GOLDFARB, DR. A. J., College of the City of New York, New York City, New York KNOWLTON, DR. F. P., 1356 Westmoreland Avenue, Syracuse, New York LEWIS, DR. W. H., Johns Hopkins University, Baltimore, Maryland LOWTHER, DR. FLORENCE DEL., Barnard College, New York City, New York MALONE, DR. E. F., 6610 North llth Street, Philadelphia 26, Pennsylvania MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pennsylvania PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania RIGGS, MR. LAWRASON, 74 Trinity Place, New York 6, New York SCOTT, DR. ERNEST L., Columbia University, New York City, New York SCHRADER, DR. FRANZ, Duke University, Durham, N. C. SCHRADER, DR. SALLY, Duke University, Durham, N. C. TURNER, DR. C. L., Northwestern University, Evanston, Illinois WAITE, DR. F. G., 144 Locust Street, Dover, New Hampshire WALLACE, DR. LOUISE B., 359 Lytton Avenue, Palo Alto, California WARREN, DR. HERBERT S., 610 Montgomery Avenue, Bryn Mawr, Pennsylvania 2. REGULAR MEMBERS ABELL, DR. RICHARD G., 7 Cooper Road, New York City, New York ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York ADOLPH, DR. EDWARD F., University of Rochester School of Medicine and Dentis- try, Rochester, New York ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota ALLEN, DR. M. JEAN, Department of Biology, Wilson College, Chambersburg, Pennsylvania 30 MARINE BIOLOGICAL LABORATORY ALLEN, DR. ROBERT D., Department of Biology, Princeton University, Princeton, New Jersey ALSCHER, DR. RUTH, Department of Physiology, Manhattanville College, Purchase, New York AMATNIEK, DR. ERNEST, Department of Neurology, College of Physicians and Surgeons, New York City, New York AMBERSON, DR. WILLIAM R., Woods Hole, Massachusetts ANDERSON, DR. J. M., Department of Zoology, Cornell University, Ithaca, New York ANDERSON, DR. RUBERT S., Medical Laboratories, Army Chemical Center, Mary- land (Box 632, Edgewood, Maryland) ANDERSON, DR. T. F., Institute for Cancer Research, Fox Chase, Philadelphia 11, Pennsylvania ARMSTRONG, DR. PHILIP B., Department of Anatomy, State University of New York College of Medicine, Syracuse 10, New York ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory, Oak Ridge, Tennessee ATWOOD, DR. KIMBALL C., Department of Microbiology, University of Illinois, Urbana, Illinois AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts AYERS, DR. JOHN C., Department of Zoology, University of Michigan, Ann Arbor, Michigan BAITSELL, DR. GEORGE A., Osborn Zoological Laboratories, Yale University, New Haven. Connecticut BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medi- cal School, Boston 15, Massachusetts BALLARD, DR. WILLIAM W., Department of Zoology, Dartmouth College, Hanover, New Hampshire BALTUS, DR. ELYANE, Laboratory of Animal Morphology, Brussels, Belgium BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School of Hygiene. Baltimore 5, Maryland BARD, DR. PHILLIP, Johns Hopkins Medical School, Baltimore, Maryland EARTH, DR. L. G., Department of Zoology, Columbia University, New York 27, New York EARTH, DR. LUCENA, Department of Zoology, Barnard College, New York 27, New York BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana, Illinois BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa City, Iowa BECK, DR. L. V., Department of Physiology and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh 13, Pennsylvania BEHRE, DR. ELINOR M., Black Mountain, North Carolina BENESCH, DR. REINHOLD, College of Physicians and Surgeons, New York 32, New York BENESCH, DR. RUTH, College of Physicians and Surgeons, New York 32, New York REPORT OF THE DIRECTOR 31 BENNETT, DR. MICHAEL V., Department of Neurology, College of Physicians and Surgeons, New York 32, New York BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet Briar, Virginia BERG, DR. WILLIAM E., Department of Zoology, University of California, Berkeley 4, California BERMAN, DR. MONKS, Institute for Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda 14, Maryland BERNHEIMER, DR. ALAN W., New York University College of Medicine, New York 16, New York BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne State University Col- lege of Medicine, Detroit 7, Michigan BERTHOLF, DR. LLOYD M., Illinois Wesleyan University, Bloomington, Illinois BEVELANDER, DR. GERRIT, New York University School of Dentistry, 477 First Avenue, New York 16, New York BIGELOW, DR. HENRY B., Museum of Comparative Zoology, Harvard University, Cambridge 38, Massachusetts BISHOP, DR. DAVID W., Department of Embryology, Carnegie Institution of Wash- ington, Baltimore 5, Maryland BLANCHARD, DR. K. C, Johns Hopkins Medical School, Baltimore, Maryland BLOCK, DR. ROBERT, 518 South 42nd Street, Apartment C7, Philadelphia 4, Penn- sylvania BLUM, DR. HAROLD F., Department of Biology, Princeton University, Princeton, New Jersey BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center, 444 East 68th Street, New York 21, New York BODIAN, DR. DAVID, Department of Anatomy, Johns Hopkins University, 709 North Wolfe Street, Baltimore 5, Maryland BOELL, DR. EDGAR J., Osborn Zoological Laboratories, Yale University, New Haven, Connecticut BOETTIGER, DR. EDWARD G., Department of Zoology, University of Connecticut, Storrs, Connecticut BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin 12, Texas BOREI, DR. HANS, Department of Zoology, University of Pennsylvania, Philadel- phia 4, Pennsylvania BOWEN, DR. VAUGHAN T., Woods Hole Oceanographic Institution, Woods Hole, Massachusetts BRADLEY, DR. HAROLD C., 2639 Durant Avenue, Berkeley 4, California BRIDGMAN, DR. ANNA J., Department of Biology, Agnes Scott College, Decatur, Georgia BRONK, DR. DETLEV W., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York BROOKS, DR. MATILDA M., Department of Physiology, University of California, Berkeley 4, California BROWN, DR. DUGALD E. S., Department of Zoology, University of Michigan, Ann Arbor, Michigan 32 MARINE BIOLOGICAL LABORATORY BROWN, DR. FRANK A., JR., Department of Biological Sciences, Northwestern University, Evanston, Illinois BROWNELL, DR. KATHERINE A., Department of Physiology, Ohio State University, Columbus, Ohio BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of Health, Bethesda 14, Maryland BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia BULLOCK, DR. T. H., Department of Zoology, University of California, Los An- geles 24, California BURBANCK, DR. WILLIAM D., Box 834, Emory University, Atlanta 22, Georgia BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilmington, Delaware BURKENROAD, DR. M. D., c/o Lab. Nal. de Pesca, Apartado 3318, Estofeta #1, Olindania, Republic of Panama BUTLER, DR. E. G., Department of Biology, P. O. Box 704, Princeton University, Princeton, New Jersey CAMERON, DR. J. A., Baylor College of Dentistry, Dallas, Texas CANTONI, DR. GIULLIO, National Institutes of Health, Mental Health, Bethesda 14, Maryland CARLSON, DR. FRANCIS D., Department of Biophysics, Johns Hopkins University, Baltimore 18, Maryland CARPENTER, DR. RUSSELL L., Tufts University, Medford 55, Massachusetts CARSON, Miss RACHEL, 11701 Berwick Road, Silver Spring, Maryland CASE, DR. JAMES, Department of Zoology, State University of Iowa, Iowa City, Iowa CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York Avenue, New York City, New York CATTELL, MR. WARE, Cosmos Club, Washington 5, District of Columbia CHAET, DR. ALFRED B., Department of Biology, American University, Washington 16, District of Columbia CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical School, Coral Gables, Florida CHANG, DR. JOSEPH J., Akademiestrasse 3, Physiologisches Institut, Postfach 201, Heidelberg, Germany CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton, New Jersey CHENEY, DR. RALPH H., Biological Laboratory, Brooklyn College, Brooklyn 10. New York CLAFF, DR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts CLARK, DR. A. M., Department of Biological Sciences, University of Delaware, Newark, Delaware CLARK, DR. E. R., 315 South 41st Street, Philadelphia 4, Pennsylvania CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, New York CLARKE, DR. GEORGE L., Biological Laboratories, Harvard University, Cambridge 38, Massachusetts REPORT OF THE DIRECTOR 33 CLELAND, DR. RALPH E., Department of Botany, Indiana University, Bloomington, Indiana CLEMENT, DR. A. C, Department of Biology, Emory University, Atlanta 22, Georgia COE, DR. W. R., 183 Third Avenue, Chula Vista, California COHEN, DR. SEYMOUR S., Department of Biochemistry, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania COLE, DR. KENNETH S., National Institutes of Health (NINDB), Bethesda 14, Maryland COLLETTE, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts COLLIER, DR. JACK R., Marine Biological Laboratory, Woods Hole, Massachusetts COLTON, DR. H. S., Box 601, Flagstaff, Arizona COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, New York COLWIN, DR. LAURA H., Department of Biology, Queens College, Flushing, New York COOPER, DR. KENNETH W., Department of Cytology, Dartmouth Medical School, Hanover, New Hampshire COOPERSTEIN, DR. S HER WIN J., Department of Anatomy, Western Reserve Uni- versity Medical School, Cleveland, Ohio COPELAND, DR. D. E., 5820 Hurst Street, Apartment 8, New Orleans 18, Louisiana COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine COPLEY, DR. A. L., Medical Research Laboratories, Charing Cross Hospital, 8 Ex- change Court, Strand, London W. C. 2, England CORNMAN, DR. IVOR, Hazleton Laboratories, Box 333, Falls Church, Virginia COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro- lina, Chapel Hill, North Carolina CRANE, MR. JOHN O., Woods Hole, Massachusetts CRANE, DR. ROBERT K., Department of Biological Chemistry, Washington Univer- sity Medical School, St. Louis, Missouri CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire CROUSE, DR. HELEN V., Department of Botany, Columbia University, New York 27, New York CROWELL, DR. P. S., JR., Department of Zoology, Indiana University, Bloomington, Indiana CSAPO, DR. ARPAD I., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York CURTIS, DR. MAYNIE R., University of Miami, Box 1015, South Miami, Florida CURTIS, DR. W. C., University of Missouri, Columbia, Missouri DAN, DR. JEAN CLARK, Misaki Biological Station, Misaki, Japan DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan DANIELLI, DR. JAMES F., Department of Zoology, King's College, London, England DAVIS, DR. BERNARD D., Harvard Medical School, 25 Shattuck Street, Boston 15, Massachusetts 34 MARINE BIOLOGICAL LABORATORY DAWSON, DR. A. B., Biological Laboratories, Harvard University, Cambridge 38, Massachusetts DAWSON, DR. J. A., 129 Violet Avenue, Floral Park, Long Island, New York DEANE, DR. HELEN W., Albert Einstein College of Medicine, New York 61, New York DILLER, DR. IRENE C., Institute for Cancer Research, Fox Chase, Philadelphia 11, Pennsylvania DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania DIXON, DR. FRANK J., Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh 13, Pennsylvania DODDS, DR. G. S., West Virginia University School of Medicine, Morgantown, West Virginia DOLLEY, DR. WILLIAM L., Department of Biology, Randolph-Macon College, Ash- land, Virginia DOTY, DR. MAXWELL S., Department of Biology, University of Hawaii, Honolulu, Hawaii DURYEE, DR. WILLIAM R., George Washington University School of Medicine, Department of Physiology, Washington 5, District of Columbia EDDS, DR. MAC V., JR., Department of Biology, Brown University, Providence 12, Rhode Island EDWARDS, DR. CHARLES, Department of Physiology, University of Minnesota, Minneapolis 14, Minnesota EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis, Missouri ELLIOTT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann Arbor, Michigan ESSNER, DR. EDWARD S., Department of Pathology, Albert Einstein College of Medicine, New York 61, New York EVANS, DR. TITUS C., State University of Iowa College of Medicine, Iowa City, Iowa FAILLA, DR. G., Building 203, Argonne National Laboratory, Argonne, Illinois FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France FERGUSON, DR. F. P., Division of General Medical Sciences, National Institutes of Health, Bethesda 14, Maryland FERGUSON, DR. JAMES K. W., Connought Laboratories, University of Toronto, Ontario, Canada FIGGE, DR. F. H. J., University of Maryland Medical School, Lombard and Green Streets, Baltimore 1, Maryland FINGERMAN, DR. MILTON, Department of Zoology, Newcomb College, Tulane Uni- versity, New Orleans 18, Louisiana FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Rich- mond, Virginia FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, Toronto, Canada FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto, Canada REPORT OF THE DIRECTOR 35 FORBES, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge 38, Massachusetts FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois, Urbana, Illinois FREYGANG, DR. WALTER H., JR., Box 516, Essex Fells, New Jersey FRIES, DR. ERIK F. B., Box 605, Woods Hole, Massachusetts FRISCH, DR. JOHN A., Canisius College, Buffalo, New York FURSHPAN, DR. EDWIN J., Department of Neurophysiology, Harvard Medical School, Boston 15, Massachusetts FURTH, DR. JACOB, 183 Cleveland Avenue, Buffalo, New York FYE, DR. PAUL M., Woods Hole Oceanographic Institution, Woods Hole, Massa- chusetts GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn 10, New York GAFFRON, DR. HANS, Department of Biology, Florida State University, Conradi Building, Tallahassee, Florida GALL, DR. JOSEPH C, Department of Zoology, University of Minnesota, Minneapo- lis 14, Minnesota GALTSOFF, DR. PAUL S., Woods Hole, Massachusetts GASSER, DR. HERBERT S., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York GILMAN, DR. LAUREN C., Department of Zoology, University of Miami, Coral Gables, Florida GINSBERG, DR. HAROLD S., Department of Microbiology, University of Pennsyl- vania School of Medicine, Philadelphia 4, Pennsylvania GOLDSMITH, DR. TIMOTHY H., Department of Zoology, Yale University, New Haven, Connecticut GOLDSTEIN, DR. LESTER, Department of Zoology, University of Pennsylvania, Philadelphia 4, Pennsylvania GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, Atlanta 22, Georgia GOODRICH, DR. H. B., Department of Biology, Wesleyan University, Middletown, Connecticut GOTSCHALL, DR. GERTRUDE Y., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York GRAHAM, DR. HERBERT, U. S. Fish and Wildlife Service, Woods Hole, Massachu- setts GRAND, MR. C. G., Cancer Institute of Miami, 1155 N. W. 15th Street, Miami, Florida GRANT, DR. M. P., Sarah Lawrence College, Bronxville, New York GRANT, DR. PHILIP, Department of Pathobiology, Johns Hopkins University School of Hygiene, Baltimore 5, Maryland GRAY, DR. IRVING E., Department of Zoology, Duke University, Durham, North Carolina GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New Bruns- wick, New Jersey 36 MARINE BIOLOGICAL LABORATORY GREEN, DR. MAURICE, Department of Microbiology, St. Louis University Medical School, St. Louis, Missouri GREGG, DR. JAMES H., Department of Biological Sciences, University of Florida, Gainesville, Florida GREGG, DR. JOHN R., Department of Zoology, Duke University, Durham, North Carolina GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical Col- lege, New York 21, New York GRIFFIN, DR. DONALD R., Biological Laboratories, Harvard University, Cambridge 38, Massachusetts GROSCH, DR. DANIEL S., Department of Genetics, Gardner Hall, North Carolina State College, Raleigh, North Carolina GROSS, DR. PAUL, Department of Biology, New York University, University Heights, New York 53, New York GRUNDFEST, DR. HARRY, Columbia University College of Physicians and Surgeons, New York 32, New York GUDERNATSCH, DR. FREDERICK, 41 Fifth Avenue, New York 3, New York GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn 10, New York HAJDU, DR. STEPHEN, National Institutes of Health, Bethesda 14, Maryland HALL, DR. FRANK G., Department of Physiology, Duke University Medical School, Durham, North Carolina HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St. Louis, Missouri HAMILTON, DR. HOWARD L., Department of Zoology, Iowa State College, Ames, Iowa HANCE, DR. ROBERT T., Box 108, R. R. #3, Loveland, Ohio HARDING, DR. CLIFFORD V., JR., 300 Knickerbocker Road, Tenafly, New Jersey HARNLY, DR. MORRIS H., Washington Square College, New York University, New York 3, New York HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research, 66th Street and York Avenue, New York 21, New York HARTMAN, DR. FRANK A., Ohio State University, Hamilton Hall, Columbus, Ohio HARVEY, DR. ETHEL BROWNE, Marine Biological Laboratory, Woods Hole, Massa- chusetts HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo 3, New York HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institution of Ocean- ography, University of California, La Jolla, California HAYASHI, DR. TERU, Department of Zoology, Columbia University, New York 27, New York HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesley 81, Massachusetts HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts HENDLEY, DR. CHARLES D., 615 South Second Avenue, Highland Park, New Jersey HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina, Chapel Hill, North Carolina V REPORT OF THE DIRECTOR 37 HERNDON, DR. WALTER R., Biology Department, University of Alabama, Univer- sity, Alabama HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts HESS, DR. WALTER N., 309 Aiken Street, Rock Hill, South Carolina HIATT, DR. HOWARD H., Department of Medicine, Harvard Medical School, Boston 15, Massachusetts HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio HILL, DR. SAMUEL E., 135 Brunswick Road, Troy, New York HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge 38, Massachusetts HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge 38, Massachusetts HODGE, DR. CHARLES, IV, Department of Biology, Temple University, Philadelphia, Pennsylvania HOFFMAN, DR. JOSEPH, National Heart Institute, National Institutes of Health, Bethesda 14, Maryland HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia 4, Pennsylvania HOLLAENDER, DR. ALEXANDER, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee HOLZ, DR. GEORGE G., JR., Department of Zoology, Syracuse University, Syracuse, New York HOPKINS, DR. HOYT S., 59 Heatherdell Road, Ardsley, New York HUNTER, DR. FRANCIS R., University of the Andes, Calle 18-a Carrera 1-E, Bogota, Colombia, South America HUTCHENS, DR. JOHN O., Department of Physiology, University of Chicago, Chi- cago 37, Illinois HYDE, DR. BEAL B., Department of Plant Sciences, University of Oklahoma, Nor- man, Oklahoma HYMAN, DR. LIBBIE H., American Museum of Natural History, Central Park West at 79th Street, New York 24, New York IRVING, DR. LAURENCE, U. S. Public Health Service, Anchorage, Alaska ISENBERG, DR. IRVIN, Institute for Muscle Research, Marine Biological Laboratory, Woods Hole, Massachusetts ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts JACOBS, DR. M. H., University of Pennsylvania School of Medicine, Philadelphia 4, Pennsylvania JACOBS, DR. WILLIAM P., Department of Biology, Princeton University, Princeton, New Jersey JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina JOHNSON, DR. FRANK H., Department of Biology, Princeton University, Princeton, New Jersey JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of Florida, Gainesville, Florida JONES, DR. RAYMOND F., Department of Biology, Princeton University, Princeton, New Jersey 38 MARINE BIOLOGICAL LABORATORY KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachusetts KABAT, DR. E. A., Neurological Institute, College of Physicians and Surgeons, New York 32, New York KARUSH, DR. FRED, Department of Pediatrics, University of Pennsylvania, Phila- delphia 4, Pennsylvania KAUFMANN, DR. B. P., Carnegie Institution, Cold Spring Harbor, Long Island, New York KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann Arbor, Michigan KEMPTON, DR. RUDOLF T., Department of Zoology, Vassar College, Poughkeepsie, New York KEOSIAN, DR. JOHN, Department of Biology, Rutgers University, Newark 2, New Jersey KETCHUM, DR. BOSTWICK, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts KILLE, DR. FRANK R., State Department of Education, Albany 1, New York KIND, DR. C. ALBERT, Department of Zoology, University of Connecticut, Storrs, Connecticut KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia KING, DR. JOHN W., Morgan State College, Baltimore 12, Maryland KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca, New York KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York KLEIN, DR. MORTON, Department of Microbiology, Temple University, Philadel- phia, Pennsylvania KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland 2, Oregon KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston, Illinois KOLIN, DR. ALEXANDER, Department of Biophysics, California Medical School, Los Angeles 24, California KORR, DR. I. M., Department of Physiology, Kirksville College of Osteopathy, Kirksville, Missouri KRAHL, DR. M. E., Department of Physiology, University of Chicago, Chicago 37, Illinois KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Baltimore, Maryland KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois KUFFLER, DR. STEPHEN, Department of Pharmacology, Harvard Medical School, Neurophysical Laboratory, Boston 15, Massachusetts KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York LACKEY, DR. JAMES B., Box 497, Melrose, Florida LAMY, DR. FRANCOIS, Department of Anatomy, University of Pittsburgh School of Medicine, Pittsburgh 13, Pennsylvania LANCEFIELD, DR. D. E., Queens College, Flushing, New York REPORT OF THE DIRECTOR 39 LANCEFIELD, DR. REBECCA C, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York LANDIS, DR. E. M., Harvard Medical School, Boston 15, Massachusetts LANSING, DR. ALBERT I., Department of Anatomy, University of Pittsburgh Medi- cal School, Pittsburgh 13, Pennsylvania LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts- burgh, Pennsylvania LAVIN, DR. GEORGE I., 6200 Norvo Road, Baltimore 7, Maryland LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota Medical School, Minneapolis 14, Minnesota LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford University Medical School, Stanford, California LEE, DR. RICHARD E., Cornell University College of Medicine, New York City, New York LEFEVRE, DR. PAUL G., University of Louisville School of Medicine, Louisville, Kentucky LEHMANN, DR. FRITZ, Zoologische Institut, University of Berne, Berne, Switzer- land LEVINE, DR. RACHMIEL, Michael Rees Hospital, Chicago 16, Illinois LEVY, DR. MILTON, Department of Biochemistry, New York University School of Dentistry, New York 10, New York LEWIN, DR. RALPH A., Scripps Institution of Oceanography, La Jolla, California LEWIS, DR. IVEY F., 1110 Rugby Road, Charlottesville, Virginia LING, DR. GILBERT, 307 Berkeley Road, Merion, Pennsylvania LITTLE, DR. E. P., 216 High Street, West Newton, Massachusetts LLOYD, DR. DAVID P. C., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York LOCHHEAD, DR. JOHN H., Department of Zoology, University of Vermont, Burling- ton, Vermont LOEB, DR. R. F., 950 Park Avenue, New York 28, New York LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York LOFTFIELD, DR. ROBERT B., Associate Biochemist, Massachusetts General Hospital, Boston, Massachusetts LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans- ton, Illinois DELORENZO, DR. ANTHONY, Anatomical and Pathological Research Laboratories, Johns Hopkins Hospital, Baltimore 5, Maryland LOVE, DR. Lois H., 1043 Marlau Drive, Baltimore 12, Maryland LOVE, DR. WARNER E., 1043 Marlau Drive, Baltimore 12, Maryland LUBIN, DR. MARTIN, Department of Pharmacology, Harvard Medical School, Boston 15, Massachusetts LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York LYNCH, DR. WILLIAM, Department of Biology, St. Ambrose College, Davenport, Iowa LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America, Washington 17, District of Columbia 40 MARINE BIOLOGICAL LABORATORY MACDOUGALL, DR. MARY STUART, Mt. Vernon Apartments, 423 Clairmont Avenue, Decatur, Georgia McCANN, DR. FRANCES, Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School, Philadelphia 4, Pennsylvania MCDONALD, SISTER ELIZABETH SETON, Department of Biology, College of Mt. St. Joseph, Mt. St. Joseph, Ohio MCDONALD, DR. MARGARET H., Carnegie Institution of Washington, Cold Spring Harbor, Long Island, New York McELROY, DR. WILLIAM D., Department of Biology, Johns Hopkins University, Baltimore 18, Maryland MAAS, DR. WERNER K., New York University College of Medicine, New York City, New York MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 135 Harrison Avenue, Boston, Massachusetts MANWELL, DR. REGINALD D.. Department of Zoology, Syracuse University, Syra- cuse, New York MARSHAK, DR. ALFRED, Department of Radiology, Jefferson Medical College, Philadelphia 7, Pennsylvania MARSLAND, DR. DOUGLAS A., New York University, Washington Square College, New York 3, New York MARTIN, DR. EARL A., Department of Biology, Brooklyn College, Brooklyn 10, New York MATHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College, Williamstown, Massachusetts MAYOR, DR. JAMES W., 8 Gracewood Park, Cambridge 38, Massachusetts MAZIA, DR. DANIEL. Department of Zoology, University of California. Berkeley 4, California MEDES, DR. GRACE, 303 Abington Avenue, Philadelphia 11, Pennsylvania MEINKOTH, DR. NORMAN A., Department of Biology, Swarthmore College, Swarthmore, Pennsylvania MENKIN, DR. VALY, University of Kansas City School of Dentistry, 1108 East 10th Street, Kansas City, Missouri METZ, DR. C. B., Oceanographic Institute, Florida State University, Tallahassee, Florida METZ, DR. CHARLES W., Box 714, Woods Hole, Massachusetts MIDDLEBROOK, DR. ROBERT, Institute for Muscle Research, Marine Biological Lab- oratory, Woods Hole, Massachusetts MILLER, DR. J. A., JR., Department of Anatomy, Tulane University Medical School, New Orleans 18, Louisiana MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire, Durham, New Hampshire MOE, MR. HENRY A., Guggenheim Memorial Foundation, 551 Fifth Avenue, New York 17, New York MONROY, DR. ALBERTO, Institute of Comparative Anatomy, University of Palermo, Italy REPORT OF THE DIRECTOR 41 MOORE, DR. GEORGE M., Department of Zoology, University of New Hampshire, Durham, New Hampshire MOORE, DR. JOHN A., Department of Zoology, Columbia University, New York 27, New York MOORE, DR. JOHN W., Laboratory of Biophysics, NINDB, National Institutes of Health, Bethesda 14, Maryland MORRILL, DR. JOHN B., JR., Department of Biology, Wesleyan University, Middle- town, Connecticut MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick, New Jersey MOUNTAIN, MRS. J. D., Charles Road, Mt. Kisco, New York MULLINS, DR. LORIN J., Biophysical Laboratory, Purdue University, Lafayette, Indiana MUSACCHIA, DR. XAVIER, JR., Department of Biology, St. Louis University, St. Louis 4, Missouri NABRIT, DR. S. M., President, Texas Southern University, 3201 Wheeler Avenue, Houston 4, Texas NACE, DR. PAUL FOLEY, Department of Biology, Hamilton College, McMaster University, Hamilton, Ontario XACHMANSOHN, DR. DAVID, Columbia University, College of Physicians and Sur- geons, New York 32, New York NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts NELSON, DR. LEONARD, Department of Physiology, Emory University, Atlanta 22, Georgia NEURATH, DR. H., Department of Biochemistry, University of Washington, Seattle 5, Washington NICOLL, DR. PAUL A., BMSI/USOM/P-K, APO 271, New York City, New York Niu, DR. MAN-CHIANG, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York XOVIKOFF, DR. ALEX B., Department of Pathology, Albert Einstein College of Medicine, New York 61, New York OCHOA, DR. SEVERO, New York University College of Medicine, New York 61, New York ODUM, DR. EUGENE, Department of Zoology, University of Georgia, Athens, Georgia OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania OSTERHOUT, DR. W. J. V., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York OSTERHOUT, DR. MARION IRWIN, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York PACKARD, DR. CHARLES, Woods Hole, Massachusetts PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio PARPART, DR. ARTHUR K., Department of Biology, Princeton University, Princeton, New Jersey PASSANO, DR. LEONARD M., Osborn Zoological Laboratories, Yale University, New Haven, Connecticut 42 MARINE BIOLOGICAL LABORATORY PATTEN, DR. BRADLEY M., University of Michigan School of Medicine, Ann Arbor, Michigan PERKINS, DR. JOHN F., JR., Department of Physiology, University of Chicago, Chicago 37, Illinois PERSON, DR. PHILIP, Chief, Special Dental Research Program, VA Hospital, Brooklyn 9, New York PETTIBONE, DR. MARIAN H., Department of Zoology, University of New Hamp- shire, Durham, New Hampshire PHILPOTT, MR. DELBERT E., 496 Palmer Avenue, Falmouth, Massachusetts PICK, DR. JOSEPH, Department of Anatomy, New York University-Bellevue Medi- cal Center, New York 16, New York PIERCE, DR. MADELENE E., Department of Zoology, Vassar College, Poughkeepsie, New York PLOUGH, DR. HAROLD H., Department of Biology, Amherst College, Amherst, Massachusetts POLLISTER, DR. A. W., Department of Zoology, Columbia University, New York 27, New York POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut POTTER, DR. DAVID, Department of Neurophysiology, Harvard Medical School, Boston 15, Massachusetts PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti- more 12, Maryland PROSSER, DR. C. LADD, 401 Natural History Building, University of Illinois, Ur- bana, Illinois PROVASOLI, DR. LUIGI, Haskins Laboratories, 305 East 43rd Street, New York 17, New York RAMSEY, DR. ROBERT W., Medical College of Virginia, Richmond, Virginia RAND, DR. HERBERT W., 7 Siders Pond Road, Falmouth, Massachusetts RANKIN, DR. JOHN S., Department of Zoology, University of Connecticut, Storrs, Connecticut RANZI, DR. SILVIO, Department of Zoology, University of Milan, Milan, Italy RATNER, DR. SARAH, Public Health Research Institute of the City of New York, Foot of East 15th Street, New York 9, New York RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta 22, Georgia READ, DR. CLARK P., Department of Biology, Rice University, Houston, Texas REBHUN, DR. LIONEL I., Department of Biology, Box 704, Princeton University, Princeton, New Jersey RECHNAGEL, DR. R. O., Department of Physiology, Western Reserve University, Cleveland, Ohio REDFIELD, DR. ALFRED C., Woods Hole, Massachusetts RENN, DR. CHARLES E., 509 Ames Hall, Johns Hopkins University, Baltimore 18, Maryland REUBEN, DR. JOHN P., Department of Neurology, College of Physicians and Sur- geons, New York 32, New York REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue, New York 16, New York REPORT OF THE DIRECTOR 43 RICHARDS, DR. A., 2950 E. Mabel Street, Tucson, Arizona RICHARDS, DR. A. GLENN, Department of Entomology, University of Minnesota, St. Paul 1, Minnesota RICHARDS, DR. OSCAR W., American Optical Company, Research Center, South- bridge, Massachusetts ROCKSTEIN, DR. MORRIS, Department of Physiology, New York University College of Medicine, New York 16, New York ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York ROMER, DR. ALFRED S., Museum of Comparative Zoology, Harvard University, Cambridge 38, Massachusetts RONKIN, DR. RAPHAEL R., Department of Physiology, University of Delaware, Newark, Delaware ROOT, DR. R. W., Department of Biology, College of the City of New York, New York City, New York ROOT, DR. W. S., Columbia University College of Physicians and Surgeons, De- partment of Physiology, New York 32, New York ROSE, DR. S. MERYL, Department of Biology, Wesleyan University, Middletown, Connecticut ROSENBERG, DR. EVELYN K., Department of Pathology, New York University- Bellevue Medical Center, New York 16, New York ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pittsburgh Medical School, Pittsburgh 13, Pennsylvania Rossi, DR. HAROLD H., Department of Radiology, Columbia University, 630 West 168th Street, New York 32, New York ROTH, DR. JAY S., Department of Zoology and Entomology, University of Connec- ticut, Storrs, Connecticut ROTHENBERG, DR. M. A., Scientific Director, Dugway Proving Ground, Dugway, Utah RUGH, DR. ROBERTS, Radiological Research Laboratory, College of Physicians and Surgeons, 630 West 168th Street, New York 32, New York RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden RUTMAN, DR. ROBERT J., General Laboratory Building, 215 S. 34th Street, Phila- delphia 4, Pennsylvania RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole, Massachusetts SANBORN, DR. RICHARD C., Department of Biological Sciences, Purdue University, Lafayette, Indiana SANDEEN, DR. MURIEL I., Department of Zoology, Duke University, Durham, North Carolina SAUNDERS, MR. LAWRENCE, West Washington Square, Philadelphia 5, Pennsylvania SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of Cali- fornia, Berkeley 4, California SCHARRER, DR. ERNST A., Department of Anatomy, Albert Einstein College of Medicine, 1710 Newport Avenue, New York 61, New York SCHLESINGER, DR. R. WALTER, Department of Microbiology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis 4, Missouri 44 MARINE BIOLOGICAL LABORATORY SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio SCHMITT, DR. FRANCIS O., Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts SCHMITT, DR. O. H., Department of Physics, University of Minnesota, Minneapo- lis 14, Minnesota SCHNEIDERMAN, DR. HOWARD A., Department of Zoology, Cornell University, Ithaca, New York SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, California SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massachusetts SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington, Indiana SCOTT, DR. ALLAN C., Colby College, Waterville, Maine SCOTT, DR. D. B. McNAiR, Botany Annex, Cancer Chemotherapy Laboratory, University of Pennsylvania, Philadelphia 4, Pennsylvania SCOTT, SISTER FLORENCE MARIE, Seton Hill College, Greensburg, Pennsylvania SCOTT, DR. GEORGE T., Department of Zoology, Oberlin College, Oberlin, Ohio SEARS, DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massa- chusetts SENFT, DR. ALFRED W., Woods Hole, Massachusetts SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and Surgeons, New York 32, New York SHANES, DR. ABRAHAM, Experimental Biological and Medicine Institute, National Institutes of Health, Bethesda 14, Maryland SHAPIRO, DR. HERBERT, 5800 North Camac Street, Philadelphia 41, Pennsylvania SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East Lansing, Michigan SHEDLOVSKY, DR. THEODORE, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont SICHEL, MRS. F. J. M., 38 Henderson Terrace, Burlington, Vermont SILVA, DR. PAUL, Department of Botany, University of California, Berkeley 4, California SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa City, Iowa SMITH, DR. DIETRICH C., Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland SMITH, MR. HOMER P., Marine Biological Laboratory, Woods Hole, Massachusetts SMITH, MR. PAUL FERRIS, Marine Biological Laboratory, Woods Hole, Massa- chusetts SMITH, DR. RALPH I., Department of Zoology, University of California, Berkeley 4, California SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington, Indiana SONNENBLICK, DR. B. P., Rutgers University, 40 Rector Street, Newark 2, New Jersey REPORT OF THE DIRECTOR 45 SPEIDEL, DR. CARL C., Department of Anatomy, University of Virginia, University, Virginia SPIEGEL, DR. MELVIN, Department of Zoology, Dartmouth College, Hanover, New Hampshire SPRATT, DR. NELSON T., JR., Department of Zoology, University of Minnesota, Minneapolis 14, Minnesota SPYROPOULOS, DR. C. S., Building 9, Room 140, National Institutes of Health, Bethesda 14, Maryland STARR, DR. RICHARD C., Department of Botany, Indiana University, Bloomington, Indiana STEINBACH, DR. H. BURR, Department of Zoology, University of Chicago, Chicago 37, Illinois STEINBERG, DR. MALCOLM S., Department of Biology, Johns Hopkins University, Baltimore 18, Maryland STEINHARDT, DR. JACINTO, Operations Evaluation Group, Massachusetts Institute of Technology, Cambridge, Massachusetts STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota, Min- neapolis 14, Minnesota STETTEN, DR. DE\ITT, Director in Charge of Research, NIAMD, National Insti- tutes of Health, Bethesda 14, Maryland STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College, South Hadley, Massachusetts STONE, DR. WILLIAM, Ophthalmic Plastics Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts STRAUS, DR. W. L., JR., Department of Anatomy, Johns Hopkins University Medi- cal School, Baltimore 5, Maryland STREHLER, DR. BERNARD L., Cellular and Comparative Physiology Section, National Institutes of Health, Bethesda 14, Maryland STUNKARD, DR. HORACE W., American Museum of Natural History, Central Park West at 79th Street, New York 24, New York STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena 4, California SUDAK, DR. FREDERICK N., Department of Physiology, Albert Einstein College of Medicine, New York 61, New York SULKIN, DR. S. EDWARD, Department of Bacteriology, University of Texas, South- western Medical School, Dallas, Texas SWOPE, MR. GERARD, JR., 570 Lexington Avenue, New York 22, Xew York SZENT-GYORGYI, DR. ALBERT, Institute for Muscle Research, Marine Biological Laboratory, Woods Hole, Massachusetts SZENT-GYORGYI, DR. ANDREW G., Institute for Muscle Research, Marine Biological Laboratory, Woods Hole, Massachusetts TASAKI, DR. ICHIJI, Laboratory of Neurophysiology, NINDB, Bethesda 14, Mary- land TASHIRO, DR. SHIRO, University of Cincinnati Medical College, Cincinnati, Ohio TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology, NINDB, Bethesda 14, Maryland 46 MARINE BIOLOGICAL LABORATORY TAYLOR, DR. WM. RANDOLPH, Department of Botany, University of Michigan, Ann Arbor, Michigan TEWINKEL, DR. Lois E., Department of Zoology, Smith College, Northampton, Massachusetts TOBIAS, DR. JULIAN, Department of Physiology, University of Chicago, Chicago, Illinois TRACY, DR. HENRY C, General Delivery, Oxford, Mississippi TRACER, DR. WILLIAM, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York TRINKAUS, PR. J. PHILIP, Department of Zoology, Osborn Zoological Labora- tories, Yale University, New Haven, Connecticut TROLL, DR. WALTER, Department of Industrial Medicine, New York University, College of Medicine, New York 16, New York TWEEDELL, DR. KEN YON S., Department of Biology, University of Notre Dame, Notre Dame, Indiana TYLER, DR. ALBERT, Division of Biology, California Institute of Technology, Pasa- dena 4, California UHLENHUTH, DR. EDWARD, University of Maryland School of Medicine, Balti- more, Maryland URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago, Chicago, Illinois DE VILLAFRANCA, DR. GEORGE W., Department of Zoology, Smith College, Nor- thampton, Massachusetts VILLEE, DR. CLAUDE A., Department of Biological Chemistry, Harvard Medical School, Boston 15, Massachusetts VINCENT, DR. WALTER S., Department of Anatomy, State University of New York School of Medicine, Syracuse 10, New York WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New Brunswick, New Jersey WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge 38, Massachusetts WARNER, DR. ROBERT C., Department of Chemistry, New York University College of Medicine, New York 16, New York WATERMAN, DR. T. H., Department of Zoology, 272 Gibbs Research Laboratory, Yale University, New Haven, Connecticut WEBB, DR. MARGUERITE, Department of Physiology and Bacteriology, Goucher College, Towson, Baltimore 4, Maryland WEISS, DR. PAUL A., Laboratory of Developmental Biology, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York WENRICH, DR. D. H., Department of Zoology, University of Pennsylvania, Phila- delphia 4, Pennsylvania WERMAN, DR. ROBERT, Department of Neurology, College of Physicians and Sur- geons, New York 32, New York WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut WHITAKER, DR. DOUGLAS M., Rockefeller Institute for Medical Research, 66th Street and York Avenue, New York 21, New York REPORT OF THE DIRECTOR 47 WHITE, DR. E. GRACE, 1312 Edgar Avenue, Chambersburg, Pennsylvania WHITING, DR. ANNA R., Department of Zoology, University of Pennsylvania, Philadelphia 4, Pennsylvania WHITING, DR. PHINEAS, Department of Zoology, University of Pennsylvania, Philadelphia 4, Pennsylvania WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, Pennsylvania WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts WIERCINSKI, DR. FLOYD J., Department of Biological Sciences, Dre:xU Institute of Technology, 32nd and Chestnut Streets, Philadelphia 4, Pennsylvania WIGLEY, DR. ROLAND L., U. S. Fish and Wildlife Service, Woods Hole, Massa- chusetts WILBER, DR. C. G., Medical Laboratories, Applied Physiology Branch, Army Chemical Center, Maryland WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore 18, Maryland WILSON, DR. J. WALTER, Department of Biology, Brown University, Providence 12, Rhode Island WILSON, DR. WALTER L., Department of Physiology, University of Vermont Col- lege of Medicine, Burlington, Vermont WITSCHI, DR. EMIL, Department of Zoology, State University of Iowa, Iowa City, Iowa WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry, Albert Einstein College of Medicine, New York 61, New York WOLF, DR. ERNST, Pendelton Hall, Wellesley College, Wellesley, Massachusetts WOODWARD, DR. ARTHUR A., Army Chemical Center, Maryland (Applied Physiol- ogy Branch, Army Chemical Corps, Medical Laboratory) WRIGHT, DR. PAUL A., Department of Zoology, University of New Hampshire, Durham, New Hampshire WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton, Massachusetts YNTEMA, DR. C. L., Department of Anatomy, State University of New York Col- lege of Medicine, Syracuse 10, New York YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts ZIMMERMAN, DR. A. M., Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn 3, New York ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings- ton, Rhode Island ZIRKLE, DR. RAYMOND E., Department of Radiobiology, University of Chicago, Chicago 37, Illinois ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie, New York ZWEIFACH, DR. BENJAMIN, New York University Bellevue Medical Center, New York 16, New York ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham 54, Massachusetts 48 MARINE BIOLOGICAL LABORATORY 3. ASSOCIATE MEMBERS ALTON, DR. AND MRS. BENJAMIN H. ARMSTRONG, DR. AND MRS. P. B. BACON, MRS. ROBERT BAITSELL, MRS. GEORGE BALL, MRS. ERIC BARBOUR, MR. Lucius H. BARTOW, MR. AND MRS. CLARENCE BARTOW, MRS. FRANCIS D. BARTOW, MR. AND MRS. PHILIP K. BELL, MRS. ARTHUR W. BRADLEY, MR. AND MRS. ALBERT L. BRADLEY, MR. AND MRS. CHARLES BROWN, MRS. THORNTON BURDICK, DR. C. LALOR BURLINGAME, MRS. F. A. CAHOON, MRS. SAMUEL, SR. CALKINS, MRS. GARY N. CALKINS, MRS. G. NATHAN, JR. CALKINS, MR. AND MRS. SAMUEL W. CARLTON, MR. AND MRS. WINSLOW CLAFF, DR. AND MRS. C. LLOYD CLARK, DR. AND MRS. ALFRED HULL CLARK, MRS. LEROY CLARK, MR. AND MRS. W. VAN ALAN CLOWES, MR. ALLEN W. CLOWES, MRS. G. H. A. CLOWES, DR. AND MRS. G. H. A., JR. COLTON, MR. AND MRS. H. SEYMOUR COWDRY, DR. AND MRS. E. V. CRANE, MR. AND MRS. BRUCE CRANE, MR. JOHN CRANE, Miss LOUISE CRANE, MRS. MURRAY CRANE, MR. STEPHEN CRANE, MRS. W. CAREY CROSSLEY, MR. AND MRS. ARCHIBALD M. CROWELL, MR. AND MRS. PRINCE S. CURTIS, DR. AND MRS. W. D. DANIELS, MR. AND MRS. F. HAROLD DAY, MR. AND MRS. POMEROY DRAPER, MRS. MARY C. DREYER, MR. AND MRS. FRANK A. ELSMITH, MRS. DOROTHY ENDERS, MRS. FREDERICK EWING, MR. AND MRS. FREDERIC EWING, MR. WILLIAM FAY, MR. AND MRS. HENRY H. FISHER, MR. AND MRS. B. C. FRANCIS, MRS. LEWIS H., JR. FROST, MRS. FRANK J. GALTSOFF, MRS. PAUL S. GlFFORD, MR. AND MRS. JOHN A. GlFFORD, MR. AND MRS. PROSSER GlLCHRIST, MR. AND MRS. JOHN M. GlLDEA, DR. AND MRS. E. F. GREEN, Miss GLADYS M. GULESIAN, MRS. PAUL J. HAIG, MRS. R. H. HAMLEN, MR. AND MRS. J. MONROE HARRELL, MR. AND MRS. JOEL E. HARRINGTON, MR. AND MRS. ROBERT HARVEY, DR. ETHEL B. HERRINGTON, MRS. A. W. S. HERVEY, DR. AND MRS. JOHN P. HlRSCHFELD, MRS. NATHAN B. HOUSTON, MR. AND MRS. HOWARD JEWETT, MRS. G. F. JOHLIN, MRS. JACOB M. KEITH, MR. AND MRS. HAROLD C. KING, MR. AND MRS. FRANKLIN KOLLER, MR. AND MRS. LEWIS LAURENCE, MR. AND MRS. THOMAS E. LEMANN, MRS. BENJAMIN LlNEAWEAVER, MR. THOMAS, III LOBB, MRS. JOHN LOEB, DR. AND MRS. ROBERT F. McCuSKER, MR. AND MRS. PAUL J. MCKELVY, MR. JOHN E. MARSLAND, MRS. DOUGLAS A. MARVIN, MRS. WALTER T. MAST, MRS. S. O. MEIGS, DR. AND MRS. J. WISTER MITCHELL, MRS. JAMES McC. MIXTER, MRS. W. JASON MOSSER, MRS. BENJAMIN D. MOTLEY, MRS. THOMAS NEWTON, Miss HELEN NICHOLS, MRS. GEORGE NIMS, MRS. E. D. PACKARD, MRS. CHARLES REPORT OF THE LIBRARIAN 49 PARK, MR. AND MRS. M. S. PENNINGTON, Miss ANNE H. REDFIELD, DR. AND MRS. ALFRED C. REZNIKOFF, DR. AND MRS. PAUL RIGGS, MR. AND MRS. LA \YRASON RIVINUS, MRS. F. M., JR. ROBINSON, DR. MILES RUDD, MR. AND MRS. H. W. DWIGHT SANDS, Miss ADELAIDE G. SAUNDERS, MR. AND MRS. LAWRENCE SHIVERICK, MRS. ARTHUR SINCLAIR, MR. AND MRS. W. RICHARD- SON SPEIDEL, DR. AND MRS. CARL STONE, MR. AND MRS. LEO STONE, MR. AND MRS. S. M. STRAUSS, DR. AND MRS. DONALD B. STUNKARD, MRS. HORACE W. SWIFT, MR. E. KENT SWOPE, MR. AND MRS. GERARD, JR. SWOPE, Miss HENRIETTA TOMPKINS, MR. AND MRS. B. A. WEBSTER, MRS. EDWIN S. WHITELEY, Miss MABEL W. WlCKERSHAM, MR. AND MRS. JAMES H. WlLHELM, DR. AND MRS. HlLMER J. WILLISTON, MR. SAMUEL WILLISTON, Miss EMILY WILSON, MRS. EDMUND B. WOLFINSOHN, MRS. WOLFE V. REPORT OF THE LIBRARIAN At the close of the year, the Library received currently 1717 journals, 56 new titles having been added in 1960. Of the total, the Marine Biological Laboratory subscribed to 502, received 653 in exchange and 190 as gifts. The Woods Hole Oceanographic Institution subscribed to 109, received 200 in exchange and 63 as gifts. The Laboratory purchased 121 books, received 92 complimentary copies (9 from authors and 83 from publishers), and accepted 74 miscellaneous gifts. The Institution purchased 38 books and received 9 as gifts. The total number of books accessioned totalled 334. Through purchase, exchange and gift, the Laboratory completed 12 journal sets and partially completed 13. The Institution completed two sets and partially com- pleted one. There were 3825 reprints added to the collection, of which 1749 were of current issue. At the close of the year there were 77,525 bound volumes and 216,452 reprints. The number of requests for inter-library loans increased over 1959. There were 469 volumes sent out and 57 were borrowed. About 1000 volumes were bound. Many valuable books and reprints were received from Drs. P. W. Whiting, L. H. Hyman, Walter S. Root, Roberts Rugh, Ethel B. Harvey, Irvine H. Page, Robt. F. Loeb, H. J. Humm, and the Department of Microbiology, University of Pennsylvania Medical School. The Library extends grateful acknowledgment to these generous contributors. Many duplicate books and reprints were sent to the Department of Cytology, Warsaw L'niversity, thus furthering our exchange relationship abroad. The year was an exceptionally busy one due to the increase in acquisitions, to the larger number of scientists using the library, and to several changes in the staff. Respectfully submitted, DEBORAH L. HARLOW, Librarian 50 MARINE BIOLOGICAL LABORATORY VI. REPORT OF THE TREASURER The market value of the General Endowment Fund and the Library Fund at December 31, 1960, amounted to $1,796,571 as against a book value of $1,146,393. This compares with values of $1,786,262 and $1,023,297 at the end of the preceding year. The average yield on the securities was 3.60% of the market value and 5.65% of book value. The total uninvested principal cash in the above accounts as of December 31, 1960, was $255.16. Classification of the Securities held in the Endowment Funds appears in the Auditor's report. The market value of the pooled securities as of December 31, 1960, was $333,218 with uninvested principal cash of $120.35 ; the market value at December 31, 1959, being $309,251. The book value of the securities in this account was $274,294 on December 31, 1960, compared with $257,576 a year earlier. The average yield on market value was 3.75% and 4.56% of book value. The proportionate interest in the Pool Fund Account of the various Funds as of December 31, 1960, is as follows: Pension Funds 23.979% General Laboratory Investment 53.681 Other : Bio Club Scholarship Fund 1.536 Rev. Arsenius Boyer Scholarship Fund 1.881 Gary N. Calkins Fund 1.760 Allen R. Memhard Fund 342 F. R. Lillie Memorial Fund 5.933 Lucretia Crocker Fund 6.423 E. G. Conklin Fund 1.088 M. H. Jacobs Scholarship Fund 774 Jewett Memorial Fund 572 Anonymous Gift 2.031 The special custodian account yielded an income last year of $9,619 and this amount is being reserved for capital improvements. Donations from the M. B. L. Associates for 1960 were $4,320 as compared with $4,170 for 1959. Unrestricted gifts from foundations, societies and companies amounted to $18,035. We are administering 15 grants for investigators in addition to those grants made directly to the Marine Biological Laboratory. The amounts of grants vary in accordance with the investigator's project of research. An amount of 15% based on amount expended is allowed the Laboratory as overhead. The Lillie Fellowship Fund with a market value of $88,415 and a book value of $92,789, as well as the investment in the General Biological Supply House with a book value of $12,700, is carried in the Balance Sheet, item "Other Investments." The General Biological Supply House for the fiscal year ended June 30, 1960, had a profit after taxes of $314,034 as compared to $303,300 in 1959 and $218,210 in 1958, and $123,430 in 1957. In the fiscal year 1960, the Marine Biological Labora- tory received dividends from the General Biological Supply House of $30,480 as against $30,480 in 1959 and $25,400 in 1958. REPORT OF THE TREASURER 51 Following is a statement of the auditors : To tJic Trustees of the Marine Biological Laboratory, Woods Hole, Massachusetts: \Ye have examined the balance sheets of the Marine Biological Laboratory as at December 31, 1960 and 1959, the related statements of operation expenditures, income and current fund for the years then ended, and statement of funds for the year ended December 31, 1960. Our examination was made in accordance with generally accepted auditing standards, and accordingly included such tests of the account records and such other auditing procedures as we considered necessary in the circumstances. In our opinion, the accompanying financial statements present fairly the assets, liabilities and funds of the Marine Biological Laboratory at December 31, 1960, and the results of its operations for the year then ended. Boston, Massachusetts May 31, 1961 LYBRAND, Ross BROS. & MONTGOMERY JAMES H. WICKERSHAM, Treasurer 52 MARINE BIOLOGICAL LABORATORY MARINE BIOLOGICAL LABORATORY BALANCE SHEETS December 31, 1960 and 1959 Investments 1960 19 5 Investments held by Trustee: Securities, at cost (approximate market quotation 1960 $1,796,000) $1,146,393 $1,023,297 Cash . 255 2,990 $1,146,648 $1,026,287 Investments of other endowment and unrestricted funds : Pooled investments, at cost (approximate market quotation 1960, $333,218; less $5,728 temporary investment of current fund cash) $ 268,566 $ 251,848 Other investments 137,742 132,882 Cash 10,839 13,973 Accounts recrivable 21 1,510 $1,563,816 $1,426,500 Plant Assets Land, buildings, library and equipment ( note) $3,280,059 $3,204,017 Less allowance for depreciation ( note ) 1,142,879 1,109,716 $2,137,180 $2,094,301 Construction in progress 1,455,811 776,628 Cash 82,042 3,699 Accounts receivable : 1,196 U. S. Government obligations, at cost : $275,000 Treasury bills, due 1/15/60 273,258 $3,675,033 $3,149,082 Current Assets Cash $ 77,546 $ 113,588 U. S. Government obligations, at cost : $75,000 Treasury bills, due 1/15/60 74,525 Temporary investment in pooled securities 5,728 5,728 Accounts receivable (U. S. Government, 1960, $43,443; 1959, $35,554) .. 59,889 61,629 Inventories of specimens and Bulletins 47,641 4,->,400 Prepaid insurance and other 16,778 12,953 $ 207,582 $ 313,823 $5,446,431 $4,889,405 REPORT OF THE TREASURER 53 MARINE BIOLOGICAL LABORATORY BALANCE SHEETS December 31, 1960 and 1959 Endoii-'incnt Funds 1960 1959 Endowment funds given in trust for benefit of the Marine Biological Lab- oratory $1,146,648 $1,026,287 Endowment funds for awards and scholarships : Principal $ 126,302 $ 126.193 Unexpended income 7,285 5,399 $ 133,587 $ 131,592 Unrestricted funds functioning as endowment 206,378 206,378 Retirement fund 71,449 61,640 Pooled investments accumulated gain 5,754 603 $1,563,816 $1,426,500 Plant Liability and Funds Funds expended for plant, less retirements $4,668,475 $3,832,639 Less allowance for depreciation charged thereto 1,142,879 1,109,716 $3,525,596 $2,722,923 Unexpended plant funds 82,042 276,957 $3,607,638 $2,999,880 Accounts payable 67,395 149,202 $3,675,033 $3,149,082 Current Liabilities and Funds Accounts payable . $ 41,106 $ 46,154 Unexpended research grants 51,726 131,146 Unexpended balances of gifts for designated purposes 9,663 10,799 Current fund . 105,087 125,724 $ 207,582 $ 313,823 $5,446,431 $4,889,405 Note The Laboratory has since January 1, 1916, provided for reduction of book amounts of plant assets and funds invested in plant at annual rates ranging from \% to 5% of the original cost of the assets. 54 MARINE BIOLOGICAL LABORATORY MARINE BIOLOGICAL LABORATORY STATEMENTS OF OPERATING EXPENDITURES, INCOME AND CURRENT FUND Years Ended December 31, 1960 and 1959 Operating Expenditures 1960 1959 * Research and accessory services $ 250,578 $ 222,624 Instruction 219,234 79,396 Library and publications 61,462 58,190 Direct costs on research grants 182,899 221,436 $ 714,173 $ 581,646 Administration and general 70,037 63,947 Plant operation and maintenance 117,980 113,454 Dormitories and dining 162,713 154,405 Additions to plant from current income 78,654 23,448 $1,143,557 $ 936,900 Less depreciation included in plant operation and dormitories and dining above but charged to plant funds 48,086 46,604 $1,095,471 $ 890,296 Income Research fees $ 56,408 $ 50,242 Accessory services (including sales of biological specimens, 1960, $48,817, 1959, $66,742) 151,109 141,022 Instruction fees 23,905 21,395 Grants for instruction and research training 185,571 40,478 Library fees, Bulletin subscriptions and other 35,174 25,225 Reimbursements and allowances for direct and indirect costs on research grants 221,197 246,326 Dormitories and dining income 105,086 106,424 $778,450 $ 631,112 Gifts used for current expenses 48,300 36,590 Grants used for current expenses 143,018 107,500 Investment income used for current expenses 105,066 100,432 Total current income $1,074,834 $ 875,634 Excess of operating expenditures over current income $ 20,637 $ 14,662 Current fund balance January 1 125,724 140,386 Current fund balance December 31 $ 105,087 $ 125,724 * 1959 amounts have been reclassified for purposes of comparison. REPORT OF THE TREASURER 55 MARINE BIOLOGICAL LABORATORY STATEMENT OF FUNDS Year Ended December 31, 1960 Balance Gifts and Invest- Used for Other Balance Jan.l, Other ment Current Expendi- Dec. 31, 1960 Receipts Income Expenses titres 1960 Invested funds . . $1,426,500 $ 142,749 $110,875 $103,074 $ 13,234 $1,563,816 Unexpended plant funds . $ 276,957 556,456 9,619 760,990 82,042 Unexpended research grants $ 131,146 470,366 549,786 $ 51,726 Unexpended gifts for designated purposes . $ 10,799 48,355 48,300 1.191 $ 9,663 Current fund $ 125,724 20,637 $ 105,087 $1,217,926 $120,494 $721,797 $775,415 Gifts $ 603,616 Grants for research, train- ing and support .... 470,366 Net gain on sales of securities 125,620 Appropriated from current income and other . . . 18,324 $1,217,926 Expended for construction of new building .... $760,990 Scholarship awards 3,185 Payments to pensioners .... 10,049 Other 1,191 $775,415 56 MARINE BIOLOGICAL LABORATORY MARINE BIOLOGICAL LABORATORY SUMMARY OF INVESTMENTS OF ENDOWMENT FUNDS December 31, 1960 Securities held by Trustee : General endowment fund : % of Market Cost Total Quotations Investment % of Income Total 1060 U. S. Government Securities .... $ Corporate bonds 35,164 572 187 3 .6 Q $ 36,619 ^47653 2.5 373 $ 1,272 18984 Preferred stocks 84,778 8 Q 70 138 48 3370 Common stocks 263,894 27 6 813 572 554 29474 $ 956,023 100 .0 $1 ,467,982 100.0 .$ 53,100 General educational board endowment fund : U. S. Government securities $ 31,060 16 $ 32434 99 $ 1 205 Other bonds 94888 40 8 88450 269 3480 Preferred stocks 26745 14 24058 73 1063 Common stocks 37,677 Q 183,647 55.9 5,972 $ 190,370 100 .0 $ 328.589 100.0 $ 11,720 Total securities held by Trustee $1,146,393 $1,796,571 Investments of other endowment and un- restricted funds : Pooled investments : $ 137,742 Total investments of other en- dowment and restricted funds $ 412,036 Total investment income Custodian's fees charged thereto Income of current funds temporarily invested in pooled securities Investment income distributed to funds . . . $ 64,820 U. S. Government securities . . . .. . $ 1,018 .4 $ 1,048 .3 $ 180 Corporate bonds 149866 54.6 147,782 44.3 6 575 Preferred stocks 3,214 1.2 3,075 1.0 112 Common stocks 120,196 43.8 181,314 54.4 5,645 $ 274,294 100.0 $ 333,219 100.0 $ 12,512 Other investments : U S Government securities .. $ 7,000 $ 981 Other bonds 47,971 1,675 Preferred stocks 3,728 131 Common stocks 46,530 31,552 Real estate 32,513 $ 34,339 $ 46,851 $111,671 (546) (250) $110,875 REACTION TO INJURY IN THE OYSTER (CRASSOSTREA VIRGINICA) FREDERIK B. BANG Mitriuc Biological Laboratory, Woods Hole, Mass., and flic Department of Pathobioloc/y, The Johns Hopkins School of Hygiene and Public Health, Baltimore 5, Md. The comparative approach to pathology, which uses both cold-blooded verte- brates and invertebrates to advantage, was pioneered brilliantly by Metchnikov (1891) and has since been continued, somewhat sporadically, in France (Canta- cuzene, 1923) and elsewhere (Cameron, 1932; Schlumberger, 1952). There still remains a tremendous dearth of information concerning the reaction of various invertebrates to injury and infection. A partial exception to this is the study of insect pathology (Steinhaus, 1949). In a continuing study of pathological processes in the oyster we have found that the initial phase of phagocytosis of bacteria by the oyster amebocyte is often preceded by adhesion of the bacteria to the amebocyte so that the cell surface is literally covered with bacteria, and the sticking may be limited to the contact of the flagellum of the organism with the amebocyte so that the still motile bac- terium becomes anchored. Secondly, the classical cellular clot formed by the agglutination of these amebocytes may be accompanied by an extracellular clot which immobilizes bacteria. And finally, the cellular clot may lie directly observed within the vascular system of the living oyster and may be produced by the injection of an extract of oyster tissue. MATERIAL AND METHODS Most of the experiments were done on a so-called half shell preparation in which, after an edge of shell was knocked off, the shell was pried open with a knife until the adductor muscle was seen ; then, with as little trauma as possible, the adductor muscle was cut and the upper shell removed. In good preparations this meant that a portion of the mantle and the muscle was cut, and the pericardium was left intact. Such preparations (Fig. 1 ) were kept in running sea water and used during the next several days. Some of these lived as long as a week or ten days, but had by that time gradually deteriorated, showing a loss of leucocytes from the blood and progressive infection and disintegration of the muscle. Heart blood was readily obtained from them at any time, and direct examination of the various vessels of the mantle, palps and gills was satisfactory under a Zeiss dissecting microscope (40 X ). Intracardiac injections were usually done directly into the ventricle, and blood was withdrawn from the auricle. During these operations, the animals must obviously be damaged to a greater degree than were Stauber's preparations (1950) in which a window was made directly over the heart. However, they allowed direct examination of the entire gill and vascular system, and were used only as acute preparations. A limited 57 58 FREDERIK B. BANG number of observations were made on oysters in which a hole was carefully drilled near the pericardium and the shell was then picked away until the sheath was exposed. Observations on phagocytosis were made with a Zeiss phase microscope both at 500 X and 1250 X. A drop of freshly obtained blood was placed on a slide, then either a drop of bacterial suspensions from a freshly grown culture of marine bacteria was added to it, or a small portion of the colony itself was added with a loop directly to the drop of blood. The preparation was covered with a #1 cover- anterior aorta- aortic bulb FIGURE 1. Diagram of half-shell preparation of oyster. Circulatory system indicated in heavy black line. Injections were made directly into the heart (H). Observations were made principally on mantle arteries. slip, and if observations were to be continued, the entire preparation was ringed with Vaseline to prevent evaporation. Amebocytes remained viable for 12 hours or more under these conditions. RESULTS Phagocytosis The amebocyte, which has been extensively studied in this and other molluscs (Faure-Fremiet, 1927), is a granular round cell floating freely in the blood stream. On contact with glass it flattens out and moves continually over the surface of the slide. This motion under phase may clearly be seen to begin by the extrusion of a REACTION TO INJURY IN OYSTER 59 series of filamentous pseudopodia which may be resolved with high power phase microscopy (Fig. 2) and which is shown in the accompanying electron micro- scope pictures. The spread of ectoplasm, illustrated in the accompanying figures (Figs. 3, 4, 5), then flows afterward, filling up the spaces between. Granules and other portions of the cell then flow into this region. A variety of cell forms may be observed on these slides ; some of them lack granules, others contain large wavy frills of pseudopodia, other large amorphous but refractile inclusions. Since the amebocyte may both lose its granules and may ingest large amounts of material, we are unable to say whether these represent different types of cell or physiological variants of one type. Most of the cells observed during the process of phagocytosis were granular cells. One of the most remarkable facts which was observed early in the study was the absence of phagocytosis. Frequently an amebocyte was seen to approach a bacterium with its fibrous processes, then either to reverse its flow or turn aside. During the course of several hours this behavior was repeated continuously and no phagocytosis was observed. Since it is so obviously contrary to established ideas of the importance of phagocytosis, and specific studies on phagocystosis of food particles by the oyster, the observations were repeated with a number of bacteria, and it was found that excellent phagocytosis might be obtained with a certain bacterium, yet little if any phagocytosis was observed in amebocytes from the same oyster if another preparation of bacteria was introduced. Repeated attempts were made to determine whether such failure of phagocytosis was due to the strain of bacteria, or to a combination of certain bacteria with amebocytes from certain oysters. It was not possible from day to day to find a combination of amebocytes and bacteria which did not phagocytize, but no observations were repeated within a few hours of each other and it remains likely that there is an undiscovered factor important in phagocytosis which is responsible for this variation. When a successful combination was obtained, and particularly when the bac- terium used was motile, phagocytosis was usually preceded by a massive sticking of the bacteria to the amebocyte, so that the amebocyte resembled a porcupine. Ingestion followed this phase (Fig. 4). Some incidental observations on phago- cytosis by amebocytes of the marine worm, Urcchis, showed the adhesion of bac- teria to be limited to the portion of the cell which had spread out on the glass. 1 In the oyster, however, since extrusions of the cell appeared from all sides it was not possible to determine whether all portions of the amebocyte were equally sticky. The curious anchoring of motile bacteria to amebocytes which renders them unable to leave the amebocyte while they seem at the same time to have no contact with it, was explained by a fortunate electron microscope picture (Fig. ^,5). The presence of the unipolar flagellum wrapped around the filamentous pseudopodia fully explains the continual tugging and jerking at an invisible anchor. Extracellular clot formation During the summer of 1956, and in the two succeeding summers, the formation of a definite extracellular clot was observed (Fig. 6). It could be seen only under phase microscopy and seemed similar in texture and formation to a clot described 1 These observations were made originally by Mr. Stuart Krassner, to whom we are indebted for permission to include this material. 60 FREDERIK B. BANG FIGURES 2-3. REACTION TO INJURY IN OYSTER 61 by Gregoire (1952) in a variety of insect bloods. The clot was found in only about one-third of the oysters which were examined, and was not present in the same oyster at all times. Furthermore, we have been unable to determine the con- ditions under which it may be consistently formed in an individual oyster. How- ever, we were able to reproduce this extracellular clot throughout the summer months of the last three years, and have been clearly able to rule out artifacts of preparation. The clot was first noticed in slides which had been kept for continued observa- tion of phagocytosis. It tended to occur near the occasional small air bubble which was entrapped beneath the coverslip in the sealed slide. It was not present immediately but usually appeared in 15 minutes to half an hour, so that it was suspected of being a local drying phenomenon until it was seen in slides which had no appreciable air bubbles, and in sealed hanging drops and direct preparations. Attempts to relate it to the time of day, time since opening of the oyster, amount of trauma, and sex of oyster, failed. A mixture of pericardial fluid and blood did not affect the process. A higher percentage of extracellular clots seemed to lie obtained from oysters which had been forced-starved by keeping them out of the sea water, and then replacing them ; but this produced positive results in only about half of the cases. Since the clot had been originally observed in a preparation to which bacteria had been added, repeated comparisons were made in the presence and absence of the bacteria. In most cases, when the clot was obtained in the presence of the bacteria, it was also found in the control slide to which bacteria had not been added, though it was usually less extensive. It seems to have a real role in the repair of traumatized tissue, for : 1) it was found already developing in small cellular clots taken directly from the heart ; 2) it occurred predominantly around clumps of cells; 3) bacteria were immobilized by its development (Fig. 7) ; and, 4) it was obtained both immediately after opening an oyster and from some preparations which had been on the half shell as long as 24 hours. Its possible relation to cycles of feeding by the amebocytes is unknown. I ntrai'ascnlar clots There is a rapid clumping of cells when oyster blood is withdrawn in glass vessels, which is also the case with many other invertebrate bloods. Direct obser- vation of traumatized blood vessels shows the formation of the same type of cellular clot at the open end of the vessel, so that within a few minutes of cutting, the clotted cells have effectively sealed the end. Similar clumps of amebocytes are observed directly covering the cut end of the adductor muscle in our preparations, and oysters which had been repeatedly bled develop a shaggy pericarditis which consists of masses of these clumped cells. However, in oysters which have had minimal trauma and have remained in clean running sea water for several hours after being opened, the circulation may be fully effective and direct examination of the distended vascular system was possible. In such a view, the cells are seen moving to and fro, FIGURE 2. Phase micrograph of oyster amebocyte on glass, approximately 600 times. FIGURE 3. Electron micrograph of whole cell preparation of amebocyte. Filamentous pseudopods extend away from the cell edge. This and succeeding electron micrographs were made by allowing the amebocytes to spread out on a collodion film. The cells were fixed with osmium vapor, washed and the film placed on grids ; 9,000 times. 62 FREDERIK B. BANG I 1 5 FIGURES 4-5. REACTION TO INJURY IN OYSTER 63 and relatively few of them are clumped. When the circulation is sluggish they may be found lining the lower side of a vessel, but they readily move from one portion to another as the oyster is tilted. The obvious question whether a par- ticular portion of the tissue was responsible for the formation of the cellular clot was tested by making a sea- water extract of gill tissue, centrifuging the extract and injecting about 0.1 cc. of the relatively clear supernatant directly into the heart. The material immediately spread throughout the animal and an interesting series of events set in. If the oyster had relatively large numbers of cells so that the blood was milky in appearance, the first reaction was the formation of large curd- like clumps of loosely aggregated cells. These became more dense, soon ceased to flow back and forth, and within 10 to 15 minutes were stuck in tight clumps to the edge of the vessel, and the fluid itself appeared perfectly clear. The vessel frequently decreased in size, particularly if the heart happened to cease beating. In many cases, some flow back and forth in the mantle vessels continued even though there was no visible heart beat, presumably from the action of the accessory heart (Fig. 1). Within about two hours after the injection, most of the effects had worn off: the heart was beating, the blood was again flowing freely, and rela- tively few clumps were seen. Individual cells were observed flowing freely in the large vessels or moving in and out of the fine branches of the palps or the gills. When these oysters were reinjected with the original extract, an apparently full- fledged repetition of the reaction was observed. The reaction was not obtained by the injection of sea water, of suspensions of carmine, or of bacteria of several sorts, though a moderate "curdling" of the blood was seen after the injection of heavy suspensions of bacteria. India ink of two sorts was then injected in suspensions of sea water. The usual preparation of colloidal ink when injected caused prompt clumping of cells, a cessation of heart beat, and the probable development of intravascular clumps like those seen following the injection of tissue extracts. However, the black masses of material which were partially phagocytized, as described by Stauber (1950), obscured the observation. A preparation of "Pelican" India ink, which lacks the gum coating present in most commercial India inks, produced a much milder reaction (Muller, 1927a, 1927b). The particles were soon phagocytized as small particles or clumps without major changes in the circulation itself, just as carmine particles had been. DISCUSSION The capacity to react to injury, an essential function of living cells, is basic to studies in pathology. Following Metchnikov (1891), who began with a marine echinoderm embryo, the greatest attention of pathologists when studying inverte- brates has concentrated on the wandering cells or amebocytes. From a comparative pathological point of view, at least three phenomena are contained in the oyster in this one cell. These are phagocytosis of invading bacteria, inflammation, and thrombosis. Since the amebocyte is the only circulating cell of the blood in the oyster, and since cellular clots are the common mechanism of closing gaps in the FIGURE 4. Beginning phagocytosis of uniflagellate bacterium. The flagellum is coiled around several pseudopods of the amebocyte ; 14,000 times. FIGURE 5. An electron micrograph showing later stage in phagocytosis of bacterium ; 9000 times. 64 FREDERIK B. BANG FIGURE 6. Phase micrograph showing extracellular clotting of amebocytes ; 1000 times. FIGURE 7. Similar extracellular clot with bacteria involved in clot ; 1000 times. REACTION TO INJURY IN OYSTER 65 vascular system among invertebrates (Geddes, 1880; Cuenot, 1891), it is of course impossible to separate the function of inflammation whereby a white cell in vertebrates becomes adherent to a vessel wall and migrates through it, and that of the adherence of many amebocytes together to form a tight clump which blocks the free flow of blood. An ideal invertebrate in which to follow the above processes would allow direct observation of the vascular channels without trauma, and would from the bacterio- logical point of view allow for external sterilization and thus obtaining of blood without contamination by the surrounding fluid or air. In this regard the oyster and other molluscs have no external surface which may be sterilized and then punctured, and have no extension of the vascular system that may be observed without the introduction of trauma. Thus, though a variety of studies have been done on diseased oysters (Herdman and Boyce, 1899; Roughley, 1926; Stauber, 1945; Mackin, 1951; Mackin, et al., 1952), there is little direct information on the pathogenesis of any of the disease states. Phagocytosis of food material for transport through the oyster, and of particulate matter has been studied rather extensively (Yonge, 1926; Takatsuki, 1934), primarily by following the events in sequence by histological sections. Phagocytosis itself was first observed about a hundred years ago, by Haeckel (1862), \vho injected particulate dyes into molluscs so that the distribution of the vascular system might be determined. He pointed to the potential importance of the phenomenon in nutrition. Then came the disclosure by Metchnikov of the role of phagocytosis as a defense mechanism (1884, 1891). In the oyster and other molluscs the importance of the amebocyte in digestion, transfer of food, and repair, has been firmly established (Yonge, 1928; Wagge, 1955). Recently Tripp (I960), has followed the fate of several species of bacteria in oyster tissue following intra- cardiac injection of large numbers. He has shown that phagocytosis may be apparent in the cells circulating within the vessels and subsequently in the tissues. Intracellular digestion appeared to be a major mechanism of disposal of the bac- teria. In several infectious diseases of the oyster the presumptive agent is thought to be disseminated by the amebocyte (Orton, 1923). It was therefore a surprise to us to find that there was a marked variation in the phagocytosis of different preparations of bacteria by leucocytes of the same oyster. Attempts to show in- creased phagocytosis in the presence of mucus from the gill, of disintegrating crystalline style material, or of extracts of the hepato-pancreas failed. Although "surface phagocytosis" (Wood, 1951-1952) took place in the process of the flow of amebocytic protoplasm around bacterium, it was not always the explanation, for masses of bacteria were found stuck to the surface of amebocytes in most of the cases where phagocytosis was apparent. The direct adherence of the amebocyte to the bacterium itself was highlighted by the observation of the flagellar adherence of the bacteria to the amebocyte so that it was unable to escape from the amebocyte. The evolutionary need for extracellular clot formation becomes greater when the amebocytes or leucocytes have much less direct contact with each other because of the presence of large numbers of red cells. However, extracellular clot or gel formation is well developed in several invertebrates (Gregoire and Florkin, 1950; Loeb, 1910; Gregoire, 1952; Bang, 1956) in which the predominant circulating 66 FREDERIK B. BANG cells are directly involved in clot formation (Yonge, 1926). The presence of this extracellular gel, which seemed fully able to limit bacterial motion in many of the oysters which we examined, may indicate that additional advantage is to be gained from such mechanisms of thrombosis which extend beyond the cell. The possible role of this extracellular material in rendering bacteria more susceptible to phago- cytosis needs further study. The origin of this extracellular gel from the extru- sion of the many cellular granules is an obvious possibility which has not been investigated. Direct observations of the formation of the cellular clot at a point of traumatic rupture of a vessel, the accumulation of great numbers of these cells on the heart when it is exposed to sea water by opening the pericardium, and the accumulation of amebocytes at the cut edge of the adductor muscle, led to the question as to the effect of tissue extracts. It was soon found that a fresh crude sea-water extract of ground gill tissue, when injected directly into the heart, caused a rapid clumping of cells and the tight adherence of these cells to the vessel wall, so that the circula- tion was greatly slowed or stopped. Injection of sea water, of bacterial suspensions, and of carmine, failed to cause similar marked effects. Thrombosis accompanied by phagocytosis was rapidly produced by the injection of certain preparations of 1mm. FIGURE 8. Diagram of observations of mantle artery: (1) shows the clear appearance of the vessel under normal conditions. Individual amebocytes may be seen poorly and are not indicated here. (2) Beginning clumping amebocytes within the vessel. They are loosely clumped and move rather freely in the vessel. (3) Amebocyte clumps which have contracted into tight balls of thrombus and are adherent to the vessel wall. REACTION TO INJURY IN OYSTER 67 India ink, but not by a preparation which is stated to lack the shellac coating which in itself causes extensive thrombosis. Our experiments have been limited to acute short term experiments. Several other molluscs have been used in the study of chronic processes (Drew and de Morgan, 1910; Zawarzin, 1927), and the importance of epithelial tissues, mucous sheets and chronic fibrous tissue "repair" needs extensive exploration (Kedrowsky, 1925; Labbe, 1929). SUMMARY 1. In vitro phagocytosis of marine bacteria by fresh oyster leucocytes, though readily demonstrable in most cases, was by no means an invariable phenomenon. When it occurred, it was frequently accompanied by a massive sticking of bacteria to the leucocytes. The flagellar portion of the bacterium might be so caught by the amebocyte that the bacterium was unable to escape, even though the body was not in contact with the amebocyte. 2. An irregular but repeated formation of an extracellular clot is described as seen in vitro by phase microscopy. Reasons for believing that it is a true phe- nomenon in the oyster are given. 3. Intravascular clotting or thrombosis was produced by the intracardiac injection of tissue extracts. The clotting disappeared spontaneously within two hours after the injection. LITERATURE CITED BANG, F. B., 1956. A bacterial disease of Linutlus polyphemus. Bull. Johns Hopkins Hosp., 98: 325-351. CAMERON, G. R., 1932. Inflammation in earthworms. /. Path, and Bact., 35: 933-972. CANTACUZENE, J., 1923. Le probleme de I'immunite chez les invertebres. C. R. Soc. Biol. (75th ami.) : 48-119. CUENOT, L., 1891. fitude sur le sang et les glandes lympathiques dans le serie animate. Arch, de Zool. E.vp. Gen.. 19: 13-90. DREW, G. H., AND W. DE MORGAN, 1910. The origin and formation of fibrous tissue produced as a reaction to injury in Pcctcn maximus, as a type of Lamellibranchiata. Quart. J. Micr. Sci., 55: 595-610. FAURE-FREMIET, E., 1927. Les amibocytes des invertebres a 1'etat quiescent et a 1'etat actif. Arch. d'Anat. Micr., 23: 99-173. GEDDES, P., 1880. On the coalescence of ameboid cells into plasmodia and the so-called coagu- lation of invertebrate fluids. Proc. Roy. Soc. London, Ser. B, 30 : 252-254. GREGOIRE, CH., 1952. Sur le coagulation du sang de Limulus polyphemus (Arachnida). Arch. Intern, de Physiol, 60: 97-99. GREGOIRE, CH., AND M. FLORKIN, 1950. Blood coagulation in arthropods. Plivsiol. Comp. et. Oecol, 2: 126-139. HAECKEL, E., 1862. Die Radiolarien. Berlin : Geo. Reimer. Pp. 104-106. HERDMAN, W. A., AND R. BOYCE, 1899. Oysters and disease. Lancashire Sea Fisheries Mem. #1. KEDROWSKY, B., 1925. Reactive Veranderung in den Geweben der Teichmuschel (Anodonta sp.) bei Einfuhrung von sterilem Zelloidin. I'irchow's Arch'w., 257: 815-845. LABBE, A., 1929. Reactions experimentales des Mollusques a 1'introduction de stylets de celloi- dine. C. R. Soc. Biol.. 100: 166-168. LOEB, L., 1910. t v ber die Blutgerinnung bei Wirbellosen. Biochem. Zeitschr., 24: 478-495. MACKIN, J. G., 1951. Histopathology of infection of Crassostrea virginica by Dermocystidiwn marinum. Bull. Alar. Sci. Gulf and Caribb., 1 : 72-87. MACKIN, J. G., P. KORRINGA AND S. H. HOPKINS, 1952. Hexamitiasis of Ostrca cdulis and Crassostrea virginica. Bull. Mar. Sci. Gulf and Caribb., 1 : 266-277. 68 FREDERIK B. BANG METCHNIKOV, E., 1884. t)ber eine Sprosspelzkrankheit der Daphnien. Vir chow's Archiv., 96: 177-195. METCHNIKOV, E., 1891. Lectures on the comparative pathology of inflammation. Trans, by F. A. Starling and E. H. Starling. London : Keagan Paul, Trench, Trubner and Co. MULLER, G. L., 1927a. Normoblastosis produced by India ink. /. Exp. Med., 45 : 399-410. MULLER, G. L., 1927b. Polycythemia, normoblastosis and erythrocytic hyperplasia of the bone marrow produced by gum shellac. /. Exp. Med., 45 : 753-770. ORTON, J. H., 1923. Summary of an account of investigations into the cause or causes of the unusual mortality among oysters in English oyster beds during 1920 and 1921. /. Mar. Biol. Assoc., 13: 1-23. ROUGHLEY, T. C, 1926. An investigation of the cause of oyster mortality on George's River, New South Wales, 1924-25. Proc. Linn. Soc. N. S. Wales, 51 : 446-491. SCHLUMBERGER, H. G., 1952. A comparative study of the reaction to injury: the cellular response to methycholanthrene and to talc in the body cavity of the cockroach (Peri- planeta amcricana). Arch. Path., 54: 98-113. STAUBER, L. A., 1945. Pinnotheres ostreum, parasites on the American oyster, Ostrca (Gryphaea) virginica. Biol. Bull, 88: 269-291. STAUBER, L. A., 1950. The fate of India ink injected intracardially into the oyster, Ostrea virginica (Gmelin). Biol. Bull, 98: 227-241. STEINHAUS, E., 1949. Insect Pathology. McGraw-Hill Book Co., New York. TAKATSUKI, S., 1934. On the nature and function of the amebocytes of Ostrea ednlis. Quart. J. Micr. Sci., 76 : 377-436. TRIPP, M. R., 1960. Mechanisms of removal of injected micro-organisms from the "American oyster Crassostrca virginica (Gmelin). Biol. Bull., 119: 273-282. WAGGE, L. E., 1955. Amebocytes. Int. Rev. Cyt., 4: 31-75. WOOD, W. B., 1951-1952. Studies on the cellular immunology of acute bacterial infections. Harvey Lectures., pp. 72-98. YONGE, C. M., 1926. Structure and physiology of the organs of feeding and digestion in Ostrea edulis. J. Mar. Biol. Assoc., 14: 295-386. YONGE, C. M., 1928. The absorption of glucose by Ostrea edulis. J. Mar. Biol. Assoc., 15: 643-658. ZAWARZIN, A., 1927. tiber die reactiven Veranderungen des Epithels bei der Einfuhrung eines Fremdkorpers in den Alantel von Anodonta. Zeitschr. f. Mikr. Anat. Forsch., 11: 215-282. THE OBLIGATE COMMENSAL CILIATES OF STRONGYLOCEN- TROTUS DROBACHIENSIS : OCCURRENCE AND DIVISION IN URCHINS OF DIVERSE AGES; SURVIVAL IN SEA WATER IN RELATION TO INFECTIVITY C. DALE BEERS Department of Zoology, University of North Carolina, Chapel Hill, North Carolina, and the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine Seven species of ciliated protozoa have been reported from the alimentary tract of the sea urchin Strongylocentrotus drobachicnsis (O. F. Miiller) in the coastal waters of Mt. Desert Island, Maine (Powers, 1933a). Three of them are holo- trichs which have no known free-living congeners and are restricted to echinoid hosts. They are Entodiscus borealis (Hentschel, 1924) Madsen, 1931 ; Madsenia indomita (Madsen, 1931) Kahl, 1934; and Biggaria gracilis (Powers, 1933) Kahl, 1934. In the words of Kirby (1941, p. 921), such ciliates "may be supposed to have evolved in the shelter of these hosts" and they are thus regarded as obligate commensals. The relation of the remaining four to their host is not entirely clear, owing to inadequate study. Powers (1933a, p. 119) regards them "as chance or vagrant ciliates, which, after being engulfed with food, are able to survive" and multiply as entozoic commensals. Two of the four are holotrichs, namely, Plagio- pyla minuta Powers, 1933, and Cyclidium stercoris Powers, 1935 ; one is a hypo- trich which Beers (1954) identified as Euplotes balteatus (Dujardin, 1841) Kahl, 1932, and the final and least common is an undetermined species of the peritrich Trichodina. Reference may be made to Beers ( 1948) for further details concerning the taxonomy of the ciliates. In order to avoid the constant repetition of the unwieldy binomial Strongylocentrotus drobachiensis, the terms "urchin" and "urch- ins" are substituted in the following pages and refer without exception to this echinoid. To return to the three obligate commensals, which are the subject of the present study, Power (1933a) notes that adult urchins at Mt. Desert Island are almost invariably infected with them and indeed may harbor them in almost incredible abundance. In the summer of 1947, Beers (1948) extended Powers' investigations by making a quantitative study of the occurrence and morphogenetic condition of the ciliates in 182 urchins, the tests of which varied in diameter from 30 to 60 mm. All the urchins were infected with E. borealis and M. indomita, and 181 of them with B. gracilis. Counts of the ciliates in fresh samples of enteric fluid showed that the vast majority of the urchins harbored infections of each species that varied in intensity from "moderate" (M) to "heavy" (H), M meaning 50-500 individuals of the species per 0.1 ml. of fluid and H meaning 500-1000 or more per 0.1 ml. The remaining infections were designated as "light" (L), meaning fewer than 50 individuals of a species per sample. A regional distribution of the ciliates was also 69 70 C. DALE BEERS noted, in that E. borealis occurred primarily in the stomach (inferior spiral or intestine of some authors), M. indomita in the intestine (superior spiral or large intestine ) , and B. gracilis in the rectum. However, the foregoing distribution of E. borealis and M. indomita prevailed as a rule only in well-fed urchins ; in inade- quately fed urchins they tended to shift aborally and in extreme cases of hunger to commingle with B. gracilis. The factors that were responsible for the regional distribution of the ciliates were unexplained. In any flourishing population of ciliates, whether free-living or associated in any way with a host, one might reasonably expect to find at almost any time a significant percentage of individuals that are dividing. It is therefore remarkable that dividing specimens of E. borealis and M. indoinita are extremely rare, even in ciliate populations of great density. With reference to the division of E. borealis, Powers (1933b, p. 130) comments as follows: "A study of about 600 specimens fixed during the day gave but three individuals showing any signs of fission." In 1947 the writer made a special effort to find dividing specimens of E. borealis and M. indomita in the 182 urchins that have been mentioned. The urchins were collected and examined without delay at practically all hours of the day and night, but only six of them revealed dividing individuals of E. borealis. Concerning M. indomita, neither Madsen (1931) nor Powers (1933a) mentioned its division, and the task of finding dividing specimens was especially difficult. In the 88 urchins that were examined in July, only one dividing individual was found ; in 94 studied in August, dividing specimens were found in only three. It was concluded that division in E. borealis and M. indoinita was a periodic phenomenon : that long intervals of non-divisional life alternated with brief periods of intense divisional activity. In retrospect it became apparent that both Powers and the writer, in their efforts to find dividing ciliates, inadvertently restricted their studies to rela- tively large mature urchins, in which the ciliate populations were already well- established and probably somewhat stabilized. It will be seen in the following pages that when some of the younger urchins are examined, divisional stages can be found in abundance. Turning finally to B. gracilis, it was evident that this ciliate differed markedly in its reproductive activities from the preceding two. Of the 181 infected urchins, all contained dividing specimens, and there was thus no evidence of long periods of non-divisional life. The relation of the size of the urchins to the condition of their respective ciliate infections was not considered in the earlier study (Beers, 1948). Actually, there is often great diversity of size in an aggregation of urchins on a rocky ledge or in a tide pool. For example, urchins taken by the writer from a single tide pool at Long Ledge, Mt. Desert Island, on July 10, 1960, varied in diameter from 8 to 65 mm. To some extent these differences merely reflected different rates of growth, but Grieg (1928) concluded that size (diameter of test) is a fairly reliable measure of the age of the urchins. Basing his studies on urchins taken from the Folden Fjord and the Bals Fjord of Norway, and on other materials, he concluded that the following relations of size to age prevail, at least in a general way : diameter 0.5 mm., metamorphosis just completed; 1-2.5 mm., "the same year-group" as the foregoing, meaning urchins in their first summer of life ; 56 mm., about 1 year old; 15 mm., about 2 years old; 24 mm., 3 years old; 40 mm., 4 years; 50 mm., 5 years; 60 mm., 6 years; 78 mm. (the largest specimen), "probably about 8 years CILIATES OF STRONGYLOCENTROTUS 71 old." In the region of Mt. Desert Island, the spawning of urchins begins in February and ends in April. Since the urchins of northern Europe have a similar spawning period (Mortensen, 1943, p. 211), there is little doubt that Grieg's esti- mates of age are equally applicable to Mt. Desert Island urchins. The present paper is a record of further observations on E. borcalis, M. indomita and B. gracilis as found in 152 urchins taken at Mt. Desert Island in the summer of 1960. It is based on seven collections or small populations of urchins, each of which consisted of specimens of as many different sizes (age-groups) as were available at the respective sites of collection. The study concerns in particular the following aspects of the biology of the ciliates. (1) Their occurrence and morphogenetic condition (whether dividing or not) in "small" urchins, meaning urchins 8-14 mm. in diameter and presumably about 1.5 years old. (A minimal size of 8 mm. was fixed solely by the unavailability of any urchins of smaller size.) This aspect attempts to answer these questions: At what age do urchins become infected with the respective ciliates ? Once established in the urchin, do the ciliate populations build up immediately or does a delay ensue following their ingestion by the host ? (2) Their occurrence and morphogenetic condition in "larger" urchins, mean- ing urchins 15-65 mm. in diameter and representing five age-groups, namely, 2.5 to 6.5 years, in increments of one year. This aspect attempts to answer these questions : Do the infections become progressively more intense (ciliates more plentiful) as the urchins increase in age? Is the division of the ciliates, in par- ticular that of E. borealis and M. indomita, correlated in any way with the age of the urchins? (3) Their morphogenetic condition throughout a population of urchins. That is to say, does division of the ciliates occur simultaneously in all the urchins of a population or does it affect only certain age-groups or random individuals? (4) Their capability to survive in sea water outside the body of the urchin, bearing in mind that cysts are unknown in all echinoid ciliates and that young urchins undoubtedly become infected by the ingestion of the usual trophic forms ; thus, such survival affects directly their transmission from urchin to urchin. MATERIAL AND METHODS Of the seven collections of urchins, three were taken at low tide from the rocks of Emery Cove Ledge on July 2, 13 and 24. The remaining four were taken from four different tide pools at Long Ledge on July 10, August 8 and 26, and September 1. Each collection consisted of about 40 individuals. Of the urchins of each collection, 10 to 15, representing as many age-groups (sizes) as were available, were opened and examined without delay on the day of collection, and a like number was examined on the following day. The remaining ones were excluded from consideration, since it seemed advisable to use only urchins that were relatively recently collected. The total number of individuals of each species of ciliate was actually counted in the small urchins, but this procedure was usually impracticable with reference to larger urchins, in view of the enormous numbers of ciliates in them. Thus, 0.05-ml. or 0.1 -ml. samples of enteric fluid were taken from these urchins, and 72 C. DALE BEERS the number of ciliates of each species was estimated in the samples. If the size of the urchin permitted, five 0.1 -ml. samples were taken from the stomach, five from the intestine, and two from the rectum. In 0.1-ml. samples, the three degrees of infection that have been defined were again distinguished with reference to each species. In 0.05-ml. samples, half the aforementioned numbers of individuals was employed to distinguish the respective degrees of infection. With reference to the survival of the ciliates in pure sea water, details of the procedure will follow. RESULTS 1. Occurrence and morphogenetic condition of the ciliates in small urchins (diame- ter of test, 8-14 mm.} Unfortunately, only nine urchins of this size were available for study. Never- theless, it is believed that they furnish information that is significant (Table I). TABLE I Total numbers of ciliates of three species in each of nine small urchins (age about 1.5 years) taken at Long Ledge, Mt. Desert Island, in 1960 Diameter of test in mm. Date collected Entodiscus borealis Madsenia indomita Biggaria gracilis 8 July 10 8 August 26 8 September 1 9 July 10 3 1 9 August 26 9 12 2 9 September 1 1 8 12 August 8 10 8 1 13 August 26 26 15 4 14 July 10 9 28 2 Since urchins attain a diameter of 5-6 mm. at the end of one year and of 15 mm. at the end of two years, it is assumed that these nine urchins emerged as plutei in February or March of 1959 and were thus about 1.5 years old in the summer of 1960. A very careful examination of the contents of the digestive tracts of three S-mm. urchins revealed no ciliates whatsoever, although the digestive tract of each was well filled with algal food. A similar examination of three 9-mm. urchins that were collected on the same dates as the foregoing revealed only small numbers of ciliates, though M. indomita was absent in one of them and B. gracilis in another. None of the ciliates was dividing. Evidently these three urchins, at the time in their second summer of life, were in the process of acquiring their respective ciliate infections. Finally, an examination of three urchins that had diameters of 12, 13, and 14 mm., respectively, showed ciliates of all three species in each urchin. On the average, these urchins contained two to three times as many individuals of each species as the 9-mm. urchins, even though the infections with B. gracilis were extremely light. Again, no dividing specimens were observed. CILIATES OF STRONGYLOCENTROTUS 73 2. Occurrence and morphogenetic condition of the ciliates in larger urchins (di- ameter of test, 15-65 mm.} Urchins 15-23 nun. in diaui. Twelve urchins of this size, assumed to be about 2.5 years old and thus in their third summer of life, were available for study. Whereas the urchins of the preceding age-group (1.5 years) were either uninfected or at best only lightly infected, all the urchins of the present group were infected with the three ciliates, and about half of the infections qualified either as moderate or heavy. The status of the respective infections in the 12 urchins was as follows : E. borealis, 1 H, 5 M, 6 L; M. indomita, 1 H, 4 M, 7 L; B. gracilis, 6 M, 6 L. Thus, a marked increase in the intensity of infection was clearly demonstrable in the 2.5-year-old urchins. It is evident that such an increase could have come about either by the ingestion of additional individuals or by the division of those already ingested. Manifestly, no comment can be made concerning the ingestion TABLE II Incidence of division of three species of ciliates in urchins of five different age-groups taken at Ml. Desert Island, summer 1960. All the urchins were infected with the three species. Number (and percentage) of urchins that contained Number of Diameter of test Approximate age dividing ciliates of species indicated examined in mm. years Rntodiscus Madsenia Biggaria borealis indomita gracilis 12 15-23 2.5 9 (75.0) 7 (58.3) 12 (100) 32 24-39 3.5 2 (6.3) 1 (3.1) 32 (100) 35 40-49 4.5 2 (5.7) 2 (5.7) 35 (100) 37 50-59 5.5 3 (8.1) 3 (8.1) 37 (100) 27 60-65 6.5 2 (7.4) 1 (3.7) 27 (100) of ciliates during the one-year interim, but it is significant that a remarkably high percentage of the 2.5-year-old urchins contained dividing individuals, showing conclusively that the respective ciliate populations were undergoing rapid augmen- tation by binary fission. The data concerning division in these urchins are sum- marized in Line 1 of Table II, reference to which shows that E. borealis was dividing in 9 of the 12 urchins, M. indomita in 7 of them, and B. gracilis in all of them. Furthermore, dividing specimens were relatively abundant, the incidence amounting to about one in every 25-50 individuals of each species. With reference to the division of E. borealis and J\l. indomita, it may be said now for purposes of emphasis that in none of the remaining age-groups was there such a high per- centage of urchins that contained the two ciliates in division. Concerning B. gracilis, it has been pointed out that this ciliate differs in its reproductive activities from the aforementioned two, in that long periods of non-divisional life are absent. Thus, B. gracilis was dividing in all 12 urchins. Urchins 24-39 mm. in diam. Urchins of this size, assumed to be about 3.5 years old, were available in almost unlimited numbers at both collecting sites, as were indeed those of all succeeding age-groups. Of 32 urchins of this size that were examined, all were infected with the three ciliates, as were all the urchins of the age-groups subsequently to be discussed. The respective degrees of infec- 74 C. DALE BEERS tion among the 32 hosts were as follows : E. borealis, 20 H, 10 M, 2 L; M. indomita, 18 H, 11 M, 3 L; B. gracilis, 7 H, 23 M, 2 L. In terms of percentages, 91 to 94 f> of the urchins harbored infections of each species that qualified as moderate to heavy. Thus, these urchins were distinctly more heavily infected than those of the preceding two groups, and it will be seen, when older age-groups are considered, that the infections had now attained their maximal intensities. The findings relative to division are summarized in Table II, Line 2. Of the 32 urchins, only two contained dividing specimens of E. borcalis, and even in them division was somewhat sparse and affected no more than one specimen in every 100. In spite of an exceptionally thorough examination of the samples, dividing individuals of M. indomita could be found in only one of the urchins (a different one from the foregoing two). In accordance with expectations, B. gracilis was dividing in all the urchins of the group. Urchins 40-49 mm. in diam. Thirty-five urchins of this size, assumed to be about 4.5 years old, were examined. The respective degrees of infection follow : E. borealis, 15 H, 17 M, 3 L; M. indomita, 17 H, 16 M, 2 L; B. gracilis, 5 H,27 M. 3 L. Again, 91 to 94% of the urchins harbored infections that varied from moderate to heavy. Data relative to the occurrence of division are summarized in Table II, Line 3, where it is seen that only two urchins contained dividing specimens of E. borealis and a like number (actually another two) those of M. indomita. All contained dividing specimens of B. gracilis. Urchins 50-59 mm. in diam. An examination of 37 urchins of this size, assumed to be about 5.5 years old, yielded the following degrees of infection: E. borealis, 12 H, 22 M, 3 L; M. indomita, 19 H, 16 M, 2 L; B. gracilis, 12 H. 21 M, 4 L. With reference to each of the species, 90 to 95% of the urchins had infections that varied in intensity from moderate to heavy. The data concerning the incidence of division, summarized in Table II, Line 4, show that three of the urchins had dividing specimens of E. borealis and three (one of the foregoing plus two others) had M. indomita in division. As usual, all the urchins contained dividing forms of B. gracilis. Urchins 60-65 mm. in diam. Twenty-seven urchins of this size (age about 6.5 years) were examined. Their respective degrees of infection were the follow- ing: E. borealis, 13 H, 12 M, 2 L; M. indomita, 15 H, 11 M, 1 L; B. gracilis, 9 H. 16 M, 2 L. Again, with reference to each species, moderate to high infections comprised more than 90% of the total. Of the 27 urchins, two harbored divisional stages of E. borealis and a third one contained At. indomita in division (Table II. Line 5), whereas B. gracilis was dividing, as expected, in all of them. 3. MorpJiogenetic condition of the ciliates in the respective collections of urchins It has been mentioned that the urchins of the present study comprised seven collections, each of which may be regarded as a small population ; each at least is believed to be a fairly representative sample of a natural population. And it has been shown, within the limits of the available material, (1) that urchins 8-14 mm. in diameter (age 1.5 years) may or may not be infected, but that if infected, they contain no dividing ciliates (Table I) ; and (2) that all urchins 15-23 mm. in diameter or larger (2.5 years of age or older) are infected with the ciliates, that B. gracilis is constantly dividing in all of them, but that E. borealis and M. indomita CILIATES OF STRONGYLOCENTROTUS 75 can be found in division in only a limited, though variable, number of them (Table II). However, the data concerning the division of E. borealis and M. indoniita in certain urchins of ages 2.5-6.5 years, as presented in Table II, tell nothing about the distribution of these particular urchins in the respective collections or population samples. Thus, one may ask : If E. borealis and J\I. indoniita are dividing in most of the urchins of one age-group of a collection for example, the 2.5-year group are they also dividing in a like percentage of urchins of the remain- ing age-groups of the same collection? This aspect can be adequately presented by considering in detail the compo- sition of two typical collections of urchins of ages 2.5-6.5 years and the condition of the two ciliates therein. The collections are those taken at Long Ledge on July 10 and August 8. The results are presented in Table III, in which the left column under the headings beginning "No. of urchins" refers to the collection of TABLE III Incidence of division of two species of ciliates in two collections of urchins taken at Long Ledge, Mt. Desert Island, on July 10 and August 8, 1960. All the urchins were infected with both ciliates. No. of urchins examined Range in size of urchins in mm. Approximate age of urchins in years No. of urchins that contained dividing ciliates of species indicated Entodiscus borealis Madsenia indomita 5 4 15-23 2.5 4 3 4 3 5 5 24-39 3.5 1 1 5 5 40-49 4.5 2 5 5 50-59 5.5 2 1 1 5 4 60-65 6.5 1 1 July 10, the right to that of August 8. Line 1 shows that five urchins of the size and age indicated were taken on July 10 and four on August 8. Of the five, four contained E. borealis in division and four had M. indoniita in division. (Three of the five contained dividing individuals of both species.) Of the four urchins taken August 8, three had E. borealis and three had M. indoniita in division. (Two had both species.) If the nine urchins are considered as a group, seven of them or 77.7% contained dividing individuals of E. borealis and seven contained M. indomita in division. What was the condition of the two ciliates in the remaining age-groups of the two collections ? Was division as widespread in the urchins of these groups ? The answer is conclusively in the negative, as shown in the remaining four lines of Table III. For example, Line 2 shows that five 3.5-year-old urchins of each collection were examined. Only one urchin of each collection contained E. borealis in division; in none of the ten was M. indomita dividing. The urchins of the remaining age-groups revealed essentially similar findings (Lines 3-5). Thus, division in E. borealis and M. indomita, when it occurs in a population of urchins, does not necessarily affect uniformly all the urchins of the different age-groups of the population. 76 C. DALE BEERS 4. Survival of the ciliates in sea zvater and its relation to infectivity It has been pointed out that cysts are unknown in ciliates of echinoids and that young hosts undoubtedly acquire their faunules by the ingestion of trophic forms that escape among the fecal pellets. This conclusion implies that echinoid ciliates can live in sea water outside the body of the host, although information on their survival is meager. Powers (1933b, p. 123) states that specimens of E. borealis when transferred to sea water "appear normal" and "live for various lengths of time," and he was able to keep specimens in hanging-drop preparations at 7 C for periods that varied from 15 to 23 days. It is doubtful that the survival of a large entozoic ciliate in the restricted confines of a small hanging drop reveals anything of special significance about its survival under natural conditions, and Powers himself states that the animals seemed "merely to exist." Since the capability of echinoid ciliates to survive in sea water is inseparably related to the infection of new hosts, a study of the survival of the three entocommensals of 5. drobachiensis was undertaken, but the procedure differed radically from that of Powers. The sea water was taken from Frenchman Bay (mean annual salinity, 31.8) well beyond the intertidal zone and was passed through Whatman No. 43 filter paper to remove the predatory or unwanted plankters. Each of the three species was dealt with separately in the following manner, as illustrated by E. borealis. A clean pre-cooled Syracuse watch glass was placed on the stage of a dissecting binocular and filled with 10 ml. of sea water (temperature 15 C., approximately that of Salisbury Cove sea water in the summer of 1960). Then, about 75 specimens of E. borealis from a recently collected urchin were placed in the watch glass near its right margin. The ciliates usually dispersed rapidly, so that many of them soon arrived in relatively pure sea water at the left margin of the watch glass, whereupon 25 of them were transferred by means of a small pipette to 1 ml. of fresh sea water in a Columbia culture dish (square plate-glass depression slide, measuring 42 mm. on a side). The culture dish was placed in a covered Stender dish which was outfitted as a small moist chamber and kept in a tray of running sea water to maintain the temperature at 15 C. The condition of the ciliates was observed and recorded at the end of 6, 9, 24, 48, 72, and 96 hours, reckoning from the beginning. The experiment as just described was repeated some 20 times, using ciliates from more than a dozen different urchins. The method was decidedly superior to the use of hanging-drop preparations, in that the ciliates were first allowed to wash themselves relatively free of intestinal materials and were then transferred to 1 ml. of fresh sea water, which is a relatively large volume for only 25 ciliates. In most of the experiments the final culture dishes were exposed to the natural light of the laboratory, but in some they were kept in darkness (Stender dishes painted black on the outside) except during the brief intervals of observation. Since the histories of the cultures were identical, there was no evidence that moderate illumination was detrimental to the ciliates or that darkness was beneficial. The procedure that has been described for E. borealis was likewise employed with M. indomita and B. gracilis. To facilitate comparisons, the results obtained with 300 individuals of each species, representing 12 culture-dish experiments, will be considered (Table IV). CILIATES OF STRONGYLOCENTROTUS 77 E. borealis. Upon transfer to sea water, the ciliates, in agreement with Powers' findings, showed little or no heightened irritability and suffered no observable distortion in shape. At the end of 6 hours, 297 of the original 300 were present in the cultures, and at end of 9 hours, 296. It is likely that the death and dis- appearance of a few resulted from injuries that accompanied the process of washing and transfer. At the end of 24 hours, 281 were present (survival, 93.7%). Some were swimming normally and others were creeping on the bottom of the dish or against the surface film. However, the many food vacuoles which they originally contained had disappeared, and thus the cytosome was relatively transparent. At the end of 48 hours, 256 (85.3% of the original number) were still present, but they were distinctly smaller, quite transparent, very slow of movement, and evi- dently much weakened from lack of food. During the succeeding 24-hour period TABLE IV Survival of three species of urchin ciliates in sea water. Total number of individuals of each species at beginning of experiment was 300. Hours cited are reckoned from the beginning. Ciliate Kntodiscus borealis Madsenia indomita Biggaria gracilis No. of survivors after 6 hours 297 298 217 9 hours 296 298 103 24 hours 281 295 48 hours 256 272 72 hours 17 36 96 hours the animals suffered drastic mortality, since only 17 (5.7%) remained at the end of 72 hours. These few survivors were much smaller than formerly and were barely able to swim. At the end of 96 hours, there were no survivors. M. indomita. Unlike E. borealis, this ciliate when transferred to sea water displayed greatly heightened irritability, for the animals swam rapidly and errati- cally. However, their intense activity subsided within 5 to 10 minutes, and with no ill effects, to judge by their survival. In general, the results paralleled those obtained with E. borealis, although there were slightly more survivors throughout the first three days. At the end of 24 hours all the food vacuoles had disappeared from the cytoplasm, but the animals were still swimming normally. At the end of 48 hours they were considerably diminished in size and were very transparent, and their locomotion was extremely sluggish. Again, a high mortality occurred during the third 24-hour period, such that only 36 were present after 72 hours. No survivors remained at the end of 96 hours. B. gracilis. The outcome of the experiments with this ciliate was entirely unexpected. Upon transfer to sea water, B. gracilis swam rapidly and quite erratically, as if the medium were distinctly unfavorable. Of the original 300 speci- mens, only 217 (72.3%) were present after 6 hours, and at the end of 9 hours this number was reduced to 103. Many of these were vacuolated and clearly 78 C. DALE BEERS abnormal in structure, and the remains of others were visible in the culture dishes. Since nearly all the survivors contained food vacuoles, the many deaths among the animals could not be attributed to starvation, but must have resulted from the properties of the medium. At the end of 24 hours there were no survivors. DISCUSSION A comprehensive investigation of the relation of the three ciliates to their host in the Mt. Desert Island region would require a study of urchins of practically all sizes taken during all the months of the year. Unfortunately, such a study has not been feasible, and the present one is admittedly incomplete. Nevertheless, the results are of special interest and are fully adequate, it is believed, to support the conclusions that are advanced in the following sections. 1. Acquisition of infections by young urchins and the delayed onset of division The absence of ciliates in 8-mm. urchins (age about 1.5 years) indicates that young urchins do not acquire their infections during their first summer of life, or even during the first year. The presence of relatively small numbers of ciliates in urchins 9-14 mm. in diameter (age likewise about 1.5 years, but no doubt some- what older than the foregoing) indicates that the urchins first acquire their ciliates during their second summer when they are at least 9 mm. in diameter and about 1.5 years old. It might reasonably be assumed that all urchins would become infected not long after metamorphosis and that all would contain fairly dense populations of ciliates by the middle of their second summer. Actually, at least four factors mili- tate against the early acquisition of infections by young urchins at Mt. Desert Island. The first three are of general occurrence ; the fourth is to some extent peculiar to the region of the Island. They are the following. (1) The ciliate losses that accompany the extrusion of fecal pellets are relatively small, to judge by earlier experience (Beers, 1948), as if each ciliate resists dislodgement from its preferred segment of the gut. Thus, urchin ciliates are extremely scarce and very difficult to find in the waters of the urchin's natural habitat, though they can be found with no difficulty in the bottom sediments of an aquarium that is well-stocked with urchins. (2) The period of survival of the ciliates in a healthy condition in sea water outside the body of the host is relatively short, varying from 6 to 48 hours. Although the ciliates tend to adhere loosely to any substratum and to creep upon it. thereby facilitating to some extent their ingestion by a new host, the length of time available for their chance discovery and ingestion by an urchin is distinctly limited. (3) Some of the ciliates are no doubt destroyed by predators. (4) Tidal extremes are great, the mean tidal range being 10.6 ft. (3.23 m.) at Salisbury Cove. Thus, enormous quantities of water ebb and flow twice daily over the urchins and undoubtedly carry away many of the extruded ciliates. In view of the existence of these inimical factors, it is perhaps not so surprising to find that the infection of the young urchins is appreciably delayed. The absence of dividing ciliates in urchins 9-14 mm. in size suggests that the respective ciliate populations do not undergo augmentation by cell division imme- diately after the infection of the host, but are increased during the second summer CILIATES OF STRONGYLOCENTROTUS 79 only by the ingestion of additional specimens. Significant augmentation by division appears to be delayed until the third summer, when the urchins are about 2.5 years old. 2. Establishment of t!ic ciliate populations; division of the ciliates in urchins of diverse ages and in populations of urchins Whereas none of the infected 1. 5-year-old urchins contained dividing ciliates. * o it has been seen that of 12 infected urchins of age 2.5 years, 9 had E. borealis in division, 7 had M. indomita, and all had B. gracilis. These findings indicate, as has been said, that it is during the third summer of the urchin's life that the respec- tive ciliate populations first experience augmentation by division, resulting in the establishment of populations of maximum density. Once the populations of E. borealis and M. indomita are established, infrequent eruptions of divisional activity seem adequate to maintain them in the host ; thus, relatively few (3.1 to S.\ c / f ) of the older urchins (age 3.5 to 6.5 years) harbor them in division. The factors that are responsible for the seemingly long intervals of non-divisional life and the occasional, intense outbreaks of division are unex- plained, as has been said. It has been seen that division in the two cannot be correlated with the age of the host ; it seems to occur randomly in older urchins, irrespective of their age. Neither does their division affect en masse the individuals of an urchin population, even though the urchins seem to be living under similar conditions. Various possibilities present themselves by way of explanation, for example : ( 1 ) There is an inherent rhythm of long frequency in the reproductive activities of the ciliates. (2) Division is correlated, either qualitatively or quanti- tatively, with the food of the urchin and with the nature of the intestinal flora. Although sea-weeds are the preferred food of 6". drobachiensis, it is actually omnivo- rous, and the nature of the intestinal contents is somewhat unpredictable. Thus, urchins of a collection from one and the same tide pool at Long Ledge were found to have fed on a variety of materials. Some contained principally filamentous green algae in their alimentary tracts; others, bladder wrack (Fucns and the like) and sea lettuce (Ulva*) ; still others, calcareous algae; and finally some contained non- descript materials that seemed to consist of barnacle remains and bottom sediments. To what extent these diverse food materials affect the ciliate fauna has not been ascertained. (3) Division is correlated with the physiological state of the urchin, though practically nothing can be said at present concerning this point. Of the foregoing possibilities, the second would seem to be the most readily amenable to experimental analysis, and it is planned that progress in this direction will be attempted within the near future. The probable significance of the constant and uninterrupted division of B. gracilis is mentioned below. 3. Survival of the ciliates in sea zvater in relation to infectivity It has been seen that under the conditions of the experiments both E. borealis and M. indomita can tolerate pure sea water for about 48 hours. Evidently this interval of time, assuming that it also prevails under natural conditions, is adequate to insure the eventual ingestion of a sufficient number of individuals to perpetuate the two species in the host. 80 C. DALE BEERS Much in contrast with the two-day survival of the foregoing species is the seeming incapability of B. gracilis to tolerate sea water longer than 6-12 hours. Although little can be said with certainty in explanation of this pecularity, certain aspects of the autecology of B. gracilis seem worthy of mention. Of all the species of ciliates that occur in S. drobachiensis at Mt. Desert Island, B. gracilis is the only one that is primarily an inhabitant of the rectum. In this disadvantageous site, it is expelled in greater numbers than any of its confreres (Beers, 1948). But it is also the only one of the ciliates, with the exception of Enplotes balteatus, which is probably nothing more than a facultative commensal (Beers, 1954), that is con- stantly dividing within the urchin. Thus, its loss in greater numbers is offset by frequent division, and its continued survival in the host is reasonably assured. In sea water outside the body of the host, B. gracilis experiences a further disadvantage from the standpoint of survival, in that it has relatively little tolerance for sea water. But it is lost in greater numbers from its host, as has just been said. The escape of larger numbers of individuals into the external world would seem to compensate adequately for the briefer period of survival of each ; thus, a relatively constant number of individuals is presumably maintained in the external environment, where they can be ingested by new hosts. Though vulnerable to excessive losses both within the urchin and without, B. gracilis nonetheless maintains itself by the agency of constant division. SUMMARY 1. The first part of the study concerns certain relationships of the ciliates Entodiscus borealis, Madsenia indomita and Biggaria gracilis to their host, the sea urchin Strongyloccntrotus drobachiensis. It is based on an examination of 152 urchins taken at Mt. Desert Island, Maine, in the summer of 1-960. The respec- tive ages of the urchins are estimates based on size (diameter of test). The second part concerns the survival of the ciliates in sea water, since their survival is insepa- rably related to the infection of new hosts. 2. Nine urchins measuring 8-14 mm. in diameter (age 1.5 years) were either uninfected or very lightly infected, and none of the ciliates was dividing. Urchins evidently acquire their ciliates at this age (second summer). 3. All the urchins of the remaining age-groups were infected with all 3 ciliates. Of 12 urchins that measured 15-23 mm. in diameter, all contained dividing speci- mens of B. gracilis, 9 contained dividing individuals of E. borealis, and 7 contained M. indomita in division. The results indicate that the respective ciliate popula- tons build up rapidly to maximal densities in the third summer of the urchin's life (age about 2.5 years). 4. The remaining urchins were assigned by size to 4 age-groups. The number of urchins in each group, their range in size, and their estimated ages follow : 32 urchins, 24-39 mm., 3.5 years; 35, 40-49 mm., 4.5; 37, 50-59 mm., 5.5; 27, 60-65 mm., 6.5. All the urchins harbored dividing specimens of B. gracilis; thus this ciliate remains in constant division once infection is well established. But in each group only a small percentage of the urchins (3 to 8%) contained dividing specimens of E. borealis and M. indomita. Thus, their division, though evidently cyclical, could not be correlated with the age of the urchins. 5. In a natural population of urchins, the division of E. borealis and M. indomita does not affect simultaneously any large percentage of the urchins. Except in 2.5- CILIATES OF STRONGYLOCENTROTUS 81 year-old urchins, it appears to occur randomly. Since the urchins of a population practice dissimilar food habits, it is possible that division is correlated with the nature of the food and the subsequent intestinal flora. 6. In pure sea water most specimens of E. borealis and M. indoinita can survive about 48 hours, and their death is due to starvation. Individuals of B. gracilis can survive no longer than 6-12 hours, and death does not result from starvation but seemingly from the properties of the medium. It is suggested that the constant voiding of B. gracilis among the fecal pellets of the host compensates for its rela- tively brief period of survival in sea water. LITERATURE CITED BEERS, C. D., 1948. The ciliates of Strongylocentrotus drb'bachicnsis: Incidence, distribution in the host, and division. Biol. Bull., 94: 99-112. BEERS, C. D., 1954. Plagiopyla minuta and Euplotes baltcatits, ciliates of the sea urchin Strongylocentrotus drobachiensis. J. Protosool., 1 : 86-92. GRIEG, J. A., 1928. The Folden Fjord. Echinodermata. Tromso Mus. Skrijtcr, 1 (Part 7) : 1-12. KAHL, A., 1932. Urtiere oder Protozoa. I: Wimpertiere oder Ciliata (Infusoria). In Dahl : Die Ticrwclt Deutschlands. Lief. 25, 399-650. Jena: Verlag von Gustav Fischer. KAHL, A., 1934. Ciliata entocommensalia et parasitica. In Grimpe u. Wagler : Die Ticnvelt dcr Nord- und Ostsce. Lief. 26, Teil II. C4, 147-183. Leipzig: Akademische Ver- lagsgesellschaft. KIRBY, H., 1941. Relationships between certain protozoa and other animals. In Calkins and Summers: Protozoa in Biological Research, 890-1008. New York: Columbia Uni- versity Press. MADSEN, H., 1931. Bemerkungen iiber einige entozoische und freilebende marine Infusorien der Gattungen Uronema, Cyclidium, Cristigera, Aspidisca und Entodiscus gen. nov. Zoo/. Anz., 96: 99-112. MORTENSEN, T., 1943. A monograph of the Echinoidea. III. 3. Camarodonta. II. Echinidae, Strongylocentrotidae, Parasaleniidae, Echinometridae. Copenhagen : C. A. Reitzel. POWERS, P. B. A., 1933a. Studies on the ciliates from sea urchins. I. General taxonomy. Biol. Bull., 65: 106-121. POWERS, P. B. A., 1933b. Studies on the ciliates from sea urchins. II. Entodiscus borealis (Hentschel) (Protozoa, Ciliata), behavior and morphology. Biol. Bull., 65: 122-136. POWERS, P. B. A., 1935. Studies on the ciliates of sea-urchins. A general survey of the infesta- tions occurring in Tortugas echinoids. Carnegie lust, of Wash., Publ. 452: 293-326. OBSERVATIONS ON THE RESPIRATION OF THE SABELLID POLYCHAETE SCHIZOBRANCHIA INSIGNIS R. PHILLIPS DALES Bedford College, University of London, London N.1V. 1 Schisobranchia insignis Bush lives in tough fibrous tubes, mostly 10-20 cm. long and 510 mm. in diameter, attached to the underside of floating wharves, to pilings and to rocks on the Pacific northwest coast of America (Fig. 1. A). It may also be dredged from muddy bottoms. The dense crown of orange, purple or grey branched filaments, which is used both for feeding and for respiration, may be expanded beyond the opening of the tube for long periods when the worm is undisturbed. For shorter periods the worm lies wholly within the tube. Worms also irrigate their tubes by waves of muscular contraction of the body wall which may pass in either direction. Irriga- tion occurs when the crown is expanded as well as when the worm is retracted within the tube. That the crown of all sabellids is used for feeding may readily be confirmed by simple observation, but its importance in respiration appears to vary from one species to another. Zoond (1931) found a 63% fall in oxygen uptake after ampu- tation of the crown in Bispira rolutacornis (Montagu) and Fox (1938) found the same decrease when Sabclla spallansanii (Viviani) was similarly treated. On the other hand, Wells (1952) found that bisected Sabclla f>ai'oiiina Savigny showed no significant fall in total rate of oxygen uptake of the two parts, but that My.ricola infundibulum Renier did, there being a sharp drop in total uptake when bisected, and the posterior part giving relatively lower values than those of Sabclla paroniiia. He concluded that in Sabclla, while the current caused by the crown provides for the crown's own respiratory needs, it is the irrigation current which is of importance to the rest of the body. Myxicola, on the other hand, does not irrigate its tube and is wholly dependent on the crown which functions not only in feeding but as a gill. These differences suggested that it might be of interest to investigate the activities of another sabellid under conditions as natural as possible. The importance of the crown in respiration has only hitherto been assessed by the drastic procedure of amputation, and the rate of oxygen uptake has never been measured with the rate of water transport through the crown. Consequently, I have made measurements of oxygen uptake by the worm when expanded and when wholly withdrawn within the tube. The volumes of water passed (1) through the tube and (2) through the crown have also been measured, and the percentage utilization of oxygen by the crown and by the remainder of the body estimated under normal circumstances. All measurements have been at 12-13 C. All the observations were made on animals from wharves in the vicinity of Friday Harbor. Washington. I am glad of this opportunity to thank Dr. Robert L. Fernald and the Staff of the Friday Harbor Laboratories of the University of 82 RESPIRATION OF SCHIZOBRANCHIA 83 Washington for their hospitality and help. I also wish to thank Professor H. Munro Fox, F.R.S., for helpful criticism of this paper. \Yorms were stripped of their own tubes and accommodated in pieces of trans- parent plastic or transparent rubber tubing of suitable length and diameter. Such tubes reveal the activities of the worm, readily enable the tube to be linked to recording apparatus, and permit measurement of oxygen uptake under nearly M chamber transparen plastic tubin D FIGURE 1. A, appearance of colony of Schizobranchia in nature; B, apparatus used to record irrigation; C, irrigation prevented; D, expansion prevented. normal conditions. The work of Hyman (1932) and Fox (1938) emphasises the importance of simulating natural conditions as far as possible. Worms used in the experiments had been acclimatised to plastic or rubber tubes for at least one or two weeks. EXTENSION AND WITHDRAWAL To obtain some idea of the amount of time spent by the worm with the crown expanded, and the amount of time passed wholly withdrawn within the tube, worms 84 R. PHILLIPS DALES accommodated in plastic tubes were attached to a recording apparatus (Fig. 1, B) similar to that used by Wells (1951) for Sabella. The apparatus was immersed in a tank through which a circulation of sea water was maintained and in which the water level (1) remained constant. By adjusting the size of the capillary leak (a) the movement of the worm could be recorded on a slowly revolving kymograph by the lever actuated by changes in the level of the water in the float chamber (f). By selecting a larger capillary which allowed a more rapid flow 7 than could be maintained by the worm irrigating under normal conditions, it was possible to adjust the capillary so that the float would be affected only by relatively rapid movements of the whole body, as in extension or withdrawal. Two typical traces, each of 12 hours duration, made by different worms are shown in Figure 2. It will FIGURE 2. Continuous record of expansion and withdrawal by two different worms. Duration of each trace, 12 hours. Read from left to right. Upward spikes represent withdrawal within the tube, downward spikes represent extension from the tube with expansion of the crown. Extension is more gradual than withdrawal, as is shown by the stepped trace. The horizontal parts of the trace represent the times when the worm remained with the crown expanded beyond the opening of the tube. be seen that extension and withdrawal occur at intervals of some regularity. The long period when the crown is expanded following extension after midnight was common to many worms and records. It may have been due merely to absence of stimulation by workers in the laboratory, although many other sabellids have been seen to expand their crowns more at night (Mclntosh, 1922, Fox, 1938). It will be noticed that each period of expansion exceeded each period spent wholly with- drawn. The time spent retracted on any one occasion did not exceed 10-15 min- uts, while periods of expansion were 20-60 minutes or longer. The interpretation of the records was confirmed by frequent observation. IRRIGATION Each tube, though firmly attached by mucus at or near the base to wharves or pilings, has one or more small openings 1 mm. or so in diameter near the hind end (Fig. 1, A). Mucus can be secreted through these to regain attachment, or new apertures made as occasion demands, and the orientation of the tube somew ? hat changed, as Fox (1938) observed in Sabella spallanzanii. Apart from these possi- bilities, which enable a small amount of re-orientation within the clump of animals so that each has room to expand the crown, Schizobranchia is completely sessile and RESPIRATION OF SCHIZOBRANCHIA 85 is unable to turn around in the tube except on its own longitudinal axis. Unlike Sabclla, however, it rarely does so. Irrigation of the tube is effected by muscular swellings passing down the body, most commonly from head to tail. Occasionally the direction of irrigation is reversed. These activities may occur when the crown is expanded or when the worm is withdrawn into the tube. The volume of the tube containing a worm of average size (2 grams fresh weight) is about 1.5 ml., the volume of such a tube empty being about 3.5 ml. Some idea of the irrigation rate may be obtained by injecting a suspension of carbon into the tube by means of a hypodermic syringe, and observing the rate of travel of the particles along a horizontally fixed graduated tube sealed on to the hind end. Under otherwise normal conditions the fluid in the tube may be completely renewed in 30-60 seconds of activity. A B C FIGURE 3. A-C, continuous record of irrigatory activity of a single worm over a period of 36 hours. Read from left to right. Each line represents 12 hours. Further explanation in text. A continuous record of irrigation may be obtained with the apparatus already described by attaching a finer capillary at (a), such that the flow into or from the tank causes a slight rise or fall of 1-3 mm. to occur in the float chamber. A pressure difference of this magnitude may easily be recorded, but is unlikely to be great enough to modify the behaviour of the worm. A record of such activity is shown in Figure 3, C. The details of such traces were interpreted by watching worms from time to time while the trace was being made. In the records elevation represents irrigation headwards ; depression, tailwards. Irrigation will be seen to be somewhat irregular in rate, but to be fairly continuous. The volume passed can be calculated, knowing the dimensions of the capillary and the rest of the apparatus ("Wells and Dales, 1951), or may be determined empiri- cally. The average rate was found to be 0.3-0.5 ml./min. for a 2-gram (fresh weight) worm. Wells (1952) found a similar rate for Sabella pavonina of compa- rable weight. By inserting a small bung into the opening of a worm's tube attached to the recording apparatus, extension of the worm and irrigation could be stopped. If the period of closure did not exceed 10-15 minutes (Fig. 3, A : 1 ; Fig. 3, B : 3) normal activity was resumed after release. If this period was exceeded (20-45- 86 R. PHILLIPS DALES minute closure) as in Figure 3, A: 2; Figure 3, B: 1, 2, release was followed by very vigorous irrigation, as much as 0.75 ml. min. being passed for an hour or more by a 2-gram worm. By connecting another piece of tubing to both the open end of a plastic tube in which a worm had been accommodated and attached to a recording apparatus (Fig. 1, B), and to the jet from the float chamber instead of the capillary leak, the circulation could be closed without preventing the worm from irrigating. Under 0-20 c u X O 0.10 008 006 004 0.0?. 2.0-4.0 ce.Ot/l. O 6.0-8.0 A 60-8.0 fellow,n S 41. /, at less tKan 1.0cc/L. A 8.0-140 (b= I2-I5C) 10 5-0 6.0 2.0 >0 40 FresVi Weio^t FIGURE 4. Rate of oxygen uptake under different external oxygen concentrations. RESPIRATION OF SCHIZOBRANCHIA 87 normal conditions worms underwent short bursts of "testing" activity, driving first in one direction and then in the other, and on release continued vigorous irrigation for some hours. Wells (1951) found that Sabclla spallanzanii responded similarly. In Figure 3, B : 3, a hurst of testing irrigation in a headward direction was in prog- ress on release, and this direction was maintained for the following two hours, the worm gradually returning to its normal behaviour pattern. OXYGEN UPTAKE The rate of oxygen uptake was measured by the modification of Fox and Wingfield (1938) of the well-known Winkler technique. The rates of uptake made under different oxygen concentrations plotted against total fresh weight are pre- sented in Figure 4. Uptake was measured in closed bottles after a period long enough to ensure accurate estimation, but normally not so long that the quantity of oxygen in the bottle was reduced to not less than -15 cc./l. or the quantity of carbon dioxide increased to a level at which the rate might be affected. Bottles of approximately 270 ml. capacity were used and most measurements were made after a 1-4-hour period, according to the size of the animal. Worms thoroughly acclimatised to plastic tubing were used, as measurements so made might be expected to be closest to those under entirely natural conditions; Fox (1938) found that the oxygen uptake in Sabclla spallanzanii was 20-30% lower in worms freshly deprived of their tubes. Each animal was therefore enclosed in a bottle large enough to ensure normal activity ; worms were able to expand the crown, to withdraw and to irrigate. The bottles were occasionally inverted to ensure thorough mixing ; most of the worms were acclimatised to being disturbed and their behaviour appeared to be affected only momentarily. All the determinations were made at 1213 C. (a) Response to raised or lowered o.vygen content of the water The normal rate of oxygen uptake could be maintained by the worm when the oxygen content of the water was lowered to 2 cc./l., either gradually by the animal itself over an extended period in a closed bottle, or by bubbling nitrogen through the water before the experiment. When the oxygen content \vas slowly reduced by the animal itself, the rate of uptake was significantly reduced between 1.3 2.5 cc. O 2 /l. If enclosed in a small chamber so that the rate of pulsation of the branchial vessels could be observed, the normal rate of 9-10 pulsations/min. (13C.) fell off rapidly below about 2.5 cc. O 2 /l., and ceased altogether around 1.3 cc. O 2 /l. Fox (1938) found the same effect in Sabella spallanzanii. The rate of oxygen uptake was not affected by raising the oxygen content of the water, similar values being obtained up to 14.0 cc. O 2 /l. It is interesting, how- ever, in confirmation of the findings of Fox and Taylor (1955), that worms were not adversely affected by these high concentrations, and survived indefinitely in the laboratory circulation which had a high oxygen content (7-8 cc. O 2 /l.) owing partly to the action of the pumps. These sabellids are indeed usually found in habitats with a good circulation of water where the concentration of oxygen is likely to be high. Fox (1932) and Ewer and Fox (1940) have shown that the chlorocruorin of Sabclla spallanzanii blood is adapted for oxygen transport only at 88 R. PHILLIPS DALES high outside concentrations, and C. Manwell (private communication) has found the same in Schisobranchia insignis. (b) Uptake of oxygen zvhen extended When the crown was removed, and after the animal had recovered from the operation for a day or two, the oxygen uptake by the rest of the body was measured ; the value obtained was about 25% that of the normal animal. With normal animals irrigation, and hence normal respiratory exchange across the body wall, could be stopped by plugging the hind end of the tube (Fig. 1, C). Observation suggested that such worms remained extended more continuously, and measurement of oxygen uptake in closed bottles showed that the values obtained were not significantly different from those of normally irrigating worms. In other TABLE I Rate of oxygen uptake under normal conditions, when irrigation is prevented (as in Fig. 1, C), and when confined within the tube (as in Fig. 1, D) Worm number Total fresh weight O2 uptake under normal conditions (cc./g./hr.) O2 uptake without irrigation (Fig. 1. C) O> uptake under forced withdrawal (Fig. 1, D) 16 0.460 0.1920 0.1790 0.0782 17 0.520 0.2330 0.2266 0.0819 18 0.600 0.2210 0.2165 0.0759 19 1.205 0.1132 0.1568 0.0463 20 0.565 0.2212 0.2298 0.0696 21 0.765 0.1684 0.1851 0.0855 22 2.100 0.0916 0.1114 0.0316 words, normal oxygen uptake can be maintained by the crown alone (i.e., without uptake through the body wall), though it may have to remain expanded to do so. Wells (1952) found that crownless Sabclla paronina perished if unable to irrigate. (c) Uptake of oxygen when withdrawn When withdrawn within the tube the crown is not well displayed for respiratory exchange or feeding, as the numerous branched filaments are tightly rolled together. By fitting a narrower tube to the opening (Fig. 1, D) extension could be prevented but irrigation continued. Under these conditions oxygen uptake was reduced, usually to about 40% of the normal value. On release, worms remained extended for some time. Values obtained for seven worms in which measurements of oxygen uptake were made under normal conditions, with irrigation prevented, and when wholly withdrawn, are compared in Table I. RATE OF FILTERING BY THE CROWN When the crown is extended water flows through the filaments as a result of ciliary activity. A measure of the volume of water strained through the crown may be obtained by the use of colloidal graphite suspensions, since the particles coming into surface contact are removed from suspension by mucus. Removal of particles is exponential if the system remains constant in volume and the particles RESPIRATION OF SCHIZOBRANCHIA 89 remaining in suspension are evenly distributed (Dales, 1957). Particles may be ingested or rejected, but in either case are removed from suspension. The rate at which unit volume is cleared of particles may be calculated by measuring the de- crease in density at known intervals against controls (J^rgensen, 1949). The filtering rate in ten experiments was calculated over a three-hour period using worms of 0.5-2.0 grams fresh weight at 12-13 C. The mean rate of filtering was 70.7 ml./g./hr. This is of the same order of magnitude as has been found in other sabellids (Dales, 1957). DISCUSSION All these observations suggest that the life of Scliisobranchia insignis is very similar to that of Sabclla spallansanii. Both species may be found in tubes open at the hind end attached to rocks and wharves. Both irrigate their tubes with equal facility in either direction, and pause in this activity for periods rarely exceeding 10 minutes. It is difficult to assess the part played by the crown in supplying the respiratory needs of the rest of the body since, as Wells (1951, 1952) has pointed out, the needs of the crown itself are high owing to its activity. The vascular supply to the crown may be of service both in conveying oxygen away and in conveying nutrients to the ciliated epithelium and other tissues of the crown. The ability to continue to live, and to regenerate the crown when this is amputated, provided that the worm is able to irrigate its tube, suggests that the crown is not essential, although under normal circumstances it may well supply part of the body's needs. Schizobranclua cannot autotomize the crown as Sabella does, so that decapita- tion results in a more serious loss in the total blood volume and perhaps a more unusual derangement of metabolism than in Sabclla. While some individuals did regenerate their crowns, many died under laboratory conditions ; Sabella seems better adapted for this contingency. The crown is also relatively larger in Schiso- branchia, and the reduction in oxygen uptake to 25% of the normal value may well be partly due to the loss of that part of the uptake accounted for by the crown itself. When the worm is retained in the tube (or when, in nature, the worm is wholly withdrawn) the irrigation current alone supplies oxygen to the animal and removes carbon dioxide and other waste products. Under these conditions the oxygen uptake is 40% of that when extended. While the crown is not then expanded and the animal's need for oxygen may be somewhat less, the cilia on the crown do not cease to move, and the muscular contractions causing irrigation of course continue. It could be argued that if crownless worms can continue to live, providing that they are able to irrigate, and that the oxygen uptake of crownless worms is 25% of what it was before decapitation, then the requirement of oxygen for maintaining irrigation may be met by 25% of the normal total oxygen uptake. Other activities may well be interrupted after decapitation, but the major part of the remaining 75% of the normal oxygen uptake may thus be accounted for by the activity of the crown itself. That worms wholly withdrawn (Fig. 1, D) have an oxygen uptake of 40% of the value for expanded though not continuously irrigating worms (Fig. 1, C) suggests that perhaps a value approaching 60% of the total oxygen uptake is due to the activity of the crown alone. While the circulation of the blood from the crown can supply the respiratory needs of the rest of the body if irrigation is not possible, under normal circumstances it need not do so. Uptake 90 R. PHILLIPS DALES of oxygen will ensure, through the intermediacy of the vascular system, a supply of oxygen to all parts at all times, for activity of the crown and irrigation are independent activities. Pulsation of the branchial vessels was observed by Fox (1938) and Wells (1951) in Sabclla to cease after some time when totally enclosed in a small chamber or in a tube, and cessation may be seen also in ScJiizobrancliia. As already noted, pulsations occur under normal circumstances at a rate of about 10/min. at 13 C., but these cease altogether when the oxygen content of the water has fallen to about 1.3 cc. C>2/1. Fox (1938) suggested that this effect may be due to accumulation of carbon dioxide, but as Wells (1951) points out, this is unlikely to occur under normal conditions owing to irrigation. On the other hand, if the worm is wholly withdrawn and, while ceasing to irrigate, its uptake of oxygen remains at 40% of its normal value (0.1 cc. O 2 /hr. for a 2-gram worm), the oxygen contained in the 1.5 ml. of water within the tube would be used up in 15 minutes. The possibility that the factor which ends a short rest from irrigation might be lack of oxygen or accumulation of carbon dioxide should not, therefore, be dismissed. In Scliizo- branchia, however, pauses were never observed to be as long as this, and in any case when the irrigatory waves cease the oxygen requirement should be less. In addition, there should be sufficient oxygen in the blood to provide for such brief pauses as occur (Ewer and Fox, 1940) and it seems more likely from the work of Wells (1951, 1955) that the resumption of irrigation is spontaneous. The measurement of filtration rate showed that 70 ml. of water/g./hr. was moved across the filaments by the activity of the crown cilia, while the normal irri- gation rate through the tube was about 12 ml. g./hr. While it would be unwise to draw too close a comparison, these figures suggest that the crown is in fact achieving more effectual work in water transport than the body when irrigating, so that it is not surprising to find that the total oxygen uptake is reduced to 40% when the crown is not expanded. The utilization of oxygen from the water passed through the crown may be obtained from the filtration rate (70 ml./g./hr.) and the rate of oxygen uptake (0.05 cc. O 2 /g./hr.). Under laboratory conditions (external oxygen content of 7.0 cc. Oo/l. ) this can be estimated at about 10%. When the worm is wholly withdrawn the oxygen consumption falls, as we have just noted, to 40% of the normal value or 0.02 cc. Oo/g./hr., which is withdrawn from only 12 ml., giving a utilisation of about 24%. The rather low utilisation by the crown rather suggests that the flow is maintained more for feeding than for respiration. Wells (1951) suggested that in Sabclla pavonlna feeding was at least a possi- bility when the crown is withdrawn. While this may be so in S. f>ai'oniiia, which has a singularly delicate and "open" crown, it seems far less likely to occur in Schizo- brancJiia in which the crown is more complicated, much branched, and closely furled when contracted. The results discussed here suggest that ScJrizobrancIiia is able to maintain its respiratory needs when withdrawn within the tube, and that it emerges to feed in response to some spontaneous mechanism such as Wells (1955) has described in other polychaetes. This ability to meet the demand for oxygen by irrigation when withdrawn within the tube is a factor with obvious survival value. It is interesting that sabellicls such as My.ricola (Wells, 1952) and Chonc, which do not irrigate their tubes, have exceptionally well developed giant fibre systems and retraction responses. This should increase their chances of survival, for not only are these RESPIRATION OF SCHIZOBRANCHIA 91 worms dependent on their crowns for respiratory exchange but the crowns must, therefore, be more constantly displayed. SUMMARY 1. Observations on the life of a sabellid Scliizobraiichia insignis have been made under conditions resembling as far as possible those found in nature. 2. The amount of time spent with the cro\vn expanded and the amount passed wholly withdrawn within the tube have been measured, and the utilisation of oxygen under these two conditions estimated. 3. The volume of water passed through the crown for respiratory and feeding purposes, as well as the volume pumped through the tube, have also been measured, and the part played by each in respiratory exchange discussed. It was found that about 70 ml./hr./g. animal (fresh weight) is passed through the crown by the action of the filamentary cilia, and the volume pumped through the tube is about 12 ml./hr./g. 4. Utilisation of oxygen by the crown is relatively low (10%) ; utilisation by the whole worm when withdrawn is about 24%, and the large volume strained by the crown is probably related to the food requirements rather than to the respiratory needs of the worm. 5. It is suggested that the oxygen taken up by the crown is largely utilised in its own activity although it can, and does, provide for the needs of the rest of the body during pauses in irrigation when expanded. LITERATURE CITED DALES, R. P., 1957. Some quantitative aspects of feeding in sabellid and serpulid fan worms. /. Alar. Biol. Assoc., 36 : 309-316. EWER, R. F., AND H. M. Fox, 1940. On the function of chlorocruorin. Proc. Roy. Soc. London, Scr. B, 129: 137-153. Fox, H. M., 1932. The oxygen affinity of chlorocruorin. Proc. Roy. Soc. London, Scr. B, 111: 356-363. Fox, H. M., 1938. On the blood circulation and metabolism of sabellids. Proc. Rov. Soc. London, Scr. B, 125: 554-569. Fox, H. M., AND A. E. R. TAYLOR, 1955. The tolerance of oxygen by aquatic invertebrates. Proc. Roy. Soc. London, Scr. B, 143 : 214-225. Fox, H. M., AND C. A. WINGFIELD, 1938. A portable apparatus for the determination of oxygen dissolved in a small volume of water. /. E.vp. Biol., 15 : 437445. HYMAN, L. H., 1932. Relation of oxygen tension to oxygen consumption in Nereis virens. J. E.\-p. Zoo/., 61 : 209-221. J0RGENSEN, C. B., 1949. The rate of feeding by Mvtilns in different kinds of suspension. /. Mar. Biol. Assoc., 28: 333-344. MclNTOSH, W. C., 1922. A Monograph of the British Marine Annelids, 4, London (Ray Soc.). WELLS, G. P., 1950. Spontaneous activity cycles in polychaete worms. /. E.rp. Biol. Sym- posium IT. Physiological mechanisms in animal behaviour : 127-142. WELLS, G. P., 1951. On the behaviour of Sabclla. Proc. Roy. Soc. London, Ser. B, 138: 278-299. WELLS, G. P., 1952. The respiratory significance of the crown in the polychaete worms Sabclla and My.ricola. Proc. Roy. Soc. London, Scr. B, 140: 70-82. WELLS, G. P., AND R. P. DALES, 1951. Spontaneous activity patterns in animal behaviour: the irrigation of the burrow in the polychaetes Chactoptcrus variopedatus Renier and Nereis d'rccrsicolor O. F. Muller. /. Mar. Biol. Assoc., 29 : 661-680. ZOOND, A., 1931. Studies in the localization of respiratory exchange in invertebrates. II. The branchial filaments of the sabellid, Bispira z'oluticornis. J. E.rp. Biol., 8: 258-266. PRELIMINARY INVESTIGATION ON THE PHYSIOLOGY AND ECOLOGY OF LUMINESCENCE IN THE COPEPOD, METRIDIA LUCENS 1 CHARLES N. DAVID AND ROBERT J. CONOVER Harvard College, Cambridge, Mass., and Woods Hole Oceanographic Institution, Woods Hole, Mass. Since the advent of the photomultiplier tube with greatly increased sensitivity to low light intensity, it has been possible to measure marine luminescence quanti- tatively. Luminescent flashes have been found to be far more prevalent at all depths in the sea than had generally been suspected (Clarke and Backus, 1956; Clarke and Breslau, 1959, 1960; Clarke and Hubbard, 1959; Clarke and Wertheim, 1956; Boden and Kampa, 1957, 1958; Kampa and Boden, 1957). Attempts to identify the source of this flashing, using tl^e luminescence camera built by Breslau and Edgerton (1958), suggest that most of the luminescence is produced by planktonic organisms less than a centimeter long (Clarke and Breslau, 1959; Clarke, personal communication). Certain planktonic species whose luminescence has been in- vestigated do not show spontaneous luminescence in the laboratory. Probably some of the luminescence which has been'-measured at sea may be artificially stimulated by the unavoidable motion of the photometer suspended from a research vessel. However, Kampa and Boden (1957) have concluded that some luminescence ap- pears to be "natural" or "spontaneous." Bioluminescence in a small planktonic animal has been examined particularly with a view toward evaluating its potential as a source of luminescence in the natural environment and determining the significance of the luminescence for the organism. The calanoid copepod, Metridia lucens, was the animal chosen. This copepod was recognized a luminescent by Boeck (1865) who described the species. Several additional workers have made microscopic or field observa- tions on the luminescent Copepoda (Dahl, 1893, 1894; Kiernik, 1908; Vanhoffen, 1895; Giesbrecht, 1895), but very little experimental work has been done. In the present work preliminary investigation of certain physical properties of the luminescent emission and of the physiology of the luminescent mechanism has been attempted, in addition to the experiments designed to ascertain what ecological significance luminescence may have for this copepod. The authors are indebted to Dr. George L. Clarke for his advice and criticism in planning the work and in the preparation of the manuscript. The authors also wish to express their thanks to Dr. W. D. McElroy, Dr. James F. Case, Dr. Edward R. Baylor and members of the staff of the Woods Hole Oceanographic Institution for their cooperation and assistance. 1 Contribution No. 1183 from the Woods Hole Oceanographic Institution. Research sup- ported by National Science Foundation Grants 3838 and 8913. 92 LUMINESCENCE OF A MARINE COPEPOD 93 MATERIALS AND METHODS The copepods used in the experiment were obtained in Cape Cod Bay about 3-5 miles northeast of the mouth of the Cape Cod Canal in 20-30 m. of water. Col- lections were made on two occasions, July 7 and August 16, 1960. with a %-meter #00 plankton net towed near the bottom. The Metridia were isolated from the catch and maintained in the laboratory approximately 40 animals to 1000 ml. of food culture. Laboratory cultures of the diatom Tlialassiosira fluriatilis were used as food diluted 1 : 20 by volume with millipore-filtered sea water from Cape Cod Bay. This gave a concentration of 6000 to 10,000 cells/ml, in the final food culture. CARBON ELECTRODE OBSERVATION CHAMBER SALT AGAR PLUG FIGURE 1. Electrode chamber used for stimulation of Metridia. For details see text. Either streptomycin or penicillin (50 mg./l.) was added to inhibit bacterial growth. For specific experiments, smaller groups of Metridia were kept in proportionately smaller volumes of food medium. All groups of animals were kept in a darkened refrigerator at 5-7 C. Measurements of luminescence were made in a "black box" consisting of a tar- paper covered wooden frame built on top of a table. A large opening on one side of the box covered with a black cloth sleeve and drawstring permitted the investi- gator's head to remain inside the box for observations or to monitor recording and stimulating instruments outside the box. The measurements of luminescence were made with the portable bathyphotom- eter designed and built by Breslau (1959), which employs a RCA 5819 photo- multiplier tube with 1200 v. battery power and a transistor amplifier circuit. Ex- perimental material was placed directly in front of the photomultiplier about 18 cm. from the sensitive surface. A Texas Instruments, Inc. single-channel, strip- chart recorder ("Recti/Riter") was used to record intensity (in yu.w./cm. 2 ) against time during each flash. 94 CHARLES N. DAVID AND ROBERT J. CONOVER Since the Mctridia do not generally luminesce spontaneously in the laboratory, mechanical or electrical stimulation must be applied to study the characteristics of the flashing. In order to standardize the stimulus delivered to the animals, a simple electrode chamber was constructed as shown in Figure 1. The device was cut out of a piece of Incite and the connecting holes between the two side chambers and the central chamber were filled with 3 c /c agar made with millipore-filtered sea water. For experiments, carbon electrodes wired to a pulse regulator were placed in the side chambers and the whole device was filled with cooled sea water to com- plete the circuit. Metridia, either individually or in groups, were then placed in the central chamber for stimulation. All stimulation was performed with alternating current controlled through an electronic switch and a continuously adjustable autotransformer, Variac Type W10MT. The switch regulated the duration of pulses to one-tenth of a second and the interval between pulses to two-tenths of a second. Although slightly sensi- tive to changes in salinity and temperature, the current was regulated accurately to one-tenth of an ampere. The chart speed of the recorder was varied for different experiments. The slower speed (6 in. hr. ) was used to record the frequency and intensity of flashes. The faster speeds (6 or 12 in./min.) were used when a measure of total luminescent flux (area under intensity curve) or the duration of a flash was required. DESCRIPTION AND DISTRIBUTION OF METRIDIA LUCENS Mctridia litccns is a medium-sized copepod, virtually colorless in the living state. Its size varies between 2.4-3.0 mm. for females and 1.8-2.5 mm. for males. Although Metridia luccns is a common copepod of temperate and boreal water, very little is known regarding its seasonal abundance or life-history. In the Gulf of Maine, Bigelow (1924) noted an increased abundance in the spring and again in September and October. Bigelow (1924) and Clarke (1933, 1934) observed extensive diurnal vertical migrations in this species. In the waters off the coast of Ireland it also seemed to have a period of maximum abundance in May and a smaller period of increase in the fall (Farran, 1920). During the spring it has been reported to be responsible for brilliant phosphorescence on the Irish coast (Farran, 1903, in Bigelow, 1924). Little is known regarding the internal anatomy of the copepods. Among the calanoicls only Calaints finiiiarchicns has been studied in detail (by Lowe, 1935). It is presumed that in the general features of its morphology Mctridia does not differ greatly from Calami s although there doubtless are certain differences in structural detail. The only light-sensitive organ in most copepods, including Metridia, is a single naupliar eye. It seems very doutbful that this organ can have any role in behavior requiring recognition of other organisms because it cannot form images. However, it can presumably detect intensity gradients (as in vertical migration) and possibly the plane of polarization of incident light. Luminescent glands The earliest workers recognized that the luminescence produced by Mctridia was primarily external. Boeck (1865), who described Mctridia luccns, noted that LUMINESCENCE OF A MARINE COPEPOD 95 the light seemed to be produced in the head region and also from the abdomen. Vanhoffen (1895), working with the larger Jl/. longa, observed luminescence dis- tributed over most of the thorax as well as the head. In addition to the external secretion, he also felt that some light was produced internally which indicated the position of the secretory glands. The authors observed that Mctridia liicens seemed to produce luminescence, when stimulated electrically, chiefly from the anterior part of the head and from the region of the caudal rami. The separation of these two regions was sufficiently distinct that the light produced persisted sometimes as two discrete points for some seconds. \ FIGURE 2. Mctridia litccns: left, a dorsal view; right, a lateral view. Arrows indicate the general regions of the body where luminescent glands were found. Due to the kindness of Dr. Robert Hessler, some histological preparations of Mctridia luccns, fixed in Zenker's and stained with hematoxylin and eosin were available for study of the glands and their distribution. Figure 2 shows the regions of the body seen microscopically to produce luminescence in observations on living animals. Glands were located in the histological preparations in most of these places with definite concentrations on the anterior surface of the head and on the posterior portion of the abdomen. The glands varied in shape somewhat depending on their location in the body. Those in the urosome had a long connecting duct between the glands and the external pore while those in the thorax opened directly to the outside through a 96 CHARLES N. DAVID AND ROBERT J. CONOVER short duct. In several cases masses of dark material which might be the lumi- nescent substance were observed in these ducts. Sewell (1932, 1947) describes the presence of external pores on the cuticle, presumably associated with glandular structures, in several groups of copepods including Metridia. It is not certain, however, that these are the openings to CM 2 o ICT 2 icr 3 10 -4 10 -5 10 10 -6 -7 10 10 -3 -4 10 -5 10 -6 10 -7 A B SEC 10 t 20 30 t 40 50 60 10 20 30 FIGURE 3. Luminescence of single Metridia when stimulated in the electrode chamber. Arrows along the time scale indicate instant of stimulation (0.7 amp.). Chart speed is 6 in./min. A shows curve for an animal tested six hours after capture ; B shows curve for an animal tested after being kept in the laboratory for one month. luminescent glands, particularly since such structures are found in several genera not presently known to be luminescent, notably Eucalanns and Tcmora. The dis- tribution of these pores has not been worked out in detail for Metridia lucens. Physical characteristics of the luminescence The luminescent emission of Metridia lucens is generally a bright flash of vary- ing duration. According to Harvey (1952), luminescence in copepods results from the simultaneous discharge of substrate and enzyme into the surrounding LUMINESCENCE OF A MARINE COPEPOD Q7 medium ; presumably the immediate peak emission occurs at the instant of initial contact between the reacting substances in the presence of oxygen. Generally a gradual decay follows as enzyme and substrate diffuse away into the medium, or perhaps as the substrate is used up. The absolute intensity of the highest peak of the luminescent emission is in doubt because of the relatively slow response time of the equipment used. Further- more, the maximum emission intensity varied to some extent for individual animals. However, the maximum intensity measured for Metridia was 1.2 X 10~ 3 /AW./cm. 2 550 600 425 482" 580 FIGURE 4. Spectrum of luminescent emission at C. from crushed animals. at the working distance of 18 cm. (Fig. 3). With groups of 5 Metridia, there was an additive effect giving maximum intensities of up to 4.5 X 10" 3 . The intensity of the response decreased after successive stimuli. The duration of individual responses varied even more widely than the maxi- mum intensities, and ranged from 3 seconds to 50 seconds for all luminescent responses with intensities between 10~ 3 and 10' 4 /AW ./cm 2 . There was no apparent relationship between the intensity of a luminescent emission and its duration. For example, two Metridia, exactly similar in laboratory history, both gave responses of 3 >: 10~ 4 /AW./cm. 2 , one emission having a duration of 10 seconds, the other a duration of 50 seconds. If responses of a lower maximum intensity than 10~ 4 /AW./cm. 2 are considered, durations as short as 1 second have been measured, particularly at the end of fatigue experiments when the Metridia had already re- sponded to 10 or 15 electrical stimuli. 98 CHARLES N. DAVID AND ROBERT J. CONOVER Through the kindness of Dr. W. D. McElroy at the Marine Biological Labora- tory, Woods Hole, Massachusetts, it was possible to measure the spectrum of Metridias luminescent emission (Fig. 4). The apparatus used was an Aminco spectrophotofluorometer in circuit with a drum recorder ("x-y" recorder) and an oscilloscope. Because of the rapid decay in intensity of Metridia's luminescence, it was necessary to cool a number of the animals in crushed ice in order to slow down the enzyme reaction producing the luminescence. Then, by immediately crushing the animals in a small test tube directly in the spectrophotofluorometer, the luminescence remained at one intensity long enough to record the entire spectrum. The peak of the spectrum for Aletridia is around 482 m/x and is therefore similar to that of Cypridina and certain other luminescent Crustacea (Nicol, 1960). The curve is slightly skewed toward longer wave-lengths with about half the spectral energy falling in the range between 440 nip. and 525 nip.. The entire spectrum lies between 425 nip. and 580 in/*. This spectrum with its peak at 482 nip. coincides closely with the wave-lengths having maximum transmission through clear, oceanic sea water (Clarke, Chap. 6, 1954). Experiments on physiology In order to determine whether laboratory culture had any effect on the lumi- nescence of Mctridia, freshly captured specimens and some which had been main- tained in the laboratory for a month were repeatedly stimulated until failure to respond to two successive stimuli indicated the onset of fatigue. A representative experiment shown in Figure 3 indicates that the maximum intensity and the rate of fatigue were not markedly different for the two specimens. The difference in flash duration is not significant considering the wide range of variation shown by this characteristic. To study the effect of strength of the stimulating pulse on the luminescence, the current was increased from .3 amp. to .7 amp. which caused a significant increase in the intensity of the luminescence and in the number of responses to stimuli. However, pulses stronger than .7 amp. did not cause further increase in lumines- cence intensity but seemed to reduce the number of successive responses. Variations in the duration of the pulse over the range tested (.10-1.0 second) had little effect on the intensity or number of successive responses. However, short intervals, i.e., 3 seconds, between pulses induced two or three times as many successive re- sponses as were observed using longer intervals between pulses, i.e., 1045 seconds. The effect of previous light- or dark-adaptation was tested with separate groups of animals kept at about 5 C. in the dark, in the light, and in a room exposed to diurnal light changes. The experiment was begun at 1700 on August 3 and the luminescence produced by each group was tested on August 5 and again on August 8 between 1000-1300. No statistically detectable difference was found between the three sets of animals. In another experiment twenty animals kept in a water bath at 5 C., where they were exposed to daily light variation, were tested at night (2330-0030) and during the day (1300-1400). In the case of a few animals the day-time response was somewhat lower than at night but there is no evidence in any of the data for a marked inhibition of luminescence by light or for a daily rhythm. LUMINESCENCE OF A MARINE COPEPOD Having established the fact that the experimental techniques used had no appreciable effect on the luminescent response of Metridia, two more physiological experiments \\ere performed. The first was designed to investigate an observation by the authors that animals which fed poorly still luminesced as vigorously as 10 MILLISECONDS 10 MILLISECONDS 20 MILLISECONDS FIGURE 5. Lag time between stimulus and luminescent response. In all four cases the stimulus was 150 v./5 msc. represented by the break in the smooth horizontal trace. Downward deflection of the jagged upper trace (lower trace in D) represents luminescence measured by the photomultiplier. The lag times were : A, 8 msc. ; B, 7 msc. ; C, 8 msc. D shows the same type of measurement but includes more of the intensity curve and the stepwise rise to maximum intensity. animals that fed well. Two groups of animals were set up, one fed on the regular culture medium and the other starved in millipore-filtered sea water. After one week, single stimulus tests were performed. On the basis of the total area under the intensity vs. time curve, the results showed no difference between the two 100 CHARLES N. DAVID AND ROBERT J. CONOVER groups. However, when maximum intensity was considered, the results indicated statistically (Wilcoxen Ranked Sum) better luminescence for the starved group. After the second week, repeated stimulus experiments were conducted on the two groups. Single stimulus data showed no statistical difference in the intensity of the response between the fed and starved groups, nor did the number of suc- cessive responses to repeated stimuli show a significant difference. At the end of the third week, however, experiments did demonstrate that the fed Metridia had a stronger luminescent response and the same group was able to respond to the electric stimulus a greater number of times than the starved animals. In another series of experiments the length of time from the beginning of a stimulus to the beginning of a response (the lag time) was measured. For the necessary guidance and equipment to make these measurements, the authors are indebted to Dr. James F. Case at the Marine Biological Laboratory, Woods Hole, Mass. Single animals \vere tested in a small cell consisting of a 3-cm. piece of glass tubing with agar plugs and silver electrodes at either end. The electrodes were connected to a Grass S4 stimulator and the luminescence was measured with a RCA 931 A photomultiplier in circuit with an oscilloscope. An automatic camera photographed the oscilloscope screen to record the results. Using a stimulating pulse of 150 v. for 5 or 10 msc., the Metridia demonstrated a lag time (at room temperature) of 8-10 msc. to the beginning of the luminescent response and a lag time of 15-24 msc. to the maximum intensity of the response. The time to maximum intensity varied widely, depending on whether or not the rise to maximum intensity was direct or in a step-wise fashion, the latter giving lag times as long as 60 msc. (see Fig. 5). By observing the Metridia through a microscope during these experiments it was noticed that for a stimulus of 10 msc./150 v. usually both head and tail luminesced while for a stimulus of 5 msc./150 v. only the organs in the head region responded. Experiments on behavior Because it has often been suggested that luminescence functions as an escape mechanism for marine animals that luminesce by means of an extracellular dis- charge, the authors decided to investigate the behavior of Metridia in the presence of a predator. A series of experiments was conducted in the dark, in which possible planktonic predators on Metridia were placed individually with 10 Metridia in 600-ml. beakers. The species tested were: Paraenchaeta norvegica (Copepoda), ParatJietnisto (Eiitlicmisto} gaudichandii (Amphipoda), and euphausiids, Eu- phausia krohnii, Thysanoessa incrmis, Nematoscelis mcgalops, and Meganyctiphanes norvegica. Each experiment \vas continued for at least two days and counts of the number of Metridia present were made at intervals of 12 to 16 hours. Only in the case of the two euphausiids, Thysanoessa and Meganyctiphanes, was there any predation on the Metridia. Although not every individual tested fed on Metridia with the same rapacity, MeganyctipJiancs was by far the most successful predator. The best predators among the animals tested were then chosen for further examination. The predator was placed in a 600-ml. beaker with 10 Metridia and this beaker was placed in the black box in front of the photometer. A cool water bath was used to keep the temperature in the experimental vessel between 10-12 C. The LUMINESCENCE OF A MARINE COPEPOD 101 photometer response was recorded at slow speed (6 in./hr.) so that each flash gave a spike indicating maximum intensity and time of occurrence. Analyzing the results of these experiments was complicated by the fact that both prey (Me- tridia) and predator (Meganyctiphanes') were luminescent. However, compari- son of the characteristics of Mctridia and Meganyctiphanes luminescence records when the animals were stimulated electrically showed that high intensity responses ICT icr 3 I0' 4 ICr 5 10 -6 -7 10 * io- 2 10 -3 10 -4 10 -5 10 10 -6 -7 (CONTINUED) SEC 10 20 30 40 f. 50 60 10 20 30 FIGURE 6. Luminescence of a single Meganyctiphanes norvegica when stimulated in the electrode chamber. Arrows along time scale indicate instant of stimulation. Chart speed is 6 in./min. The stimulus (0.7 amp.) was as strong as any ever used for Mctridia. Low inten- sity of luminescence is notable in comparison to Mctridia's bright flash. were almost surely due to Mctridia. Even using a maximum stimulus (1.1 amp.), Mcganyctiplianes never produced a response higher than 1 : : 10~ 4 /xw./'cm. 2 and it was generally much lower. Furthermore, the luminescent emission of Mega- nycti phones was usually a prolonged irregular glow (Fig. 6). The Mctridia, by contrast, always gave a single flash that appeared as a perpendicular spike on the slow-speed record (see Fig. 7). Using these two criteria, a reasonable inter- pretation of the records could be made. 102 CHARLES N. DAVID AND ROBERT J. CONOVER io-* I0' s * ID' 6 ID' 7 1600 HOURS 1700 1800 2 o *v S a. IO" 10- 10- ICT 8 ID' 5 5 u 510- ID' (CONTINUED) ll 1 1 1900 2000A A (CONTINUED) 2100 A 2200 2300 io- s IQ-" io- s ID' 7 (CONTINUED) 1 III * i ML , . i V 0200 A 0300 \l 0600 A FIGURE 7. Record of behavior experiment 3 (see Table I). At the chart speed of 6 in./hr., luminescent flashes appear as spikes indicating maximum intensity and time of occurrence. Solid triangles indicate successful predation under interpretation outlined in text. The decreasing background intensity between 1600 and 1900 hours is due to the setting sun which reduced the ambient light in the laboratory. The increased background at 2045-2100 and 2150-2300 hours was caused by lights in the laboratory used to monitor the recorder. LUMINESCENCE OF A MARINE COPEPOD 103 A sample record from an experiment with both prey and predator present is show in Figure 7. All the experiments are summarized in Table I. Experiments 6 and 7 in the table show quite clearly that the two species when separated from each other ordinarily do not produce any spontaneous luminescence. Only a single weak flash (4 X 10~ 7 ju,w./cm. 2 ), which may have been caused by some accidental mechanical stimulus, was observed for the group of Metridia alone. The Mega- nyctiphanes alone produced no luminescence at all. This corroborates Mauchline's (1959) observation that Meganyctiphanes does not luminesce spontaneously in the laboratory except during the breeding season (Dec.-Feb.). On the other TABLE I Summary of behavior experiments. The table shows the interrelationship between luminescence and predation in Metridia lucens. For detailed explanation see text and Figure 8. Predator No. of Metridia Number of luminescent responses Expt. Date Total time No. (hrs.) No. Species In expt. Eaten Above 10~ 7 jiw./cm. 2 Above 10-< /iw./cm. 2 1 8/12-13 15 1 Meganyctiphanes norvegica 9 8 30 15 2 8/17-18 8 1 Meganyctiphanes norvegica 10 3 17 3 3 9/14-15 16 1 Meganyctiphanes norvegica 10 10 33 14 4 9/16-17 13.5 1 Meganyctiphanes norvegica 11 5 41 21 5 9/18 10.5 1 Meganyctiphanes norvegica 10 4 23 1 6 8/13-14 15.75 10 1 7 8/14-15 9.5 1 Meganyctiphanes norvegica 8 8/11-12 7.25 2 Parathemisto gaudichaudii 10 1 9 8/19-20 15.5 1 Parathemisto gaudichaudii 9 10 8/18-19 15.5 2 Thysanoessa inermis 10 1 3 11 8/24-25 15 1 Nematoscelis megalops 10 1 hand, when the two species were placed in the same container, considerable lumi- nescence was observed and some Metridia were eaten (experiments 1-5). Since most of the flashes showed up on the record as single spikes, some with an intensity greater than 10~ 4 /xw./cni. 2 (see Fig. 7), it was concluded that the copepod was primarily responsible for the display. On the original records (copied in Fig. 7), it was possible to distinguish two kinds of single spikes, ones representing only a single luminescent flash and ones where several tracings were actually superimposed. This latter kind represented several flashes of different intensities which occurred within an interval short enough (30-40 seconds) to prevent their resolution at the slow chart speed. Sometimes this multiple-flash sequence was spread out over a longer period of time and the smaller flashes were resolved on the record (e.g. 0230 hours in Fig. 7). The number of multiple-flash sequences was, in almost every case, exactly equal to the number of Metridia eaten. These sequences presumably represent a Metridia s capture (large flash) and subsequent struggle to escape (small flashes). The 104 CHARLES N. DAVID AND ROBERT J. CONOVER remaining spikes on the record (caused by single flashes) are presumed to represent successful escapes by Metridia. In order to determine if the mere mechanical disturbance of another organism in the container could cause luminescence, groups of Metridia were tested with several other species in the container placed in front of the photometer. A large ParatJieinisto (Eutheinisto) gaudichandii, a vigorously swimming hyperiid amphi- pod, did not induce any luminescence w r hen placed with Metridia nor did it eat any (Table I, experiments 8 and 9). Similar results were obtained with the euphausiid Nematoscelis megalops (experiment 11). When TJiysanoessa inennis was used (experiment 10) a few flashes were produced and a single Metridia was eaten during the experiment. A further test of the effect of mechanical stimula- tion was made by vigorously stirring the water in a beaker containing Metridia. Considerable disturbance was necessary before any flashing occurred and even the most energetic agitation elicited a maximum response of only 6 X 10~ 5 /xw./cm. 2 , less than one tenth of the highest responses shown in Table I and Figure 7. Direct observation of predation was also attempted in order to determine the nature of the luminescence stimulus. An infra-red-sensitive "sniper-scope" (Ed- mund Scientific Co.) was used with the infra-red source and a focusing lens placed behind the experimental beaker so that the animals appeared in opaque profile against a light background. The small Metridia were not always visible with this optical arrangement but some individuals were seen to be carried toward the euphausiid by the currents set up by the larger animal's pleopods. Sometimes the Metridia would dart away before reaching the Meganyctiphanes, but at other times the copepod would seem to come in contact with the euphausiid before darting away. On a few occasions the euphausiid started off as though in pursuit, but the actual act of capture was never observed. These observations are in general agreement with those of Mauchline (1959) who found Meganyctipliancs capable of filter-feeding on organic detritus and even sucking into the "food basket" individual copepods (Paraeuchaeta norvegica) and Sagitta by lateral-ventral movements of the thoracic limbs. The animal can also seize larger objects by raptorial movements of the appendages but in the laboratory "no hunting or stalking of prey takes place" (Mauchline, 1959). DISCUSSION Over the years there has been considerable speculation regarding the role of bioluminescence in the life of various marine organisms. In higher marine forms, luminescence has been found associated with either mating behavior, feeding mecha- nisms, or defense. Among planktonic species, however, there is less agreement as to its functional significance. Besides the three interpretations given above, it has been suggested that this phenomenon may often be coupled with other life processes in lower animals and therefore might have no function of its own (Russell and Yonge, 1928; Harvey, 1929). It has also been suggested that luminescence in planktonic and sessile creatures may serve as a "burglar alarm," thereby revealing a predator to its own enemies along the food chain (Burkenroad, 1943). From the results of the behavior experiments with Metridia, it is apparent that there is some relationship between luminescence and the act of predation. Since the exact nature of the stimulus is still unknown, it is impossible to determine LUMINESCENCE OF A MARINE COPEPOD 105 positively which, if any, of the above hypotheses is applicable. Nevertheless, some of the possibilities may be eliminated. Any functional use of luminescence involving species recognition, such as mating display or warning systems to other individuals of danger, is doubtful because Metridia probably does not have an adequate image-forming eye. Of the remaining speculations presented above, the authors currently feel that the defense mechanism is the one most consistent with the experimental results. However, Burkenroad's hypothesis is not specifically ruled out. The reasons for favoring the idea of an escape mechanism arise from : ( 1 ) certain of the physical and physiological characteristics of Metridia's luminescent emission, and (2) a unique pattern of behavior associated with luminescence in this copepod. The maximum intensity of Metridia's luminescence is surprisingly brilliant. At the working distance (18 cm.) used in this study the flash was of the same order of magnitude as that of certain coelenterates and of the crustacean Euphausia pacified, and greater than that of the teleost Myctoplutm punctatum, all measured at 1 cm. (Nicol, 1960). The duration of the flash is long and its spectral composition is similar to the spectrum of the transmission of light through sea w r ater with the maximum of the two curves at nearly the same \vave-length. It has also been shown that Metridia has an extremely short lag time between stimulus and response. The animal recovers quickly after stimulation and fatigues rather slowly on re- peated stimulation, even after several weeks without food, suggesting that the ability to luminesce is important enough to the organism to be maintained under adverse conditions. All these characteristics of Metridia's luminescence, both physical and physiological, would certainly be selectively advantageous to the animal if its luminescence functioned as an escape mechanism. The most significant evidence for the defense mechanism hypothesis, however, comes from observations of the behavior of single Metridia stimulated in the electrode chamber. On stimulation a point of luminescence was immediately pro- duced and then in the majority of cases the animal appeared to dart off into the dark, leaving a bright luminescent spot at its original position and sometimes a trail of tiny luminescent specks that soon disappeared. Although the animal itself could not be seen during this reaction, the agitation of the water gave a clue to its behavior and its new position could be verified by passing a second electrical stimulus through the water and observing the new location of the resulting lumi- nescent flash. The original luminescent emission remained a more or less discrete point of light for some seconds after stimulation. Such a behavior pattern appears to the authors to indicate the manner in which Metridia escapes from Meganyctiplwnes. Although the precise role that lumines- cence plays in this escape mechanism is still unknown, two speculations are possible. The luminescent emission may startle the attacker, interrupting its feeding pro- cedure, or it may merely function as an attractive decoy. In either case, the Metridia's rapid departure from the spot where it had luminesced would complete the escape. The possibility that luminescence only occurs when the Metridia is actually captured is not entirely eliminated. More definitive proof must await the elucidation of the specific stimulus that induces luminescence. Nevertheless the evidence pre- 106 CHARLES N. DAVID AND ROBERT J. CONOVER sented here indicates that luminescence functions on the behavioral level as an escape mechanism for Metridia. It would then seem probable that luminescence, which is of such widespread occurrence in the oceans, may well have survival value in defense against predation in some similar manner for many other animals of the plankton. SUMMARY 1. Skin glands believed to be the source of luminescence were found on the anterior portion of the head, on the last thoracic segment, and on the posterior margins of each segment of the abdomen. 2. The maximum intensity of the luminescent flash was 1.2 X 10~ 3 /AW. /cm. 2 (at 18 cm.). The flash rose rapidly to peak intensity and then decayed slowly. The total duration of the flashes with peaks greater than 10~ 4 //.w./cm. 2 ranged from 3 to 50 seconds. 3. The peak of the luminescence spectrum occcurred at 482 mp. and the curve fell off to one-half the maximum value at 440 m/x, and 525 m/x. 4. The ability of Metridia to luminesce on stimulation was found to be largely unaffected by prolonged laboratory culture. Starvation had little effect on the luminescence for the first three weeks and there was never any inhibition by previous light- or dark-adaptation. 5. With an increase in the strength of the electric stimulus from 0.3 amp. to 0.7 amp., the intensity of the luminescent flash was found to increase. With pulses stronger than 0.7 amp. no change in intensity was recorded but the number of successive responses to repeated stimuli was reduced. Duration of the pulse had little effect on the intensity or the number of successive responses. 6. Metridia showed a lag time of 8-10 msc. to the beginning of the luminescent response. The lag time to the peak of the luminescent response varied from 20 to 60 msc. 7. There was no spontaneous luminescence produced by groups of Metridia under conditions of constant darkness. However, the presence of certain plank- tonic predators, most notably Meganycti phones norvegica, caused a brilliant display of luminescence. The number of flashes attributable to Metridia was always greater than the number of Metridia eaten by the predator. There was little evi- dence that the luminescent euphausiid, MeganyctipJianes, flashed spontaneously either in the presence or absence of its prey. 8. Observations on the behavior of Metridia during and just after luminescence suggest that the flashing may be involved in an escape mechanism, but the precise effect of the light on the predator has not been determined. LITERATURE CITED BIGELOW, H. B., 1924. Plankton of the offshore waters of the Gulf of Maine. Bull. U. S. Bur. Fish,, 40: pt. II, 509 pp. BODEN, B. P., AND E. M. KAMPA, 1957. Records of bioluminescence in the ocean. Pacific Sci., 2: 229-235. BODEN, B. P., AND E. M. KAMPA, 1958. Lumiere, bioluminescens et migrations de la couche diffusante profande en Mediterranee occidentale. Vie et Milieu, 9 : 1-10. BOECK, A., 1865. Oversigt over de ved Norges kyster iagttage Copepoder henhorende til Calanidernes Cyclopidermes og Harpactidernes familier. Fork. Vidcnsk. Selsk. Krist., 7: 226-282. LUMINESCENCE OF A MARINE COPEPOD 107 BRESLAU, L. R., 1959. The portable bathyphotometer. Unpublished manuscript. Reference No. 59-28, Woods Hole Oceanographic Institution. BRESLAU, L. R., AND H. E. EDGERTON, 1958. The luminescence camera. /. Bio!. Photogr. Assoc., 26: 49-58. BURKENROAD, M. D., 1943. A possible function of bioluminescence. /. Mar. Res., 5: 161-164. CLARKE, G. L., 1933. Diurnal migration of plankton in the Gulf of Maine and its correlation with changes in submarine irradiation. Biol. Bull., 65 : 402-436. CLARKE, G. L., 1934. Further observations on the diurnal migration of copepods in the Gulf of Maine. Biol. Bull., 67: 432-455. CLARKE, G. L., 1954. Elements of Ecology. John Wiley and Sons, New York. CLARKE, G. L., AND R. H. BACKUS, 1956. Measurements of light penetration in relation to vertical migration and records of luminescence of deep-sea animals. Deep-Sea Res., 4: 1-14. CLARKE, G. L., AND L. R. BRESLAU, 1959. Measurements of bioluminescence off Monaco and Northern Corsica. Bull. lust. Occanogr., Monaco, 56: (1147): 31 pp. CLARKE, G. L., AND L. R. BRESLAU, 1960. Studies of luminescent flashing in Phosphorescent Bay, Puerto Rico, and in the Gulf of Naples using a portable bathyphotometer. Bull. Inst. Occanogr. Monaco, 57 (1171) : 32 pp. CLARKE, G. L., AND C. J. HUBBARD, 1959. Quantitative records of the luminescent flashing of oceanic animals at great depths. Limnol. Oceanogr., 4 : 163-180. CLARKE, G. L., AND G. K. WERTHEIM, 1956. Measurements of illumination at great depths and at night in the Atlantic Ocean by means of a new bathyphotometer. Deep-Sea Res., 3: 189-205. DAHL, F., 1893. Pleuromma, ein Krebs mit Leuchtorgan. Zoo/. Ans., 16: 104-109. DAHL, F., 1894. Leuchtende Copepoden. Zoo/. Ans., 17 : 10-13. FARRAN, G. P., 1920. On the local and seasonal distribution of the pelagic Copepoda of the southwest of Ireland. Publ. Circ., Cons. Perm. Int. Explor. Mer, No. 73, 30 pp. GIESBRECHT, W., 1895. t)ber das Leuchten der pelagischen Copepoden und das tierische Leuchten in allgemeinen. Mitt. Zoo/. Sta. Neapel, 11: 631-694. HARVEY, E. N., 1929. Phosphorescence. Encyclopedia Brittanica, 14th ed., p. 117. HARVEY, E. N., 1952. Bioluminescence. Academic Press, New York. KAMPA, E. M., AND B. P. BODEN, 1957. Light generation in a sonic-scattering layer. Deep- Sea Res., 4 : 73-92. KIERNIK, E., 1908. t)ber einige bisher unbekannte leuchtende Tiere. Zoo/. Anz., 33 : 376-380. LOWE, E., 1935. The anatomy of a marine copepod Calanus finmarchicus (Gunnerus). Trans. Roy. Soc. Edinb., 58: 561-603. MAUCHLINE, J., 1959. The biology of the euphausiid crustacean, Meganyctiphanes norvegica (M. Sars). Proc. Roy. Soc. Edinb., 67: 141-179. NICOL, J. A. C., 1960. The Biology of Marine Animals. Sir Isaac Pitman, London. RUSSELL, F. S., AND C. M. YONGE, 1928. The Seas. Frederick Warne, London. SEWELL, R. B. S., 1932. The Copepoda of Indian seas. Calanoida. Mem. Indian Mns., 10: 407 pp. SEWELL, R. B. S., 1947. The free-swimming planktonic Copepoda. Systematic account. Set. Repts., John Murray Expd., 8 : 303 pp. VANHOFFEN, E., 1895. Das Leuchten von Metridia longa Lubb. Zoo/. Atiz., 18 : 304-305. THE PHYSIOLOGICAL CONTROL OF WATER INGESTION IN THE BLOWFLY 1 V. G. DETHIER AND D. R. EVANS Zoological Laboratories, University of Pennsylvania, Philadelphia 4, Pa., and Department of Biology, The Johns Hopkins University, Baltimore 18, J\Id. Because of their small size and terrestrial habitat, insects constantly face a pressing problem in water conservation. Recognition of this fact has stimulated many investigators to study routes and mechanisms of water loss and adaptations for its prevention (rf., Edney, 1957). Some of these studies have dealt with behavioral adaptations, such as humidity preferences, that decrease water loss. With regard to the uptake of water, however, little is known apart from observa- tions of direct water uptake through the integument at very high humidities by some insects. One isolated study of a fly (sp. ?) by Bolwig (1953) showed a negative correlation between response to water by drinking and the vapor pressure of the blood. Otherwise there appears to be no experimental work on the control of drinking by insects (Leclerq, 1946; Edney, 1957). Accordingly, experiments were undertaken to reveal the factors underlying thirst and water ingestion in the blowfly, Phortnia regina Meigen. METHODS The blowfly was chosen for study because a considerable body of knowledge relating to its sensory physiology and feeding exists. The flies employed were taken from a culture maintained in these laboratories since 1947. Flies were desiccated or humidified by storage in a sealed vessel containing calcium chloride or water, respectively. Measurement of the water intake of individual flies was carried out by surgically removing the crop after drinking was complete and weighing it. Preferences and volumes consumed of test solutions over periods of 24 hours and longer were measured by the method of Dethier and Rhoades (1954). Injections of fluid into the haemocoel were carried out as before (Evans and Dethier, 1957). Flies were designated water-positive or -negative on the basis of a uniform response to three tests being obtained ; a positive response consisted of proboscis extension upon tarsal contact with water. Bleeding was accomplished by cutting off the prothoracic legs close to the thorax and expressing the blood by gentle compression of the thorax. RESULTS Sensory control of drinking A fly that has been deprived of water will respond to it in a variety of ways : it will orient from a distance to a locus of high humidity ; it will extend its proboscis 1 This work was aided by National Science Foundation Grants G-6015 and G-5927 and by National Institutes of Health Grant E-2358. 108 WATER INGESTION IN THE BLOWFLY 109 in response to stimulation by water vapor ; it will extend its proboscis in response to water applied to the tarsi or labellum ; it will open the labellar lobes and com- mence sucking in response to water applied to the labellum. After a period of time which is related to the extent of previous water deprivation, drinking will cease and the fly will become refractory to further stimulation by water. The sense organs on the mouthparts whose stimulation initiates drinking are the same chemoreceptive hairs that respond to sugars (Dethier, 1955). Of the three sense cells that innervate the hair, one is a mechanoreceptor, one responds preferentially to salts, and the other to sugars. Wolbarsht (1957) reported that distilled water applied to certain of the hairs caused a firing of nerve impulses in both chemosensory cells. Each discharged at an initially high rate but adapted rapidly (one had often ceased firing at the end of thirty seconds). TABLE I The effect of desiccation on water and sugar consumption by the blowfly. Each value is based upon tests with thirty individual flies. The figures in parentheses represent ranges. Experiment number Treatment of flies Av. \vt. (mg.) of fly minus wings Av. duration (sec.) of sucking of each solution presented successively HjO 0.1 M sucrose 1.0 M sucrose 1 Three-day-old flies fed once on 0.1 M sucrose, starved 24 hrs., then desiccated 24 hrs. 12.1 (10.2-16.9) 24 (6-52) 46 (23-73) 35 (17-62) 2 Three-day-old flies fed once on 0.1 M sucrose, starved 24 hrs., then desiccated 24 hrs. 11.0 (9.7-17.0) 54 (40-90) 3 Three-day-old flies fed once on 23.4 0.1 M sucrose, starved 24 hrs., then humidified 24 hrs. (19.6-27.7) 38 (20-60) As has been reported to be the case for the ingestion of sugar solutions (Dethier et al., 1956), not only the initiation but also the maintenance and termination of water ingestion are dominated by the input of the taste receptors. Water ingestion is driven by the input, and adaptation finally terminates it. This control can be demonstrated by stimulating one side of the labellum until the receptors adapt, whereupon drinking ceases, and then stimulating the remaining receptors, where- upon drinking resumes. In addition to this primary control of drinking by chemosensory input, other factors are involved in the ingestion of water and nutrients. Conditions modifying drinking Three conditions were considered as possibly modifying drinking behavior : namely, starvation and feeding, unacceptable contaminants, and desiccation. Each of these was investigated in turn. 110 V. G. DETHIER AND D. R. EVANS Starvation and feeding. The average life span of Phonnia in the absence of food is three days ; accordingly, experiments on starvation were perforce limited to this period. The daily intake of water of fourteen individual flies was measured. No consistent change in intake was noted over this period. In another experiment designed to control the effects of desiccation, thirty flies were starved 24 hours and then placed in a humidifier for 24 hours. A control sample was placed in a desiccator. The results are summarized in experiments two and three of Table I. The humidified flies did not drink even though they had starved 48 hours. In a more drastic experiment, flies were kept in the humidifier until the last had died of starvation five days later. All remained negative to water till the end. It can be concluded that starvation does not induce drinking as long as water loss is prevented. TABLE II The effect of Nad an water intake by the blowfly. Each value is based on tests with ten individual flies. The figures in parentheses represent ranges. Materials available to the fly Mean volume of water consumed in 3 days 0*1.) Mean volume of NaCl consumed in 3 days (id.) Mean total fluid intake in 3 days 0l.) Water (no food) 29 (12-39) 29 Water and dry sucrose 16 (12-61) 16 0.1 M XaCl and dry sucrose 23 (9-36) 23 0.5 M NaCl and dry sucrose 19 (12-38) 19 0.5 M NaCl, dry sucrose, and water 12 (5-18) 6 (4-7) 18 1 M NaCl and dry sucrose 12 (3-24) 12 1 M NaCl, dry sucrose, and water 14 (6-18) 4 (0-5) 18 2 M NaCl and dry sucrose 3 (0-12) 3 2 M NaCl, dry sucrose, and water 15 (3-24) 4 (0-7) 19 In order to test whether or not dry food as the only source of nutrition causes an increase in drinking, the daily water intake of individuals of two groups was measured over a three-day period. One group of flies had free access to water but had no food. The other group had free access to water and to a lump of sugar. The results are summarized in the first two lines of Table II. Contrary to expectations, the ingestion of food did not bring about increased drinking even though part of the process of eating solid food involves dissolving it in saliva. Whether the reduction of water intake in the presence of sugar is real is not known. As a variant of the preceding, the experiments described in Table III can equally well be done on flies that have been made water-positive by storage in a sealed vessel in contact with anhydrous glucose. They have the opportunity to feed continuously, and yet become positive to water after a time. The mechanism is very likely the same as that of storage with calcium chloride, but the latter is a better desiccant. Contaminants. To test the effect of unacceptable taste stimuli on drinking, several series of experiments involving the addition of sodium chloride to water were undertaken. In one experiment, flies were kept in individual cages equipped with two pipettes, one of which contained water, the other a salt solution. The salt solutions paired with water ranged in concentration from 10~ 5 M to 5 M. WATER INGESTION IN THE BLOWFLY 111 The volume of each solution imbibed was measured each day. Results are sum- marized in Figure 1. from which it can be seen that the volume of salt solution drunk decreases as the concentration increases. Concurrently, the quantity of water drunk increases so that the total fluid intake is approximately constant. Under more rigorous conditions where the fly was provided with a salt solu- tion as its only source of fluid, higher concentrations (as judged by the volume imbibed) were tolerated than when water also was present (Table II). As the Q O or - 08 LU CL cr CJ o: LJ a. Q LU CO o o LJ ^ .06 .04 02 V \r o / ,0 -5 -4 -3 -2 LOG MOLAR CONG. -i FIGURE 1. Volume of different concentrations of sodium chloride ingested per fly per 24 hours in a two-choice situation. Solid line, sodium chloride. Broken line, water. concentration of salt was increased, however, the amount of fluid imbibed decreased. In still another series of experiments, 60 flies were tested for their responses to salt solutions applied to the tarsi, placed in a desiccator with a supply of dry sugar for food, then re tested periodically until death. The results obtained with 1.0 M NaCl illustrate the trend of events. Before being placed in the desiccator none of the flies gave any response to this solution. As water loss increased, they first would extend the proboscis when the tarsi were stimulated but would not open the lobes of the labelluin ; later they would open the labellar lobes but not drink ; still later they would drink for a few seconds ; finally, the drinking time 112 V. G. DETHIER AND D. R. EVANS would increase. In short, as desiccation increased, the rejection threshold of the tarsi to salt rose followed by a rise in the rejection threshold of the mouthparts. Desiccation. Implicit in all of the foregoing experiments is the idea that water loss powerfully affects drinking. The experiments summarized in Table I demon- strate the effect of desiccation for 24 hours on response to water. They show further that the state of water balance also affects the amount of liquid food ingested. Since desiccated flies take more liquid food than do humidified flies, the response is clearly directed toward the acquisition of water. TABLE III The effect of injections on responses of the blowfly to water Experiment number Number of flies Response before treatment Injectedf Per cent negativ after treatment 1 35 + 2.5 /il. water 6 2 52 + 8 jul. water 85 3 27 + 2 jul. water 7 2 jul. water 22 2 jul. water 70* 4 26 + 3 jul. water immediately 58 at 10 minutes 58 at 60 minutes 54 5 120 3 n\. 2 M glucose 82* 6 116 7 jul. 2 M glucose 96* 7 29 2.4 jul. 4X saline 100 8 53 + 3 jul. 4X saline 55 9 69 + 3 ,ul- 2 M glucose 55 10 48 + 3 jul. 2 M glucose in saline 56 11 40 + 6 jul. 2 M glucose in saline 85 12 92 + 4 jul. mineral oil, moribund 66 at 15 min. 13 14 + Fed 2 M glucose 100 0-60 minutes * Responded subsequently to 0.1 M sucrose. f Water indicates distilled water ; the saline was Bodenstein's 10 ; 4 X indicates saline four times more concentrated; exps. 1-9 from Evans (1961). The control of water responsiveness A series of injection experiments was undertaken to assess the effect of blood osmotic concentration on the responsiveness of flies to water (Table III). Injec- tions of water rendered positive flies negative to water (exps. 1-4). The percentage made negative was a function of the volume injected (exps. 1-4). The effects of repeated injections were additive (exp. 3), and the effect was immediate (exp. 4). Next it was found that injections of even huge volumes of highly concentrated solutions did not produce responsiveness to water (exps. 5-7). But these same hypertonic injections could abolish water responsiveness (exps. 8-11), indicating that volume and not osmotic or dilution factors was the significant feature. Even mineral oil, before its toxic effects were apparent, blocked responsiveness to water WATER INGESTION IN THE BLOWFLY 113 (exp. 12). Ingestion of nearly saturated sugar solutions (exp. 13) abolished the water responses. These results suggested blood volume or pressure as the agent regulating water responsiveness ; but since responsiveness had not been induced in any case, the effects could have been unspecific even though responsiveness to sugar was not affected in the few cases tested (exps. 3, 5, 6). Consequently, it was crucial to reduce blood volume and thereby induce water responses. The crude procedure of cutting off the abdomen with crop did not make flies positive ; however, bleeding did. If a population of flies was desiccated until some responded to water, bleeding made most of the remainder (45 of 53) responsive to water. Vigorous responses were obtained as quickly as a fly could be tested after the bleeding (a few seconds). Bleeding did not alter the response of already responsive flies (12 of 12). And in the case of flies given water to satiation, bleeding induced water responses in only a few (4 of 13). Apparently there is a threshold such that blood volume must be reduced below some particular level before the treatment is effective. In another series of experiments, the recurrent nerve was cut in 80 flies which were responsive to water before the operation and an additional 50 flies that were satiated. Sixty per cent of the former became bloated; 50% of the latter. Removal of the corpora allata in 30 flies produced no abnormal water intake. Removal of the median neurosecretory cells of the brain from 60 flies caused bloat- ing in four. Where bloating occurred, the behavior pattern was characteristic. Flies imme- diately began drinking and continued to drink intermittently over a period of one to two hours. Thereupon no further drinking occurred although the proboscis was repeatedly extended. DISCUSSION As in the case of the ingestion of sugars (Dethier et al., 1956), the act of drinking appears to be controlled primarily by sensory input. Stimulation of tarsal or labellar taste receptors by water elicits reflexive extension of the proboscis and sucking. When adaptation (central and peripheral) has proceeded to some level, ingestion is terminated ; the stimulus becomes ineffective. The behavioral threshold of the fly to water, however, varies as a function of water balance. Specifically, water responsiveness can be abolished by injections into the haemocoel and can be induced by bleeding. The chemical nature and the concentration of injected material do not matter; the effect is related only to the volume and can be altered by injection or bleeding. Since these treatments are fully effective as quickly as can be tested, neural mediation of the effect is indicated, probably via a mechanoreceptor that signals distension or pressure. Accordingly, the input of the mechanoreceptor must act somewhere to set the level of taste threshold to water. The behavioral threshold to water is also affected by cutting the recurrent nerve immediately anterior to the hypocerebral ganglion. Flies undergoing this operation became bloated on water. Dethier and Bodenstein (1958) reported that flies in which the recurrent nerve had been cut became bloated on sugar solutions. They interpreted this effect as interference with the elevation of sugar threshold that normally follows sugar ingestion. Evans and Barton Browne (1960) confirmed 114 V. G. DETHIER AND D. R. EVANS the fact of hyperphagia following recurrent nerve section but found that the sugar threshold still rose in the normal fashion. Incidental observations suggested to them that the effect might be due to a hypersensitivity to water. Day (1943) and Thomsen (1952) had observed polydypsia in some flies after removal of the corpus allatum. However, allatectomy frequently involves a variable degree of injury to the recurrent nerve which could account for the low incidence (ca. 10%) of bloating observed by these workers. We have not been able to produce polydypsia as a result of allatectomy. Removal of the medial neurosecretory cells of the brain also sometimes results in bloating, but this does not necessarily imply a hormonal mechanism any more than a neural one since these cells are in neural connection with the recurrent nerve. Sensory control '(i.e., sensory input to drive and adaptation to stop) of drinking still operates in polydypsic flies so that an operated fly becomes bloated through repeated rather than continuous drinking. It was observed that operated flies kept in contact with water for long periods of time no longer imbibed any even though they continued to respond feebly for more than 24 hours. If such flies were presented with sugar, they resumed vigorous sucking until the crop and abdomen burst. There is obviously in the fly bloated on water a strong pressure opposing further intake. The water stimulus is not intense enough to produce effective sucking, but sugar, a stronger acceptable stimulus, can still produce effective sucking. This suggests that back-pressure was responsible for blocking continued imbibition of water. Since the behavioral threshold to water is affected by bleeding, by injection, and by cutting the recurrent nerve, the simplest explanation is that the recurrent nerve carries the neurons that signal blood volume or pressure and set water sensitivity. Elucidation of this point will have to await further knowledge of the finer details of structures innervated by the recurrent nerve and of connections with other parts of the nervous system. At this point evidence obtained earlier regarding the control of ingestion of sugars bears on the possible mechanisms and greatly complicates the interpretation. On the basis of much evidence, Dethier (1955) postulated that each of the two chemosensory neurons in the receptor subserved one of the taste qualities, acceptance and rejection. According to this hypothesis, water and sugar would be ingested because they stimulate the one neuron, and salts would be rejected because they stimulate the other (termed, respectively, 5 and L by Hodgson and Roeder, 1956). Subsequent electrophysiological studies have supported this view in general. Sugars do activate primarily the ^ fiber and salts the L fiber (Hodgson and Roeder, 1956). Furthermore, Wolbarsht (1958) and Tateda and Morita (1959) have shown that neither fiber exhibits appreciable spontaneous discharge, and Wolbarsht (1958) showed that there is no reason to believe that electrical responses of the two receptor cells influence one another. Consequently, the hypoth- esis explaining the qualities of taste is compatible with this evidence : discharge of the 5" fiber initiates and drives feeding, and discharge of the L fiber inhibits the feeding reflex somewhere beyond the sense cell level. Taste thresholds to sugars vary with feeding and starvation, and the mechanism has been studied in some detail (Dethier ct ol., 1956; Evans and Dethier, 1957; Dethier and Bodenstein, 1958; Evans and Barton Browne, 1960). All of the evi- WATER INGESTION IN THE BLOWFLY 115 clence suggests that it is the presence of sugar solution in the foregut (exclusive of the crop) that sets the level of the sugar threshold. The detector in the foregut and the intervening processes have not yet been elucidated, but it was suggested (Evans and Dethier,, 1957) that the final effect was to inhibit centrally the effect of 5" fiber discharge. The problem now arises as to how taste thresholds to water and sugar can be independently regulated as the present results show that they are. Now it should be pointed out that the neural explanation of the two taste qualities is not really so simple as the discussion above and some of the literature suggests. Some data will be cited that show the unexpected complexities that have emerged from electrophysiological studies. Wolbarsht (1957) reported that both 5" and L fibers respond to distilled water. In addition to the two chemosensory cells, there is a third sense cell associated with the socket of a chemoreceptor hair (Dethier, 1955) that Wolbarsht (1958) has shown to be a mechanoreceptor acti- vated by motion of the hair. The distal process of the cell does not enter the hair (Dethier and Larsen, personal communication) and therefore chemicals applied to the hair tip would not be expected to stimulate it. It is known that bending of a hair can evoke proboscis extension in a very starved fly (Dethier, 1955). Hodg- son and Barton Browne (1960) reported that bending of a hair influences, albeit unpredictably. the electrical response of the L and S fibers to chemicals. The ex- periments dealing with ingestion of salt solutions place limitations on hypothetical interpretations of the water threshold mechanism. Since the sensory input clue to water can drive the ingestion of more and more concentrated salt solutions as the flv is made more dehvdrated, the sensorv effects of water are balanced against those * / j o of salts, just as are the stimuli, sugar and salt. In view of the data presented it seems to us that the hypothesis that water and sugar act on the same neuron is no longer tenable. Evidence of the existence of a specific water receptor is now being sought. LITERATURE CITED BOLWIG, N., 1953. On the variation of the osmotic pressure of the haemolymph in flies. 6". Afr. Ind. Chcm., June. DAY, M. F., 1943. The function of the corpus allatum in muscoid Diptera. Biol. Bull., 84: 127-140. DETHIER, V. G., 1955. The physiology and histology of the contact chemoreceptors of the blowfly. Quart. Rev. Biol, 30: 348-371. DETHIER, V. G., AND D. BODENSTEIN, 1958. Hunger in the blowfly. Zcitschr. Tierpsvchol., 15: 129-140. DETHIER, V. G., D. R. EVANS AND M. V. RHOADES, 1956. Some factors controlling the in- gestion of carbohydrates by the blowfly. Biol. Bull.. Ill: 204-222. DETHIER, V. G., AND M. V. RHOADES, 1954. Sugar preference-aversion functions for the blowfly. /. E.vp. Zool, 126: 177-204. EDXEY, E. B., 1957. The Water Relations of Terrestrial Arthropods. Cambridge Monographs in Experimental Biology 5, Cambridge University Press. EVANS, D. R., 1961. Control of the responsiveness of the blowfly to water. Nature (in press). EVANS, D. R., AND L. BARTON BROWNE, 1960. The physiology of hunger in the blowfly. Amcr. Mdl. Nat., 64: 282-300. EVANS, D. R., AND V. G. DETHIER, 1957. The regulation of taste thresholds for sugars in the blowfly. /. Ins. Physiol, 1 : 3-17. HODGSON, E. S., AND L. BARTON BROWNE, 1960. Electrophysiology of blowfly taste receptors. Anat. Rec., 137 : 365. 116 V. G. DETHIER AND D. R. EVANS HODGSON, E. S., AND K. D. ROEDER, 1956. Electrophysiological studies of arthropod chemo- reception. I. General properties of the labellar chemoreceptors of Diptera. J. Cell. Comp. Physiol., 48: 51-76. LECLERCQ, J., 1946. Des insectes qui boivent de 1'eau. Bull. Ann. Soc. Ent. Beige., 82 : 71-75. TATEDA, H., and H. MORITA, 1959. Initiation of spike potentials in contact chemosensory hairs of insects. I. The generation site of the recorded spike potentials. /. Cell. Comp. Physiol., 54: 171-176. THOMSEN, E., 1952. Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blow-fly, Calliphora erythrocephala Meig. /. Exp. Biol., 29 : 137-172. WOLBARSHT, M. L., 1957. Water taste in Phormia. Science, 125: 1248. WOLBARSHT, M. L., 1958. Electrical activity in the chemoreceptors of the blowfly. II. Re- sponses to electrical stimulation. /. Gen. Physiol., 42: 413-428. WOLBARSHT, M. L., AND V. G. DETHIER, 1958. Electrical activity in the chemoreceptors of the blowfly. I. Responses to chemical and mechanical stimulation. /. Gen. Physiol., 42 : 393-412. FLIGHT AND SWIMMING REFLEXES IN GIANT WATER BUGS HUGH DINGLE Department of Zoology, University of Michigan, Ann Arbor, Michigan Loss of substrate contact or tactile stimulation initiates a "classic" flight reflex in insects (Fraenkel, 1932; Chadwick, 1953). Either one or both factors can operate to elicit the reflex ; flight ceases when the legs again make contact with the substrate. When giant water bugs were removed from substrate contact, they did not fly, but instead swam. If they stopped, they would begin again with direct tactile stimulation. In short, they appeared to swim in those situations in which terrestrial insects fly. Although a few of the water bugs eventually flew, they did so only after a considerable period ; during this time they were swimming. This study is an attempt to analyze the swimming and flight reflexes of these giant water bugs. MATERIALS AND METHODS Two species of giant water bug were used, Lcthocerus americanus and Benacus griseus. The bugs were captured by light trap (a sheet and a Mercury Vapor bulb, General Electric H100 L4) between April and September, 1960, on the Edwin S. George Reserve, the wildlife reserve of the University of Michigan, Livingston County, Michigan. A total of 60 animals were used ; they were kept in the labora- tory on a diet of small fish. Giant water bugs are large (about 4.5 to 6.5 cm. long) dorso-ventrally flattened predaceous insects. The forelegs are raptorial with enlarged femora and bear only a single tarsal claw ; the middle and hind legs are adapted for swimming ; they are flattened and bear hairs so arranged as to be raised during the power stroke of the leg and depressed during the forward stroke. The swimming legs have the usual two tarsal claws. Respiration is accomplished with two retractable tubes which protrude from the posterior end of the abdomen (Fig. 4). In the analysis of swimming and flight reflexes, the bugs were suspended from an applicator stick using a mixture of paraffin, beeswax, and resin to attach the stick to the prothorax. They were then placed in the air stream of a wind tunnel and given a stick to hold which served as a contact stimulus for the legs. The wind tunnel was made from wood and light cardboard and included a cardboard honey- comb baffle to cut down turbulence which, as determined by smoke, was slight ; the diameter of the tunnel mouth was 10 cm. For wind a fan was used, the speed of which could be controlled by a rheostat. Wind speed was calibrated with a Taylor Briam's Type Anemometer (No. 3132) ; it ranged up to 7.0 m./sec. In certain experiments small jets of water or air, which were directed by attaching a glass tube to a rubber hose, were used ; no attempt was made to measure the velocity of these. 117 118 HUGH DINGLE SWIMMING Loss of substrate contact almost invariably elicited swimming movements. Tbe rate and duration of these movements varied. The initial rate for 19 bugs in quiet air ranged from 120 to 320 strokes per minute with an average of 206; the duration ranged from 6 seconds to more than 180 seconds with an average of 51 seconds. This swimming response was clearly distinguishable from haphazard movement ; the forelegs were carried forward of the head, and in intense swimming they were stretched forward almost full length. The abdomen was raised, and the middle and hind pairs of legs were usually protracted and retracted (see Hughes, 1952, for definitions) simultaneously and not alternately as reported by Lauck (1959) for a different species. Although alternation was never observed, it was noted that the two pairs were sometimes not quite simultaneous. The two legs of each pair operated simultaneously as reported by Lauck. Swimming could be stopped by giving the bug a stick to hold. Contact with any one tarsus was sufficient ; when the bug made contact, the ipselateral leg reached for and grasped the stick. Swimming also ceased with contact on other parts of the leg, e.g. tibia and femur, especially if tension was applied; Diakonoff (1936) reports similar results in a flying cockroach. Sometimes, however, the bug dropped the stick or "walked" off it and continued to swim. If the stick was removed care- fully, leaving the legs folded under the body, the bug usually remained motionless. Swimming in such a situation could be initiated by gently lowering the legs until they were outstretched. Bugs also stopped swimming on occasion when they presumably saw the stick in front of them, reaching out and seizing it with the forelegs. Touching any part of the forelegs resulted in attempts to grasp the stick. In experiments testing the effect of increasing wind velocity, the bugs were holding a stick which was removed at each higher velocity ; it was returned when the bug stopped swimming. After 30 seconds the velocity was increased by about 1 m./sec. and the process repeated. Rate and duration of swimming increased up to a point and then decreased ; this decrease will be discussed in greater detail below. The lowest wind velocity measured, 0.5 m./sec., was sufficient to increase rate and duration in 50% of the bugs ; for the remainder higher velocities were needed. Twenty per cent of the bugs reached their maximum rate at 1.6 m./sec.; maxima were attained up to 6.7 m./sec. Maximum durations occurred from 0.5 to 7.0 m./sec., the total range used in these experiments. Except for one bug which gave a brief burst of strokes at around 400 minute, the greatest rate of swimming ob- served was 320 strokes/minute which was reached by half the animals ; they could not be induced to swim faster. If wind was blown on an animal from the side, it often responded with compensatory movements of the legs on the opposite side. Figure 1 shows rate and duration with increasing wind speed for three repre- sentative bugs. If the bug was holding an object, wind alone initiated swimming and consequent dropping of the stick in 25% of the cases. Usually, however, swimming occurred only when wind was combined with loss of substrate contact. Ordinarily loss of contact was the significant stimulus, but often the few bugs that would not swim with just loss of contact could be induced to do so if wind was simultaneously applied. A bug that had been swimming, but had stopped, would start again when wind was applied. REFLEXES IN GIANT WATER BUGS 119 In addition to loss of contact and wind, direct tactile stimulation, e.g. of the abdomen, and vibration or movement of the bug while suspended also caused swim- ming. Any movement, whether up and down or to and fro, and any vibration, caused either by tapping the stick to which the bug was attached or pounding the 70 300- in O E 250 5 V) u_ o UJ 5 cr ZOO ISO 100 \ OPENED WINGS FLIGHT POSITION OPENED WINGS -0 / ---o o X ^FLIGHT POSITION 2345 WIND VELOCITY ( m /sec) FIGURE 1. Graphs of rate and duration of swimming plotted against wind velocity for three representative bugs. Circled points indicate beginning of first noticeable flight preparation movements ; these were not observed in one animal. table with the fist, elicited the swimming. Fraenkel (1932) reports that flight in 1'cspa, Calliphora, Apis, Schistocerca, etc. resulted from a blow on the abdomen, and Diakonoff (1936) found that cockroaches flew if allowed to fall, a phenomenon he termed a "fall reflex.' 120 HUGH DINGLE Not too surprisingly, suspended bugs also swim when placed in water, although the swimming is very quickly adapting, lasting only a few seconds. Swimming can be further induced by directing a current at the bug, moving the animal through the water, or by taking the animal out of the water, but again the swim- ming is quickly adapting. By far the most rapid swimming comes when the bug is allowed to touch some object with its forelegs which it then attempts to grasp. This too adapts, but after a longer time. Swimming also follows on occasion when the bug presumably sees an object in front of it. A water jet directed at the bug from one side causes some compensatory movements of the legs on the opposite side. The same results are observed when the bug is rotated through the water in a small circle; this phenomenon was also recorded by Hughes (1958) in Dytiscus. Sense ore/cms mediating swimming Fraenkel (1932) found that his insects would not stop flying when their tarsi were removed, which led him to believe that a receptor sensitive to contact was located there. Diakonoff (1936), however, was unable to find sensilla on the tarsal claws of the cockroach and found that in addition to the tarsi, stimuli on the tibiae, femora, and even coxae could stop flight. Since water bugs swam on loss of substrate contact, presumably a mechanism similar to that eliciting flight in the above cases is involved. It was found that swimming ceased either with tarsal contact or with stimuli on the tibiae or femora. Touch receptors seem to be implicated in the instance of more rapid swimming when the bug touches an object with its forelegs. The leading edges of the femora of these bugs are covered with an extensive sensory area, and this area when touched is especially apt to elicit increased swimming. The exact nature of these receptors and others at the same spot affords a promising line of future investigation. Specific receptors, eliciting swimming in response to either air or water currents, have not been located. With the forehead, eyes, hair beds behind the eyes, hair beds at the bases of the fore femora, and hair beds at the junction of the pro- and mesothorax covered individually or all together with paraffin, the swim reflex did not appear to be hindered in any way. Although the antennae were not removed completely, located as they are in grooves under the eyes, the bugs still began to swim in currents after an operation to destroy the brain, indicating that neither the antennae nor for that matter any other head receptors innervated by the brain are mandatory for the initiation of this swimming. It is suspected that swimming in response to current can be initiated by any of several receptors located on the body. Certainly the body possesses many groups of hairs located at various joints and articulations, and that several of them may be "current receptors" is indicated by the fact that a bug will swim in a current coming from virtually any direction. When the bugs are in water, however, there do seem to be specific sense organs which initiate swimming. The first hint of such receptors came while a bug whose nervous connectives had been severed between the pro- and mesothoracic ganglia, the cut being made just posterior to the forelegs, was being studied. Such an insect loses all muscle tone posterior to the cut, and the legs hang limply. When this bug was put in water, the legs began to protract and retract slowly and rhyth- mically with enough force to give the bug some forward momentum. Further observation revealed that this swimming commenced only when the legs had floated REFLEXES IN GIANT WATER BUGS 121 up so as to be extended almost laterally from the body. Swimming was also observed when the bug was held upside down and the legs were in almost the same position as when floating, but this was never more than a few strokes. In attempts to locate more closely the receptors responsible for this swimming response, the leg segments and joints from all four swimming legs were removed successively with the following results : (1) Removal of first tarsal segment and joint between the two tarsal segments bug swam, but kept legs rather sharply bent at tibio-femoral joint. (2) Removal of second tarsal segment and tibio-tarsal joint bug swam with shorter and more rapid strokes. (3) Removal of tibia and tibio-femoral joint bug swam with short, rapid, and choppy strokes that were not well co-ordinated. These results seemed to indicate that the receptors responsible for the swimming response were located somewhere proximal to the tibia. Because of the flotation of the legs which seemed to be necessary, the location was suspected to be at either the coxo-trochanteral or femoro-trochanteral joint ; the former location appeared to be the more likely. Hair beds are located on the trochanters at this joint just distal to the trochanteral condyles (Fig. 2). When the legs hung down as they did when the bug was suspended, these hair beds were covered by membranous cuticular folds present on the coxae ; when the legs floated in water, the hair beds were uncovered. In bugs with the connectives severed between the pro- and mesothoracic ganglia, the trochanteral hair beds on various legs were burned with a hot needle. If these were destroyed on all four swimming legs, the bugs showed no response when placed in water ; if the hair beds on the middle legs were destroyed, the hind legs still swam, with the converse true if the hind leg hair beds were burned. In a bug lacking the hair bed on one middle leg, the other three legs swam in the usual fashion while the operated leg gave strokes on each alternate stroke of the rest; with the hair beds on three legs burned, only the single intact (hind) leg gave swimming strokes, and these were slower than previously. In bugs with the central nervous system intact, when the hair beds were destroyed on all four swimming legs, walking was more or less as usual, but the bugs seemed to have difficulty gaining traction on surfaces where normal animals had no difficulty. In both water and air, swimming strokes were short and jerky; in air, swimming proved also to be more difficult to induce than in normal individuals. These hair beds thus appear to be intimately involved with swimming and co-ordination of leg movements. The trochanteral hair beds are apparently excited by the cuticular folds which cover them when the legs hang do\vn or are folded beneath the body. As the legs float up when the bug is in water, these folds roll back progressively until the hair beds are uncovered when the legs are extended laterally. Presumably, then, when stimulation of the hair bed by the cuticular fold ceases, the leg begins to swim. Possibly direct contact with water prompts the swimming movements to some extent although this is not the only factor since inverted bugs with severed con- nectives also swim. Pringle (1938) described three hair plates on the leg of the cockroach, including one at the coxo-trochanteral joint, which he believed were also stimulated by a cuticular fold ; the hair plates were incompletely adapting. 122 HUGH DINGLE Because of the location and action of these sense organs, Pringle considered them "position" receptors. The action of the hair beds on the legs of the giant water hug seem to have an analogous function, i.e. registering the position of the legs until they finally reach swimming position, whereupon the swimming reflex is triggered. This proposed action of the hair beds helps to explain some aspects of the bugs' behavior. As mentioned earlier, a suspended bug tends not to swim when its legs are folded under the body as when grasping a stick. This lack of response would, on the above explanation, be due to the covering of the hair beds by the cuticular folds. In nature the bugs cling to submerged vegetation ; if they were torn free, the resultant flotation of the legs would provoke swimming and lead to regaining of foothold. tr 1-hb FIGURE 2. Ventral view of the coxo-trochanteral joint. The coxa and trochanter have been depressed dorsally as they would be if the leg were floating to expose the trochanteral condyles and hair beds. When the leg hangs down, the cuticular fold covers these two struc- tures ; the fold rolls back as the leg floats up. tr, trochanter ; ex, coxa ; thb, trochanteral hair bed ; tc, trochanteral condyles ; cf, cuticular fold. Vision also seems to affect swimming. If a suspended bug is rotated through the water in a tight circle, the inside legs show compensatory movements that oppose the direction of rotation. In a bug with its eyes covered, the compensatory move- ments are so reduced as to be almost negligible. Hughes (1958) found reduction in the compensatory movements of a rotated Dytiscus when the eyes were covered. FLIGHT Pre-flight behavior in giant water bugs follows a fairly elaborate and somewhat varied pattern. The first sign is usually scraping of the hind legs over the wings and depression of the abdomen. There then follows twitching of the legs, which in the more advanced stages can be quite violent ; this twitching is often accompanied by "shrugging" movements in which the pterothorax and abdomen are moved rapidly anterior-posterior at the articulation between the pro- and mesothorax. The wings can, at least from the author's observations, be opened at any stage of these preparations. REFLEXES IN GIANT WATER BUGS 123 This rather extensive pre-flight behavior is apparently necessary because of a ball and socket mechanism which locks the wings to the pterothorax (Lauck, 1959) ; this mechanism is illustrated in Figure 3. The ball protrudes posteriorly from the dorsal margin of the mesepimeron and inserts into the socket on the costal margin of the hemelytron ; the mesal border of the clavus matches the wing grooves on the postnotum. In order to open the wings, the bug must first release the ball and socket mechanism, which is probably accomplished, according to Lauck, by a combination of contractions of the third axillary muscle and the tergo-sternal prsc epm epm 3 ro sw sw FIGURE 3. Lcthoccnts: views of pterothcrax and hemelytron to show position of wing ball (wb) and wing socket (ws). The posterior margin of the clavus (cl) fits along the wing groove (wg). A: Pterothorax with wings on left side removed, prsc, prescutum ; ph, phragma ; sc, scutum; sc-scl, scuto-scutellum ; pN, postnotum; epm, epimeron; wb, wing ball; wg, wing groove ; T, tergite of abdomen. B : Ventral aspect of left hemelytron. ws, wing socket ; co, corium ; cl, clavus ; me, membrane ; we, wing clip. C : Diagram showing wing locking mechanism, he, hemelytron ; ra, respiratory apparatus ; ab, abdomen ; sw, swimming leg. Arrow points anteriorly. A and B redrawn from Lauck (1959) by permission of the publishers. Not drawn to same scale. muscles which levate the wings. The various violent leg twitchings, depressions of the abdomen, and oscillations of the body characteristic of the pre-flight behavior are apparently the result of attempts by the animal to get the wings unlocked. There is, however, another possible reason for the pre-flight movements. Krogh and Zeuthen (1941) note that lamellicorn beetles "pump" before flight; they measured the rise in temperature of the muscles during "pumping" and found that not until the temperature was at least 32 C. would the beetles fly. The flight tem- perature varied from 32 to 37. Poor fliers like the beetles needed higher body temperatures to fly than did sphingid moths which are quite active fliers. Since giant water bugs are relatively poor fliers, it is possible that the pre-flight movements raise the body temperature enough to fly. 124 HUGH DINGLE In spite of the extensive pre-flight behavior in most animals used, only a few actually flew ; of 44 suspended bugs, four flew while four more opened their wings, but did not fly. Several others showed a tendency to assume the flight position, but never reached the stage of opening the wings. The flight position is shown in Figure 4. The swimming legs are carried folded flat against the underside of the body, although not in this illustration ; the abdomen is depressed ; and the respiratory apparatus is fully extended and held erect. Those bugs that did fly were, with one exception, suspended for five minutes or longer and most of the time in winds of greater than 6 m./sec. Weis-Fogh (1956) found that in locusts wind speeds of greater than 2 m./sec. were necessary to initiate flight. FIGURE 4. (1) Suspended bug holding drinking straw as substrate contact. (2) Swim- ming bug; swimming legs are approximately at the end of the backstroke. (3) Bug in flight position; note position of swimming legs and respiratory apparatus compared with (2). (4) Bug with wings open ; again note position of swimming legs and respiratory apparatus. Once a bug had flown, the threshold for further flight or wing opening was lowered considerably. Flight could be stopped by bringing the bug into contact with the substrate and could usually be initiated again if the animal was suspended. If flight was not induced by suspension, it could then be initiated by putting the bug into a wind. The contact-loss of contact mechanisms is presumably similar to those mentioned above when discussing swimming. The stimulation of flight by wind is of some interest. It was found that a jet of compressed air delivered through a bit of glass tubing was most effective in promoting flight or wing opening (in bugs that had already flown or opened their wings ) when it was aimed directly at the bugs' heads from in front. In these bugs the wings invariably opened while the air jet was blowing on the head and would close when it was removed. If the area of the head above the beak and between the eyes was covered with paraffin, the response disappeared ; it reappeared when the paraffin was removed. This was true for all 8 of the bugs tested. Partial REFLEXES IN GIANT WATER BUGS 125 covering of the forehead with paraffin did not abolish the response ; so long as part of it was exposed, the response was maintained. Examination of a bug's head under the dissection microscope revealed that the area being considered was covered with fine hairs, virtually invisible to the naked eye, which are presumably responsible for the initiation of wing opening or flight when stimulated by air currents. Weis- Fogh (1956) found 5 paired groups of wind-sensitive hairs on the head of the locust which were sufficient for both the initiation and maintenance of flight, but were not necessary for either. Aside from the hair beds, flight in the locust could be initiated by loss of tarsal contact, which was also found to be true with giant water bugs, and could be maintained by wind on the moving wings, which was not observed in this study. In both bugs and locusts the direction of the wind im- pinging on the hair beds was not particularly important. INTERACTION OF FLIGHT AND SWIMMING There seems to be little doubt that the initial response of these insects to loss of substrate contact is swimming. As previously mentioned, with increased wind speed both rate and duration of swimming increased up to a point, which varied from one bug to another, and then decreased. At first it was thought that this was due to fatigue or adaptation, but careful observation of the bugs' behavior revealed that the most likely possibility was the inhibition of swimming by the pre-flight activities even in those bugs, the most usual, in which neither flight nor wing opening ever occurred. In the latter cases, however, the bugs often did assume flight position with the legs, abdomen, and respiratory apparatus (Fig. 4). Reduc- tion of swimming also occurred when the bugs were given successive bursts of wind at a constant speed (5.9 m./sec.), although it was not so marked. THE CENTRAL NERVOUS SYSTEM The anatomy of the giant water bug central nervous system reflects the general anatomy and habits of the bug. The sub-oesophageal and prothoracic ganglia are fused into one ganglion located between and slightly anterior to the bases of the coxae of the raptorial prothoracic legs which are innervated from this ganglion. The meso- and metathoracic ganglia are also fused into a common structure located between the bases of the mesothoracic legs. This ganglion innervates all four swimming legs and the wings. The brain and circumoesophageal connectives appear to be grossly similar to those of other insects. A bug with its brain destroyed (using a hot needle) moved about apparently quite normally. Closer observation, however, revealed certain rather distinctive abnormalities. For instance, when walking about, a brainless bug tended to lose its balance and fall over on its back when stepping over small objects; once on its back it had considerable difficulty righting itself, often being unable to do so. An intact animal would, when placed on its back, bridge up with its forelegs and give a hard kick with the middle and hind legs on one side pivoting over on the tip of the abdomen; a brainless bug, on the other hand, was unable to bridge as high with the forelegs or to use the swimming legs effectively to flip over. When placed in wind, the brainless bugs differed from the normal in two ways. First, they would swim for much longer periods, usually showing no signs of slowing 126 HUGH DINGLE down ; and second, they accepted a stick and thus ceased swimming much more readily. Roeder (1937) and Roeder ct al. (1960) note that the praying mantis also exhibits hyperactivity with the brain destroyed, walking until exhaustion. Bugs with only half the brain destroyed carried out the classic maneuver of circling to the intact side. Severing the connectives just behind the forelegs resulted in loss of tone in the swimming legs, but the legs continued to swim when the animal was placed in water, as noted above ; the forelegs often twitched for a time after the cut was made. DISCUSSION The fact that when the trochanteral hair bed on one mesothoracic leg was destroyed, that leg swam on the alternate strokes of its counterpart seems to indi- cate transfer of impulses from one side of the mesothoracic ganglion to the other. Rowe (1960) has shown electrically that such intraganglionic transfer occurs, while several authors (e.g. Diakonoff, 1936; Ten Gate, 1941; and Hughes, 1957) have behavioral evidence for it. Destroying the hair beds on both of a pair of swimming legs resulted in loss of activity of that pair while the other two con- tinued to swim. Thus, as was the case with Pringle (1940), the author was unable to demonstrate transfer of a reflex from one thoracic ganglion to another even though the meso- and metathoracic ganglia are, in this case, fused. Roeder (1937; see also Roeder, 1953) proposed a model for the operation of the insect central nervous system ; in this model the brain exercises inhibitory control over locomotion, in view of the locomotor hyperactivity of brainless insects. Since giant water bugs are also hyperactive when brainless, they appear consistent with Roeder's model. Bugs whose connectives had been severed posterior to the fused sub-oesophageal and prothoracic ganglia lost all muscle tone in the swimming legs, but because of their fusion, it was not possible to separate the two ganglia functionally. There is some evidence from studies on cockroaches (Diakonoff, 1936; Ten Gate, 1941; Chadwick, 1953) that the prothoracic ganglion may be essential for normal co-ordination. If Hemiptera are secondarily aquatic, then the swimming reflex of aquatic forms like the giant water bugs may be considered a modification of the flight reflex of exclusively terrestrial insects. The reflexes, under natural conditions, would be triggered by similar sets of circumstances. A floating water bug, for instance, is free of substrate contact, and a swimming reflex might result, particu- larly since the usual habit of the bug is to cling to floating vegetation. A falling terrestrial insect, on the other hand, is also free of substrate contact and generally flies. The two situations of floating and being air-borne are essentially the same, and the reflexes of a particular insect, be they swimming or flying, are modifications to suit the particular medium. The escape responses are similarly modified. Strong tactile stimulation, espe- cially of the abdomen (Fraenkel, 1932), causes terrestrial insects to leap off the substrate and fly. In the aquatic bugs tactile stimuli or vibrations result in violent swimming whether the animal is in water or suspended in air. But if the swimming reflex is a modification of the flight reflex, why then do the water bugs sometimes fly? There appear to be two major possibilities. First, the body posterior to the articulation of the pro- and mesothorax of a bug REFLEXES IN GIANT WATER BUGS 127 suspended in air hangs down at a rather sharp angle ; in water this part of the body is buoyed up. Diakonoff (1936) reports that movement at the pro-meso- thoracic articulation of the cockroach results in a "fall reflex" that elicits flight and is apparently due to stimulation of the numerous receptors at the articulation. A similar mechanism may stimulate flight in giant water bugs. Second, when the bugs are suspended in wind, the hair beds on the head, which have been shown to be receptors concerned with flight, are stimulated. This stimulation, if strong enough or if summation occurred, would presumably overcome the swimming reflex and elicit flight. One would predict, on the assumption that swimming with lack of substrate contact is a modification of a flight reflex, that it would be a fairly general adaptation among aquatic insects. This prediction appears to be largely true. Hughes (personal communication) has observed the swimming reflex in Hydro- pliilits and Dytiscns, and the author has found it in gyrinids, hydrophilids, dytiscids, corixids, and the genus Belostoina, as well as in the giant water bugs discussed here. Further investigations of the phenomenon in these groups are now in progress. The author wishes to thank Drs. L. B. Slobodkin and D. M. Maynard for their encouragement and for reading and criticising the manuscript. Dr. F. C. Evans kindly permitted the use of the E. S. George Reserve for light trapping, Mr. D. F. Owen supplied many of the animals used, and Mr. John Alley took the photographs in Figure 4. A special debt is owed to the author's wife, Geraldine Dingle, who has read the manuscript several times ; most of her many suggestions and criticisms have been included. This research was carried out while on a National Science Foundation Co- operative Graduate Fellowship. SUMMARY 1. Giant water bugs swim when suspended free of the substrate. This situation contrasts with that of terrestrial insects which fly when freely suspended. Swim- ming can be stopped by returning contact to the bugs. 2. Suspended bugs respond to wind with a general increase in rate and duration of swimming, followed by a decrease in both. 3. When bugs are in water, swimming is stimulated by a hair bed located on the trochanter at the coxo-trochanteral joint. These hair beds seem to be stimulated by cuticular folds which cover them when the legs hang down, but roll back and leave them uncovered when the legs float, resulting in swimming. 4. Flight or wing opening occurred with 8 of 44 suspended bugs. A hair bed on the head functions in both the maintenance and initiation of flight in response to wind. 5. The bugs possess an elaborate pre-flight behavior which is apparently necessary to unlock a ball and socket mechanism attaching the wings to the ptero- thorax. This pre-flight behavior inhibits swimming and causes the decline in rate and duration mentioned in (2) above. 6. In the central nervous system the sub-oesophageal and prothoracic ganglia are fused, as are the meso- and methathoracic ganglia. There is behavioral evi- 128 HUGH DINGLE dence for transmission of impulses across a ganglion, but not from one ganglion to another, even though the ganglia are fused. 7. There is evidence that the swimming reflex is a general phenomenon ; appar- ently it is an aquatic modification of the flight reflex. LITERATURE CITED CHADWICK, L. E., 1953. The flight muscles and their control. In : Roeder, K. D., Insect Physiology. New York, Wiley. Pp. 648-655. DIAKONOFF, A., 1936. Contributions to the knowledge of the fly reflexes and the static sense in Periplaneta americana L. Arch. Necrl. Physiol., 21 : 104-129. FRAENKEL, G., 1932. Untersuchungen iiber die Koordination von Reflexen und automatisch- nervosen Rhythmen bei Insekten. I. Die Flugreflexe der Insekten und ihre Koor- dination. Zcitschr. vergleich. Physiol., 16 : 371-393. HUGHES, G. M., 1952. The co-ordination of insect movements. I. The walking movements of insects. /. Exp. Biol, 29: 267-284. HUGHES, G. M., 1957. The co-ordination of insect movements. II. The effect of limb ampu- tation and the cutting of commissures in the cockroach (Blatta orientalis). J. Exp. Biol., 34 : 306-333. HUGHES, G. M., 1958. The co-ordination of insect movements. III. Swimming in Dytiscus, Hydrophilus, and a dragonfly nymph. /. Exp. Biol., 35 : 567-583. KROGH, AUGUST, AND ERIK ZEUTHEN, 1941. The mechanism of flight preparation in some insects. /. Exp. Biol., 18: 1-10. LAUCK, DAVID R., 1959. The locomotion of Lcthoccrus (Hemiptera, Belostomatidae). En-t. Soc. Amer. Annals, 52 : 93-99. PRINGLE, J. W. S., 1938. Proprioception in insects. III. The function of the hair sensilla at the joints. /. Exp. Biol., 15: 467-473. PRINGLE, J. W. S., 1940. The reflex mechanism of the insect leg. /. Exp. Biol., 17: 8-18. ROEDER, K. D., 1937. The control of tonus and locomotor activity in the praying mantis (Mantis religiosa L.). J. Exp. Zool., 76: 353-374. ROEDER, K. D., 1953. Reflex activity and ganglion function, hi : Roeder, K. D., Insect Physi- ology. New York, Wiley. Pp. 463-487. ROEDER, K. D., L. TOZIAN AND E. A. WEIANT, 1960. Endogenous nerve activity and behaviour in the mantis and cockroach. /. Ins. Physiol., 4 : 45-62. ROWE, E. C., 1960. Activity of single nerve cells in an insect thoracic ganglion. Anat. Rec., 137: 389. TEN CATE, J., 1941. Quelques remarques a propos de 1'innervation des mouvements locomo- toires de la Blatte (Periplaneta americana L.). Arch. Neerl. Physiol., 25: 401 109. WEIS-FOGH, T., 1956. Biology and physics of locust flight. IV. Notes on sensory mechanisms in locust flight. Phil. Trans. Roy. Soc., Ser. B, 239: 553-585. ION REGULATION IN TETRAHYMENA 1 PHILIP B. DUNHAM 2 AND F. M. CHILD Department of Zoology, University of Chicago, Chicago 37, III. Fresh water animals maintain their cells hyperosmotic to their environment (Prosser ct al., 1950). In higher animals this is accomplished by specialized organs or tissues (e.g. the vertebrate kidney, frog skin, and the anal papillae of dipteran larvae) which adjust the osmotic level of body fluids to a level isosmotic with the cells. Lower invertebrates (protozoa, sponges and coelenterates) have no osmotically regulated body fluids. Therefore all cells in fresh-water representatives of these groups have the problem of continuous water influx, and must have osmoregulatory ability (Kitching, 1954). A high potassium content relative to their medium is characteristic of all living cells that have been investigated. Most cells are richer in potassium than sodium, and there is often less sodium in cells than in the medium. Evidence has been offered that in such fresh-water animals as Hydra and Spirostonimn, inorganic ions are readily available for exchange with the environment (Lilly, 1955 ; Carter, 1957). Table I lists potassium and sodium levels in several fresh-water invertebrates. Therefore regulation of body volume in such forms involves regulation of ions as well as water. This paper reports an investigation of ionic regulation in Tctraliymena pyri- jonnis, a fresh-water ciliate. A remarkable ability to maintain a high potassium concentration as well as a lower sodium concentration in very dilute medium was found. Evidence for a sodium extrusion mechanism was also found. These findings will be discussed in terms of a model system for ion regulation in Tctra- hyincua, and in terms of relevance to similar problems in other animals. METHODS AND MATERIALS TctroJiyuiena pyriformis, strain W, was grown axenically in 2% proteose- peptone medium (hereafter called normal medium), the ion content of which is indicated in Table II. One-liter Roux culture bottles containing 500 ml. of medium were innoculated with 5 ml. of a culture in log phase of growth. After four days' growth at 22-25 C, the cells were concentrated approximately ten-fold by gentle centrifugation. After experimental treatment, which involved either dilu- tion of cell suspension in normal medium with distilled water or increasing medium 1 The methods reported in this paper were worked out initially by the junior author with the assistance of Miss Marina Dan. The results and hypotheses are the work of the senior author who gratefully acknowledges the help and criticism of Dr. H. B. Steinbach during the course of this study. The work has been supported by National Science Foundation grant G-4526 and U. S. Public Health Service grant RG-6879. 2 Trainee in United States Public Health Service Training Grant Program 2G-150. 129 130 PHILIP B. DUNHAM AND F. M. CHILD TABLE I K and Na (or osmolar) concentrations of some fresh water invertebrates. (See Willmer (1958) for a table of osmolar concentrations of some protozoans determined by a variety of methods.) Organism Concentration Method of determination Literature source Acanthamoeba (distilled water washed) K 26.9 (meq./l. cells) Na 14.3 elemental analysis Klein (1959) Spirostomum K 7 (meq./l. cell water) Na 1 equilibration with isotopic tracer Carter (1957) Tetrahymena (in 2% proteose- peptone) K 31.7 (meq./l. cells) Na 12.7 elemental analysis Dunham and Child (present report) Spongilla (summer) osmolarity equivalent to 27 meq. NaCl/1. cell water vapor pressure determination Zeuthen (1939) Pelmatohydra (whole animal) K 14.4 (meq./l. cell water) Na 2.7 equilibration with isotopic tracer Lilly (1955) Tubifex (whole animal) K 27.0 (meq. /kg. wet Na 23.4 weight) elemental analysis Dunham (unpublished) Anodonta (muscle) K 10.6 (meq. /kg. wet Na 5.2 weight) elemental analysis Hayes and Pelluet (1947) concentration by adding NaCl or KC1, cell suspensions were reconcentrated when necessary so that 10 ml. gave a packed cell volume of about 0.2 ml., as determined by centrifugation at a relative centrifugal force of 1600 to constant volume (10 minutes in 10-ml. Kolmer tubes). Dry weight of cells was determined by drying to constant weight at 60 C. Cell counts were made in a hemocytometer. Cells were extracted for ion analyses by suspending them in dilute acetic acid (1 drop glacial acetic acid in 10 ml. water), heating near boiling for 5 minutes and allowing them to stand for one hour. K and Na analyses were made with a Coleman model 21 flame photometer. Preliminary Cl analyses were made with an Aminco-Cotlove chloride titrator. Analysis of nitric acid digests of residues after extraction indicated that more than 98% of intracellular K, Na and Cl was extracted. Intracellular cation concentrations are expressed in meq./l. cells, after appropriate correction for extracellular space as determined by use of radioactive iodinated serum albumin (Risa) added immediately prior to centrifugation. Total exchangeability and kinetics of intracellular K and Na were determined using trace TABLE II A' and Na concentrations of Tetrahymena in 2% proteose-peptone (normal) medium. K, Na, and Cl concentrations of normal medium. Standard errors and number of determinations are given. Cells K Na Medium K Na Cl meq./l. cells 31.650.43 (44) 12.681.3S (44) mM 4.750.13 (60) 36.50.13 (70) 28.71.02 (6) ION REGULATION IN TETRAHYMENA 131 amounts of the appropriate radioisotope, K 42 or Na 24 , obtained from Oak Ridge National Laboratories as chlorides in HC1 solution, and neutralized with NaOH before use. Counts per minute of wet samples were determined with an end- window counter or a Nal-Tl crystal scintillation well counter. Per cent exchanges of intracellular K and Na were calculated from the specific activities of the cells and of the medium. 40 30 CT O> E oC ~ 10 OD Ko'.mM t 5 10 20 30 FIGURE 1. K and Na content of Tctrahymena in normal and diluted media. Ordinate: cellular concentrations of K and Na (meq./l. cells); abscissas: concentrations of K and Na in the medium (MI A/). Open circles: Ki ; solid circles: Nai. Points for Ki and Nai in normal medium (see arrows) are averages of 44 determinations; vertical lines delimit the total range of the determinations in normal medium. RESULTS Table II shows the K and Na content of cells in normal medium, and the K, Na and Cl content of normal medium. The volume and weight of an average cell in normal medium were 1.83 X 1(H ^1. and 1.97 X 1O 2 /*g., respectively, as determined from cell counts and packed cell volumes (corrected for Risa space). Dry weight of cells in normal medium was determined to be 19.4% of wet weight, so cells are 80.6% water. The percentage of Risa space in a volume of packed cells in normal medium was 15% (eight determinations ranging from 14% to 18%, SE = 0.57). This value was not significantly different for cells equilibrated with medium diluted eight-fold. 132 PHILIP B. DUNHAM AND F. M. CHILD 40 t initial 20 40 60 80 100 120 TIME IN MINUTES 140 160 180 300 FIGURE 2. Changes in cellular K and Na in Tetrahymena after dilution of normal medium. Ordinate: cellular K and Na concentrations (meq./l. cells); abscissa: time (minutes). Open symbols: Ki ; solid symbols: Nai. Initial Ki and Na ( (diamonds) are averages of values in normal medium. Dilutions were made at zero time. The extents of the dilutions of normal medium in the three experiments were as follows : triangles, 6-fold dilution ; circles, 8-fold dilution; squares, 13-fold dilution. In order to demonstrate how intracellular K and Na are maintained over a range of medium concentrations, cells were allowed to equilibrate for at least 30 minutes in various dilutions of normal medium from two-fold to over 100-fold, the extreme dilution involving several distilled water washes. Figure 1 shows 120 - 120 140 160 180 200 220 Normal K Normal Na K orNa :mM FIGURE 3. K and Na content of Tetrahymena in media concentrated with KC1 or NaCl. Ordinate: cellular concentrations of K and Na (meq./l. cells); abscissas: concentrations of K and Na in the media. Open circles: Ki ; solid circles: Na,. a and b : ordinate intercepts of the linearly increasing portions of the Na and K curves, respectively. Points for Ki and Nai in normal and diluted media are taken from Figure 1. ION REGULATION IN TETRAHYMENA 133 final intracellular concentrations of K and Na (Ki and Na t ) plotted against medium concentration (K and Na ). K, and Naj are quite constant over the range of medium dilutions investigated : average K 5 = 25.4 meq./l. cells and average Nai = 5.0 meq./l. cells; Ki/Naj = 5.1. Figure 2 shows the results of three experiments in which changes of Kj and Nai were followed after six-fold and greater dilutions of normal medium. Changes in Na; take place within the first 30 seconds, after which Nai is constant. Kj decreases very slowly after medium dilution, so it is difficult to assign an equilibrium level. Therefore it was arbitrarily decided that Ki values in cells in dilute medium more than 30 minutes would be reported with the reservation that time for equilibration may involve a matter of days. (In one experiment, K } in cells in medium diluted 20-fold for two days was about half normal KI.) Rates of decrease of IM in cells in dilute medium were never greater than 10% per hour, and generally were much slower. 2OO - o> o cr o> 50 100 TIME IN MINUTES 160 FIGURE 4. Changes in volume and Na content of Tetrahymena after increasing the NaCl concentration of normal medium. Ordinates : cell volume (open circles); Na content per unit number of cells (crosses) ; Na content per unit volume of cells (solid circles). Abscissa: time (minutes). NaCl concentration of normal medium was increased by 176 inM at zero time. Ki per unit number of cells at 160 minutes was not significantly different from initial Ki. Cells were equilibrated for 30-120 minutes in media made more concentrated than normal in either K or Na (added as chlorides). Figure 3 shows Kj and Na 4 values from these experiments plotted against K and Na , respectively. Both curves are linear above certain medium cation concentrations, with values of 0.51 for the slope of Ki./K,, above 11 inM K and 0.48 for the slope of Nai/Na above 20 mM Na . Below 20 mM Na , Na s is constant at 5.0 meq./l., whereas below 11 mM K , the K curve is roughly sigmoid. The kinetics of net influx of cation and the concomitant water movements were investigated by subjecting cells to sudden large increases in K or Na . Changes in packed cell volume, cation concentration per unit volume of cells, and cation concentration per unit number of cells were followed. Figures 4 and 5 show the results of subjecting cells to increases above normal of 176 mM NaCl and 137 mM KC1, respectively. In both cases there is a large initial influx of the 134 PHILIP B. DUNHAM AND F. M. CHILD elevated cation (shown by increase in //,eq./10 7 cells) and large efflux of water (4-5% cell shrinkage in each case) in the first 1.5 minutes. K t per cell increases initially about 1.75 times, and subsequently increases slowly to 4.5 times the initial level by 200 minutes, accompanied by water re-entry. Na 4 per cell increases initially to 6 times initial level, and subsequently slowly increases to 13.7 times initial level at 160 minutes, with water re-entry. So equilibration in both cases is fast, but much faster in high Na medium. When Na t increased, final Kj per cell was not significantly different from the initial value, and likewise for Na } per cell after 200 minutes when Kj increased. This lack of reciprocal changes means that increases in KI and Nai are accompanied by proportionate increases in 100 - 100 TIME IN MINUTES 200 FIGURE 5. Changes in cell volume and K content of Tetrahymcna after increasing KC1 concentration of normal medium. Ordinates : cell volume (open circles); K content per unit number of cells (crosses) ; K content per unit volume of cells, solid circles. Abscissa: time (minutes). KC1 concentration of normal medium was increased by 137 mM at zero time. per unit number of cells at 200 minutes was not significantly different from initial some anion, or decreases in some other cation, if electroneutrality is preserved within the cells. (In a few experiments in which both K and Na were increased, both Kj and Nai increased in the same way as when studied singly, as described.) Preliminary analyses of Cl content of cells show that Cl does not balance increases in KI or Naj. Figure 6 shows Clj values in cells in normal, dilute, and high NaCl and KC1 media. Clj is quite constant in media ranging from a 2-fold dilution (14 mM C1 ) up to 123 mM C1 (99 mM K ) and 73 mM C1 (83.5 mM Na ) : for 18 determinations, Clj averaged 6.4 meq./l. cells, ranging from to 13.2 meq./l. One set of determinations at 150 mM C1 (163 mM Na ) showed Qi as high as 37 meq./l., but Na ; was 78.5 meq./l. in this case. For this one deviant value, only one-half of Nai could be balanced by Cl, all other determinations showing no relationship at all between Clj and K 4 or Na^ Preliminary experiments were done on the washout of high K or high Na content of cells transferred to normal medium after one hour's equilibration in ION REGULATION IN TETRAHYMENA 135 tu _ 30 ^.20 - E 5 10 4 !_J!___! A O 4 i AA i 8 i i i 1 t 20 t 40 60 80 CI : mM 100 120 140 FIGURE 6. Cl content of Tctrahymcna in normal, diluted, and high NaCl and KC1 media. Ordinate: cellular concentrations of Cl (meq./l. cells) ; abscissa: Cl concentration of the media. Triangles: Ch in normal and diluted media; open circles: Ch in high KC1 media; solid circles: Ch in high NaCl media. Arrow indicates Cl concentration of normal medium. Hori- zontal line indicates average of all Ch values in medium Cl concentrates ranging from 17 mM to 123 mM. high K or Na medium. Cells were equilibrated in normal medium with the KC1 concentration increased by 65 mM. Ten minutes after washing with normal medium, Ki had decreased from 64 meq./l. to 57 meq./l. , and after 45 minutes to 48 meq./l. Initial Kj was 33 meq./l. Therefore a portion of elevated cellular K washed out much more slowly than it can be increased. This observation is consistent with the slow decrease of Kj from cells after dilution of normal medium. Cells were also equilibrated for one hour in normal medium plus 170 mM NaCl, then washed with normal medium. Ten minutes after washing, Na } was only slightly above normal Na ; , indicating that Na s is easily washed out of cells, as a portion of Na; is after dilution of normal medium. TABLE III Kinetics and extent of exchange of cellular K and Na with normal and diluted media containing trace amounts of K 42 or Na 24 . Each horizontal row of data represents at least two experiments with replicate determinations. Medium cone. (mM) Total cell cone, (meq./l. cells) Time for maximum exchange (minutes) Time for i maximum exchange (minutes) Exchangeable cell content (meq./l. cells) Unexchange- able cell content (meq./l. cells) Exchange- able fraction K, normal medium 4.75 31.6 180 30 29.2 2.4 92.5% K, diluted medium 0.83 27.4 180 30 25.0 2.4 91.0% Na, normal medium 36.5 12.7 120 <1 11.2 1.5 88% Na, diluted medium 3.0 5.5 120 3 3.25 2.25 59% 136 PHILIP B. DUNHAM AND F. M. CHILD Table III summarizes the results obtained when cells were exposed for 5 or more hours to normal and diluted media containing trace amounts of K 42 or Na 24 . Cellular K and Na are largely available for exchange with medium K and Na, and exchange is rapid, particularly the exchange of Na. However, small amounts of both K and Na do not exchange in a 5-hour period, although the exchange reaches a maximum level by three hours for IM and two hours for Na^ The amounts of unexchangeable IM and Na ; do not change significantly with medium dilution. DISCUSSION That Tctrahymena is capable of maintaining a high cellular K content relative to the K content of normal and diluted media is evident from Figure 1 and Table II. Ratios of Kj/Ko are of the order of 100 and higher in very dilute medium. Tetra- Jiynnena also retains a small amount of Na in very dilute medium. Sizable portions of cellular K and Na are exchangeable with the medium, as shown in Table III. Therefore K and Na are not retained in the cells by an impermeable membrane. Table III also shows, however, that small and constant amounts of both K and Na are unexchangeable. Since net changes are also evi- dent, exchange diffusion cannot be responsible for the ready exchange of isotopes. A system of internal binding sites with specific affinity for K is suggested to explain active K maintenance by Tctrahymcna. Na may be retained by a similar mechanism. In addition, a Na extrusion mechanism is proposed. Na in Tetrahymena is best explained in terms of a formal model involving compartmentalization of Nai. Two components of Na$ are constant, i.e. do not vary with Na . One, 1.9 meq./l., is unexchangeable with the medium. The second, 3,1 meq./l. , is held constant, but is rapidly exchangeable. Forty-eight per cent of cell volume is free "Na space," and is available to a mobile Na component which is freely diffusible, and proportionate to Na . However, this mobile component is maintained 20 meq./l. of water less than Na by Na extrusion. Below Na there is no mobile Na component and Na; is constant at 5 meq./l., but at 20 mAI Na the Na extrusion mechanism is operating maximally and mobile Nai begins increasing with Na . Since the other two Na components are constant, total Nat increase is linear and represents only the mobile component. There are no net exchanges between any of the compartments. These compartments can be visualized as physiological entities only, since no morphological significance can be attached to them. This model is suggested by the following points of evidence : ( 1 ) Na extrusion is indicated first, by the constant level of Na 4 up to 20 inM Na , and second, by the negative intercept on the ordinate axis of the linearly increasing portion of the Naj/Nao curve (indicated in Figure 3). (Permeability of the cells to Na precludes passive exclusion of Na.) (2) The mobility of Na^ above the constant 5 meq./l. is apparent from the rapid equilibration of cells with high Na medium, and the rapid washout of Na from cells both when normal medium is diluted and when high Na cells are washed in normal medium. (3) The linearity of the Nai/Na curve above 20 mM Na and the mobility of Nai above 5 meq./l. allow one to conclude that the slope of the Na curve, 0.48, represents the fraction of cell volume occupied by the mobile Na component, or "Na space." ION REGULATION IN TETRAHYMENA 137 (4) The magnitude of the gradient effected by maximum Na extrusion is pro- portional to the magnitude of the negative intercept (about 5 meq./l. cells) cor- rected for the constant amount of retained Nat (5 meq./l. cells), or 10 meq./l. cells. Since the Na space is 48%, the difference in Na concentration between medium and cell water is 10/0.48 = 20.8 meq./l. water. This value should and does corre- spond closely to the medium Na concentration at which the Na extrusion mecha- nism becomes saturated. This saturation concentration, the "threshold" of Na, increase, is analogous to the threshold of glucose excretion in renal tubules at the concentration of maximum glucose reabsorption (Shannon and Fisher, 1938), which was also interpreted as representing saturation of an accumulating mechanism. (5) The evidence for the unexchangeable and exchangeable but constant com- ponents of cellular Na has already been presented. The nature of the preservation of electroneutrality in the cells upon Na or K entry is not at all clear. There is definitely no reciprocal relationship between Na and K. There is little intracellular Cl, even upon large increases in the medium of either NaCl or KC1. These results also obviate explaining active retention of K in terms of a Donnan equilibrium resulting from Na extrusion, unless Cl is also specifically excluded from the cells, which seems unlikely at present. If a Donnan situation obtained, the relationship Kj/K = Cl /Cli would be expected in hold in any medium, and it obviously does not. With increasing medium KG. Kj/Ko decreases to less than 1, while Cl /Cli increases to as high as 22. The nature of the Cl exclusion is suggested here to be electrostatic rather than a matter of specificity or impermeability. Possibly cellular K and Na are associated with fixed anionic groups more or less strongly, depending on the ion and the medium concentration of the ion. Steinbach (1947) suggested that K is always associated with organic components which occupy spaces unavailable to Cl. This still does not explain the preservation of electroneutrality. This problem is under investigation. The KI/KO gradients in normal and diluted media are definite evidence for specific K retention. The rapid and nearly complete exchange of K ; and the rapid net increase in KI with K make membrane impermeability or any mem- brane involvement unlikely explanations for K retention. In media with K higher than normal, the slope of the Kj/K curve is steep up to 1 1 inM K , above which KI increases less sharply with K , and in a linear fashion. The slope of this portion of the curve is 0.51, close to the slope of the Nai/Na curve (0.48), suggesting that this increasing KI is occupying a cellular space iden- tical with the "Na space." However, a portion of this increased cellular K does not readily wash out of the cells, and is not nearly as mobile as high cellular Na. Cells equilibrate less rapidly with high K medium than with high Na medium. Increased Ki is not accompanied by reciprocal Na changes or by Clj increase, as noted above. These considerations suggest that the increase in K t involves association with previously "empty" K binding sites in the "Na space." (Steinbach (1940) reported K increase without Na decrease or Cl increase in Phascolosoina muscle. He suggested (1947) that vertebrate skeletal muscle behaves as though there were a limited number of groups capable of binding K which are normally saturated, whereas heart and invertebrate muscle are normally not saturated with K.) The slower rise of K, 138 PHILIP B. DUNHAM AND F. M. CHILD with K greater than 11 mM and the linearity of this rise suggest that the actively maintained, or bound, K is at a maximum above 11 mM K . Then the intercept of the linear portion of the Ki/K curve at the ordinate axis, 43 meq./l. (shown in Figure 3), should be the level of maximum actively maintained K. In cells equili- brated in high K medium, and washed in normal medium, Ki fell to 48 meq./l. after 45 minutes, a concentration comparable to the ordinate intercept of the linear KI/K O . This constitutes additional evidence for the maximum saturation of bound K. Initially it would appear that K maintenance in Tctrahymena, because of the sigmoid shape of the Ki/K curve below 11 mM K , does not fit elementary Langmuir adsorption theory (applicable to Michaelis enzyme kinetics), which it should if a system of binding sites with a saturation level is invoked. However, it is likely that this is not the true shape of the curve. The K washout experiment described above shows that K i; although it can be rapidly increased, can be only slowly washed out. Since the initial K in the experiments involving changing K was always that of normal medium, an inflection in the Ki/K curve is expected at the K of normal medium, and it is observed (see Figure 2). Therefore the explanation of the sigmoid shape of the curve lies not in the nature of the K retention mechanism, but in the experimental procedure. Then the real relation- ship between actively maintained K and K should fit a Langmuir adsorption iso- therm, and a binding sites mechanism for K retention is consistent with the data. No comparable obscuring factor exists in the case of Na s since Na apparently washes in and out of cells with equal facility in the range of medium concentra- tions investigated. The Na retention system is apparently saturated at a very low Na . The data suggest compartmentalization of K as well as Na : unexchangeable K (2.4 meq./l.), exchangeable but bound K (maximum about 43 meq./l.), and freely diffusing K, with no threshold K . (See Cowie, Roberts and Roberts, 1949, for a discussion of compartmentalization of K in E. coli.) The hypothesized ion regulatory machinery of Tctrahymena, shown to be con- sistent with the data, consists, first, of a system of internal binding sites which specifically accumulate and retain K ; second, a system for retention of a constant, low level of Na, and third, a Na extrusion mechanism. Cl plays little role in ion balance in Tctrahymena. Cellular K and Na are separable into three components : unexchangeable, exchangeable but bound, and mobile components. The Na extrusion mechanism may facilitate water removal, and therefore may be associated with the contractile vacuole. The water economy of Tctrahymena may be analogous to that of other fresh-water animals, in that Tctrahymena may not be capable of secreting pure water, but water removal may be facilitated by ion secretion (cf. Prosser et al., 1950). The relationship of Na retention to Na extrusion and/or K retention, cannot be decided from the data. Retained Na may represent lack of complete specificity of the K binding sites, but in this case a reciprocal relationship between actively retained K and Na would be expected. The retained Na might be a reservoir necessary for vacuolar function. This possibility is consistent with the rapid Na turnover indicated by rapid exchange, but cannot be easily resolved with the constancy of the retained Na relative to Na . A third possibility is a specific protoplasmic requirement for a low, constant Na level, for which there is no ION REGULATION IN TETRAHYMENA 139 evidence here and little precedent. (Hydra (Lenhoff and Bovaird, 1960) and Chilomonas (Pace, 1941) have possible specific Na requirements.) The specificity of K retention in Tetrahymena indicates a protoplasmic K re- quirement. (Kidder ct al. (1951) demonstrated a nutritional requirement for K in Tetrahymena.} The similarity of levels of K in Tetrahymena and the other animals listed in Table I suggests a minimum requisite protoplasmic level of K. It is often held that high cellular K is only a reflection of a Donnan equilibrium resulting from Na extrusion (see Hodgkin, 1951). Carter (1957) attributes K maintenance in Spirostomum to Na exclusion. This has been shown here not to be so in Tetrahymena, and Robertson (1957) has shown that in a number of ani- mals, including some marine invertebrates, a portion of cellular K cannot be accounted for by a Donnan equilibrium, but must be due to specific K retention. Steinbach (1947) suggests that cellular K is regulated relative to a constant proto- plasmic composition rather than to serve an osmoregulatory function. No doubt in marine animals and vertebrates, with their relatively high ionic content, some cellular K is held non-specifically to preserve electroneutrality. Cellular K levels vary considerably, particularly among fresh-water animals, and probably reflect the ability to regulate body fluids, and to an extent the activity of the animal. Therefore in vertebrate cells, there may be a K component reflecting a Donnan equilibrium plus a component serving a specific protoplasmic role. Since evolu- tion of basic cellular mechanisms is generally conservative, a similarity between K retention mechanisms in Tetrahymena and other animals is an attractive possibility. Tetrahymena affords a system for studying this mechanism without high ion con- centrations and large Donnan effects, which would obscure specific K retention in other animals. Akita (1941) reported data on Na, K and Cl contents of Paramecium which were comparable to the data presented above on Tetrahymena. SUMMARY 1. The K and Na content of Tetrahymena pyriformis has been determined, and the mechanisms of ionic regulation were investigated. 2. The main findings were : K and a small amount of Na are maintained in very dilute medium. Cellular K and Na are readily exchangeable with K and Na of the medium. However, small, constant amounts of each are unexchangeable. Cells rapidly equilibrate with media high in K or Na. High K washes out of cells slowly, whereas Na enters and washes out of cells with equal facility. There is no reciprocal relationship between cellular K and Na. Tetrahymena contains little Cl. Increases in cellular K or Na are not accompanied by increases in Cl. 3. The results are interpretable according to the following proposals : K is specifically accumulated and retained by a system of internal binding sites with a saturation level. Na is probably retained by a separate mechanism. There is also a Na extrusion mechanism which has no relationship with K or Na retention. Cellular K and Na are compartmentalized into three components : unexchangeable, exchangeable but bound, and freely diffusible components. LITERATURE CITED AKITA, Y. K., 1941. Electrolytes in Paramecium. Mem. Fac. Sci. Agric., Taihoku hup. U., 23 : 99-120. 140 PHILIP B. DUNHAM AND F. M. CHILD CARTER, L., 1957. Ionic regulation in the ciliate Spirostomwn ambiguum. J. Exp. Biol., 34: 71-84. COWIE, D. B., R. B. ROBERTS AND I. Z. ROBERTS, 1949. Potassium metabolism in E. coli. I. Permeability to sodium and potassium ions. /. Cell. Comp. Physiol., 34 : 243-257. HAYES, F. R., AND D. PELLUET, 1947. Electrolytes in mollusc blood and muscle. /. Mar. Biol. Assoc., 26 : 580-589. HODGKIN, A. L., 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Rev., 26: 339-409. KIDDER, G. W., V. C. DEWEY AND R. E. PARKS, 1951. Studies on the inorganic requirements of Tctrahymcna. Physiol. Zool., 24 : 69-75. KITCHING, J. A., 1954. Osmoregulation and ionic regulation in animals without kidneys. Symp. Soc. Exp. Biol., 8 : 63-75. KLEIN, R. L., 1959. Transmembrane flux of K 42 in Acanthamocba. J. Cell. Comp. Physiol., 53 : 241-258. LENHOFF, H. M., AND J. BOVAIRD, 1960. The requirement of trace amounts of environmental sodium for the growth and development of Hydra. Exp. Cell Res., 20 : 384-394. LILLY, S. J., 1955. Osmoregulation and ionic regulation in Hydra. J. Exp. Biol., 32: 423-439. PACE, D. M., 1941. The effects of sodium and potassium on metabolic processes in Chilomonas paramccium. J. Cell. Comp. Physiol., 18 : 243-255. PROSSER. C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. J. WULFF, 1950. Com- partive Animal Physiology. W. B. Saunders Co., Philadelphia. ROBERTSON, J. D., 1957. Osmotic and ionic regulation in aquatic invertebrates. In : Inverte- brate Physiology. B. Scheer, ed., University of Oregon Publications, Eugene, Oregon, pp. 229-2*46. SHANNON, J. A., AND S. FISHER, 1938. The renal tubular reabsorption of glucose in the normal dog. Amcr. J. Physiol, 122 : 765-774. STEINBACH, H. B., 1940. The distribution of electrolytes in Phascolosoma muscle. Biol. Bull. 78: 444-453. STEINBACH, H. B., 1947. Intracellular inorganic ions and muscle action. Ann. N. Y. Acad. Sci., 47 : 849-874. WILLMER, E. N., 1958. Some further factors affecting the metaplasia of an amoeba (Naeglcria (intbcri). J. Embryol. Exp. Morph., 6: 187-213. ZEUTHEN, G., 1939. On the hibernation of Spongilla lacustris (L.). Zeitschr. vergl. Physiol. 26: 537-547. FURTHER STUDIES ON ALLOCENTROTUS FRAGILIS, A DEEP-SEA ECHINOID 1 ARTHUR C. GIESE Department of Biological Sciences, Stanford University, Stanford, California In a previous paper some data on the natural history and breeding of a deep- sea echinoid, Alloccntrotus jragilis, were presented (Boolootian ct al., 1959). Further studies are reported here, primarily to define more clearly the breeding season of the species, as well as to get further information on its nutrition. BREEDING SEASON The breeding season in the previous study appeared to coincide with winter (December to March) but remained uncertain because storms on Monterey Bay during the critical period interfered with collecting at the time the boat was avail- able. For the present study the urchins were collected from the same beds and by the same methods as previously, and the gonad index (the ratio of gonad volume to wet weight times 100) was used to estimate the breeding condition of the speci- men as before. The breeding activity has now been followed for almost three years, and although the data for each year are incomplete, pieced together for the entire period in Figure 1, they give support to the notion that a single breeding season occurs in this species. Perhaps several periods of spawning and redevelopment of eggs occur in a given individual of this species but monthly sampling does not give information on this point. However, the appearance of germinal vesicles in spawned-out ovaries suggests just this. It proved impossible to keep specimens in healthy condition in the laboratory for more than about a month, although it was noticed that the red-spot "disease" was much less frequent in animals kept in aquaria in the dark (Araki, personal communication). It may prove possible to keep the animals in the laboratory for a longer time once the most favorable con- ditions are discovered. More decisive evidence for an annual cycle is obtained from a study of teased pieces of the gonads and from attempts to fertilize the eggs. Such a study indicates that although the gonad index may be high and sperms may appear in September, October and November, the eggs are almost all in the germinal vesicle stage, each with a large nucleus. Such eggs do not mature after shedding and in no case are they fertilized on addition of active and presumably mature sperm. In December most of the females had ripe eggs and only occasional germinal vesicles were seen among them. The eggs fertilized and developed into normal plutei. The same was found to be true during February and March. Some of the females examined 1 1 am indebted to Messrs. George Araki, Peter Glynn and Joseph Balusteri for collecting the Allocentrotus; to Messrs. John Lawrence and James Stanley for help with some of the chemical determinations ; and to Mr. Albert Towle for help with some of the respirometric determinations. 141 142 ARTHUR C. GIESE had few eggs but those eggs which remained in the ovaries fertilized and developed normally, suggesting that they were only a remnant, the bulk of the gametes having been released. During the period immediately following the breeding season, few females had eggs and germinal vesicles again became apparent in some. Thereafter, all the teased gonads examined microscopically appeared indeterminate as to sex. Apparently the tissue had entered a resting stage. The gonads remain indeter- X LU Q 4 Q 1957 1958 l959 I960 GERMINAL * VESICLES FERTILE EGGS INDETERMINATE N D F M MONTH M FIGURE 1. Reproductive cycle of Allocentrotus fragilis as measured by the gonad index (size of gonad relative to body weight) and presence and ripeness of the gametes. The gametes were studied closely only during 1959-60. minate for several months but at the end of summer sperms can be seen and small germinal vesicles again make their appearance, long before the gametes form. Active sperms are present over a much wider span of time than mature eggs. NUTRITION Since the intestines of Allocentrotus brought in from the field are sometimes devoid of food, and at other times have very little, it would appear that the urchins may go for long periods of time without food. This seems likely since defecation may continue for a week or more in the laboratory, indicating slow digestion of an ample meal. In the previous study, on only one occasion were we fortunate enough STUDIES ON A DEEP-SEA URCHIN 143 to obtain specimens richly charged with food when a diatom bloom occurred in the area. In the present study many collections yielded animals relatively full of diatoms, presumably because of similar blooms. On November 7, 1960, a collection was made nearer the edge of the urchin bed in an attempt to get smaller specimens for a study of respiration. The intestines of these specimens were rilled with bites out of large algae : green, red and brown. The fragments of algae were irregular and much larger than the balls of diatoms illustrated in the previous paper. In the foregut the algae were undigested and had little of the gelatinous material around them. In the hindgut more fully digested algae were enclosed in the mass of gelatinous material within which were TABLE I Chemical composition of gonads and gametes of Allocentrotus fragilis in per cent of dry weight Tissue Condition Lipid Non-protein N Protein Glycogen Testis gravid 14.54 15.34 13.08 3.26 3.62 4.24 28.70 30.25 31.93 0.36 Testis starved animal 18.6 17.7 Testis spent 12.36 3.66 36.13 Sperm 3.60 4.22 38.7 Gonad indeterminate 14.50 17.85 3.65 3.66 23.88 27.05 0.83 Ovary gravid 17.79 14.61 15.01 3.40 4.40 2.94 34.09 32.47 27.39 0.69 Ovary starved animal 20.8 20.0 Ovary spent 12.83 3.17 20.87 Eggs 18.07 2.44 28.68 many bacteria, much as previously described in Strongylocentrotus purpuratus (Lasker and Giese, 1954). Fecal pellets collected from animals which had been in the laboratory aquaria overnight retained their shape and looked more like the rounded pellets previously described from A. fragilis. The algal fragments in many of them were almost completely digested, only colorless pieces remaining. In only one previous collection of Allocentrotus had individuals with pieces of larger algae (Cladophora) in the gut been obtained, all the others having diatoms and fragments of various minute materials present at the bottom of the sea. The November 7th collection followed rough seas which may have torn algae from the rich offshore beds nearby, making them available to the urchins. When Allocentrotus, kept in the laboratory without food for a month, were 144 ARTHUR C. GIESE dissected, they were found to be free of intestinal contents. Since in some col- lections urchins were observed free of food, it is likely that in their natural environ- ment they are occasionally unable to get food for a comparable period of time. A store of nutrients is therefore necessary to maintain the urchins between the sporadic droppings of material from the surface waters. The sea urchin has three major organs in which storage might occur : gonads, intestine and body wall. The latter (hereafter called the test) consists of the test proper, the epidermis, the tissues of the coelomic lining and the water vascular system attached to it. Biochemical analyses 2 TABLE II Chemical composition of intestine, intestinal contents, and shell of sea urchins in per cent of dry weight Tissue Condition Lipid Non-protein N Protein Allocentrotus fragilis Intestine well-fed 28.88 2.17 35.38 26.81 3.13 31.22 23.24 2.77 38.84 starved 20.6 Contents fresh meal 8.28 1.66 22.83 feces (rectal) 1.87 ' 0.33 7.15 Test well-fed 1.79 0.22 5.66 starved 0.9 0.8 - Diatoms* entire 8 28.1 Strongylocentrotus purpuratus Foregut well-fed 12.28 3.84 39.33 Hindgut well-fed 12.52 3.30 33.94 Contents fresh meal 3.88 0.34 10.70 Strongylocentrotus franciscanus Gut well-fed 22.2 18.7 * From Pease, 1932; 63.2% carbohydrate present in diatoms. indicated that the fragile urchin, like the purple sea urchin (Giese et al., 1958), stores considerable lipid and a small amount of glycogen in its tissues, as seen in the data in Tables I and II. Protein, the main structural constituent of protoplasm, is also present in considerable quantity, as expected in any tissue. It would appear that the main reserve food is lipid, glycogen being a minor reserve. Since relatively little sugar appears in the body fluid, the latter finding is perhaps not surprising. 2 The methods employed were like those described elsewhere (Giese et al., 1958). Many of the measurements were done in triplicate ; later only duplicates were run since the repeats were so much alike. STUDIES ON A DEEP-SEA URCHIN 145 The data in Table I show that considerable lipid is present in the gonads of Allocentrotus, a bit more in the gravid than in the spent ones. There is more lipid in the eggs than in the ovary but relatively little in the sperms taken alone. With- out comment, the data in Table I do not express fully the meaning of the changes in the organic content of the gonads of the animals during the breeding season. The gonad index varies by a factor of at least 5 (Fig. 1), the maximal variability being 9-fold. Therefore, the lipid content per unit dry weight of a gonad is not a true measure of the reserves, since the shrunken gonad of a spent or indeterminate gonad may be only one-fifth the size of the gravid gonad. The total lipid present in the gonad of a gravid animal would be at least 5 times as great as in a spent animal. TABLE III Wet and dry -weight of tissues and tests* Tissue Wet wt. Dry wt. % solid % water GI** % body fluid Allocentrotus fragilis Whole 35.6 3.8 10.6 89.4 2.06 58 Whole 66.9 6.25 9.4 90.6 Whole 96.6 11.38 11.8 88.2 2.63 62 Gonad (cf) 2.54 0.51 20 80 Gut 2.1 0.66 31.3 68.7 Lantern 1.75 1.06 60 40 Test 22.7 7.45 32.8 67.2 Strongylocentrotus purpuratus Whole 98.3 32.55 33.2 66.8 8.6 22.3 Whole 64.6 24.5 38 62 10.83 30.3 Gonad ( 9 ) 7 1.45 20.6 79.4 Gut 2.64 0.95 36 64 Lantern 2.2 1.6 73 27 Test 3.2 20 61 39 * Some data for Strongylocentrotus are given for comparison to Allocentrotus. Note the more massive skeleton in Strongylocentrotus, its lesser water content, and the lesser amount of body fluid. ** GI refers to gonad index (defined in the introduction). Lipid is stored in quantity in the intestine and body wall. Analyses indicate that as much as 29% of the dry weight of the gut may consist of lipid, but only about 2% of dry weight of the test and its tissues consists of lipid. While the per cent lipid content of the test appears small, it must be remembered that the test forms a considerable part of the entire dry weight of the urchin (Table III). An urchin which when wet weighs 96.6 grams, weighs only 11.38 grams when dry, including the body fluid salts, indicating that 88.2% of its wet weight is water. The dried gonads weigh 0.51 gram and the intestines, washed free of gut contents and dried, about 0.66 gram. The dry test and lantern weigh 9.5 grams. According to the data in Table II about 1/20 of the test (5.66%) is protein, which is probably an approximate measure of the amount of tissue present. Therefore, 146 ARTHUR C. GIESE about 5.66% X 9.5 grams, or 0.54 gram of tissue, is probably present in the test. The amount of tissue in the test is thus probably equal to, or greater than that in the gut (no account was taken of the other organic constituents in the above cal- culations). It is possible that some protein forms a network in the test, in the interstices of which the salts are deposited, since the echinoderm test is supposedly a mesodermal structure like vertebrate bone (Hyman, 1955). To compare the relative stores of lipids in gonad, test tissue, and gut, it is only necessary to multiply the amount of each tissue by its content of lipid. In this respect it appears that 9.5 grams X 1.79% or 170 mg. are stored in the test and lantern, 0.5 grams X 15.5% or 77 mg. are stored in the gonad, and 0.66 grams X 26.8% or 176 mg. are stored in the gut of an animal which weighs 96.6 grams wet weight. This shows that in regard to its storage of lipid, the test and intestine may be of equal importance in an animal of intermediate gonad index (2.63) such as the one tested here. For an animal with an index of 5 the amount of lipid stored in the gonad would be increased by a factor of almost 2, making it about equal to the gut or test. For an animal of low index (1.5 to 0.8) the amount of lipid stored in the gonad would be reduced by a factor of 1/2 or 1/3, and the stores in the intestine and the tissues of the body wall would then be of even greater importance. Since so much lipid is stored in the intestinal walls, it seems most likely that the food is either the source of the lipid or that the lipid is manufactured from carbohydrates or protein in the diet. The gut contents vary in lipid, a relatively fresh meal of diatoms containing 8.28% of lipid, whereas a well-digested mass of material contains only 1.87% of lipid (Table I). Pease (1932) lists the lipid content of diatoms as 8% of the dry weight. a It therefore seems likely that the lipid probably is obtained from the diatoms and other food eaten by the urchin. The lipid is probably digested and stored, some of it in the gut, some in the other tissues. In this respect it is interesting that another animal feeding upon diatoms, the sipunculid worm Phascolosoma agassisi, also has large stores of lipid in its gut, about 25% of the dry weight (A. Towle, personal communication). On the other hand, the data in Table II show that the purple sea urchin, Strongylocentrotus purpuratus, which feeds upon larger algae, stores only half as much lipid in its gut as AllocentrotHs. Nonetheless, lipid is prominent and a fairly large amount is present in the intestinal pellets, 3.88% of the dry weight in a fresh meal in the intestine. It is surprising in this regard that 6\ franciscanus, which has a diet much like 5\ purpuratus, has much more lipid stored in its intestine (Table II). Algae may at times also accumulate considerable lipid (Milner, 1953; Fogg and Collyer, 1954). Perhaps S. franciscanus has a diet richer in lipids than S. pur- puratus. How the nutrient gets from the gut to the other tissues is not known at present. Lipid may pass out as small droplets of fatty material like the chylomicrons of mammals or it might be carried out and distributed by wandering cells. Lipid is present in the body fluid but the exact amount has not been determined for lack of adequate methods. 3 Diatoms in culture do not always have this much lipid. According to Barker (1935) diatoms in a culture in the laboratory first synthesize carbohydrates during photosynthesis, the ratio of oxygen production to carbon dioxide consumption being unity. However, as the diatoms age they accumulate oil which is visible in droplets. STUDIES ON A DEEP-SEA URCHIN 147 Upon starvation, stored nutrients are utilized. This was most evident in the shrinkage of the gonads of starved animals. Seven Allocentrotus starved for a month in an aquarium supplied with running sea water showed somewhat shrunken gonads at least the index for the animals at the time of collection on November 6 was 3.2 a month later one would expect it to have risen to about 4.7 ; instead the gonad index for the starved animals was 2.5 on December 7. Similar shrinkage had previously been noted for the purple shore urchin (Lasker and Giese, 1954). It is probable that the nutrients stored in the gonads had been resorbed. While it seemed likely that the lipids were preferentially utilized, biochemical analysis revealed that per unit weight lipid increased in amount in the gonad, although it decreased both in total quantity in the animal and per unit weight in the body wall and the intestine. While the increase in lipid content per unit weight in the gonad may appear paradoxical, it is to be expected if one or more of the other nutrients in the gonad are utilized at a more rapid rate than the lipids. Since the total bulk of the gonad shrinks and the only other major organic material present in the gonad TABLE IV Respiration of tissues of Allocentrotus* Tissue Qo^liS /il./mg./hr. R.Q. Water content per cent Number of experiments Test 0.076 0.58 65.6 2 Testis 0.570 0.92 80.4 3 Ovary Gut 0.088 0.383 0.65 0.57 80.8 79.2 8 10 * Note the high respiratory rate for the testis and gut as compared to the ovary and test. is protein, proteins are probably being selectively metabolized in the gonads during the period of starvation. Wilber (1947) has described similar results after starva- tion of Phascolosoma gouldii. RESPIRATION A few studies were made of the respiration of Allocentrotus tissues, primarily with a view of determining how it compared with other marine animals. The respiratory quotient was determined to ascertain, if possible, what types of foods were being used by the urchins. The data are given in Table IV. It is at once apparent that the rate of tissue respiration is comparable, per unit wet weight, to that for other sea animals (Nicol, 1960, p. 152). Of greater interest is the respiratory quotient characterizing the respiration of the sea urchin. If lipid is of importance in the economy of the sea urchin, a respiratory quotient of about 0.7 might be expected. If the urchin uses carbohydrates or mixtures of these with proteins and lipids, the respiratory quotient should be higher, approaching 1.0 when only carbohydrate is utilized. Determination of the respiratory quotient of entire animals proves difficult because at the end of an experiment it is necessary to liberate the carbon dioxide which is trapped in the buffering system of the sea water bathing the urchins. Since to do this the sea urchins must be removed from the vessel, the extra manipulations 148 ARTHUR C. GIESE may permit the sea water to equilibrate with the air. Furthermore, the urchins shed some spines, pedicellariae or other skeletal pieces containing lime salts. Con- sequently, it is necessary to filter such water through bolting cloth to get rid of the calcareous materials. This involves still another step during which equilibration of the bathing water can occur with air, further vitiating the correction. The R.Q. for an entire animal small enough to fit into a Warburg flask was 0.7, suggesting lipid utilization. However, if any carbon dioxide had accumulated in the sea water during respiration, the manipulations preceding addition of acid and measurement might have liberated it, favoring a lower R.Q. value. Therefore, the data cannot be considered satisfactory since the method is unsatisfactory. To by-pass this difficulty the gonad and gut tissues of the urchins were removed from animals, washed in sterile sea water, and the pressure changes measured manometrically in the presence of KOH in one series, and in the absence of KOH in another. In the latter case 3 N sulfuric acid was contained in the side arms, and at the end of the experiment the acid was added to liberate the excess carbon dioxide contained in the sea water surrounding the tissues. The data for respira- tion of the tissues in Table IV are therefore more satisfactory than those for the entire animal. Excepting the testes, the R.Q. for the tissues is between 0.6 and 0.7, definitely suggesting lipid metabolism. Since the experimental deficiencies mentioned for the studies on the entire animal do not apply, the data on tissues are more convincing than those for the entire animal. Presumably the respiration of the entire animal is the sum of the respiration of its various parts (Field ct ol., 1939). Consequently, one might suppose that the data for the tissues are appli- cable to the entire sea urchin. The high R.Q. for the testes could result from utilization of carbohydrate along with some other nutrients. DISCUSSION The present study suggests that lipid may play a significant role in the economy of Allocentrotus. The sea urchin has a considerable supply of lipid in its usual diet of diatoms. It stores considerable lipid in its intestine and gonad and some even in the tissues adherent to the skeleton. Furthermore, the stores of lipids decrease in amount when the sea urchin is starved for a month. The amount of lipid is thus closely related to the nutritive state of the animal. This has proven to be the case in other echinoderms from this area (Giese, 1959) and in other regions as well (unpublished). The small amount of glycogen found in tissues of Allocentrotus suggests that either some other kind of carbohydrate is stored in this urchin or else that carbo- hydrate plays a minor role here. Glucose does not increase the respiration of gut or gonadal tissue here, just as it failed to do in tissues of a purple sea urchin. The respiratory quotient for the tissues studied gut, test and ovary is about 0.6 to 0.7. This indicates that some lipid is being used for respiration ; that is, it is being metabolized. Addition of glucose does not change the R.Q. It thus appears possible that lipid is being used preferentially for metabolism although it is more likely that added sugar fails to stimulate respiration because it fails to enter the tissues. In view of the apparent reliance of Allocentrotus on lipids, its occasional eating of large algae red, brown and green which have little lipid, but much polysac- STUDIES ON A DEEP-SEA URCHIN 149 charide, is interesting. Either Allocentrotus uses only the readily digestible ma- terials in the algae or it has enzymes to utilize some polysaccharides, as does the purple sea urchin (Huang and Giese, 1958; Eppley and Lasker, 1959). The algae in the fecal pellets of Allocentrotus feeding on large algae were rather completely digested, suggesting that more than the lipids are being utilized. It would be interesting to know whether the urchin itself has such enzymes, and secondly, whether the monosaccharides obtained are stored as polysaccharides in the urchin or are converted to lipid. The observation that Allocentrotus takes in large algae indicates that it is more resourceful than had been previously considered (Boolootian ct al., 1959). In the laboratory it failed to eat the large algae but apparently it does so when it gets them in nature. While Allocentrotus can withstand starvation at least a month, and probably longer, judging from the healthy appearance of the specimens starved (in the dark) for over a month, in nature this is probably not the rule since dissected animals usually had at least a few pellets in the gut. Specimens choked with pellets of algae at the time they were collected had nothing whatsoever in the gut. No clear-cut correlation between the breeding season and the availability of food has been observed in the monthly collections. An annual reproductive cycle is suggested by the present study but the stimulus to the development of the gonads still remains elusive. SUMMARY 1. Additional data on the size of the gonads relative to the body and the presence and ripeness of gametes and their maturity were gathered for a year on the deep-sea echinoid, Allocentrotus jragilis. 2. The data indicate that the breeding season is an annual cycle with a maximum gonadal size in January and February, accompanied by the presence of mature eggs and sperm. 3. The sea urchins in one collection were found to have fed on large algae, the pellets resembling those of the intertidal sea urchin, Strongylocentrotiis purpuratus. 4. A considerable quantity of lipid is found to be stored in the wall of the gut, less in the gonad, and still less in the body wall (per unit dry weight). Total amount of stored lipid is largest in the gut, next in the test, and least in the gonad. 5. The usual diatom diet of the sea urchin contains much fat; the algae tested in one series contain considerably less. 6. The respiratory quotient of the gut, ovary and test of the sea urchin was found to be about 0.6 to 0.7, suggesting utilization of lipids. The R.O. for the testis was 0.92. 7. Some comparisons are made between Allocentrotus and the purple intertidal urchin, Strongylocentrotus purpuratus. LITERATURE CITED BARKER, H. A., 1935. Photosynthesis in diatoms. Arch. Mikrobiol., 6: 141-156. BOOLOOTIAN, R. A., A. C. GIESE, J. S. TUCKER AND A. FARMANFARMAIAN, 1959. A contribution to the biology of a deep-sea echinoid Allocentrotus fragilis (Jackson). Biol. Bull., 116: 362-372. 150 ARTHUR C. GIESE EPPLEY, R. W., AND R. LASKER, 1959. Alginase in the sea urchin, Strongylocentrotus pur- puratus. Science, 129: 214-215. FIELD, J., II, H. S. BELDING AND A. W. MARTIN, 1939. An analysis of the relation between basal metabolism and summated tissue respiration in the rat. /. Cell. Comp. Physiol, 14: 143-157. FOGG, C. E., AND D. M. COLLYER, 1954. Accumulation of fats as a characteristic of certain classes of algae. Congr. intern, hot. P/u is, !\' I '* .1 *'*.~fcv'"' v ^ * t| >* Wv L t t ff / uf m- <- ^ ". rs. - , , " ' ,.**' 2^ *"\ FIGURES 1-5. Daily photographic sequence of autograft (left) and homograft (right) scales on the first through fifth days after transplantation at 28 C. The row of three homo- SCALE HOMOGRAFT SURVIVAL 165 RESULTS Temperature effect Groups of fish each bearing autografts and homografts were maintained at temperatures of 7, 14, 21, and 28 C. All except the 7 C. group, which was kept in a refrigerator, were in circulating sea water. The scale grafts were inspected daily and photographed at frequent intervals to determine as precisely as possible the time of melanophore fragmentation. The lower the temperature the greater variation there was in the end point. At 28 C., incipient breakdown of pigment cells was detectable two days after operation, but not until the third day had all melanophores been destroyed. At this temperature, the reaction is relatively abrupt. Homotransplants of fish at 21 C. underwent pigment cell disintegration on the fifth and sixth days after grafting. Those maintained at 14 C. required 14 to 16 days to break down. At 7 C. the pigment cells of the homografts remained intact for 26 days at which time it was necessary to terminate the experiment. In all groups of fish, the autograft scales remained healthy indefinitely. Sf>lcncctoin\' In four fish, the spleens were removed via a ventral incision on the day prior to scale grafting. Four controls were subjected to sham operations. In all fish, controls and experimentals, the homografts broke down on the third day. The results are consistent with those of Vogel (1940) who noted that splenectomy failed to protect skin homografts in Rana pipiens from destruction. Hypophysectoiny Animals were deprived of their pituitaries, or subjected to sham operations, two days before scales were grafted. This operation did not enhance the survival of homografts. In 12 hypophysectomized fish and 13 controls, the homografts exhibited breakdown of pigment cells on the third day. Trypan blue Four experimental animals received scale grafts on the day of the first intra- peritoneal injection of 0.1 ml. of \% trypan blue in distilled water. Injections were repeated daily. Control fish similarly grafted were injected intraperitoneally with 0.1 ml. distilled water daily. On the third day there was complete break- down of pigment cells in the homografts of both control and experimental groups, despite the fact that the treated fish had become intensely stained with dye. Xitclcic acid antagonists The injection of substances which inhibit nucleic acid synthesis proved to be very successful in protecting the homograft scales from the antibody response of graft scales exhibited pigment cell breakdown on the third day, with subsequent disappearance of the pigment granules. Autografts remained intact throughout. 10 X. FIGURE 6. A normal, expanded, scale melanophore showing typical binucleate condi- tion. 1000X. FIGURES 7-9. Appearance of scales before, during and after onset of homograft re- action. 100 X. 166 RICHARD J. GOSS the host. In two separate experimental series in which a total of eight fish were given 1 mg. of 5-fluorouracil each daily, starting on the day of operation, the homografts showed no signs of pigment cell breakdown as long as the fish sur- vived. This dose, although effective, was at the same time toxic and resulted in the deaths of animals after two to six days. Nevertheless, four fish still alive on the fifth day possessed intact scale homografts, and it would appear that their transplants would have survived longer had the hosts lived. Similar experimental series on eight fish injected with 2 mg. of 5-fluorodeoxyuridine (FUDR) likewise resulted in protection of the homografts for up to eight days, by which time five of the animals had died as a result of the toxic effects of the drug. Injection of 2 mg. of 6-mercaptopurine into each of four fish bearing homograft scales afforded protection for four days at which time the injections were discontinued. Thereafter, melanophores were gradually destroyed until only one intact homograft scale re- mained on the seventh day, when the experiment was terminated. From these results it is clear that near-lethal doses of these drugs effectively prolong the survival time of homografts. In the cases of all three substances, when the doses were reduced to 1/100 of the above levels, no protection whatever was observed. Adrenal cortical hormones Daily intraperitoneal injection of a 1 mg. suspension of cortisone acetate to grafted fish starting on the day of operation had no beneficial effect on the survival of the homografts. On the third day there was complete breakdown of all scale pigment cells, probably due to inadequate doses of cortisone. More potent prepa- rations of cortical hormones, however, proved to be more effective. Injections of 2 mgf. of 6-fluorohvdrocortisone actetate on the day of transplantation and on o ^ J r the two days thereafter resulted in the survival of homografts for four days, at which time about half of all the pigment cells were undergoing fragmentation. This nevertheless marks a definite delay in the destruction of the grafts. In a third experiment, 2 mg. of delta- 1 -by clrocortisone sodium succinate were injected daily through the second day after operation. In these fish, no pigment cell break- down was observed on the third day, an incipient destruction was noted on the fourth, and, after five days, disintegration was well progressed in all except one animal in which the homografts remained intact. This compound, therefore, exerted a distinct protective effect. Antibiotics Chloramphenicol sodium succinate was tested at two dose levels. Daily injection of 1 mg. per animal intraperitoneally had no detectable effect on the survival time of the homografts. Similar injections of 10 mg. chloramphenicol, however, resulted in survival of homograft pigment cells beyond the third day. On the fourth day there was breakdown of melanophores in the grafts of one fish, and on the next day extensive, but still not complete, disintegration of foreign pigment cells had occurred. All were destroyed by the sixth day. Tetracycline hydrochloride was likewise tested at two different dosages. Injec- tion of 0.1 mg. per fish gave no protective effect. Administration of 1 mg. of tetra- cycline per day through the third day resulted in prolonged survival of homograft SCALE HOMOGRAFT SURVIVAL 167 pigment cells. In three fish the grafts were destroyed on the fourth day, and in three others they broke down on the fifth day. In two animals the pigment cells of homograft scales were still intact on the seventh day when the experiment was terminated. These antibiotics, therefore, interfere with the immunological response of the host against foreign grafts. Amino acid analogues Six different analogues of amino acids were tested for possible interferences with the homograft reaction. In general, they proved to be rather toxic and not very effective in protecting the foreign scale grafts from destruction by the host. In all cases, at least two dose levels were tried, the larger usually representing the limits of solubility in 0.1 ml. distilled water. Two analogues of serine were TABLE I Summary of effects of antimetabolites, administered to hosts, on survival of scale homografts at 28 C. Dose Survival time Substance injected (IP) (mg. /fish/day) (days) Controls 3 5-fluorouracil 0.01 3 1.0* 5 + 5-fluorodeoxyuridine 0.02 3 2.0* 6-8 6-mercaptopurine 0.2 3 2.0 5-7 Cortisone 1.0 3 6-fluorohydrocortisone 2.0 4-5 Delta-1-hydrocortisone 2.0 5 Chloramphenicol 1.0 3 10.0 4-6 Tetracycline 0.1 3 1.0 4-7 /3-2-thienylserine 1.0 3 5.0 3-4 DL-/3-phenylserine 1.0 3 5.0* 3-4 DL-a-CH'j phenylserine 1.0 3-4 3.3 3-4 /3-2-thienylalanine 1.0 3 5.0 4 DL-|S-phenyllactic acid 1.0 3 2.5* 3 5.0* 4 10.0* Ethionine 1.0* 3-4 3.3* 5 * Lethal dose. 168 RICHARD J. GOSS used. /?-2-thienylserine and DL-/3-phenylserine were injected in doses of 1 mg. and 5 mg. into groups of four fish. The smaller dose in all cases was ineffectual ; the larger dose resulted in survival of homograft pigment cells for four days. None of these doses was lethal except in one fish which died on the third day following injections with 5 mg. of DL-/?-phenylserine. Analogues of phenylalanine included DL-a-CH 3 phenylalanine which was administered in doses of 1 mg. and 3.3 mg. per day to groups of four fish. In all cases, foreign pigment cell breakdown was initiated on the third day but was not complete until the fourth. There was no detectable difference between the effects of the two doses utilized, nor did either dose prove to be lethal. /3-2-thienylalanine was injected in doses of 1 mg. and 5 mg. daily. Only the larger dose prolonged the survival of scale homografts (to the fourth day). DL-/3-phenyllactic acid, also an analogue of phenylalanine, was given in four different doses to four groups of fish. Doses of 1 mg. and 2.5 mg. failed to protect the homografts from destruction on the third day. A single injection of 5 mg. to another group of fish on the day of transplantation killed two fish the next day, but enabled the homografts of the remaining two animals to survive to the fourth clay before being destroyed. Administration of 10 mg. of this com- pound proved lethal to all fish within one day. A final analogue, ethionine, was tested at levels of 1 mg. and 3.3 mg. The lesser dose permitted homografts to survive until the fourth day ; 3.3 mg. per day through the second day after trans- plantation resulted in survival of homografts for two days beyond the controls. The latter dose, however, was lethal to three out of four fish by the fourth day after operation, at which time the foreign pigment cells were still intact. The one fish still alive on the fifth day exhibited complete breakdown of its homograft melanophores at that time. Ethionine therefore was considerably more effective in enhancing the survival of homografts than were the other five amino acid ana- logues tested. DISCUSSION The immunological reaction leading to homograft destruction has been divided into three phases (Billingham, Brent and Medawar, 1956), involving the release of graft antigens (afferent phase), the production of antibodies (central phase), and the reaction of antibodies with the graft (efferent phase). Although inter- ruption at any one of these levels would insure homograft survival, it is the central phase which is most amenable to experimentation. The process of antibody pro- duction may in turn be partitioned into subsidiary processes, leading from the initiating influence of the antigen on antibody-producing cells (induction or adapta- tion phase) to the eventual fabrication of antigen-specific antibodies (production phase). The method by which antibodies are formed is essentially a problem of protein synthesis with the added prerequisite that the protein antibody be capable of reacting specifically with the antigen originally responsible for initiating its formation. The synthesis of such specific proteins necessarily involves the participa- tion of a system by which the nature of the specificity can be communicated from the antigen to the molecular architecture of the antibody concomitant with its synthesis. There is reason to believe that nucleic acids constitute such a communi- cation system. This is substantiated by the dependent relationships of protein synthesis on RNA and of specific RNA synthesis on DNA. If this system is to remain sufficiently labile to adapt to new modes of protein synthesis (e.g., specific SCALE HOMOGRAFT SURVIVAL 169 antigen-stimulated antibody production) it is necessary to assume that new specific types of RNA molecules can be synthesized on demand. This requirement may be taken to indicate that RNA synthesis is necessary for the production of specific proteins. Schweet and Owen (1957) have postulated that antigen reacts with BNA which in turn makes specific RNA, and that the RNA acts as template in giving rise to specific antibodies. It is not surprising, therefore, that analogues of purines (6-MP) and pyrimi- dines ( 5-fluorouracil and 5-fluorodeoxyuridine), which interfere with nucleic acid synthesis, likewise arrest antibody production and thus result in the prolonged survival of homografts on treated hosts. Studies on 8-azaguanine. a purine ana- logue, have shown that it also inhibits nucleic acid synthesis (Skipper et a!., 1951) and antibody production ( Malmgren, Bennison and McKinley, 1952; Button, Button and George. 1958). Berenbaum (1960) demonstrated that 6-MP also inhibits the production of antibodies, and Schwartz and Bameshek (1960) and Meeker ct al. (1960) have reported the protection of skin homografts in rabbits by the administration of 6-MP. It is generally acknowledged that many antibiotics exert their growth-limiting effects by inhibiting protein synthesis, either directly or indirectly. Chloram- phenicol, for example, has been noted to prevent the synthesis of BNA (Brakulic and Errera, 1959; Schneider, Cassir and Chordikian, 1960), RNA (Gros and Gros, 1956; Webster, 1957), and protein (LePage, 1953; Smith, 1953; Pardee and Prestidge, 1956; Webster, 1957; Gale, 1958) in bacteria and mammalian tissues. Because of such manifold effects of chloramphenicol, and probably other antibiotics as well, their interference with antibody production and the homograft reaction is not unreasonable. Amino acid analogues, in so far as they have been tested, were generally less effective in protecting homografts from destruction than were the other agents already discussed. There is evidence that [3-2- and /?-3-thienylalanine inhibit anti- body formation in the rat (Ferger and du Vigneaud, 1949; Wissler et al., 1956). and thymidine uptake in BNA is inhibited by /3-2-thienylalanine and ethionine (Schneider, Cassir and Chordikian, 1960). Amino acid analogues are generally agreed (Matthews, 1958; Shive and Skinner, 1958) to act either by preventing protein synthesis via interference with the utilization of natural ammo acids or by becoming incorporated themselves into proteins, thus displacing their normal counterparts. Of the amino acid analogues studied in the present investigation, at least ethionine and /3-2-thienylalanine have been shown to act in the latter fashion (Levine and Tarver, 1957; Munro and Clark, 1958; Munier and Cohen, 1959). Structurally defective proteins would be expected not to be biologically inactive unless the incorporated analogues occupied an indispensable position. Since con- siderable portions of protein molecules are known to be functionally superfluous, the relative ineffectiveness of amino acid analogues in promoting homograft sur- vival may find an explanation along these lines of reasoning. The ability of cortisone to protect homografts from immunological destruction is too well known to require elaboration (Morgan, 1951; Billingham, Krohn and Medawar, 1951; Krohn, 1954; Medawar and Sparrow, 1956; Scothorne, 1956; Hamer and Krohn, 1959). This hormone also depresses antibody production (Germuth and Ottinger, 1950; Kass and Finland, 1953; Berglund, 1956) and 170 RICHARD J. GOSS inhibits nucleic acid synthesis (Skipper ct al., 1951). It has been claimed that these effects of cortisone are augmented by its interference with the release of antigens during the afferent phase of the homograft reaction (Billingham, Krohn, and Medawar, 1951; Medawar and Sparrow, 1956; Scothorne, 1956). In view of the well documented evidence in favor of the efficacy of cortisone in suppressing the homograft reaction, plus the demonstrated effectiveness of the more potent preparations (6-fluorohydrocortisone and Delta- 1-hydrocortisone), the failure of cortisone to enhance the survival of scale homografts in the present experiments may reasonably be ascribed to insufficient dosages. With reference to the mode of action of the various agents found effective in promoting extended survival of homografts, it could be argued that such results might be attributed to nonspecific toxicities rather than to effects directly related to the inhibition of antibody synthesis. Although some of the drugs tested proved to be fatal at effective doses, there is little reason to conclude that their efficacy resulted directly from their lethality per sc. The majority of the compounds which prolonged homograft survival manifested no other toxic effects during the period of treatment. Moreover, in the case of DL-/3-phenyllactic acid, a dose of 2.5 nig. was lethal without being effective in precluding the homograft reaction. Additional treatments not reported here have also failed to interfere with foreign tissue rejec- tion at otherwise lethal doses. Thus, while inhibition of nucleic acid or protein synthesis may be fatal, other kinds of toxicity need not interfere with immunological mechanisms. The accumulated evidence supports the contention that homograft rejection may be subject to a moratorium in the absence of the successful synthesis of nucleic acids and/or proteins. On the basis of the limited number of compounds tested, there is reason to expect that numerous other agents with comparable physiological properties might exert similar influences. Granted that there are numerous factors which inhibit antibody production and thus actually or potentially interfere with the homograft reaction, it remains to be demonstrated conclusively whether such effects are permanent or temporary. In their investigations of the beneficial effects of 6-MP on skin homograft survival in rabbits, Meeker ct al. (1960) noted that sustained treatment was necessary to insure continued survival of the grafts. In the present experiments, a comparable conclusion seems to be indicated, for despite the survival of scale homografts in treated hosts beyond the control period, eventual though dilatory breakdown was the rule. Notwithstanding these preliminary observations, it remains as a theoretical possibility that a specific tolerance might be conferred upon an adult host exposed to a foreign antigen by selectively inacti- vating those antibody-synthesizing pathways specifically stimulated by the antigen. If, as Burnet (1959) contends, antibody-producing clones are descended from specific cells stimulated to proliferate by exposure to antigen, then the application of treatments designed to render such cells vulnerable to destruction or inactivation at this critical period should, perforce, result in an animal subsequently tolerant to the original antigen. Alternatively, if antibody production can continue irrespec- tive of whether or not the involved cells are stimulated to proliferate, specific tolerance could be realized only by permanently and selectively incapacitating the biochemical pathways by which the specifically stimulated antibodies are synthe- sized. To achieve this without doing violence to any other mechanism of protein synthesis will be a challenging enterprise. SCALE HOMOGRAFT SURVIVAL 171 The author extends his thanks to Miss Marsha Rankin for technical assistance, and to Drs. David A. Karnofsky and Charles E. Wilde, Jr. for their generous donations of base analogues and amino acid analogues, respectively. SUMMARY 1. At 28 C., the melanocytes on scale homografts in Funditlus are destroyed in three days by the immunological response of the host. This reaction is slower to occur at progressively lower temperatures, but is not adversely affected by splenectomy or hypophysectomy of the host, nor by daily injections of trypan blue. 2. Survival of homografts was enhanced by daily intraperitoneal injections of base analogues, potent preparations of adrenal cortical hormones, antibiotics and amino acid analogues. 3. These results are taken to indicate that the inductive and productive phases of antibody formation are particularly vulnerable to agents which interfere with protein and/or nucleic acid synthesis. LITERATURE CITED BERENBAUM, M. C., 1960. Effect of cytotoxic agents on antibody production. Nature, 185 : 167-168. BERGLUND, K., 1956. Studies on factors which condition the effect of cortisone on antibody production 1. The significance of time of hormone administration in primary homolysin response. Acta Path. Micro. Scand., 38: 311-328. BILLINGHAM, R. E., L. BRENT AND P. B. MEDAWAR, 1956. 'Enhancement' in normal homografts with a note on its possible mechanism. Transpl. Bull., 3 : 84-88. BILLINGHAM, R. E., P. L. KROHN AND P. B. MEDAWAR, 1951. Effect of cortisone on survival of skin homografts in rabbits. Brit. Mcd. J ., 1: 1157-1163. BRENT, L., J. B. BROWN AND P. B. MEDAWAR, 1959. Skin transplantation immunity in relation to hypersensitivity reactions of the delayed type. /;; : "Biological Problems of Graft- ing," Colloque de 1'Universite de Liege, 12 : 64-78. BURNET, M., 1959. The Clonal Selection Theory of Acquired Immunity. Vanderbilt Univer- sity Press, Nashville, Tennessee. DRAKULIC, M., AND M. ERRERA, 1959. Chloramphenicol-sensitive DNA synthesis in normal and irradiated bacteria. Biochiin. Biopliys. Acta, 31 : 459-463. BUTTON, R. W., A. H. BUTTON AND M. GEORGE, 1958. Effect of 8-azaguanine on antibody synthesis in vitro. Nature, 182 : 1377-1378. FERGER, M. F., AND V. DU VIGNEAUD, 1949. Antiphenylalanine effect of /32-thienylalanine for the rat. /. Biol. Chan., 179: 61-65. GALE, E. F., 1958. The mode of action of chloramphenicol. In: Ciba Foundation Symposium on "Amino Acids and Peptides with Antimetabolic Activity," (Wolstenholme and O'Connor, eds. ), pp. 19-34. GERMUTH, F. G., AND B. OTTINGER, 1950. Effect of 17-hydroxy-ll-dehydrocorticosterone (compound E) and of ACTH on Arthus reaction and antibody formation in the rabbit. Proc. Soc. Exp. Biol. Mcd., 74: 815-823. GORER, P. A., 1960. Interactions between sessile and humoral antibodies in homograft reactions. In: Ciba Foundation Symposium on "Cellular Aspects of Immunity," (Wolstenholme and O'Connor, eds.), pp. 330-347. GROS, F., AND F. GROS, 1956. Role des aminoacides dans la synthese des acides nucleiques chez Eschcrichia coli. Biochim. Biophys. Acta, 22: 200-201. HAMER, J. B., AND P. L. KROHN, 1959. The influence of ACTH and cortisol upon skin homografts in the rat. /. Endocrinol., 18: 85-88. HILDEMANN, W. H., 1956. Scale transplantation in goldfish (Carassius auratits). Transpl. Bull., 3 : 67-68. HILDEMANN, W. H., 1957a. Early onset of the homograft reaction. Transpl. Bull., 3 : 144-145. 172 RICHARD J. GOSS HILDEMANN, W. H., 1957b. Scale homotransplantation in goldfish (Carassius auratns). Ann. N. Y. Acad, Sci., 64: 775-791. HILDEMANN, W. H., 1958. Tissue transplantation immunity in goldfish. Immunol., 1 : 46-53. HILDEMANN, W. H., AND R. HAAS, 1960. Comparative studies of homotransplantation in fishes. /. Cell. Comp. Physiol, 55: 227-233. KASS, E. H., AND M. FINLAND, 1953. Adrenocortical hormones in infection and immunity. Ann. Rev. Microbiol, 7: 361-388. KROHN, P., 1954. Transpl Bull., 1: 20-21. LAWRENCE, H. S., 1960. Some biological and immunological properties of transfer factor. In: Ciba Foundation Symposium on "Cellular Aspects of Immunity," (Wolstenholme and O'Connor, eds.), pp. 243-279. LEPAGE, G. A., 1953. Effects of chloratnphenicol on incorporation of glycine-2-C 14 into mam- malian tumor cell proteins and purines. Proc. Soc. E.vp. Biol. Mcd., 83 : 724-726. LEVINE, M., AND H. TARVER, 1957. Studies on ethionine. III. Incorporation of ethionine into rat proteins. /. Biol. Chcm.. 192: 835-850. MALMGREN, R. A., B. E. BENNISON AND T. W. McKiNLEY, JR., 1952. The effect of guanazolo on antibody formation. /. Nat. Cancer lust., 12 : 807-818. MATTHEWS, R. E. F., 1958. Biosynthetic incorporation of metabolic analogues. Pharmacol. Rev., 10: 359-406. MEDAWAR, P. B., AND E. M. SPARROW, 1956. The effect of adrenocortical hormones, adreno- corticotrophic hormone and pregnancy on skin transplantation immunity in mice. /. EndocrinoL, 14: 240-256. MEEKER, W. R., JR., R. M. CONDIE, R. A. GOOD AND R. L. VARCO, 1960. Alteration of the homograft response by antimetabolites. Ann. N. Y. Acad. Sci., 87: 203-213. MORGAN, J. A., 1951. The influence of cortisone on the survival of homografts of skin in the rabbit. Surge ry, 30 : 506-515. MUNIER, R., AND G. N. COHEN, 1959. Incorporation d'analogues structuraux d'aminoacides dans les proteines bacteriennes au cours de leur synthese in vivo. Biochim. Biophys Acta, 31 : 378-391. MUNRO, H. N., AND C. M. CLARK, 1958. The action of thienylalanine on protein and ribo- nucleic-acid synthesis in liver slices. Biochim. Biophys. Acta, 27 : 648-649. PARDEE, A. B., AND L. S. PRESTIDGE, 1956. The dependence of nucleic acid synthesis on the presence of amino acids in Eschcrichia coli. J. Bacterial., 71 : 677-683. SCHNEIDER, J. H., R. CASSIR AND F. CHORDIKIAN, 1960. Studies on the incorporation of thymidine into DNA by rat-liver homogenates in vitro. Biochim. Biophys. Acta, 42: 225-229. SCHWARTZ, R., AND W. DAMESHEK, 1960. The effects of 6-mercaptopurine on homograft reactions. /. Clin. Invest., 39 : 952-958. SCHWEET, R. S., AND R. D. OWEN, 1957. Concepts of protein synthesis in relation to antibody formation. /. Cell. Comp. Physiol.. 50 (Suppl. 1) : 199-228. SCOTHORNE, R. J., 1956. The effect of cortisone on the cellular changes in the regional lymph node draining skin homograft. Transpl. Bull., 3: 13-14. SHIVE, W., AND C. G. SKINNER, 1958. Metabolic antagonists. Ann. Rev. Biochem., 27: 654-678. SKIPPER, H. E., J. H. MITCHELL, JR., L. L. BENNETT, JR., M. A. NEWTON, L. SIMPSON AND M. EDISOX, 1951. Observations on inhibition of nucleic acid synthesis resulting from administration of nitrogen mustard, urethane, colchicine, 2,6-diaminopurine, 8-aza- guanine, potassium arsenite, and cortisone. Cancer Res., 11:145-149. SMITH, G. N., 1953. The possible modes of action of chloromycetin. Bacterial. Rev., 17: 19-29. TRIPLETT, E. L., AND S. BARRYMORE, 1960. Tissue specificity in embryonic and adult Cymato- gastcr aggrcgata studied by scale transplantation. Biol. Bull., 118: 463-471. VOGEL, H. H., JR., 1940. Autoplastic and homoplastic transplantation of skin in adult Rana pipicns Schreber. /. E.rp. ZooL, 85: 437-474. WEBSTER, G. C., 1957. Inhibition of polynucleotide metabolism by inhibitors or protein synthesis. Arch. Biochem. Biophys., 68: 403-411. WISSLER, R. W., L. F. FRAZIER, K. H. SOULES, P. BARKER AND E. C. BRISTOW, III, 1956. The acute effects of beta 3 thienylalanine in the adult male albino rat ; observations on nitrogen balance, antibody formation, and tumor growth. Arch. Path., 62: 62-73. THE LIFE-CYCLE OF PORPHYRA TENERA IN VITRO HIDEO IWASAKIi Haskins Laboratories, 305 East 43rd Street, Nczc York 17, N. Y. Cultivation of the red sea- weed Porpliyra tcncra was started in Japan several centuries ago. It is now the largest industrial cultivation of any marine product. Despite this success, more knowledge of the life-cycle and innate potencies of Porpliyra is needed to improve methods of cultivation to bring them under a control comparable to that achieved in land agriculture. At present this goal is unrealizable to its fullest extent. Mass-scale use of artificial fertilizers on 50 square miles of bays is uneconomical. But improvements in production and control of seeding, genetic improvement of the plant in respect to greater production and resistance to parasites, and perhaps extension of the growth period of the thallus, seem attainable goals. Several obstacles have slowed research on the life-cycle and potencies of Por- pliyra. Until a few years ago only one part of the life-cycle was known : mysteri- ously, the bays abound in monospores in the autumn ; these monospores, collected on bamboo or cord nets, develop into the edible, leafy thallus which is periodically harvested until March, when it fruits and disintegrates while producing carpospores. What was happening to the carpospores, and the origin of the monospores, were unknown. Those mysteries were solved after Drew (1949) discovered that the carpospores of another species, Porph\ra uuibilicalis, germinated into a filament which, in enriched sea water, produced a flimsy, sickly mat. The fact that the germ tubes produced by the carpospores are very similar to those of fungal spores, and that the older filaments of the mat are generally abnormal in appearance sug- gested to her that a specific host or a special substrate are needed for normal growth. Indeed several molluscan shells and even egg shells proved an excellent substrate. The filamentous thallus grows well in the shells, forming colonies identical with Concliocclis rosca ; C. rosca is obviously merely a phase of the life- cycle of Porpliyra. Kurogi (1953) and Tseng and Chang (1954) found that the carpospores of Porpliyra tcncra behaved similarly. Kurogi (1953) studied the growth of the carpospores of Porpliyra uuibilicalis pro.v., P. snborbicnlata, P. pscudolincaris and P. tcncra ; these form "'ConcJwcclis" colonies in the shells which can hardly be told from one another. In Kurogi's cultures the "ConcJwcclis" phase cultured on glass slides produced monosporangiate branches but not free mono- spores. However, from ConcJwcclis in oyster shells Kurogi (1953) obtained monospores which produced germlings of the leafy thallus. The complete life-cycle of Porpliyra was now known. This discovery renewed interest in the biology of Porpliyra, especially the conditions for growth (Iwasaki and Matsudaira, 1958) and production of monospores (Kurogi and Hirano, 1956). 1 Present Address : Tohoku University, Faculty of Agriculture, Department of Fisheries, Sendai, Japan. 173 174 HIDEO IWASAKI Obstacles to speedy progress were: (a) inability to grow the Conchocelis phase outside of the shells in free conditions; and (b) inability to cultivate in the labora- tory, out of season, the two growth phases of Porphyra which in nature are strictly seasonal (autumn- winter for the thallus phase and spring-summer for the Concho- celis phase). As mentioned, Drew and Kurogi had grown the Conclwcclis phase in enriched sea water on glass slides. Although the growth of Conchocelis was poor, Drew (1954) mentioned (p. 193) ". . . . that such free-living filamentous growths can be maintained and continue to grow indefinitely provided the culture solution is renewed regularly"; this seemed promising. Drew obtained with P. uinbilicalis only filamentous Conchocelis growth on glass slides and no monosporangia were formed, while the four species of Porphyra (including P. umbilicalis pro.v.) studied by Kurogi produced monosporangia. The discrepancy between these results implied that good growth and fruiting of the Conchocelis phase in the free-living conditions might be obtained under different cultural conditions and with better media. Another reason for trying again to grow the Conchocelis phase of P. tenera in vitro was the success of Hollenberg ( 1958 ) in obtaining on glass slides in liquid media minute filamentous Conclwcelis-\ike plants of P. perjorata which produced "spo- rangia branchlets" and fertile "conchospores." PRELIMINARY EXPERIMENTS The original materials brought from Japan were a few sterilized oyster shells which had been inoculated in March, 1959, with carpospores produced by natural- grown thalli. Colonies of Conchocelis developed normally in the shells kept in Woods Hole sea water enriched with nitrate, phosphate, and EDTA, (medium SWI, Table I) indicating the suitability of Atlantic sea water for Porphyra tenera. TABLE I Enriched sea water media SWI SWI I Filtered sea water 1000 ml. 1000 ml. KNO 3 72.2 mg. ( = 10 mg. N) 72.2 mg. ( = 10 mg. N) KH 2 PO 4 8.8 mg. (=2 mg. P) 4.5 mg. ( = 1 mg. P) Na 2 -glycerophosphate .5H 2 O 10.5 mg. ( = 1 mg. P) Fe-EDTA (1:1 chelation) 0.5 mg. (as Fe) 0.5 mg. (as Fe) "Tris" buffer* 500 mg. 500 mg. pH 8.0-8.2 8.0-8.2 * Tris (hydroxymethyl) amino methane (Sigma Company). The nutrient solution was changed fortnightly ; the shells were periodically cleaned with cotton to eliminate epiphytic diatom growth and kept in subdued, continuous fluorescent light 2 (10-30 foot-candles) at 13-15 C. In September, 1959, some shells were broken in pieces and thin flakes containing one Conchocelis colony were thoroughly wiped clean of epiphytes with cotton and also by repeated dipping into 1.5% agarized enriched sea water containing antibiotics. The flakes were 2 "Cool white." LIFE-CYCLE OF PORPHYRA TENERA 175 then inoculated into various artificial marine media and in enriched sea water, and kept in continuous subdued light at 13-15 C. At the end of December, 1959, in two tubes of medium ASP 2 NTA and in a tube of SWI + 5 ng.% indolacetic acid, a few young thalli appeared at the bottom of the test tube and on the shell flakes. Simultaneously in two tubes of SWI. tufts of free Conchocclis were growing out from the shell flakes. Later on (February-April, 1960), free Conchocclis colonies, attached to the bottom of the test tube or to the shells, appeared in the two ASP 2 NTA tubes and the SWI + IAA. The young thalli and the free Conchocelis employed in the subsequent experiments were derived from these 5 original cul- tures, which are unialgal, but accompanied by bacteria and yeasts. Microbial con- tamination, though permanent, was minimal in all the media employed because of the lack of organic substrates and because aseptic techniques were employed throughout. IN VITRO CULTURE OF FREE-LIVING CONCHOCELIS PHASE Origin of free Conchocclis Strains of free-living Conchocclis were obtained in several ways: (1) from the free Conchocelis growing out of the shell flakes in SWI medium; (2) directly from carpospores released by mature thalli collected in Japan, shipped to New York (March, 1960), and germinated in liquid media; (3) from carpospores produced by thalli grown in artificial media /;; vitro. At first, on the assumption that a substrate might be somehow advantageous to the Conchocclis phase, tufts of Conchocclis filaments, cut from the free growth on shell flakes in SWI, were transferred into biphasic media. To simulate the conditions in shells, the solid phase (10 ml. ASPI medium + agar 1.5%) was enriched with 0.1 % CaCCX, 0.01 % chondroitin, or both; the liquid phase consisted of 5 ml. either of ASP7 or SWI; the Conchocclis tufts were implanted in the agar at the interphase. All these combinations allowed good growth at 13-15 C. and continuous subdued light and at 18-20 C. and 10 hours daily. In 2-3 months, from an initial tuft 1 mm. in length, spherical colonies of 0.5-1 cm. were obtained; later, new colonies formed at the interphase or on the glass wall. Growth was almost entirely in the liquid phase and in all the different combinations, indicating that a solid substratum rich in CaCO., or protein is unnecessary. Further experi- ments were done in liquid media to determine the best cultural conditions for free growth in liquid media. Once some of these conditions were known, it became possible to germinate directly in liquid media carpospores collected from thalli grown either in nature or in vitro. Thalli of P. tcncra collected in Matsukawa-ura inlet were shipped to Xew York in March, 1960. Following the method suggested by Professor Y. Yamada of Hokkaido University, the thalli were put between pads of absorbent cotton wet with sea water and shipped in Thermos bottles; this method avoids rotting and gives good survival. Upon their arrival in New York, the thalli were placed in enriched sea water and produced carpospores. The collected carpospores were washed several times in sterile sea water by means of capillary pipettes, and 3-5 carpospores were inoculated in test tubes containing 10 ml. of 3 types of enriched sea water (AS\Y8 ; SWI, SWII) and 9 artificial media (ASM, ASPI, 176 HIDEO IWASAKI TABLE II Artificial media composition (w./v.) ASPl ASP2(NTA) ASP6 ASP7 ASP12(NTA)* Distilled water 100 ml. 100 ml. 100 ml. 100 ml. 100 ml. NaCl 2.4 g. 1.8 g. 2.4 g. 2.5 g. 2.8 g. MgSO 4 -7H,O 0.6 g. 0.5 g. 0.8 g. 0.9 g. 0.7 g. MgCl 8 -6H.O 0.45 g. 0.4 g. KC1 0.06 g. 0.06 g. 0.07 g. 0.07 g. 0.07 g. Ca (as C1-) 40 mg. 10 mg. 15 mg. 30 mg. 40 mg. NaNO, 10 mg. 5 mg. 30 mg. 5 mg. 10 mg. K 2 HPO 4 2 mg. 0.5 mg. K 3 PO, 1.0 mg. Na 2 -glycerophosphate 10 mg. 2 mg. 1.0 mg. Na 2 SiO 3 -9H 2 O 2.5 mg. 15 mg. 7 mg. 7 mg. 15 mg. Na 2 CO 3 3 mg. Fe (as Cl) 0.05 mg. BIJ 0.02 M g. 0.02 Mg- 0.05 /xg. 0.1 Mg- 0.02 Mg- Biotin 0.1 Mg- Thiamine 10 Mg- Vitamin mix 8** 0.05 ml. 0.1 ml. Vitamin mix S3** 1 ml. 1 ml. PII Metals**** 1.0 ml. 3 ml. 3 ml. 1 ml. SI I Metalsf 1 ml. P8 Metalsff 1 ml. Tris buffer 0.1 g. 0.1 g. 0.1 g. 0.1 g. o.i g. Nitrilotriacetic acid (10 nig.) 7 mg. (10 mg.) pH 7.6 7.8 7.4-7.6 7.8-8.0 7.8-8.0 * Developed by L. Provasoli for tropical species of dinoflagellates. ** One ml. of Vitamin mix 8 contains: thiamine HC1, 0.2 mg. ; nicotinic acid, 0.1 mg. ; putrescine 2HC1, 0.04 mg. ; Ca pantothenate, 0.1 mg. ; riboflavin, 5 Mg- ; pyridoxine 2HC1, 0.04 mg. ; pyridoxamine 2HC1, 0.02 mg. ; />-aminobenzoic acid, 0.01 mg. ; biotin, 0.5 Mg- ; choline H citrate, 0.5 mg. ; inositol, 1.0 mg. ; thymine, 0.8 mg. ; orotic acid, 0.26 mg. ; Bi 2 , 0.05 Mg- ; folic acid, 2.5 Mg- ; folinic acid, 0.2 Mg- *** One ml. of Vitamin mix S3 contains: thiamine HC1, 0.05 mg. ; nicotinic acid, 0.01 mg. ; Ca pantothenate, 0.01 mg. ; />-aminobenzoic acid, 1 Mg- ; biotin, 0.1 Mg- ; inositol, 0.5 mg. ; folic acid, 0.2 Mg- ; thymine 0.3 mg. **** One ml. of PII metal contains: ethylenediamine tetracetic acid, 1 mg. ; Fe (as Cl), 0.01 mg. ; B (as H 3 BO 3 ), 0.2 mg. ; Mn (as Cl) 0.04 mg. ; Zn (as Cl), 0.005 mg. ; Co (as Cl), 0.001 mg. f One ml. of SI I metals contains: Br (as Na), 1.0 mg. ; Sr (as Cl), 0.2 mg. ; Rb (as Cl), 0.02 mg. ; Li (as Cl), 0.02 mg. ; I (as K), 0.001 mg. ; Mo (as Na), 0.05 mg. ft One ml. of P8 metal contains: Na 3 versenol, 3 mg. ; Fe (as Cl), 0.2 mg. ; Mn (as CD, 0.1 mg. ; Zn (as Cl), 0.05 mg. ; Co (as Cl) 0.001 mg. ; Cu (as Cl), 0.002 mg. ; Mo (as Na), 0.05mg. ; B (as H 3 BO 4 ), 0.2 mg. Versenol =hydroxyethyl-ethylenediamine triacetic acid. ASP2, ASP2NTA, ASP6, ASP7, ASP12, ASP12NTA and D; Table II). Conchocelis growth was obtained in most of these media except ASW9 and ASP2. ASPl, ASP6, ASP12NTA, ASP12 and ASW8 gave very good growth; ASP7, D, and SWII were less good ; SWI very poor. Young germlings of P. tenera (5 mm. long) cultured at 14-16 C., and illumi- nated 13 hours a day with 400-500 foot-candles of incandescent light, did not grow normally (see later) and produced carpospores from which Conchocelis colonies developed. LIFE-CYCLE OF PORPHYRA TENERA 177 Suitable media and cultural conditions for free-living growth of Conclioeelis Several artificial media and enriched sea waters permit continued growth of the Conclioeelis phase. In decreasing order, ASP12NTA, ASP2NTA, ASP12, ASP6, and ASP7 are the most suitable artificial media, and SWII and SWI the enriched sea waters. Conclioeelis cultures easily last 6 months ; the color of the colonies varies in different media : pinkish-red in ASP12NTA, ASP12, and ASP6; pale brown in ASP2NTA, dark brown in SWII, and pinkish-grey in ASM. The color is more intense in the center of the colony, probably because of the presence there of intensely pigmented monosporangial branches. The type of medium in- fluences growth rate and monosporangia formation. In decreasing order, growth was fastest in ASP12NTA, SWII and ASP7 and slower in ASP2, ASP12, and MEC3. Monosporangia were formed and monospores liberated earlier in ASP12NTA, and in decreasing order in ASP12, ASP7, ASP2, SWII, SWI. The temperature range is between 10 and 26 C. ; the optimum between 13 and 20. Single pieces of the filament of the Conclioeelis phase ("-'I mm.) transferred in new media grew into new Conclioeelis colonies vegetatively. It was possible in this way to subculture the Conclioeelis phase : 5 serial transfers (one every 2-3 months from February to December, 1960) resulted in good growth. Quite likely the Conclioeelis phase can be grown indefinitely as free-floating colonies in liquid media. The Conclioeelis colonies in test tubes of liquid media generally grew at the bottom of the tube attached to the glass wall and appeared as fuzzy balls 4-10 mm. in diameter (Fig. 1). In larger containers, where they grow free-floating in the medium, they were stellate, often reaching a diameter of 10-15 mm. (Fig. 2). The Conclioeelis phase can be grown, but poorly, also on agar slants in screw- cap tubes. At the beginning of this work the free-living Conclioeelis colonies were grown in subdued light (20-40 ft.c. ) to simulate natural conditions; under these conditions growth was quite slow. Later, in surveying the effect of light intensity, it was found that growth was greatly increased by higher light intensities the higher, the better (maximum tried, 350 ft. c.). Incandescent and fluorescent light were equally effective ; however, the color of the Conclioeelis was different : reddish in fluorescent light and cool brown-black in incandescent light. Continuous illumina- tion also favored growth. Under these conditions (350 ft. c. continuous fluorescent light) mass cultures were obtained in 2-liter Erlenmeyer flasks and in tall, 4-liter bottles (Fig. 3) by gradual transfer in increasingly larger containers (10 ml. inoculated into 100 ml.; 100 ml. in 1 liter, etc.). Effect of fihotoperiodisni on monosporangia and monospore production Kurogi's experiments (1959) indicated that photoperiodism may govern mono- sporangia production and monospore liberation in Conclioeelis grown in shells. The following experiment was set to test the effect of photoperiodism on free- living Conclioeelis. With fluorescent light of 150-250 ft. c., a daily photoperiod of 8-11 hours induced formation of monosporangia in 2-3 weeks and young thalli in 3-8 weeks from the time of inoculation into new media of pieces of Conclioeelis filaments (Figs. 5, 6, 7). No substantial difference was found in cultures grown 178 HIDEO IWASAKI FIGURES 1-7, 12. LIFE-CYCLE OF PORPHYRA TENERA 179 at 13-15 C. or 18-20 C. in high light (150-250 ft. c.) for a daily photoperiod of 8-11 hours. When the light intensity was reduced to 30-50 ft. c., the appearance of young thallus germlings was greatly retarded in the 8-hour photoperiod ( > 96 and < 184 days) and apparently prevented in the 11 -hour photoperiod (no leafy thalli in 180-240 days). Under continuous fluorescent light at intensities of 150-250, 60-100, and 10-20 ft. c., both at 13-15 and 20-26, neither spores nor thallus germlings were found during the two experiments which lasted, respectively, 180 and 240 days. Growth of Conchocelis filaments and colonies proceeded normally, and may, indeed, be favored by continuous light ; very good mass cultures were obtained in con- tinuous light. Intensely purple, inflated portions similar to monosporangia, appeared after a month or more in cultures of 60-250 ft. c. At that time these structures were thought to be small monosporangia. Unfortunately the obser- vations of these experiments were done at great intervals (one month or more), and through the walls of the test tubes using a dissecting microscope. Only later, when it became evident that these structures were not producing spores, was a simple experiment tried : a Conchocelis colony grown for two months in continuous light at 13-15 C. was transferred to new medium and illuminated 8 hours a day ; after 5 weeks many thallus germlings ( 5-8 mm. long ) were growing alongside the Conchocelis colony. Evidently, maturation of monosporangia, release of monospores, or both, are induced by a short photoperiod and prevented by continuous light. The sporangia produced in continuous light (250350 ft. c. ) in the mass cultures seemed to be morphologically different from the monosporangia produced under short-day conditions. Sporangia cells in continuous light have thicker walls and length of the cells is usually about half their diameter ; some cells are quadrate (Fig. 4). They are very similar to the ''plantlets" described for P. iiinbilicalis var. laciniata by Drew (1954, p. 203, Fig. 4c). On the contrary, the cells of the short-day monosporangia often have elongated cells and the appearance of monosporangia is much more twisted (Figs. 5, 6, 7) because of the lateral branches. Are the continuous-light sporangia undeveloped or abnormal monosporangia, or are they a new type of sporangium ? The evidence at hand does not exclude either possibility. The aforementioned experiment of transferring a Conchocelis colony from con- tinuous light to short-day is indicative but not conclusive ; only one observation was made 38 days after the transfer : young thalli of 5-8 mm. were found. The formation dc noz'o of true monosporangia is not excluded because under the same light and temperature conditions (exp. //, Table III) young thalli appeared between 22 and 31 days in a culture started with pieces of Conchocelis filaments (no length was noted in the protocols; they were probably 2-3 mm. long). FIGURE 1. Colonies of free-living Conchocelis in artificial medium. FIGURE 2. Free-floating Conchocclis (detail of Figure 3). FIGURE 3. Mass culture of Conchocelis in aerated 3-liter bottle, continuous light. FIGURE 4. Sporangia formed in continuous illumination. FIGURE 5. Typical monosporangia formed in short day conditions (8-11 hours daily). FIGURES 6, 7. Same detail. FIGURE 12. Young thallus germlings, and monospores. 180 HIDEO IWASAKI The hypothesis that the Conchocelis phase can produce other sporangia besides the monosporangia has already been advanced by Drew for Porphyra umbilicalis var. laciniata (1954) and Bangia juscopiirpiirea (1958) to explain Conchocelis infections in sterile shell derived from other Conchocelis-miected shells. This possibility is greatly reinforced by our experiments with P. tenera. More than 5 serial transfers were done directly from Conchocelis to Conchocelis in test tubes or in mass culture without passing through the thallus phase or carpospores : in every case, inoculating pieces (in test tubes) of Conchocelis filaments or entire colonies (in mass cultures ) led to numerous new colonies. These experiments also do not prove conclusively that the increase in the number of Conchocelis colonies is due to the production of special spores developing into new Conchocelis colonies, TABLE III Effects of short-day and continuous light conditions on Conchocelis phase* Temperature Light** period Light intensity** Appearance of sporangiaf Appearance of foliaceous thallif Remarks 13-15 C. 8 hr. 150-250 ft. c. / 19 days M. II <22 days M. <46 days >22-<3l days good growth of thalli (1-1.5 cm.) 3 cm. thalli in 96 days 30-50 ft. c. I 27 davs M. 11 <48 days M. none up to 84 days >96- <184 days discontinued at 84 days small thalli 18-20 C. 11 hr. 150-250 ft. c. 1 19 days M. 11 23 days M. >56- <84 days 31 days good growth of Concho- celis-tha\\i soon bleached 30-50 ft. c. I 19 days S2 II 23-31 daysS.? none up to 240 days none up to 184 days large Conchocelis colonies 13-15 C. continuous 150-250 ft. c. / 35 days Sl-SZ 11 31 days S1-S2 none up to 240 days none up to 184 days tt 30-50 ft. c. I 84 days SZ II 72-96 days S2 none up to 240 days none up to 184 days 20-26 C. continuous 60-100 ft. c. I 35 days SZ II 31 days S3 none up to 240 days none up to 184 days 10-20 ft. c. / 56 davs S2 // S? none up to 240 days none up to 184 days (Figs. 8-11) * Media: ASP7 and SWII. Results of two separate experiments (/ and //). M = monosporangia; SI, see Figure 4; 5.?, see Figures 8-11. ** Fluorescent : "cool white." t Days from date of inoculation. Inoculum = a small piece of Conchocelis filament. ft At 50 days a Conchocelis colony was transferred to new medium and to 8 hours of light. In a month monospores and thalli appeared. because even small pieces of Conchocelis filament, which can grow into a full new colony, could have been present. However, the short-celled sporangia produced by P. tenera in continuous light (Fig. 4) or the sporangia-like swollen cells described by Drew for Bangia (1958, Fig. 3, p. 366) may be a new type of sporangium whose spores produce a new Conchocelis colony. Other very strange structures are produced in continuous (10100 ft. c.) or 11 hours subdued fluorescent light (30-50 ft. c.) at 13-15 C. and 20-26 C. (Table III). The similarity of the latter sporangia with fungal structures is striking (Figs. 8, 9, 10, 11 ; sporangia (?) 52 of Table III). The variety of structures created in different lights and temperatures shows that P. tenera has unusual powers of adaptation. The Conchocelis phase can now LIFE-CYCLE OF PORPHYRA TENERA 181 be grown free, making the morphological observations easy. This permits a wider analysis of the unusual morphological versatility of P. tcnera as well as of the possible deviations from the normal life-cycle induced by various lights and temperatures. THE LEAFY THALLUS PHASE Some cultural conditions for the growth of the thallus had been determined previously (Iwasaki and Matsudaira. 1958, and unpublished). (1) Leafy thalli grow normally in enriched sea water (Miquel's sea water), while they are short and unhealthy when grown in filtered, unenriched inshore sea water. (2) High-intensity, incandescent light is required for normal continued growth; growth is, however, slower than in natural sunlight. Young plants grown in fluorescent light die in a few days. (3) Young plants grow normally when illuminated 8-10 hours daily but die quickly when grown in continuous light. These results were on the whole confirmed by the present investigation. Effect of media on leafy thallus t/ro'^'th Thalli (1-2 mm.) derived from monospores produced by free-living Conchocelis (Fig. 12) were grown in enriched sea water and artificial media at 1416 C., TABLE IV Thallus growth (two-month) Media Growth Color ASP1 10 X 40 mm. brown ASP2 8 X 50 mm. ASP12 20 X 40 mm. red-brown ASP12NTA 8 X 20 mm. SWI 8 X 80 mm. reddish SWII 20 X 35 mm. pale brown and illuminated 9 hours daily with 400 ft. c. of incandescent light. The experiment was done in test tubes (20 X 120 mm.) containing 10 ml. of medium ; once a month the medium was replaced aseptically with 10 ml. of fresh medium. Good normal growth was obtained in two months in some artificial media and in enriched sea water (Table IV and Fig. 13). Narrow long thalli were obtained in SWI and most artificial media, broader thalli in SWII and ASP12. The 1957 experiments were done during the season in which the thalli grow in nature (fall- winter). On the contrary, the new experiment was done between May and August. 1960, indi- cating that normal thalli can be grown out of season if the light period is suitable (8-11 hours daily ). Effect of long-day conditions on leafy thalli In retrospect, the importance of the photoperiod for Porphyra tenera might have been suspected because the two phases of the life-cycle of P. tcncra correspond so sharply to the seasons. The leafy thallus grows in the short-day seasons 182 HIDEO IWASAKI FIGURES 8, 9, 10, 11. Inflated cells (sporangia?) produced in subdued light. FIGURE 13. Two-month-old thalli grown in test tube. From left, medium SWI, SWII, ASP12, ASP1. FIGURE 14. Young thallus degenerated under long-day conditions (13 hours daily). Lower part bleached; large pigmented cells at top; Conchocclis filaments germinating from "spores." FIGURE 15. Root-like projections growing out of a young thallus grown in SWII under long-day conditions. LIFE-CYCLE OF PORPHYRA TENERA 183 (autumn-winter), the Conchocelis phase in the long-day seasons (spring-summer). Furthermore, the transition between the two phases of the life-cycle coincides with the equinox (Fig. 16). On the contrary a great part of the temperature range (7-21 C.) is common to the two phases: normal thalli grow in nature between 3-21 C. and the Conchocelis between 7 and 25 C. Therefore, only the lower j zone (3-7 C.) may be suspected to affect Conchocelis growth and the upper zone (21-25 C. ) thallus growth. These considerations, and the already known effects of continuous light on thallus growth (Iwasaki and Matsudaira, 1958) and short day on monosporangia formation (Kurogi, 1959) suggested trial of growth under long-day conditions. Five young germlings (0.5 mm.), derived from monospores of free-living Conchocelis, were inoculated in each tube of the following media: SWI, SWII, ASP1, ASP2, and ASP12. They were incubated at 14-16 C. and illuminated 13 hours daily with 400-500 ft. c. of incandescent light. The controls were grown o .c o> c o Q 15 14 13 12 I I 10 9 30 o o 0> CL o> 10 o 0> CO Jon. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan. FIGURE 16. Day length at Sendai and average sea water temperature in Matsushima Bay. under similar conditions but illuminated 8 hours daily. The control gave, as in the previous experiment, normal thalli of the narrow shape. The thalli in long-day conditions grew very slowly and very soon became thick and irregular in shape. After 20 days or more, the thalli became pale, leaving numerous scattered big reddish-colored cells. The thallus around the edges as- sumed the appearance of a callus tissue (Fig. 14). After 40 days (in ASP2) or more, spores were released. Since these spores germinated into filaments which later formed well-developed Conchocelis colonies, we assume that, at least function- ally, they are equivalent to carpospores. These events differ slightly in time and amount of growth in the various media except SWII. In SWII the thallus after 27 days started to produce root-like projections (Fig. 15) which branched out into thinner filaments; after two months the thallus became covered with Concho- celis colonies. Apparently 13 hours of daylight, which corresponds at the latitude of Sendai (39 N) to late April, already inhibits normal thallus growth and in- 184 HIDEO IWASAKI duces the formation of structures functionally equivalent to carpospores. This experiment was repeated later with similar results. In another experiment in tall covered containers (10 cm. diameter; 7 cm. high) containing 200 ml. of ASP1, young thallus buds (1-2 mm.) were grown for one month (April 22-May 25) at 14-16 and illuminated 8 hours daily with 400 ft. c. of incandescent light : the thalli, which had reached by then an average size of 3 cm. 2 , were illuminated for 10 days (May 25-June 4) with 100-200 ft. c. of incandescent light : on alternate days, 8 hours daily followed by one day in continuous light. After this period of alternating photoperiods, the culture was grown in 100-200 ft. c. and 8 hours daily of fluorescent light. In a month (July 5) big, dark cells appeared, scattered at the edges of the leafy thalli which were stunted and curled. Ten days later these cells produced germ tubes which devel- oped into Conchocelis colonies. Two months (August 5) after the alternating light treatment, many colonies of Conchocelis were growing free on the bottom of the dish and covering the stunted disintegrating thalli. After 82 days (August 25) mature monosporangia were formed and a few days later the monospores were released and produced thallus germlings. These germlings on September 10 had already reached an average size of 1 cm. 2 The complete life-cycle was obtained in 5-6 months. It is remarkable that the leafy thalli of the second generation grew normally, though slowly, in fluorescent light and at low intensities (100-200 ft. c.). This is by no means an isolated case of thallus growth in fluorescent light : all the cultures of Conchocelis grown at 13-20 C. for 8-11 hours daily of fluorescent light even- tually released monospores (1-4 months, depending upon light intensity). These spores gave rise to leafy thalli reaching 2-10 mm. before they became pale and died. The arrest in growth of these leafy thalli was probably due to lack of nutrients : the medium in these experiments was not changed monthly, as was done for the experiment on thallus growth in different media. Ability of the thalli to grow in fluorescent light may be an adaptation to utilization of fluorescent light acquired during the Conchocelis phase : the Conchocelis phase does not require incandescent light. This adaptability, whatever the cause, reflects again the great plasticity and versatility of P. tenera. DISCUSSION These results may help solve some of the problems of life-cycle and growth potencies of P. tenera. Solutions here, in turn, may improve the farming of this sea weed. The first report of the entire life-cycle of a Porphyra obtained in vitro is the one of Hollenberg (1958). He obtained from carpospores Conchocelis-\ike filaments which formed sporangia and liberated spores (16 days from carpospore germination ) . These spores in turn developed into blade-like plantlets (young thalli). Since the cultures were grown during the summer in north light, it is possible that the very rapid formation of sporangia and the poor growth of the Conchocelis phase were due to light conditions. While this paper was being written, the paper on the Conchocelis stage of P. umbilicalis by Kornmann (1960) appeared. Like us, Kornmann obtained the complete life-cycle in vitro. He started in November, 1959, with a "plantlet" (probably an immature, or abnormal mono- sporangium) cultured in Erdschreiber. The "plantlet" became fertile and made LIFE-CYCLE OF PORPHYRA TENERA 185 monospores which did not develop. Only a few cells of this structure remained vegetative and reproduced in a month another "plantlet" (without "rhizoids") which produced many monospores. Of these, only 6 germinated into leafy thalli which in a month and a half reached 1.5-2 mm. in length. The thalli formed "Ballchen" from which thin filaments grew out; the filaments, by division, produced "zweige" (his Figure 5 -- "plantlet" = monosporangia ?). From the "zweige" arose as side-branches ("seitliche Verzweigung") thin filaments which in free culture produced a confused ball of yarn (verworrene Knauel = free Conchocelis) or enveloped the original plantlet. These filaments grow also as a typical Concho- celis in calcareous shells. Unfortunately, no data are given of the light period under which the cultures were grown. From his Figures 2 and 5, the "plantlets" are very similar to mono- sporangia. If so, the thin filaments (which are Conchocelis filaments) should produce, and not be produced by the monosporangia (as Kornmann states in Figure 5 and the text). But the structure in Figure 5 could be equivalent to the sporangia which were produced in our Conchocelis colonies grown in continuous light (our Fig. 4). As mentioned, these sporangia are suspected of producing spores germi- nating into a new Conchocelis. Kornmann's light conditions seem also to be inade- quate for thallus growth because, as in our thallus cultures under long-day condi- tions, Conchocelis filaments arise from the thallus (Kornmann, Fig. 3B) or big colored cells are formed (Kornmann's "Ballchen" which can be seen at the base of the thallus of Figure 3, C) from which Conclwcelis filaments arise. Kornmann's Figure 1C represents, most likely, true monosporangia and Conchocelis filaments. Whatever the interpretation, it is seen that, both in Kornmann's and in our experiments, the life-cycle can be obtained in vitro. Detailed morphological studies are planned to solve some of the many questions ; e.g., what is the typical mor- phology of the true monosporangia of Conchocelis grown free how do they differ from those produced in shells ? What are the mysterious "plantlets" of Drew, and of Figures 1A and 2A of Kornmann are they sporangia whose spores develop another Conchocelis phase, or abnormal monosporangia? What are the deviations from the natural life-cycle in shells that develop when the Conchocelis phase is grown free and in different day-lengths and light-intensities? What are the big, dark cells formed in the degenerating thalli under long-day conditions ? The present research confirms and extends previous results on the effect of the photoperiod on P. tcncra. As mentioned, the Conchocelis phase grows in nature during the long-day seasons and the leafy thallus phase in short-day seasons. The leafy thallus phase is apparently a short-day plant: growth is arrested and the thallus degenerates when exposed to 13 hours of light daily. The Conchocelis phase is not strictly a long-day plant : in vitro it grows, but slowly, under short- day (8-hour) conditions and in subdued light. However, high light, longer day (11-hour), and especially continuous light enhance growth vigorously. The in- complete data available indicate that the photoperiod governs the formation of monosporangia and the liberation of monospores. Our in vitro experiments confirm fully the results of Kurogi (1959) obtained with Conchocelis grown in shells. He found that photoperiods of 10 and 12 hours of light (corresponding to conditions of winter, spring and autumn, respectively) induce an abundant formation of mono- spores, while 15 hours of light daily did not enhance the formation of monosporangia. 186 HIDEO IWASAKI Furthermore, the Conchocelis which were liberating monospores in 10-hour photoperiods continued for only a few days, and then stopped liberating mono- spores, when transferred to 15 hours of light; conversely the long-day (15-hour) Conchocelis began to liberate monospores after they were transferred to short-day (10-hour) conditions. Similarly the in vitro experiments on free-living Concho- celis show that short-day (8-, 11-hour) induces early formation of monosporangia and liberation of monospores. Continuous light, or subdued 11-hour photoperiods, induce the formation of interesting and different sporangia, or peculiar inflated cells in the Conchocelis filaments, whose fate and origin need further investigation. The preliminary experiments on the thallus indicate that the photoperiod also governs the formation of carpospores ; 13 hours of light daily induce cessation of growth and degeneration of the leafy thallus, followed by formation of carpospores or their physiological equivalents. Exposure of full-grown thalli to different photo- periods is now needed to define precisely the effect of the photoperiod on carpospore production. These findings emphasize the need of determining the effect of photoperiods on the life-cycle and alternation of generations in sea weeds. Foyn (1955) had observed that the northern species of Uh'a (lactuca) can grow normally in con- tinuous light, while the southern Mediterranean species (Tliurcti) dies in such conditions. This work was supported by contract NR 104-202 of the Office of Naval Re- search and by Grant G-1198 of the National Science Foundation to Dr. L. Provasoli. of Raskins Laboratories. I wish to thank Dr. Provasoli for his hospitality, advice and constant interest. SUMMARY 1. The complete life-cycle of Porphyra tcncra was obtained in vitro. 2. Chemically defined media or enriched sea water permit good growth of these unialgal (not bacteria-free) cultures. 3. Under suitable light and temperature, the complete life-cycle is completed in 5-6 months. Both the Conchocelis and the thallus phases may be grown out of season. 4. The Conchocelis phase grows well free in liquid media ; a calcareous substrate is unnecessary. Conchocelis colonies grown in liquid media when free-floating, are stellate and round, but mold-like when attached to glass walls. They are brown- black or purple-red, depending on the composition of the medium. Rapid and abundant growth of the free Conchocelis is elicited by high-light intensities. Fluor- escent light is a good light source. 5. Monosporangia formation and release of fertile monospores are induced by short-day conditions (8-11 hours daily) ; monosporangia and germinating mono- spores develop after 1-2 months from the inoculation of the Conchocelis filaments. In continuous light, Conchocelis growth is rapid but the sporangia produced are somehow different from the ones produced in short-day conditions. 6. In continuous light, the number of colonies increases rapidly after transfer to new media. This could be due to formation of new colonies from small pieces of filaments. However, even though free spores were not found, it is not excluded that ne\v Conchocelis colonies may have been derived from special spores. LIFE-CYCLE OF PORPHYRA TENERA 187 7. The Conchocelis phase was cultured for one year by transferring free Concho- cclis colonies or pieces of filaments every two months in new media. Mass cultures with good yields were obtained in continuous fluorescent light. 8. The leafy thallus, derived from monospores grown in shells, grows well and normally in artificial media, at 13-18 C. and in high intensity incandescent light of 8-11 hours daily, but not in fluorescent light. 9. A photoperiod of 13 hours daily inhibits growth of young thalli (1-2 mm.). The thalli became thick, curly, degenerate, assume a callus appearance, bleach almost completely except for scattered groups of dark-pigmented, big cells which produce spores germinating into ConcJwcclis filaments. In one type of enriched sea water (SWII), the thalli, after thickening, and while degenerating, produce rhizoid- like structures which give rise to Conclwcelis filaments. 10. In nature, the Conchocelis phase grows in the long-day seasons, the leafy thallus phase grows in the short-day seasons ; and the transition between the two phases is almost exactly at the equinox. On the contrary, no correlations exist between temperature and the phases of the life-cycle : a large temperature zone (7-21 C.) is common to the two phases. Similarly, our preliminary experiments show that the length of the photoperiod has remarkable effects on the Conchocelis and leafy-thallus phases of P. tcncra. The photoperiod governs, besides growth, the formation of the spores producing the next phase of the life-cycle. It is reasonable, therefore, to suppose that like land plants, some sea w r eeds, or phases of their life- cycle, may be long- or short-day plants. LITERATURE CITED DREW, K. D., 1949. Conchocelis in the life history of Porphvra umbilicalis (L.) Kiitz. Nature , 164: 748. DREW, K. D., 1954. Studies in the Bangioidae III. The life-history of Porphyra umbilicalis (L.) Kutz. var. laciniata (Lightf.) J. Ag. Ann. Bot. N. S., 18: 183-211. DREW, K. D., 1958. Studies in the Bangiophycidae IV. The Conchocelis-phase of Bangia juscopurpurca (Dillw.) Lyngbye in culture. Pubbl. Stas. Zool. Napoli, 30: 358-372. FOYN, B., 1955. Specific differences between northern and southern European populations of the green alga Uh'a lactnca L. Pubbl. Stas. Zool. Napoli, 27 : 261-270. HOLLENBERG, G. J., 1958. Culture studies of marine algae III. Porphvra pcrjorata. Anier. J. Bot., 45: 653-656. IWASAKI, H., AND C. MATSUDAIRA, 1958. Culture of a laver, Porphyra tcncra Kjellm. I. Preliminary research on cultural conditions. Bull. Jap. Soc. Sci. Fish.. 24: 398-401 KORNMANN, P., 1960. Von Conchocelis zu Porphyra. Hclgolander Wiss. Mccrcsuntcrs., 1 : 189-193. KUROGI, M., 1953. Studies of the life-history of Porphyra. I. The germination and development of carpospores. Bull. Tohoku Reg. Fish. Lab., No. 2: 67-103. KUROGI, M., 1959. Influences of light on the growth and maturation of Conchocelis-tiiallus of Porphyra. I. Effect of photoperiod on the formation of monosporangia and liberation of monospores. Bull. Tohoku Reg. Fish. Lab. No. 15 : 33-42. KUROGI, M., AND K. HIRANO, 1956. Influence of water temperature on the growth, formation of monosporangia and monospore-liberation in the Conchocelis phase of Porphyra tcncra Kjellm. Bull. Tohoku Reg. Fish. Res. Lab., No. 8: 45-61. TSENG, C. K., AND T. J. CHANG, 1954. Studies on the life history of Porphyra tcnera Kjellm. Sci. Sinica, 4: 375-398. RESPIRATION RATES IN PLANARIANS. III. THE EFFECT OF THYROID COMPOUNDS ON OXYGEN CONSUMPTION 1 MARIE M. JENKINS Department of Zoology, Unii'crsity of Oklahoma, Norman, Oklahoma lodinated proteins have been found throughout the invertebrate world (Roche, 1952; Gorbman ct al., 1954), primarily in the form of mono- and diiodotyrosine, although in a number of insects (Limpel and Casida, 1957) and in Miisculium, a fresh-water fingernail clam (Gorbman ct al., 1954), a high percentage of the protein-bound iodine has been shown to be in the form of thyroxine. None of these compounds has been demonstrated unequivocally to take part in physiological processes in the invertebrate animal (Goldsmith, 1949; Gorbman ct a!., 1954), but recent reports indicate the question is not settled. Wingo and Cameron (1952) found that thyroxine hampered the multiplication of a ciliate protozoan, Tetra- hyincna gcleii, but increased the rate of oxygen uptake above that of parallel control cultures. Thyroxine added to the diet of rice moth (Corcyra cephalonica) larvae is reported to have increased the oxygen consumption requirement, although thyroglobulin was without effect (Srinivasan ct al., 1955). The presence of iodinated proteins in planarians has not been investigated, but several workers have reported a positive action of thyroid compounds on physio- logical activities in this group. Castle (1928) observed that Phagocata (Planaria) vclata was attracted to and fed readily upon macerated sheep thyroid, and sub- sequently decreased in size even more rapidly than worms subjected to starvation. Goldsmith (1937), studying the effect of endocrine feeding on regeneration and growth in Ditgcsia tigrina (Planaria inaculata), noted no significant differences in the head regeneration time in the gland-fed animals, but found that thyroid-fed individuals increased in size to a lesser extent than the liver- and pituitary-fed forms. The influence of thyroxine on eye formation in Phagocata gracilis was investigated (Weimer ct al., 1938) in pieces of planarians cut at different levels and allowed to regenerate in a saturated thyroxine solution.. Once the reconstitution process had begun, the rate of eye formation was reported to be much higher for the pieces in thyroxine. No reports are available of the effect of thyroid hormones on oxygen con- sumption in planarians. Phenylthiourea, an anti-thyroid agent, has been shown, however, to exert a depressing effect on planarian respiration (Jenkins, 1961). In view of these findings an investigation was undertaken to ascertain the effect of certain thyroid compounds on respiration rates in Dugcsia dorotoccphala, a common fresh-water planarian. 1 Supported in part by grants from the National Science Foundation (G-3209), the South- ern Fellowship Fund, and the University of Oklahoma Alumni Development Fund. This study represents part of a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree at the University of Oklahoma, under the direction of Dr. Harriet Harvey. 188 THYROID COMPOUNDS AND PLANARIANS 189 DESIGN OF EXPERIMENT The planarians used in this study were large, sexually mature animals, collected from Buckhorn Springs - in Murray County, Oklahoma. They were maintained in pans of lake water, provided with an aerator, at a constant temperature of 20 C. Experimental animals were taken on the seventh day after feeding and were not fed during the course of the experiment. Compounds used for this investigation were thyroxine (T 4 ), 3,5,3'-triiodo- thyronine (T 3 ), 3 and 3,5-diiodotyrosine (DIT). In order to determine the con- centration to be used, groups of cut posterior ends of planarians were allowed to regenerate in a graded series of molar solutions of each of the compounds in lake water, and the regenerated animals examined for signs of any abnormalities. In worms in both the thyronines, eye spots were visible under a dissecting microscope by the third day, compared to the fifth day for the animals in water and in diiodo- tyrosine. It was noted, however, that the worms in the 3 X 10~ 5 M dilution of tri- iodothyronine appeared to have a slight thickening across the head behind the eyes. This was not apparent in the 2 X 10~ 5 M solution; the latter was therefore chosen as the higher concentration for the experiment. All three chemicals were made up at this concentration so their effects could be compared. In addition, a solution of half the molarity given above was used for each in order to test whether or not a more dilute solution would have an appreciable physiological effect. The controls were cultured in lake water. The procedure was similar to that employed for observing the effect of goitro- gens on oxygen consumption in planarians (Jenkins, 1961) with the following exceptions : The Latin square method was used in order to provide maximum randomization. Seven replicate experiments were performed. Each replicate experiment employed seven groups of five planarians each ; one group for each of the two concentrations of the three chemicals vised, and one water control. On the day that a replicate experiment was begun. 35 of the largest specimens in one stock pan were selected and divided randomly into the seven unit groups. For each replicate experiment, oxygen consumption determinations were made as follows : Day : Oxygen consumption was measured over a period of three hours with all seven groups of animals in water. At the close of the day's readings, each group was placed in an individual fingerbowl of water until the following day. Day 1 : The Warburg flasks were prepared with the solutions of thyroid com- pounds (or of culture water) to be used. The planarians were placed in the flasks and manometer readings were made during the first 3 ] /2 hours of exposure to the chemicals. At the termination of the day's readings, each group was placed in a fingerbowl containing the same concentration of thyroid compound as that in which oxygen consumption was to be determined during the remainder of the experiment. Further readings for periods of three hours were made on the second, fourth, and sixth days. The w 7 orms were placed in fresh thyroid compound solutions every second day. 2 Acknowledgment and thanks are due to Oscar Lowrance, owner of Buckhorn Springs property, for permission to collect the planarians. 3 Supplied through the courtesy of the Sigma Chemical Company, St. Louis, Missouri. 190 MARIE M. JENKINS RESULTS *? The oxygen consumption of each group of planarians was calculated according to standard methods (Umbreit ct aL, 1949) and the results are given in Table I. Little variation was shown among the groups in response to treatment on any one day, the normally-occurring slight downward trend shown by the controls being apparent in each of the experimental groups. The data show significantly that, TABLE I Effect of thyroid compounds on oxygen consumption of planarians \^\ M51 n\. O/gm./hr. 1 ^1 / +tl Treatments Days yew & 1 2 4 6 \*Y Water 130 128 131 120 121 Thyroxine, 2 X lO' 5 M 133 130 130 121 120 Thyroxine, 1 X 10~ 5 M 132 129 131 120 124 Triiodothvronine, 2 X 10~ 5 M 129 133 128 124 124 Triiodothyronine, 1 X 10~ 5 M 131 130 131 121 120 Diiodotyrosine, 2 X 10~ 5 M 133 132 128 123 119 Diiodotyrosine, 1 X 10~ 5 M 126 130 130 124 125 under the conditions of this experiment, there is no demonstrable effect on the oxygen consumption of the planarians. DISCUSSION Although neither iodotyrosines nor iodothyronines, which are widely distributed among invertebrates, have been found to influence physiological processes in these animals, it is obvious that there is some homeostatic regulation in organisms without a thyroid, and that metabolic processes do occur and are regulated within the animal. \Yhether thyroxine or its analogs play any part in this regulation remains to be established. The prevailing opinion at the present time is that invertebrate tissues are insensitive to the action of thyroid hormones. Findings which are not in agreement, such as moth larvae showing an increased metabolic rate when fed with thyroxine (Srinivasan et aL, 1955), have not been confirmed in other invertebrates. Although evidence of the physiological activity of thyroid hormones in metazoan invertebrates is inconclusive, there is increasing evidence that one-celled organisms respond markedly to these chemicals. The findings of Wingo and Cameron in regard to Tctrah\nnena gclcii have been mentioned above. Gutenstein and Marx (1957) have demonstrated that respiration of yeast cells (Sac char omyccs ccrevisiae} is significantly accelerated by thyroxine and inhibited by a specific thyroxine antagonist. Augmentation of oxygen consumption has also been observed in Eschcricliia coli subjected to the influence of T 3 and T 4 (Roche ct aL, 1959), although the respiratory action of the two iodothyronines does not seem bound to a metabolic transformation of the hormones. THYROID COMPOUNDS AND PLANARIANS 191 In some instances it appears that invertebrates are able to metabolize thyroid compounds in much the same manner as the tissues of vertebrates do. The hepa- topancreas of the mollusks, Mytilus galloproz'inciatis and Octopus vulgaris, has been shown to degrade T :i by deiodination and oxidative deamination, followed by oxidative decarboxylation (Covelli et aL, 1960). This is considered to be a metabolic degradation of the hormone rather than a physiological activation. The formation of thyroxine metabolites by Eschcrichia coll has also been reported (Grasbeck ct a/., 1960) and explained on the basis that both E. coli and other micro-organisms are able to oxidatively deaminate many ammo acids. The question of extrathyroidal iodine metabolism is closely related to the problem of invertebrate tissue responses to thyroid hormones. The discovery that thyroxine could be recovered from iodinated casein (Ludwig and von Mutzen- becher, 1939) was followed by early reports (Chapman, 1941 ; Morton ct a!., 1943) that newly-formed thyroxine-like compounds could be demonstrated in the tissues of thyroidectomized rats. The observation that the same limited series of iodine compounds is formed when any of a large number of proteins is iodinated, whether in the thyroid, in artificially iodinated proteins, or in the iodoproteins of inverte- brates (Reineke, 1949) appeared to confirm this idea, but it has recently been discredited (Taurog ct a!., 1960) on the basis that the concentrations of iodine used in the early experiments were far above the physiological range. The evidence to date strongly indicates that tissues of invertebrates are insensi- tive to the action of thyroxine and its analogs. The negative results obtained in the present experiment support this view. However, the continual recurrence of reports which substantiate the opposite view to some extent, such as the augmenta- tion of oxygen consumption in protistans, noted above, prevents the complete acceptance of the view that invertebrate tissues are wholly insensitive to thyroid compounds. Since most reports of a positive response of non-chordate tissues to thyroxine and its analogs are limited to those experiments in which groups of like cells are used, either as tissues from metazoans or as concentrated groups of one- celled organisms, it is possible the elucidation of the cellular metabolism of thy- roxine may bring to light principles which will aid in solving the question of regula- tion of metabolic processes in thyroidless organisms. It seems reasonable to suppose that the same fundamental pattern of metabolic regulation may be found throughout the animal kingdom. SUMMARY AND CONCLUSIONS 1. Using the Latin square method, a study was made of the effect of the thyroid compounds diiodotyrosine, triiodothyronine, and thyroxine on respiration in pla- narians. No statistically significant effect was found with any one of the three chemicals under the conditions of the experiment. 2. The regulation of metabolic processes in invertebrates is discussed briefly. The suggestion is made that the same fundamental pattern of metabolic regulation may be found throughout the animal kingdom, despite the fact that no evidence of this is demonstrated in the present paper. LITERATURE CITED CASTLE, W. A., 1928. An experimental and histological study of the life cycle of Planaria velata. J. .r/>. Zoo/., 51 : 417-482. 192 MARIE M. JENKINS CHAPMAN, A., 1941. Extrathyroidal iodine metabolism. Endocrinol., 29: 686-694. COVELLI, I., F. BLASI AND A. TOMMASELLI, 1960. Sur la degradation de la 3 : 5 : 3'-triiodo-L- thyronine par 1'hepatopancreas de la Moule (Mytilns gallof>roi'incialis L.) et du Poulpe (Octopus vulgaris L.). C. R. Soc. Biol,, 154: 1225-1228. GOLDSMITH, E. D., 1937. The relation of endocrine feeding to regeneration, growth and egg" capsule production in Planarla nwciilata. Biol. Bull., 73: 227-236. GOLDSMITH, E. D., 1949. Phylogeny of the thyroid : Descriptive and experimental. Ann. A r r- York Acad. Sci., 50: 283-316. GORBMAN, A., M. CLEMENTS AND R. O'BRIEN, 1954. Utilization of radioiodine by invertebrates, with special study of several Annelida and Mollusca. /. E.rp. Zool., 127 : 75-92. GRASBECK, R., B.-A. LAMBERG AND F. BJORKSTEN, 1960. The formation of thyroxine metabo- lites by Escherichia coli. Acta Endocrinol., 34: 113-120. GUTENSTEIN, M., AND W. MARX, 1957. Stimulation of yeast respiration by L-thyroxine. J. Biol. Chcm., 229: 599-602. JENKINS, M. M., 1961. Respiration rates in planarians. II. The effect of goitrogens on oxygen consumption. Biol. Bull.. 120: 370-374. LIMPEL, L. E., AND J. E. CASIDA, 1957. Iodine metabolism in insects. I. In vivo metabolism of radioiodide. /. Exp. Zool., 135 : 19-27. LUDWIG, W., AND P. VON MuTZENBECHER, 1939. Die Darstellung von Thyroxin, Monojod- tyrosin aus jodiertem Eiweiss. Zcitschr. physiol. Chcm., 258: 195-211. MORTON, M. E., I. L. CHAIKOFF, W. O. REINHARDT AND E. M. ANDERSON, 1943. Radioactive iodine as an indicator of the metabolism of iodine. VI. Formation of thyroxine and diiodotyrosine by the completely thyroidectomized animal. /. Biol. Chcm., 147 : 757-769. REINEKE, E. P., 1949. The formation of thyroxine in iodinated proteins. Ann. Nnv York Acad. Sci., 50 : 450-465. ROCHE, J., 1952. Biochimie comparee des scleroproteines iodees des Anthozoaires et des Spongiaires. Experientia, 8: 45-54. ROCHE, J., G. SALVATORE AND I. COVELLI, 1959. Sur le metabolisme der hormones thyroidiennes par Escherichia coli. C. R. Soc. Biol.. 153: 1116-1119. SRINIVASAN, V., N. R. MOUDGAL AND P. S. SARMA, 1955. Influence of thyroxine and thyro- globulin on rice moth larva. Science, 122 : 644-645. TAUROG, A., E. S. EVANS, G. D. POTTER AND I. L. CHAIKOFF, 1960. Failure to demonstrate extrathyroidal thyroxine formation in thyroidectomized rats on low and moderate iodine intakes. Endocrinol., 67 : 635-649. UMBREIT, W. W., R. H. BURRIS AND J. F. STAUFFER, 1949. The Warburg constant volume respirometer. In : Manometric Techniques and Tissue Metabolism. Minneapolis, Burgess Pub. Co. WEIMER, B. R., C. W. PHILLIPS AND D. M. ANDERSEN, 1938. The influence of thyroxine on eye formation in Phagocata gracilis (Haldeman). Physiol. Zool., 11: 158-167. WINGO, W. J., AND L. E. CAMERON, 1952. Effect of thyroxine upon growth and oxygen uptake of Tetrahymcna geleii (Y). Texas Rep. Biol. Med., 10: 1075-1083. HISTOPHYSIOLOGICAL STUDIES ON THE CORPUS ALLATUM OF LEUCOPHAEA MADERAE. III. THE EFFECT OF CASTRATION 1 - 2 BERTA SCHARRER AND MARIANNE VON HARNACK 3 Department of Anatomy, Albert Einstein College of Medicine, Nctt 1 York 61. Ncic York In the insect, Leucophaca maderac, and in related species reproductive processes are controlled by an intricate neuroendocrine mechanism (Scharrer, 1958a, 1959; Engelmann, 1960). Each reproductive cycle is initiated by a hormone from the corpora allata. Concurrently with alternating phases of ovarian activity and qui- escence, the corpora allata become active and inactive at regular intervals. They remain inactive as long as they are restrained by nervous stimuli from the brain. The reactivation of the corpora allata at the appropriate time is controlled by a variety of factors, among them feed-back from the reproductive organs. The exact nature of this feed-back is still unknown, but it seems that a humoral factor is involved that acts by way of the central nervous system. As a rule, corpus allatum activation occurs when the ovaries contain undeveloped eggs, except during "preg- nancy," i.e., as long as an ootheca is present in the uterus. The active corpus allatum, found in animals with growing eggs, shows a considerable increase in volume and other distinct changes in its histology as compared with the inactive gland characteristic of newly emerged or pregnant animals (Engelmann, 1957; Scharrer and von Harnack, 1958). The individual components of this neuroendocrine control system can be ana- lyzed by experimental interference at various levels, for example, by severance of the nerves restraining the corpora allata (Scharrer, 1952), or by gonadectomy (von Harnack and Scharrer, 1956). Such procedures disrupt the normal func- tional and structural periodicity of the corpora allata. In the absence of the ovaries, the corpora allata of Lcucophaca continue in a state of activity beyond the normal period. The question arises whether this con- dition surpasses the normal degree of activity of the corpus allatum cells, and whether the lack of intermittent inactivation of the gland may perhaps result in permanent structural change or even damage. The present paper deals for the most part with a morphological analysis of the corpus allatum of gonadectomized females of Lcncophaca ranging in age from emergence to senescence. The situation in males seems less interesting, since their corpus allatum fails to respond to gonadectomy as in females. 1 Supported by grants from the American Cancer Society and the U. S. Public Health Service ( Nos. C-3413 and A-3984). - The authors are grateful to Dr. Helen Wendler Deane for valuable suggestions. 3 Grantee of the American Association of University Women. 193 194 BERTA SCHARRER AND MARIANNE VON HARNACK MATERIAL AND METHODS The present study includes 154 adult females and 26 males of the roach. Leucophaca maderae, gonadectomized under varying environmental conditions in our laboratories in Cleveland, Denver, and New York between 1944 and 1959. The large majority of the females (147) were operated upon at various nymphal stages, the remaining seven shortly after they had become adults. In the ovari- ectomized nymphs, the interval between operation and emergence ranged from 1 to 152 days. In the group operated in the adult stage, the longest interval between emergence and castration was 18 days. The completeness of the gonad- ectomy was verified by examination of the excised tissues under the dissecting microscope, by testing the experimental animals individually for possible repro- duction by placing them in separate jars containing normal males, and by autopsy at the time of fixation. The condition of the accessory sex glands was also recorded ; excessive amounts of secretory material are characteristic of gonadectomized females. Fixation was scheduled at intervals ranging from an adult age of nine to 457 days. The material thus encompasses the entire adult life span. The total period after gonadectomy, therefore, ranged from a minimum of 32 to a maximum of 491 days. The majority of the animals (143) had been castrated for over 100 days. A smaller number of males was included in this study ; their corpora allata are more uniform in structure, and presumably less complex in function, than those of females. All of the 26 male castrates were operated upon as nymphs, 12 to 104 days before emergence. The adult age at the time of fixation varied between and 366 days. The total castration period thus ranged from 39 to 432 days, with all except three specimens falling in the group above 100 days. The histological procedures and the methods of quantitative evaluation of the corpora allata were essentially the same as those described in the first paper of this series (Scharrer and von Harnack, 1958). Most cases were fixed in Kelly's solution. The Halmi-Dawson "aldehyde fuchsin" method was used with the addi- tion of \Yeigert's hematoxylin as a nuclear stain. The present study includes tissues treated with the standard periodic acid-Schiff (PAS) technique. The sections were kept in the periodic acid solution and in the Schiff reagent for 10 minutes each. The duration of pretreatment with buffered saliva ranged from 30 minutes to two hours. Certain additional specimens were fixed in W% formalin, alcohol-formol-acetic acid (AFA), or Carney's solution. Among the female castrates, 57 representative cases were selected for complete quantitative analysis, i.e.. volumes of the corpora allata, nuclear numbers, and nuclear-cytoplasmic ratios were determined. In the remaining 98 animals only volumetric determinations were made. Since organ volume, accompanied by histo- logical inspection of the relative amount of cytoplasm, is a good indicator of the degree of activity of the corpora allata, these specimens serve to substantiate the conclusions drawn from the study of the more completely analyzed group. In the males, volumetric determinations were used as an indication of corpus allatum activity, since the normally low variability of their corpora allata was not significantly altered after gonadectomy. In the largest and smallest glands of this group, nuclear numbers and nuclear-cytoplasmic ratios were calculated in order to delimit the range of variability within the male castrates. CORPUS ALLATUM AND CASTRATION 195 Additional methodological matters, such as hemicastration, will he mentioned in the text. RESULTS a. Females \Yhen the volumes of the corpora allata of all gonadectomized females studied were plotted according to adult age (Fig. 1), several facts became apparent. All age groups with the exception of the youngest and the oldest showed a considerable spread in corpus allatum size. Starting from glands whose volume 64 60 56 52 48 44 136 c ~32 LJ 2 28 Q24 20 16 12 8 4 b _ a I I I I I 40 80 120 160 200 240 280 320 360 400 440 ADULT AGE (in days) FIGURE 1. Diagram showing the size variation of corpora allata of 154 ovariectomized Lcucophaca arranged according to adult age. Each dot represents the measurement of the paired glands from one individual. The arrows indicate that, with increasing age, the volumetric maxima rise stepwise to reach peak values in a mid-period (140-280 days; shaded area), and gradually decline in old age. For comparison, three maximum levels of corpus allatum size in groups of non-castrate animals are indicated on the scale : a, First activity cycle preceding first ovulation of normal controls ; b, Second activity cycle following first parturition in normal controls ; c, Response to feeding following drastic starvation. matches that of the smallest inactive glands in normal controls, through specimens appearing moderately or fully active, the range extends to organs much larger than any observed in the previous studies of this series. These supernormal values seem to be unique for gonadectomized females. They are, therefore, of primary interest, even though their number in the present series is comparatively small. A total of 26 animals surpasses the maximum size of the corpora allata characteristic of the second reproductive cycle of normal controls. 196 BERTA SCHARRER AND MARIANNE VON HARNACK Of these, seven exceed also the higher level reached by females which had been returned to a normal diet after prolonged starvation (von Harnack, 1958). The possible reasons for the scarcity of excessively high corpus allatum values as well as the great variability of corpus allatum size within age groups will be discussed below. If one follows, in consecutive age groups, the distribution of the maxima in corpus allatum size a pattern becomes apparent which is indicated by the arrows in Figure 1. Comparable to the situation in normal post-emergence females, there is, with increasing adult age, a gradual volumetric increase. However, in contrast to the intact animals with, their predictable periodic return to corpus allatum inactivity, the growth trend continues for a longer time in the ovariecto- mized animals. At about 100 days of adult age, their highest value for the corpus allatum reaches the limit typical of the second reproductive cycle of normal females. One hundred and forty days after emergence the maxima in the castrates match the still higher top level of those females which had received a normal diet after drastic starvation (von Harnack, 1958). The "giant" corpora allata, found so far exclusively in castrate females, all occur in the age group between 140 and 280 days. After this period the maxima gradually decline throughout the remaining adult life span whose termination is represented by a female killed at the high adult age of 455 days. The interpretation of these volumetric data should be based on the following considerations. Our previous studies of the activity cycles of the corpora allata of LeucopJiaea have shown a remarkable capacity of these glands to change their structure according to physiological conditions, a responsiveness which expresses itself by alternating volumetric increase and decrease. It is difficult to assess the range of individual variation, both as to the degree of response and the time required by each specimen to react in a given situation. Since this variability from individual to individual exists in normal animals kept under uniform laboratory conditions (Scharrer and von Harnack, 1958), one may expect it to be even more pronounced in experimental animals such as the starvation series (von Harnack, 1958) or in the present, in which the normal cyclic pattern of the corpora allata has been disrupted by the removal of an important target organ, the ovary. Furthermore, the value obtained for a given specimen fixed at any given age does not permit conclusions as to its past history or (theoretical) future potentiality. For example, small corpora allata in an animal of let us say 200 days of adult age may have become hyperactive early, as did those of the female fixed 148 days after emergence, and may subsequently have regressed. It is equally possible that a sample represents a slowly responding type which would have eventually reached a peak of the kind indicated by the excessively large gland fixed at the adult age of 280 days. At the time of fixation, a corpus allatum devoid of feed- back stimuli from the ovary may even have passed through more than one phase of activation. This possibility is suggested by the observation that corpus allatum implants free from central nervous control may change from inactivity to activity and vice versa (Engelmann, 1960). Certain as yet undetermined extrinsic or intrinsic conditions may modify the effect of castration on the corpus allatum (see also Doane, 1960). Finally a specimen, for reasons unknown, may have been altogether unresponsive to the abnormal situation created by gonadectomy. CORPUS ALLATUM AND CASTRATION 197 In view of all these considerations, it is not surprising that a relatively large number of cases in the present experimental series show corpora allata that lie within the normal range of volumetric variation. Theoretically, the chances of "hitting" a gland at its peak value are small, especially if the state of maximal activation were of short duration. Thus it is the occurrence rather than the frequency of abnormally high volu- metric values which is of primary significance. Their existence demonstrates the capacity of the corpus allatum of Lcncophaea to respond to abnormal physiological states which, in degree, far surpasses even that observed under drastically changed nutritional conditions. The most plausible explanation for this pronounced histo- physiological response in gonadectomized specimens seems to be the persistent lack of ovarian feed-back stimuli. The prolonged effort of the corpora allata to stimulate a target organ which is no longer present presumably results in morphological changes indicative of hyperf unction. The assumption made here, that corpus allatum volumes are not maintained at peak levels indefinitely, is supported, but not proved, by the absence of abnormally high volumetric values among all gonadectomized females of the series whose adult life span surpasses 280 clays. Since no periodic changes in afferent stimuli, like those from gonad and ootheca in normal females, can account for the inactiva- tion of the corpora allata of castrates, one may conclude that states of excessive glandular stimulation eventually subside on their own account. The next question of interest concerns the changes in nuclear-cytoplasmic ratio in relation to corpus allatum volume (Fig. 2). It may be recalled that, as a general rule established in normal adult females, the number of nuclei per unit of corpus allatum tissue decreases with increasing organ volume. One may inquire whether the same holds for the corpora allata of gonadectomized females and, if so. whether the shift in nuclear-cytoplasmic ratio continues in those female cas- trates whose corpus allatum volume surpasses the maxima of the normal physio- logical range. In all those gonadectomized females whose corpus allatum size, at the time of fixation, was within that of the normal physiological range, the relationship between organ volume and nuclear-cytoplasmic ratio compared well with that in unoperated controls. In the smallest corpus allatum from a castrate animal (3.6 million ju, 3 ) the number of nuclei per mm.- was calculated as 17,251. In a control gland of similar size (3.4 million /j. 3 ) the corresponding figure was 16,765. In both the experimental and the normal series, these figures gradually decreased with increas- ing organ size to about one fourth of the initial level. In the castrates, glands with volumes paralleling the upper limit of the normal physiological range had, for example, 4389 nuclei per mm.-', the corresponding count for normal specimens being 3770 nuclei per mm.-. Young and old gonadectomized females with corpora allata of similar size did not show differences in nuclear-cytoplasmic ratios. Thus, gen- erally speaking, the level of "activity," as determined by the histological criteria discussed so far, is approximately the same in pairs of specimens with comparable corpus allatum size, irrespective of age, or the presence or absence of ovaries. If the trend in shifting nuclear-cytoplasmic ratios were to continue at the same rate beyond the confines of the physiological size range, the relative cytoplasmic content and thus the degree of "activity" in the largest corpora allata of the gonad- ectomized series would indeed be unusual. 198 BERTA SCHARRER AND MARIANNE VON HARNACK 64 60 56 52 48 . 44 = 40 ' . 36 UJ 32 3 28 2 24 20 16 12 8 o o o O 00 O 00 o O O o o XX 24.000 i 20.000 - 16.000 ' I < 12000 ( I ( 8,000 I * 4.000 vt i <\J 14.000 - J 12.000 - X J.5 10.000 f 3 8,000 ~ X vX X X xxx X X X X v x X X "5 6.000 x x x x w x x x xx xx x x/x \ x xx x x i i i i i i r i x i I i i i i ! i i i i I I i I I 40 80 120 160 200 24O 280 ADULT AGE (days) 320 360 400 440 FIGURE 2. Diagram summarizing quantitative analysis of corpora allata of 57 samples of ovariectomized Lcucophaca selected so as to illustrate the range of variation throughout the adult life span. = volume of both corpora allata ; o = number of nuclei calculated for both glands ; x = nuclear-cytoplasmic ratio. The following data show that this is not the case. The smallest figure, 3486 nuclei per mm.-, found among the oversized corpora allata of the castrates, is not significantly lower than the minimum of the control series (3698 nuclei per mm. 2 ). Therefore, one must conclude that the increase in relative cytoplasmic content with rising corpus allatum size reaches a plateau at a volumetric level equal to the maximum of the control group. Beyond this point, there seems to be no more drastic change in nuclear-cytoplasmic ratio, a situation which is also demonstrated by the fact that the nuclear counts in gonadectomized specimens continue to rise UJ. I0 r m ~ 1 1 1 1 1 i i i i i i i i i 1 I 40 80 120 ADULT 160 AGE 200 240 ( days ) 280 320 360 FIGURE 3. Diagram showing the volumetric values of corpora allata of 27 gonadectomized males. Compared with the situation in females, no clear-cut castration effect is noted. The range of variation is small and bears no relationship to adult age. CORPUS ALLATUM AND CASTRATION 199 with increasing corpus allatum volume. They reach a maximum (18,736), which considerably surpasses that in normal controls. Thus it appears that the increase in corpus allatum volume following ovari- ectomy, once it reaches abnormal proportions, differs from the growth process signalling activation of the gland in the course of its normal functional cycles. In the normal gland, the increase in nuclear number is surpassed by that in cytoplasmic content. By contrast, both nuclear number and cytoplasmic content increase pro- portionally during the period of overgrowth of the corpora allata of castrates. In accordance with their considerable size variation, the corpora allata of castrate females differ widely in their morphology. As may be expected, they lack clear-cut signs of cyclic activity such as those that can be identified in normal females (Scharrer and von Harnack, 1958). Thus, for example, mitotic figures and pycnotic nuclei, although not frequent, occur throughout the life span with the exception of very young castrate adults. Aside from this apparent difference in periodicity, the quantitative data as well as histological features presented so far indicate that, within a comparable volu- metric range, the glands of castrates resemble those of normal controls in several ways. A small corpus allatum, be it from a newly emerged normal or an old gonad- ectomized animal, has densely packed nuclei and shows very little cytoplasmic detail. With increasing size of the corpora allata conspicuous histological changes which are related to the gradually shifting nuclear-cytoplasmic ratio occur in both the normal and the experimental groups. Cells filled with small, aldehyde ftichsin-positive granules, similar to those de- scribed in normal animals (Scharrer and von Harnack, 1958), are found in many of the castrates, especially those with medium-sized and large corpora allata. As a rule such cells are not numerous, and a careful search of serial sections is neces- sary for their detection. The youngest castrate female in which such cells were recorded had been killed 30 days after emergence. From this point on no prefer- ential age distribution of positive cases was apparent. The present study did not add anything to the understanding of the functional role of these cells. The possi- bility that they are hemocytes is being explored. More important in the corpus allatum cells of castrate as well as normal females are inclusions which stain lavender or pale green with the Halmi-Dawson method used. They are present after fixation in Helly's as well as formalin, AFA, and Carnoy's solution. In saliva-treated PAS preparations these granules stain intensely positive. This and the results with the other techniques used so far suggest that they belong to the category of glyco- or mucoproteins. Matched pairs of experimental and normal animals show the following relation- ships. In younger castrates the PAS-positive granules are not conspicuous ; they compare in number and size with those of normal controls. However, at an adult age of two months and over, the amount of this material in castrates with good-sized corpora allata significantly surpasses that in unoperated females (Figs. 6 and 7). The examination of several other tissues revealed that this phenomenon is not restricted to the corpus allatum (Scharrer and von Harnack, 1960). In the fat body, the difference between ovariectomized and normal specimens is consider- able. The wide range of possible variation in the content of PAS-positive, saliva- resistant material is illustrated by Figures 10 and 11. It should be stated, however, 200 BERTA SCHARRER AND MARIANNE VON HARNACK FIGURES 4-7. CORPUS ALLATUM AND CASTRATION 201 that not all castrates reach the degree of accumulation shown in Figure 10, while on the other hand normal controls may contain more inclusions than those depicted in Figure 11. In spite of these variations, there can be no doubt that the average amount of this material is considerably increased several months after gonadectomy. Comparable PAS-positive inclusions are also noticeable in the muscles of gonadectomized females with an adult age of over 40 days. They are particularly conspicuous in areas near the insertion of a muscle fiber in the chitinous skeleton (Fig. 12). In normal animals very few or no such inclusions are observed in the musculature. Another example is the central nervous system where castrates also show a greater amount than normal females of PAS-positive material other than glycogen. This material is seen in varying amounts within numerous ganglion cells. How- ever, the most pronounced difference occurs in a special type of neurosecretory cell (B cell) of the subesophageal ganglion which normally contains only few granules in the periphery of the perikaryon (Fig. 9). With few exceptions, in castrates this pair of cells accumulates large amounts of cytoplasmic granules which stain intensely with the PAS technique (Fig. 8). In Halmi-Dawson preparations these granules are distinguished by their green coloration. These cells have been called "castration cells" in an earlier study (Scharrer, 1955). Within the limited scope of the histological tests employed so far, the inclusions in the fat body, musculature, and nervous tissue resemble those of the corpora allata. Morpho- logically they differ in that they are larger. Sections of corpora allata with high cytoplasmic content may show varying degrees of vacuolization. Also, individual cells may appear partially separated from each other and thus more easily identified than in smaller specimens where cell boundaries can be detected only in electron micrographs. When the glands reach abnormally high volumes in castrates, the form and arrangement of individual cells undergo further characteristic changes. The orientation of cell processes toward the periphery of the gland and the probable relationship of this arrange- ment to the release of secretory material into the surrounding hemolymph in highly active normal corpora allata have already been reported (Scharrer and von Harnack, 1958). In the largest castrate specimens, many cells appear "stretched" to the point of becoming spindle-shaped. The resulting pattern of corpus allatum struc- ture is quite characteristic (compare Figs. 4 and 5). To what extent this might FIGURE 4. Section through corpus allatum of gonadectomized female of Lcitcopliaca. Adult age 148 days; volume of corpora allata 62.9 million n 3 (maximal value of series). Note characteristic pattern of spindle-shaped cells. Helly, 7 M, modified aldehyde fuchsin. X 450. FIGURE 5. Control section for Figure 4 ; same technique. Active corpus allatum of normal female fixed at adult age of 125 days; 15 days after parturition; ovarian eggs nearly mature; volume of corpora allata 18.7 million M 3 . Note irregular arrangement of cells (the existence of cell boundaries, difficult to see in the light microscope, will be demonstrated in a future publication on the ultrastructure of the corpus allatum). >C 450. FIGURE 6. Section through corpus allatum of castrate female showing numerous PAS- positive granules. In addition to these intracellular inclusions, other PAS-positive elements seen are a small part of the corpus allatum sheath (upper right corner) and intercellular material within the gland. Adult age 228 days. Formalin, 7 M, saliva pretreatment, PAS. < 1200. FIGURE 7. Control section, treated with same technique as in Figure 6, through active corpus allatum of normal female of same adult age; 10 days after parturition; development of ovarian ova well under way. Note relative paucity of PAS-positive granules. Intercellular elements are comparable to those in Figure 6. X 1200. 202 BERTA SCHARRER AND MARIANNE VON HARNACK ililK "**# 'A - V"& m * 4* MP ^j* 111 \ f W . ^* * .^ 4^3 mtir^ v / \ FIGURES 8-12. CORPUS ALLATUM AND CASTRATION 203 be a sign of "pathological" change remains undetermined. Aside from this feature, glands of extraordinary size show few cytological peculiarities not also observed in smaller glands. In our material several unoperated cases with a history of ovarian dysfunction, related to senility or of unknown origin, were of particular interest for comparison with castrates. As might be expected, these "normal" animals displayed several of the cytological features, though not always all, characteristic of the experimental group. For example, one specimen with the exceptional adult age of 599 days had abnormally large corpora allata (vol. 37 million /j. 3 , as compared with 25.5 for the maximum control value) with a normal "active" appearance except for the presence of a giant nucleus. In this case, PAS-positive granules were more abun- dant than normally, in the corpora allata as well as the central nervous system and the fat body, but not in the musculature. In another case, which had reached the adult age of 300 days without signs of reproductive activity, the number of granules was increased in the musculature as well as the corpus allatum and the fat body. In view of the castration effects just described it was of interest to study the situation after unilateral gonadectomy. Fifty-two females whose right ovary had been removed before emergence produced offspring at normal intervals but, on the average, only one-half of the number of young borne by unoperated control animals (Scharrer, 1958b). In their histology, the corpora allata of this experi- mental group resembled those of normal adult females. According to the stage in the ovarian cycle at which the animals were fixed, the structural criteria used in this study indicated varying degrees of glandular activity. The fat body, muscula- ture, and nervous tissue also showed no abnormalities. Thus, hemicastration apparently fails to lead to those morphological changes characteristic of females without both ovaries or with severe disturbances in gonadal function. b. Males The corpora allata of the 26 castrated males studied showed no drastic effects of the operation. The volumetric values in this group (range: 2.8 to 9.4 million fj. 3 ; mean: 5.5 million ju, 3 ) are only somewhat higher than those of 46 normal control specimens (range: 2.1 to 9.0 million ^. 3 ; mean: 4.9 million ^ 3 ). The total number of nuclei calculated for the smallest (3486) and the largest glands (3867) of the FIGURE 8. Section through subesophageal ganglion of castrate female showing abundance of PAS-positive granules. These occur in various ganglion cells, but are particularly concen- trated in the large B-type neurosecretory cells designated as "castration cells" ; one of these in upper left corner of Figure. Same animal and same technique as in Figures 6 and 10. X 600. FIGURE 9. Control section through same area as in Figure 8 of subesophageal ganglion of normal adult female. Note that B-type neurosecretory cell and adjacent ganglion cells con- tain only a few PAS-positive granules. Formalin, 7 /*, saliva pretreatment, PAS. X 600. FIGURE 10. Section through cephalic fat body of castrate female. Same animal and same technique as in Figures 6, 8, 12. Note numerous PAS-positive granules. < 1400. FIGURE 11. Control section for Figure 10 through cephalic fat body of normal female. Same animal and same technique as in Figure 7. In contrast to that shown in Figure 10, this specimen contains few granules. X 1400. FIGURE 12. Longitudinal section through cephalic musculature of castrate female. Same animal and same technique as in Figures 6, 8, 10. Note conspicuous accumulations of PAS- positive granules. Normal controls show only rare granules. X 560. 204 BERTA SCHARRER AND MARIANNE VON HARNACK gonadectomized series differs only little, and consequently the range of nuclear- cytoplasmic ratios is considerable (15,571 nuclei per mm. 2 in the smallest, and 5117 in the largest gland). Thus the corpora allata of castrate males resemble those of normal males and females in that the relative amount of cytoplasm is increased when the glandular volume rises. In normal adult males, fluctuations in corpus allatum volume occur during the entire adult life span (Scharrer and von Harnack, 1958). Similarly, castrates show no significant variations of "activity" as determined by organ volume and nuclear-cytoplasmic ratio during adult life (Fig. 3). Neither is there, within the range of the time intervals of the experiment, a relationship between these values and the total (nymphal plus adult) length of the castration period. The animal with the largest glands happens to have an adult age of 138 days, that with the smallest one of 273 days. It may also be of interest to illustrate the relationships between male and female castrates by selecting a comparable pair of specimens. In the male with the largest corpora allata (9.4 million /* 3 ) the total nuclear count was 3896 (5117 nuclei/mm. 2 ), in a female with corpora allata of the same size it was 3994 (6110 nuclei mm. 2 ). Inasmuch as the corpora allata of male Leucophaea show hardly any volumetric response to changing physiological conditions, either during the normal life cycle or following castration, no dramatic changes in glandular histology were expected. The glands from castrate males compare in their cellular structure with those of normal males and of females whose corpora allata are inactive or only mildly active. The amount of saliva-resistant PAS-positive material present in castrate males does not surpass that in normal males or females. This applies to the cell inclusions in the corpora allata as well as those in the fat body, musculature, and nervous tissue. DISCUSSION The present study has demonstrated significant changes in the histology of the corpora allata in response to gonadectomy in females of Lcucopliaea. By con- trast males showed at best only a very mild reaction. The latter result is in line with previous negative results in males of several other insect species (Day, 1943; Bodenstein. 1947; Johansson, 1958). The slight rise in the average volume of the corpora allata of male castrates of Leitcopliaea might be dismissed as insignifi- cant, except that it seems to parallel a similar responsiveness in normal adults. In these, a mild stimulation of the glands occurs during the post-emergence period, i.e., at a time when in normal females a much more pronounced (pre-ovulatory) activation of the corpora allata was observed (Scharrer and von Harnack, 1958). Possibly these data indicate a limited capacity in male insects to respond to changes in the internal milieu comparable to that displayed, for example, by the male mammary gland. The lack of clear-cut histological correlations between the corpora allata and gonadal activity in males of Leucophaea is not surprising because all other available data speak for the absence, in the male sex, of a physiological role of these glands in the control of reproduction (Scharrer, 1946). But even in a case, such as in the gregarious phase of Scliistoccrca, where "sexual maturation" in the male is controlled by the corpora allata, these glands seem unaffected by gonadectomy, at least as far as physiological criteria are concerned (Loher, 1960). The data in the literature are less consistent regarding the situation in female insects. Several investigators noted a volumetric increase in the corpora allata CORPUS ALLATUM AND CASTRATION 205 of castrates (Pfeiffer, 1940; Thomsen, 1942; Day, 1943; Bodenstein, 1947; Johans- son, 1958; for indirect evidence see also Vogt, 1942; Lukoschus, 1956; Doane, 1960; Gill, 1960). Studies in other species failed to reveal increased volumes fol- lowing ovariectomy (Day, 1943; Kaiser, 1949). In view of the small proportion of cases with excessively large corpora allata in the series described in the present paper and their restriction to a certain age group, it seems reasonable to suggest that in the investigations quoted above castration effects ma}- have escaped detec- tion. This was certainly the case in two previous papers dealing with Leucophaea (Scharrer, 1946; Liischer and Engelmann, 1955). On the other hand, species differences have to be taken into consideration. This refers not only to volumetric responses, but to the manner in which these come into effect. For example, in two Diptera, Callipliora (Thomsen, 1942) and Lucilia (Day, 1943), the higher volume in the corpora allata of castrates is due to an increase in cell size, but not in cell number, whereas in Leucophaea an increase in cell number, as well as cell size, in conjunction with glandular activation has been definitelv established. j The possible reasons for the low frequency of corpora allata showing pronounced castration effects in our present material have been discussed earlier in this paper. The same interpretation may apply to other species for which a considerable overlap between the volumetric values of gonadectomized and normal specimens has been reported (Thomsen, 1942; Bodenstein, 1947). Similarly, according to Doane (1960) 4 , the hypertrophy of the corpus allatum in the sterile mutant adp/adp of Drosophila is an unstable condition. These observations, in addition to the present more detailed study, make it seem unlikely that the corpora allata of ovariectomized females stay active indefinitely as was postulated for Leucophaea by Liischer and Engelmann (1955). The role of the PAS-positive, saliva-resistant granules observed in various tissues of Leucophaea is still uncertain. The discussion of their possible significance must take into account the following points : ( 1 ) Cytoplasmic inclusions whose histochemical characteristics suggest that they belong to the category of glycoproteins are not restricted to the corpora allata, but occur in a variety of other tissues. Among these, the fat body, the musculature, and the nervous system have been tentatively explored in this study. The tests applied so far are too limited to permit any conclusions regarding the extent of chemical relationship among these cell inclusions. The granules occur in both sexes. Their numbers show some individual variation, presumably in relation to different physiological states. It seems possible that some of them, e.g., in the corpus allatum or in the neurosecretory cells, are glycoprotein hormones or their precursors comparable to those of the delta cells of the vertebrate adenohypophysis. (2) Attention should be focused on the fact that these ''glycoprotein" granules increase in number and size in the corpora allata of gonadectomized females, as compared with normal controls. Concomitantly, the fat body, musculature, and 4 We wish to thank Dr. W. W. Doane, Yale University, for permitting us to read her thesis before its publication. Part of it has appeared in print since the present paper went to press. See: Doane, W. W. 1960. Developmental physiology of the mutant female sterile (2) adipose of Drosophila i/ielanogastcr. I. Adult morphology, longevity, egg production, and egg lethality. J. Exp. Zool., 145, 1-21 ; and Doane, W. W. 1960. Developmental physiology of the mutant female sterile (2) adipose of Drosophila melanogaster. II. Effects of altered environment and residual genome on its expression. J. Exp. Zool., 145, 23-41. 206 BERTA SCHARRER AND MARIANNE VON HARNACK central nervous tissue of female castrates show conspicuous deposits of this material, whereas it may be almost absent in normal animals. Furthermore, the tissues examined show a certain uniformity as to the degree of their response. A given animal with a particularly high content of inclusions in the fat body also has proportionately more of them in the other tissues involved than does another animal in which these changes are altogether less conspicuous. Another site showing castration effects of this kind is the hemolymph, but their analysis must await future study. By contrast, male castrates do not differ from intact males with respect to the amounts of this material present in the various tissues mentioned. This dif- ference between the sexes represents an additional illustration of an already known situation, i.e., the apparent absence of a corpus allatum-gonad axis in male Leu- cophaca. Thus, the data in castrate and normal males can hardly contribute to the understanding of the functional relationships among the "glycoprotein" granules in different tissues. Our interest, therefore, centers on the more dynamic situation in the females. Here, the marked increase of these inclusions after gonadectomy, as well as the parallelism in the response of several different types of tissue, suggests a common denominator. Obviously, the appearance of these castration effects is related to the absence of the ovaries. One possible explanation of the phenomenon is that the increase in the number and size of these cytoplasmic inclusions in animals without ovaries is an expression of a metabolic change (see, for example, Sagesser, 1960). Perhaps "precursor materials" (Pfeiffer, 1945), normally intended for yolk production, become de- posited in abnormal quantities in tissues of castrate females. If this were true in the case of Lcncophaca, allatectomized females, in which the ovaries and accessory sex glands are arrested (Scharrer, 1946), should show an increase in cell inclu- sions in the fat body, musculature, etc. comparable to that in castrates. Preliminary exploration of such animals, as well as female castrates subjected to prolonged starvation, suggests that the cell deposits in question are not, or at least not exclusively, surplus metabolites and that a role of the ovary more specific than that of "depository" for yolk material may be involved. Perhaps the castration effects reported are related to the absence of an ovarian hormone, an interpretation which is also strengthened by recent observations in Drosophila (Doane, 1960). The effects of gonadectomy on the corpora allata in female insects discussed here show certain parallelisms among crustaceans and vertebrates ; in these both sexes are involved. Parasitic or surgical castration in isopods, in which no organ analogous to the corpus allatum is known, leads to hypertrophy of the sinus glands (Yamamoto, 1955, Oguro, 1960). In mammals, such as the rat, the absence of the gonads elicits changes in the anterior pituitary indicative of glandular hyper- function. Among such castration effects have been reported not only hypertrophy and increased gonadotropin content, but also characteristic changes in the mor- phology and cytochemistry of the gonadotrophs resulting in "castration cells" (see Hellbaum and Creep, 1940; Ladman and Barrnett, 1956; Takewaki, 1956). It is of particular interest that these cells, like the corpus allatum cells of ovariecto- mized Lcncophaca, contain more glycoprotein than those of normal controls (Catch- pole, 1949/50; Purves and Griesbach, 1955). CORPUS ALLATUM AND CASTRATION 207 SUMMARY 1. The corpora allata of Leucophaea maderae display an impressive capacity to respond to alteration of the internal milieu resulting from ovariectomy. Com- pared with the periodic structural changes of these glands in the course of normal reproductive cycles, the morphological characteristics of the corpora allata of castrate females are quite unpredictable. There is considerable spread from low organ volumes indicating glandular inactivity to excessively high values so far unmatched in non-castrate animals. 2. Only a small proportion of ovariectomized animals have abnormally large corpora allata, and these are confined to an intermediate period of the adult life span. This suggests that their growth response lacks permanency. Its apparent cause is the persistent absence of feed-back stimuli from the ovaries. The reasons why this castration effect on the corpus allatum eventually subsides are unknown. 3. The rise in relative and absolute cytoplasmic content with increasing organ size, observed earlier in normal animals, applies equally to gonadectomized females whose corpora allata at the time of fixation did not exceed the maximum of the control values. Above this level, the growth pattern differs in that no further drastic change in nuclear-cytoplasmic ratio occurs, i.e., nuclear numbers and cyto- plasmic content increase at the same rate. 4. Compared to those of normal controls, the corpora allata of castrate females contain progressively increasing amounts of saliva-resistant PAS-positive granules, probably glycoprotein in nature. A conspicuous accumulation of comparable cyto- plasmic inclusions also occurs in various other tissues, such as the fat body, muscu- lature, and nervous system. The functional relationships of these variously located inclusions are still uncertain, but the parallelism in their frequency suggests a common denominator. Whether they accumulate because of the absence of an ovarian hormone, or merely in response to metabolic changes following castration, is as yet undetermined. 5. In contrast to females, male castrates of Leucophaea do not differ significantly from normal controls as to the volume and cytoplasmic content of their corpora allata. The same applies to the presence of PAS-positive material in various tissues. This difference between the sexes in response to castration is undoubtedly related to the absence of a corpus allatum-gonad axis in male Leucophaea. LITERATURE CITED BODENSTEIN, D., 1947. Investigations on the reproductive system of DrosophUa. J. E.vp. Zool 104: 101-151. CATCHPOLE, H. R., 1949-50. Distribution of glycoprotein hormones in the anterior pituitary gland of the rat. /. Endocrinol., 6: 218-225. DAY, M. F., 1943. The function of the corpus allatum in muscoid Diptera. Biol. Bull.. 84 : 127-140. DOANE, W. W., 1960. Corpus allatum-complex and ovarian transplantations in the mutant female sterile (2) adipose of Drosophila melanogastcr. Anat. Rec., 138 : 344. ENGELMANN, F., 1957. Die Steuerung der Ovarfunktion bei der ovoviviparen Schabe Leuco- phaea madcrae (Fabr.). /. Ins. Physiol., 1: 257-278. ENGELMANN, F., 1960. Mechanisms controlling reproduction in two viviparous cockroaches (Blattaria). Ann. N.Y. Acad. Sci., 89: 516-536. GILL, K. S., 1960. Developmental physiology of five female-sterile mutants in Drosophila melanogastcr. Anat. Rcc., 138 : 351. 208 BERTA SCHARRER AND MARIANNE VON HARNACK HARNACK, M. vox, 1958. Histophysiological studies on the corpus allatum of Leucophaea inadcrac. II. The effect of starvation. Biol. Bull., 115: 521-529. HARNACK, M. VON, AND B. SCHARRER, 1956. A study of the corpora allata of gonadectomized Leucophaea madcrae (Blattaria). Auat. Rcc., 125: 558. HELLBAUM, A. A., AND R. O. CREEP, 1940. Qualitative changes in the gonadotropic complex of the rat pituitary following removal of the testes. Amcr. J. Anat., 67: 287-304. JOHANSSON, A. S., 1958. Relation of nutrition to endocrine-reproductive functions in the milkweed bug Oncopeltus fasciatns (Dallas) ( Heteroptera : Lygaeidae). Nvtt Ulaci. Zool.: 7: 1-132. KAISER, P., 1949. Histologische Untersuchungen iiber die Corpora allata und Prothoraxdriisen der Lepidopteren in Bezug auf ihre Funktion. Arch. f. Entit'., 144: 99-131. LADMAN, A. J., AND R. J. BARRNETT, 1956. Variations of the histochemically demonstrable protein-bound sulfhydryl and disulfide groups in cells of the anterior pituitary gland under various endocrine conditions. /. Morph., 98: 305-334. LOHER, W., 1960. The chemical acceleration of the maturation process and its hormonal con- trol in the male of the desert locust. Proc. Roy. Soc. London. Scr. B., 153: 380-397. LUKOSCHUS, F., 1956. Untersuchungen zur Entwicklung der Kastenmerkmale bei der Honig- biene (Apis mcllifica L. ). Zeitschr. Morph. u. Okol. Ticrc, 45: 157-197. LUSCHER, M., AND F. ENGELMANN, 1955. t)ber die Steuerung der Corpora allata-Funktion bei der Schabe Leucophaea madcrae. Rev. Suisse Zool., 62 : 649-657. OGURO, C., 1960. Effects of castration on the neurosecretory system in the marine isopod, Tecticeps japonicus. I. Effects on the sinus gland of male animals. Annot. Zool. Jap.. 33: 37-41. PFEIFFER, I. W., 1940. Further studies on the function of the corpora allata in relation to the ovaries and oviducts of Melanoplus diffcrcntialis. Anat. Rcc., 78: Suppl., 39-40. PFEIFFER, I. W., 1945. Effect of the corpora allata on the metabolism of adult female grass- hoppers. /. Exp., Zool, 99: 183-233. PURVES, H. D., AND W. E. GRIESBACH, 1955. Changes in the gonadotrophs of the rat pituitary after gonadectomy. Endocrinology, 56 : 374-386. SAGESSER, H., 1960. tfber die Wirkung der Corpora allata auf den Sauerstoffverbrauch bei der Schabe Leucophaea inadcrac (F. ). /. Ins. Physiol., 5: 264-285. SCHARRER, B., 1946. The relationship between corpora allata and reproductive organs in adult Leucophaea inaderae (Orthoptera). Endocrinology, 38: 46-55. SCHARRER, B., 1952. Neurosecretion. XL The effects of nerve section on the intercerebralis- cardiacum-allatum system of the insect Leucophaea inaderae. Biol. Bull., 102 : 261-272. SCHARRER, B., 1955. "Castration cells" in the central nervous system of an insect (Leucophaea madcrae, Blattaria). Trans. \". V. Acad. Sci., 17: 520-525. SCHARRER, B., 1958a. Neuro-endocrine mechanisms in insects. Proceedings Second Internat. Sympos. Neurosecretion. Springer Verlag, Berlin-Gottingen-Heidelberg, pp. 79-84. SCHARRER, B., 1958b. Reproduction in insects after unilateral ovariectomy. Anat. Rec., 130: 369. SCHARRER, B., 1959. The role of neurosecretion in neuroendocrine integration. Proceedings Columbia University Sympos. on Comparative Endocrinology, Cold Spring Harbor. Wiley, New York, pp. 134-148. SCHARRER, B., AND M. VON HARNACK, 1958. Histophysiological studies on the corpus allatum of Leucophaea inaderae. I. Normal life cycle in male and female adults. Biol. Bull.. 115: 508-520. SCHARRER, B., AND M. VON HARNACK, 1960. Castration effects in the insect, Leucophaea inaderae. Anat. Rec., 138: 382. TAKEWAKI, K., 1956. Changes in the release of gonadotrophic hormones from the hypophysis of the rat after castration. Annot. Zool. Jap., 29: 129-132. THOMSEN, E., 1942. An experimental and anatomical study of the corpus allatum in the blow- fly Calliphora erythrocephala Meig. I'idcnsk. Mcdd. Dansk Nathist. Forcniny, 106: 317-405. VOGT, M., 1942. Weiteres zur Frage der Artspezifitat gonadotroper Hormone. Untersuchun- gen an Drosophila-Arten. Arch. f. Entzc., 141 : 424-454. YAMAMOTO, Y., 1955. Effect of ovariectomy on the sinus gland of the isopod crustacean, Armadillidium imlgarc. Annot. Zool. Jap.. 28: 92-99. Vol. 121, No. 2 October, 1961 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY INTRA- AND INTERSPECIFIC DIFFERENCES IN RATE OF OXYGEN CONSUMPTION IN GOBIID FISHES OF THE GENUS GILLICHTHYS * GEORGE W. BARLOW 2 Department of Zoology, University of California, Los Angeles, California Many investigators have searched for adaptive physiological differences between intraspecific units. The growing literature has been reviewed a number of times (Thorpe, 1940; Timofeef-Ressovsky, 1940; Dobzhansky, 1951, Ch. 6; Prosser, 1955, 1957; Dehnel, 1955; Moore, 1957). Recently attention has turned to physiological variation in relation to latitudinal distribution (Roscoff, Station Biologique, 1957; Vernberg 1959a, 1959b; Vernberg and Tashian, 1959) . The results from most of the past research of this type cannot be applied to studies of intraspecific divergence (Prosser, 1957). Unfortunately, the early workers either did not consider thermal acclimation (Bullock, 1955, for review), or purposely acclimated the animals to the temperatures at which they were found (e.g., Rao, 1953). Thus latitudinal and acclimation effects have not been clearly separable. Developmental rates of embryos from latitudinally separated populations (Dehnel, 1955) may be independent of latitudinal difference in environ- ment, but only if temperature at the time of yolk formation is without effect on the metabolism of the yolk-consuming embryo. To appreciate genetically adaptive divergence in physiology as a function of latitude, the animals must be acclimated to, and compared at, the same temperatures. The present study was undertaken to establish the degree of physiological di- vergence, if any, between widely separated populations of Gillichthys mirabilis Cooper. The results of an extensive morphological examination of several popula- tions of this species, and of Gillichthys seta (Ginsburg), (Barlow, 1961, and un- published data) constitute the point of departure for the program at hand. Oxygen consumption, a highly integrated metabolic phenomenon, was the physiological system chosen for the investigation. 1 A revised portion of the dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of California, Los Angeles. Pro- cedural details, and further figures and tables, are available in the dissertation. 2 Present address : Department of Zoology, University of Illinois, Urbana, Illinois. 209 Copyright 1961, by the Marine Biological Laboratory 210 GEORGE W. BARLOW MATERIAL AND METHODS Gillichthys seta doubtless is the species most closely related to G. mirabilis (Barlow, 1961). In occurrence, G. seta is restricted to the northern part of the Gulf of California. It is found only along the exposed coast in the highest intertidal zone among the streams and pools of rocky reefs. The habitat of G. mirabilis is the intertidal region of coastal sloughs. Typically, this is a muddy region exposed to air at low tide. Some of the fish remain at low tide in crab burrows, or the likes, and breathe air ; G. seta also is capable of aerial respiration. The distribution of G. mirabilis embraces several faunal regions. The northmost populations in central California co-exist with a low-boreal fauna; the southmost populations in the Gulf of California occur within a tropical Panamic region ; and the northern part of the Gulf of California, where G. mirabilis is also found, has a depauperate, mixed fauna. Thus G. mirabilis is arranged in disjunct populations over three roughly parallel, north-south, coast lines. Distribution along a north-south coast ordinarily corresponds to a temperature gradient, warmer to the south. This applies to the Pacific Coast, and in the winter to the Gulf of California. In the late summer, however, the gradient in the Gulf is either reversed, cooler to the south, or the temperatures are everywhere about the same. As a result, the annual range of mean temperatures in the Gulf of Cali- fornia increases from south to north. The climate of the Salton Sea continues this trend of increased temperature extremes to the north. In 1930 G. mirabilis was introduced into the Salton Sea. The parental stock came from San Diego Bay, just a few miles south of the slough at Los Penasquitos. The fish have become well established in the Salton Sea in spite of the many peculiar features (Carpelan, 1958) of this environment. Judging from the number of aber- rant specimens, the Salton Sea is a marginal habitat for G. mirabilis. Treatment of the animals The fish were held in sea water in 20- and 40-gallon tanks. The water was aerated, and filtered continuously, and was replaced with fresh sea water two to three days prior to each experiment. The fish were fed exclusively on fresh squid, which they ate voraciously. The number of days between feedings, as well as the feeding time, were purposely kept irregular to minimize cyclic behavior. They were not fed just prior to an experi- ment; this interval was adjusted according to the size of the fish and the acclimation temperature (at 10, intervals of seven to twelve days, and at 24, of two to four days; longer periods for longer fish), so that the fish when tested were flat-bellied, and production of fecal material was low. The minimum acclimation time was two weeks at 24 0.5, and four weeks at 10 0.05. Experience indicated acclimation was complete sooner than that, especially at 24. In changing fish from one temperature to the other, they were held for one week at the intermediate temperature of 17, as was done in the experi- ments by Brett (1952). Illumination was continuous. For both species, only male or immature fish were used. OXYGEN CONSUMPTION IN GILLICHTHYS 211 Apparatus Special chambers for the Warburg apparatus were constructed, from ground- glass fittings, to accommodate the elongate fish, and in various sizes (45-cc., 85-cc., and 250-cc.) to maintain the proper ratio between fish and chamber volumes. Reduction of the oxygen tension in the chamber was circumvented by installing an oxygen well, a collapsible bag made of No. 6, 100 gauge, Saran film, on the air inlet of the manometer. Each time the stopcock was opened to balance the manom- eter fluid, pure oxygen, at atmospheric pressure, replaced that utilized. Experimental procedure Each fish was blotted gently, then weighed in the chamber. Filtered sea water was added in an exact amount, equal to about 5% of the flask volume (enough water to wet the belly and the floor of the mouth of the fish). The flask was then flushed with oxygen, potassium hydroxide solution was quickly introduced into the cup, and the flask was affixed to the manometer. The bath was darkened and the manometers were stationary. Temperatures were controlled to within 0.025. The initial temperature was always the same as the acclimation temperature of the fish. The animals were placed in the bath in the evening, and the first reading was taken twelve hours later between 0830 and 0930 hours. To determine the adjust- ment period, however, readings were commenced 30 minutes after placing the fish in the bath. Oxygen uptake was recorded at the acclimation temperature during the thir- teenth and fourteenth hours in the chamber. The bath temperature was then steadily raised 7 during one hour. Two hours elapsed at the new temperature before the oxygen uptake was recorded during another two-hour period, the eighteenth and nineteenth hours in the flasks. Excepting the fish from Puerto San Carlos, no animal was used a second time in the same series of acclimation experiments. The respiratory rates were compared at 10 and 17 for four populations of G. mirabilis acclimated to 10. From north to south, the fish were from San Francisco Bay (Alviso Ponds), latitude 37 N., in central California; Los Peiias- quitos, latitude 33 N., in southern California; the Salton Sea, latitude 33 N. ; and San Felipe ("El Marino" slough), latitude 31 N., in the northern part of the Gulf of California. Though the Salton Sea and Los Peiiasquitos are on about the same latitude, the warmer climate of the Salton Sea is considered here to be more "south- ern." San Felipe is not much farther south than southern California but is in a region with a warmer over-all climate. Fish from the same four places were also acclimated to 24 and tested at 24 and 31. A group of fish from Puerto San Carlos, latitude 28 N., near Guaymas in the central part of the Gulf of California, was included in the latter comparison. The G. mirabilis from San Carlos were small w r hen obtained. In spite of growing well in aquaria, the distribution of sizes of the San Carlos fish included much smaller animals than in the other samples. Consequently they were not included in the analysis of co-variance within the species G. mirabilis. Comparisons between G. 212 GEORGE W. BARLOW mirdbilis and G. seta were based on experiments with G. mirabilis from San Carlos, and G. seta from Puertecitos, latitude 30 N. Treatment of the data The decision to accept or reject the stated statistical hypothesis was based on the 5% critical level in order to minimize type II errors (Hoel, 1954, p. 33). Acceptance of a hypothesis such as equal means does not imply the populations in question were proven to be the same. The mean rate of oxygen consumption was employed throughout. Minimum rates were only 3% to 5% below the mean. Regression lines were fitted by least squares to the logarithms of the experi- mental data. It was convenient to express this rectilinear relationship as oxygen consumption per unit weight, instead of per total weight. The slope is then nega- tive, as indicated in the expression : Rate == a Wt~ 6 , where a is the rate when weight (Wt) equals one, and b is the regression coefficient. 200 100 70 e> o o 40 20 24 V \ ~. \ I 12 15 HOURS FIGURE 1. Time to steady rate of oxgen consumption for Gillichthys mirabilis from Los Penasquitos. Solid lines connect means and dashed lines connect ranges. Individual rates were adjusted to the eight-gram intercept using regression coefficients of 0.28 for the 10 group and 0.56 for the 24 group (n = 5) ; the procedure is discussed in Roberts (1957). OXYGEN CONSUMPTION IN GILLICHTHYS 213 RESULTS Uniformity of results The rate of oxygen consumption remained the same from month to month in a given population. Fish from the Salton Sea, tested in January, April, and May, showed no perceptible differences. And fish from San Carlos re-tested after pe- riods of growth, continued to manifest rates appropriate to their earlier determined regression lines (Fig. 4). The oxygen added to the chambers at the beginning of each experiment ap- parently did not disturb the fish. Controls with air, instead of oxygen, metered into the chambers showed no differences. At 10 the initial rate of oxygen uptake was high, but reached a constant level within nine to ten hours (Fig. 1). The adjustment time at this temperature was independent of the size of the fish relative to the volume of the chamber. The initial rate was also high at 24. For all fish in small or medium size cham- bers, adjustment was achieved within six to ten hours (Fig. 1). The adjustment time in the largest chambers, however, varied depending on the ratio of the fish size to chamber volume. In the large chambers oxygen consumption was reason- ably regular within eight to ten hours if the fish were larger than 15 grams. Be- cause of this, only fish equal to or larger than 20 grams were used in large chambers at 24. Wells (1935a) investigated the adjustment time of G. mirabilis, but in water instead of air. The results of his four experiments show excellent agreement with TABLE I Relation of oxygen consumption to body weight in Gillichthys mirabilis and G. seta Temperature 10 C. 17C. Parameter n a -b n a -b G. mirabilis San Francisco 46 36.9 0.186 34 68.6 0.138 Los Penasquitos 53 43.7 0.277 43 86.2 0.286 Salton Sea 46 31.0 0.160 36 62.6 0.193 San Felipe 35 57.1 0.346 35 79.5 0.247 Temperature 24 C. 31 C. Parameter n a -b n a -b G. mirabilis San Francisco 36 254 0.577 36 300 0.526 Los Penasquitos 49 256 0.560 40 331 0.530 Salton Sea 36 310 0.626 36 419 0.622 San Felipe 34 246 0.568 34 351 0.613 San Carlos 54 248 0.451 42 318 0.487 G. seta Puertecitos 24 177 0.289 24 289 0.257 214 GEORGE W. BARLOW TABLE II Results of co-variance tests on differences in rate of oxygen uptake in Gillichthys mirabilis and G. seta Entries refer to G. mirabilis unless stated otherwise. F = F ratio, d.f. = degrees of freedom, 0.95 = 5% critical value Tempera- ture F d.f. Fo.95 Decision Hypothesis: means same after adjustment for body weight San Francisco/Los Penasquitos/Salton 10 C. 8.50 3,175 2.66 Reject Sea/San Felipe San Francisco/Los Penasquitos/Salton 17 C. 6.58 3,143 2.67 Reject Sea/San Felipe San Francisco/Los Penasquitos/Salton 24 C. 1.93 3,150 2.67 Accept Sea /San Felipe San Francisco/Los Penasquitos/Salton 31 C. 5.46 3,141 2.67 Reject Sea /San Felipe G. mirabilis (San Carlos) /G. seta (Puertecitos) 24 C. 28.6 1,76 3.97 Reject G. mirabilis (San Carlos) /G. seta (Puertecitos) 31C. 1.07 1,63 3.99 Accept Hypothesis: Same slope within the groups San Francisco/Los Penasquitos/Salton 10 C. 2.65 3,172 2.66 Accept Sea /San Felipe San Francisco/Los Penasquitos/Salton 17 C. 3.58 3,140 2.67 Reject Sea/San Felipe San Francisco/Los Penasquitos/Salton 24 C. 0.68 3,147 2.67 Accept Sea /San Felipe San Francisco/Los Penasquitos/Salton 31C. 2.25 3,138 2.67 Accept Sea /San Felipe G. mirabilis (San Carlos) /G. seta (Puertecitos) 24 C. 7.65 1,74 3.97 Reject G. mirabilis (San Carlos) /G. seta (Puertecitos) 31 C. 12.6 1,62 4.00 Reject the curves in Figure 1, when recalculated in cc. O 2 /kg./hr. and plotted on semi- logarithmic graph paper. For all groups, the mean rate of oxygen consumption remained consistent over a 12-hour period after adjustment, and no cyclic phenomena were evident. During the readings the rate of uptake of each fish fluctuated, resembling fluctuations noted for other fishes by Spoor (1946) and by Pritchard (1955). Influence of sise on oxygen consumption As is so often the case (examples in Dehnel, 1960), the smaller the animal, the greater its oxygen consumption per unit weight. In G. mirabilis and in G. seta this relationship between size and metabolism is more pronounced than in most fishes, the values of b being large (see Discussion). Moreover, both inter- and intraspecific differences in the estimate of b were found in the genus Gillichthys. The slopes of the lines at 10 were nearly the same for the various populations of G. mirabilis, though the differences approached the 5% level of significance (Tables I, II). The slopes for the populations steepened from the northmost ( 0.19) to the southmost (0.35), excluding the Salton Sea group (Table I, Fig. 2), but these differences were of doubtful significance. OXYGEN CONSUMPTION IN GILLICHTHYS 215 7 SAN FRANCISCO SALTON SEA SAN FELIPE 20 - 4 7 10 20 GRAMS 40 70 100 FIGURE 2. Relation between oxygen consumption and body weight of Gillichthys mirabilis from San Francisco, Los Penasquitos, Salton Sea, and San Felipe at 10 (closed circles) and 17 (open circles). 216 GEORGE W. BARLOW 200 200 o o SAN FRANCISCO 100 CL X 70 40 200 100 70 40 20 10 LOS PENASQUITOS SALTON 7 10 GRAMS 20 40 70 100 FIGURE 3. Relation between oxygen consumption and body weight of Gillichthys mirabilis from San Francisco, Los Penasquitos, and Salton Sea at 24 (closed symbols) and 31 (open symbols) ; in small and medium chambers (circles), and in large chambers (triangles). Cham- ber size not indicated for San Francisco. At 17 the slopes for the various populations were significantly different (Table II), though the relationship between them was similar to that observed at 10 (Table I, Fig. 2). The value of b was low for fish from San Francisco and Salton OXYGEN CONSUMPTION IN GILLICHTHYS 217 1000 700 400 200 or I o o 400 - 200 100 70 40 h 20 I ! \ \ I I I I I I I I . SAN CARLOS 31 24 SAN FELIPE i i i i i i J I 0.4 07 1.0 2 47 GRAMS 10 20 40 FIGURE 4. Relation between oxygen consumption and body weight of Gillichthys mirabilis from San Felipe at 24 (closed symbols) and 31 (open symbols) ; in small and medium cham- bers (circles), and in large chambers (triangles) ; and of G. mirabilis from Puerto San Carlos, at 24 (closed symbols) and 31 (open symbols), in small and medium chambers (not indi- cated) ; tested on 21 Feb., 1958 (circles), 26 March, 1958 (triangles) and 15 May, 1958 (squares). Sea, and higher for those from San Felipe and Los Penasquitos. Thus the increase in temperature sharpened the differences but did not alter their direction. No trend with latitude was noted for the slopes at 17. The values of the constant b were statistically uniform at 24 (Tables I, II; 218 GEORGE W. BARLOW Figs. 3 and 4). The fish from San Carlos were not included in the statistical analysis, but the slope of their data (0.45) was lower than that of the four prin- cipal populations. Even the stress of a higher temperature, 31, failed to reveal significant differ- ences between the slopes (Tables I, II ; Figs. 3 and 4). The value of b for the San Carlos fish (0.49) diverged from the other groups in the same direction as at 24 in that it was lower again. Comparisons of the slopes obtained from G. seta and from G. mirabilis (San Carlos), showed that the two species differed at 24 and at 31 (Tables I, II ; Figs. 4 and 5). The separation at 31 was particularly noteworthy because at this tem- perature the mean rates of oxygen consumption (see below) were not significantly different for the two species. Both at 24 and at 31, the constant b for young G. mirabilis (0.45 and 0.49) was closer to that of G. seta (0.24 and 0.26) than were the slopes for larger G. mirabilis (0.56 to 0.63, and -0.53 to 0.62). Compared to adult G. mirabilis, the slope was lower for smaller G. mirabilis, and lower still for G. seta. Mean rates of oxygen consumption At 10 the mean rates of oxygen consumption of the four populations of G. mirabilis were significantly different (Table II, Fig. 2) ; one regression for all the data also was not statistically acceptable (statistical parameters for this test are not indicated in Table II ) . This divergence apparently was caused by the smaller fish. Within the experiments at 17, the mean rates of oxygen consumption of the four populations were not the same, nor was one regression adequate (Table II, Fig. 2 ) . The divergence, however, seemed to be independent of fish size. The means of the rates of oxygen consumption at 24 (not including the San Carlos sample) showed no differences, and the hypothesis of one regression line was just acceptable (Table II, Figs. 3 and 4). The larger fish from San Carlos fell on about the same regression as did the fish from the four other populations, whereas the smaller San Carlos fish seemed to lie slightly below this regression. Under stress of a higher temperature, 31, differences arose between the mean rates of consumption of the four populations, and one regression also was statis- tically unsatisfactory (Table II, Figs. 3 and 4). No trend between the means of populations was discernible. The rates of the smaller fish from San Carlos once more appeared to lie below the regression for the other groups. The oxygen uptake of G. mirabilis from San Carlos was compared with that of G. seta from Puertecitos. At 24 the means were significantly different, but at 31 the average rates were the same (Table II). Influence of temperature on oxygen consumption Four sets of O 10 comparisons were made for respiratory rates. Two categories involved the acute changes 10 to 17, and 24 to 31. Another category was the comparison of rates at 10 and at 24 after acclimation, the chronic changes. Fi- nally, the rates for fish acclimated to 10 but tested at 17 were contrasted with the rates for fish acclimated to 24 but tested at 31. These relationships are sum- marized in Table III. Q 10 was used only as a descriptive device. OXYGEN CONSUMPTION IN GILLICHTHYS 219 TABLE III Temperature coefficients (<2io) for Gillichthys mirabilis and G. seta 10 C. to 17 C. 24 C. to31C. 1 g. 10 g. 1 g. 10 g. G. mirabilis San Francisco 2.42 2.84 1.27 1.50 Los Penasquitos Salton Sea 2.64 2.73 2.56 2.45 1.45 1.45 1.59 1.57 San Felipe San Carlos 1.60 2.22 1.66 1.43 1.43 1.47 G. seta Puertecitos 2.01 2.24 10 C. to 24 C. 17C. to31C. 1 g- 10 g. 50 g. 1 g. 10 g. 50 g. G. mirabilis San Francisco 3.97 2.09 1.33 2.87 1.52 0.97 Los Penasquitos Salton Sea 3.53 5.17 2.22 1.61 2.40 1.41 2.62 3.89 1.75 1.32 1.92 1.17 San Felipe 2.84 1.97 1.52 2.89 1.58 1.04 Rates used in the calculations of the Q 10 values were obtained from intercepts of the regression lines. Most comparisons were made at the 10-gram intercept be- cause this value was near the median of the size ranges for most samples. The temperature coefficients at the one-gram intercept, too, were listed to facilitate com- parisons with other published results. Vagaries of the slope due to chance can be magnified by comparisons made outside the size distribution of the fitted lines, and may lead to erroneous conclusions. When the temperature was raised from 10 to 17, the Q 10 of fish acclimated to 10 had a value greater than 2.0 in all populations at the 10-gram level. This was appreciably higher than the O 10 's recorded for animals acclimated to 24. The O 10 's for G. mirabilis (10-gram fish) from San Felipe were almost always the lowest for that species. The other Gulf population of G. mirabilis, from San Carlos, also had a commensurately low coefficient for the one temperature interval at which it was tested. Thus, the G. mirabilis from the Gulf of California had lower temperature coefficients than those from the Pacific Coast, or Salton Sea. Within the north to south sequence of populations in California (San Francisco, Los Penasquitos, and Salton Sea), the predominant trend was higher coefficients to the south. But for the acute change in temperature among the 10 acclimation groups, the trend was just the opposite. In general, the values for Los Penasquitos and Salton Sea were closer to one another than to that for San Francisco, partic- ularly for acute changes. Gillichthys seta clearly had a higher temperature coefficient than any population of G. mirabilis at 24 (Table III). No G. seta were tested at 10. An additional set of experiments was performed to establish whether or not the 220 GEORGE W. BARLOW high chronic Q 10 's observed in the cold-acclimated fish could be ascribed to the removal of cold depression. The three populations of G. mirabilis from California acclimated to 10 were tested at 3.5 intervals from 10 to 27.5. The program was the same as described in Material and Methods. Cold depression was not a factor in the temperature response of the cold- acclimated fish. As seen in Figure 6, the Q 10 's remained approximately the same between 10 and 24. Prosser (1958) has presented a classification of types of metabolic responses to temperature changes in relation to cold- and warm-acclimation. To provide such a comparison, data from a warm-acclimated (24) group of G. mirabilis from San Francisco were included in Figure 6. This curve, however, has been improvised from two different experiments, and requires an explanation. The points representing rate of oxygen consumption at 24 and 31 were derived from the lines fitted to the results of the standard experiment (Fig. 3), and are reliable. The values for 17 and 34.5 were obtained as follows. The fish in the res- pirometers were slowly (eight hours) cooled from 24 to 17, held at 17 four hours, recorded two hours, then tested at 3.5 intervals up to 34.5, following the usual program. The values at 17 regularly fell near the projected 24-31 line. Warming the water to 20.5 and 24, on the other hand, resulted in pronounced individual differences. These seemed to be of two types: (1) some of the fish manifested high Q 10 's, followed by a decrease in rate at a higher temperature, then an increase again at a still higher temperature, whereas (2) the others showed what could be called the anticipated regular increment. I have exercised my judgment and selected out those data that I believe to be reliable, i.e., all the recordings from the San Francisco fish taken at 17 and 34.5, but none in between. The change in Q 10 revealed by a comparison of the two kinds of curves in Figure 1000 700 = 400 \ O O 200 O 100 G. seta ai 0.2 0.4 0.7 LO GRAMS 4.0 7.0 10 FIGURE 5. Relation between oxygen consumption and body weight of Gillichthys seta from Puertecitos at 24 (closed circles) and 31 (open circles). OXYGEN CONSUMPTION IN GILLICHTHYS 221 100 70 V. O X. o 40 o 20 \o 10 13.5 17 20.5 24 TEMPERATURE C 27.5 31 34.5 FIGURE 6. Influence of cold- and warm-acclimation on metabolic responses of Gillichthys mirabilis to temperature changes. Each point represents the mean rate of oxygen consumption for ten fish, weighing from 4 to 10 grams. Individual rates were adjusted to the 8-gram inter- cept using the appropriate values of b listed under 10 and 24 in Table I ; the procedure is discussed in Roberts (1957). Acclimation to 10 : circle = San Francisco population, triangle = Los Peiiasquitos population, square = Salton Sea population. Acclimation to 24 : X = San Francisco population. 6 would be classified in the system proposed by Prosser (1958) as Type III B. rotation counter-clockwise with cold acclimation. Clockwise rotation evidently is more common than counter-clockwise (Prosser, 1958). The curves for warm- and cold-acclimated fish intersect between 17 and 20.5. The curve (not shown) for the warm-acclimated fish from Los Penasquitos intersects the curves for cold- acclimated animals at 20.5. Evidently little or no translation is involved. This would be more apparent if the warm-acclimated fish had been tested over a lower temperature range. In- cidentally, above 34 the oxygen uptake in many of the 24-acclimated fish decreases, and between 35 and 37 many die (further experiments not reported here). Influence of size on temperature coefficients There was no apparent difference in Q 10 between large and small G. mirabilis for acute temperature changes. But the temperature coefficients after acclimation were influenced greatly by the size of the fish involved. The chronic Q 10 's were strikingly higher in the smaller fish (Table III ; Figs. 2 to 4). Apparently acclima- tion was more effective in larger fish. The 50-gram fish, acclimated to 24 and tested at 31, commonly had lower respiratory rates than fish of the same size acclimated to 10 and tested at 17. The size of the fish, therefore, is important in chronic, but not acute, metabolic adjustments. DISCUSSION Evidence in fishes for intraspecific differentiation of adaptive physiological sys- tems has received infrequent but notable attention. Heuts (1947, 1956) described GEORGE W. BARLOW the physiological adaptations to salinity and temperature in the embryos of three races of the stickleback, Gasterosteus aculeatus. Minamori (1957, and earlier papers cited there) studied in detail various aspects of developmental physiology in races of the spinous loach, Cobitus taenia. The races were arranged in a series according to temperature adaptation. The forms ecologically adapted to higher temperatures, tolerated higher temperatures and developed more slowly at inter- mediate temperatures than did the cold-adapted forms. Hart (1952) investigated geographic variation in heat tolerance in twelve species of fresh-water fishes. In three species, only, significant differences were apparent between subspecies. In field experiments, Wohlschlag (1957) discovered the sympatric, but ecologically separate forms of an arctic whitefish, Coregonus sardinella, had different rates of oxygen consumption. But these "forms" of Coregonus probably would be con- sidered distinct species by Svardson (1953). Guppies, Lebistes reticulatus, from inbred lines had different susceptibilities to high temperatures (Gibson, 1954). Under extreme environmental conditions some genotypes of carp, Cyprinus (sp. ?), were more viable than others (Kirpichnikov, 1945). Differences between populations, after acclimation, usually are attributed to genetic dissimilarities. The evidence for this thesis is indirect, but consistent (ref- erences in Introduction, and Fry, 1957a). The role of the environment in modify- ing the degree of reversibility, however, is not known. The temperatures associated with early development allegedly can alter the thermal resistance of guppies, Lebistes reticulatus (Gibson, 1954). The thermal resistance of fish reared at 25 and at 30 was similar, but those cultured at 20 were less resistant. At the lowest temperature only two female guppies could be induced to give birth, and at the highest temperature mortality was appreciable. The fish reared at the high, and especially at the low, temperatures could have been selected for by the effects of the thermal environment. Other experiments in the same paper demonstrated different heat resistances in different genetic lines. Males and females also had dissimilar tolerances, and the sex ratios in the experiments on thermal history were not given. The influence of the early thermal experiences on the metabolism of adult poikilotherms remains, then, an open question. The results reported here, nonetheless, are interpreted as though metabolic dif- ferences between fully acclimated animals from naturally occurring populations, indicate heritable dissimilarities. Influence of size on oxygen consumption Although the regression coefficients relating oxygen consumption to weight were not especially informative at the intraspecific level, they were most revealing for species relationships. The slope of the regression for the small species, G. seta, obviously was lower than that for the adults of G. mirabilis (24 acclimation). The disparity was less apparent when the data from adult G. seta were compared with those for sub-adult G. mirabilis. As noted below, the value of b (i.e., b) tended to decrease at small sizes in G. mirabilis (acclimated to 24 only) . Morphologically, adult G. seta retain many characteristics which in G. mirabilis are juvenile (Barlow, 1961). The low regression coefficients for oxygen uptake in G. seta might also represent a retention of a juvenile feature. Von Bertalanffy (1951) discussed the relation between regression coefficients OXYGEN CONSUMPTION IN GILLICHTHYS 223 and phylogeny. He opined that some higher taxonomic categories could be char- acterized by the value of b. Apparently this does not hold for gobiid fishes since the slopes differ between species, and change with acclimation as well. Certain difficulties were encountered with the data obtained from fish acclimated to 24. The relationship between the logarithms of oxygen consumption and body weight was not strictly rectilinear over the size range of the experimental animals. There was a tendency for the negative value of b to decrease slightly in the smaller G. mirabilis, and also in the large adults, though less so. As Zeuthen (1955) suggested, it may be best to regard b as a tangent to some more complicated curve. To complicate matters, the value of b in G. mirabilis acclimated to 10 was relatively constant over the entire size range of the experimental animals. The low temperature of 10 is a normal winter temperature for G. mirabilis, but surely it is suboptimal for this species in terms of activity and growth; the fish seem well suited to 24. Possibly, the low temperature of 10 effects a physiological com- pensation for the unnatural continuous illumination. Or, at 10 metabolism is less affected by growth and activity. The values of b obtained from the cold-acclimated fish are more in accord with most of those reported for other fishes. Zeuthen (1953) suggested that 0.22 probably is a representative value for b among fishes. Fry (1957b) cited 0.10 to 0.20 as usual values for b, but also noted five instances where values of from 0.33 to 0.50 had been reported. The value of b, 0.50 to 0.60 for G. mirabilis acclimated to 24, then, is higher than that usually considered typical for fishes. The value of b (0.15) reported by Scholander et al. (1953), was derived from a composite of several species. The data for some of the species, however, appear to show a slope greater than 0.15 (Abudefduf sa.ratilus, Scants croicensis). Wohlschlag (1957) recorded a slope of 0.60 for a coregonid fish, though he attributed the high value to the small size of the sample. Recent investigations into other animal groups have revealed values of b well above 0.33 predicted from the surface volume relationship (references in Zeuthen, 1953; Mann, 1956; Dawson and Bartholomew, 1956; Vernberg, 1959a; Dehnel, 1960; Velma Vance, personal communication). Mean rates of oxygen consumption Physiological responses to stress may reveal differences when measurements within the temperature range to which the animals are adjusted might fail (Prosser, 1957). The means of the rates of oxygen consumption in G. mirabilis were the same only at 24, which probably lies within its optimal temperature range. Dif- ferences were apparent when the temperature was raised to 31. The lowest temperature, 10, must have been stressful: activity was greatly reduced, although social behavior, frontal display by males, was observed. Fish acclimated to 17 were only slightly less active than those at 24. Nonetheless, the sudden 7 in- J o J crease in temperature from 10 to 17 probably stressed the metabolic system ot the cold-acclimated fish. While differences in mean rates of oxygen consumption at a given temperature appeared to indicate divergences between populations of G. mirabilis, the direction of the departures seemed to be irregular. At 10, only, there may have been evi- dence for higher metabolic rates to the south in smaller fish. This was particularly noticeable in the animals from San Felipe, that had been tested in late spring. The 224 GEORGE W. BARLOW other populations had been examined during the winter. But if there had been a residual seasonal adjustment, the rates for the San Felipe group should have been low in comparison to the winter groups. As seen, just the opposite occurred. The oxygen consumption of G. mirabilis is similar whether the fish are in or out of water. Though not measured in water, the rates reported here for aerial respiration of G. mirabilis agree with those found by Wells (1935a) for respiration in water of the same species, when allowances are made for differences in thermal acclimation. The rate of oxygen uptake of the Hungarian mud-minnow, Umbra lacustris (kramerif), also is the same whether entirely aquatic, or a combination of aquatic and aerial (Geyer and Mann, 1939). The rates of oxygen uptake in Gillichthys mirabilis and G. seta accord with the values reported for other species of air-breathing Gobiidae. The rates at 24.5 for Periophthalmus vulgaris (seven grams) and P. dipus (16 and 17 grams) given by Schottle (1931) coincide nicely with the regression relating oxygen consumption to weight for G. mirabilis. The rate for P. schlosseri (110 grams) was about 100% higher than would have been expected for G. mirabilis of that size, but agreed well with the extension of the regression for G. seta. The data of Schottle were reduced by 50% before making the comparisons because she recorded the rates during the first hour; the values for G. mirabilis decreased by about this amount during the 12-hour adjustment period. Influence of temperature on oxygen consumption The responses to temperature changes seemed to be of more adaptive significance than the absolute level of oxygen consumption. While the mean rates showed no interpretable differences, the disposition of the Q 10 's suggested a meaningful pattern. The temperature coefficient data infer the following arrangement of populations : G. mirabilis from the north and central regions of the Gulf of California are alike, but differ from the other groups considered by having consistently lower Q 10 's; G. mirabilis from San Francisco Bay are similar to, but somewhat apart from, the other two populations in California in that their Q 10 is highest at low temperatures, but relatively low at high temperatures ; G. mirabilis from the Salton Sea and Los Penasquitos show a correspondence when responses to acute changes are compared, but are dissimilar when temperature coefficients of acclimated rates are considered. Temperature coefficients generally are higher with adaptation to higher tempera- tures (but see Dehnel, 1960) : northern species or forms usually have lower co- efficients than their southern counterparts (Rao and Bullock, 1954; Minamori, 1957; Heuts, 1956; Tashian, 1956; Dehnel. 1955). In some respects G. mirabilis was just the opposite : warm-acclimated fish had low Q 10 's in all the groups, and cold-adapted ones had high Q 10 's. A further inconsistency was that the Q 10 's were higher in northern populations when the fish were cold-acclimated. Certain data, however, were in accord with the hypothesis of higher Q 10 's in more southern populations. Warm-acclimated G. mirabilis from the Pacific Coast and Salton Sea had higher Q 10 's to the south. But the two Gulf of California populations had even lower coefficients than the northmost Pacific Coast population, San Francisco. Certainly the Gulf habitats experience much higher maximum temperatures and a greater range, too, than do those in San Francisco Bay. The temperature patterns in the Gulf and on the Pacific Coast are not equatable. OXYGEN CONSUMPTION IN GILLICHTHYS 225 The most obvious assumption is that the low sensitivity to temperature changes observed in G. inirabilis from the Gulf of California is an adaptation to the more variable climate there. Yet G. seta from the upper Gulf lives in a habitat subject to even greater thermal oscillations ; and the Q 10 's for G. seta are higher than for any population of G. inirabilis under the same conditions. This may not be a fair comparison because it involves another species with different behavior and ecology. The adaptive significance of the nature of the Q 10 's in G. inirabilis, and in G. seta, cannot be appreciated until more information is at hand, both with regard to the general and the specific aspects of the problem. It might be presumed, though, that within the species G. inirabilis low sensitivity to temperature change is an adaptation to higher temperatures, and high sensitivity an adjustment to lower temperatures. This is at variance with the conclusions of Scholander ct al. (1953) which were based on interspecific comparisons, and also with the hypothesis put forth by Rao and Bullock ( 1954 i. Influence of size on temperature coefficients The few available data for fishes indicate that size affects Q 10 in different ways depending on the particular species, and probably on the experimental procedures. The temperature coefficient was said to be independent of size in goldfish, Carassius auratiis (Fry and Hart. 1948). Within a given race of the loach, C obit is taenia, temperature coefficients of embryos were independent of egg volume (Minamori, 1957). At low temperatures size had no role in the determination of Q 10 's in the killifish, Fiindnlus parripinnis, but the smaller fish were more sensitive at higher temperature intervals (Wells. 1935b; re-evaluated on the basis of relative changes). In another cyprinodontid fish. Crcnichthys baileyi, from a warm spring, small in- dividuals were more sensitive to a temperature drop than were large ones (Sumner and Lanham, 1942). In the brook trout, Salreliiuis fontinalis. size had little in- fluence on the response to chronic changes in temperature when standard metabolic rates were compared, except at the highest temperature interval where small fish were more responsive. When active, however, the smaller trout had higher tem- perature coefficients than the larger ones (Job, 1955). Measured by opercular rates, large carp, Cyprians carpio, were more responsive to long term (chronic) temperature changes than were small individuals (Meuwis and Heuts, 1957). In Gillichthys iiiirabilis, and in G. seta, temperature coefficients were not size- dependent in short term (acute) temperature changes. The relatively greater in- crease in respiratory rate of small G. inirabilis acclimated to 24 as contrasted to 10 may indicate that large G. inirabilis are metabolically more homeostatic than small, though only when allowed time to adjust. Meuwis and Heuts (1957) noticed a similar relationship in the carp, except the smaller fish, not the larger, were more homeostatic. Comparison of physiological and morphological findings The two species involved, G. inirabilis and G. seta, are distinct but difficult to distinguish from one another (Barlow, 1961). Ichthyologists, unless experienced with this group, frequently have difficulties separating them. In many respects G. seta represents an extension of the morphological cline manifested by the widely 226 GEORGE W. BARLOW distributed populations of G. mirabilis (Barlow, unpublished data). Physiolog- ically, however, the two species diverge where their distribution overlaps, the Q 10 's and the regression coefficients, b, showing marked character displacement. Within the species G. mirabilis, the greatest structural differences between popu- lations were for the most part discernible only by means of statistical analysis. The aberrant Salton Sea population, of course, was an exception, but need not disturb the thesis being put forward here. Furthermore, the morphological investigation involved the analysis of many characters, such as measurements of several body parts, counts of the various fins and the vertebrae, and extent and nature of squama- tion. Among the multitude of possible physiological parameters, only oxygen con- sumption was studied. Considering this, it is fair to say the magnitude of intra- specific variation in oxygen uptake was commensurate with that observed in the morphological study. The pattern of divergence, the "species structure" of G. mirabilis, became ap- parent only when many intervening populations, in this case 17, were compared morphologically. It is questionable if much sense could have been made of the morphological data from only those five populations analyzed experimentally. Indeed, before the series of populations had been investigated, three nominal species had been proposed for the two under discussion, and the situation was not at all clear (Barlow, 1961 ). Beyond this, it might be pertinent to ask if the experiments were designed properly to reveal meaningful differences in the respiratory rates. Perhaps more informative data could be obtained by acclimating the animals to a controlled regime of oscillating temperatures, simulating the natural situation. After all, the most obvious thermal dissimilarity in the locales involved is the range of temperatures experienced by each, not the mean. Constant temperature, not to mention illumina- tion, is an unreal circumstance for the animals ; the absence of these modulating influences might even be inimical to certain metabolic processes. In closing, the lack of differences in the nature of the oxygen uptake at 24 should be emphasized. From numerous field observations, I believe this tempera- ture is near the thermal optimum for the species G. mirabilis throughout its geo- graphic range. The similarities at 24, therefore, might have been expected. The dissimilarities in the populations were manifested as the temperature departed from this value. The environment of each population evidently is unique in its combina- tion of various temperatures (especially maxima and minima), photoperiod, salinity, and so on. Probably the singular features of each habitat have stamped their char- acter on the gene pool of the population, effecting a slight, though significantly adaptive, divergence in the metabolic response to temperature. I take pleasure in acknowledging the helpful suggestions of George A. Barthol- omew, Thomas W. James, and Henry W. Thompson concerning the preparation of this article. I should also like to thank Velma Vance for stimulating discussions, and Gerta M. Barlow for preparing many of the illustrations. I am grateful to C. Ladd Prosser and Betty I. Roots for their criticism of the final manuscript. The research and writing were done under the guidance of Boyd W. Walker, and I am deeply indebted to him for his encouragement and material aid. OXYGEN CONSUMPTION IN GILLICHTHYS 227 SUMMARY 1 . Rate of oxygen uptake was determined for moist fish in a Warburg apparatus. Fish acclimated to 10 were tested at 10 and 17 ; those acclimated to 24 were tested at 24 and 31. Five populations of G. mirabilis were investigated (San Francisco Bay in central California, Los Penasquitos in southern California, San Felipe in the northern Gulf of California, Puerto San Carlos in the central Gulf, and the Salton Sea). One population of G. seta (from the upper Gulf) was studied at 24. 2. Rate of oxygen consumption per unit weight was plotted against body weight. The regression coefficient, b, differed between populations for G. mirabilis though not in an interpretable fashion. The value of b was low at 10 and 17 (0.14 and -0.35 ), and high at 24 and 31 (-0.45 and -0.63). The slopes were much lower for G. seta at 24 and 31 (-0.29 and -0.26) than for G. mirabilis. 3. The mean rates of oxygen uptake were different for the populations of G. mirabilis at all temperatures except 24. The average rate for G. seta at 24 was lower than that for G. mirabilis. but the rates were about the same for both species at 31. 4. The temperature coefficients, Q 10 , varied from population to population of G. mirabilis in what seemed to be a pattern. The coefficients were lower in groups from the Gulf of California at almost all temperature intervals. Within the Pacific Coast and Salton Sea populations, the O 10 's for the northern population were highest at the low acclimation temperature, and lowest at the high acclimation temperature. For all groups, the O ir( 's were highest when cold-acclimated (2.2 to 2.8), and lowest when warm-acclimated (1.4 to 1.6). The coefficient for warm-acclimated G. seta was high (2.0 ). 5. There was no apparent difference in temperature coefficients between large and small G. mirabilis for acute temperature changes. The smaller fish manifested appreciably higher O 1( ,'s than did the larger fish, however, when rates were com- pared after acclimation. 6. The degree of intra- and interspecific differences in the nature of the oxygen consumption was roughly of the same order of magnitude as the morphological differences reported in another article. LITERATURE CITED BARLOW, G. \Y., 1961. Gobies of the genus Gillichthys, with a comment on the sensory canals as a taxonomic tool. Copeia, in press. VON BERTALAXFFY, L., 1951. Metabolic types and growth types. Amcr. Xat., 85: 111-117. BRETT, f. R., 1952. Temperature tolerance in young Pacific salmon, genus Oncorhynchns. J. Fish. Res. Bd. Can.. 9: 265-323. BULLOCK, T. H., 1955. Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev., 30: 311-342. CARPELAN, L. H., 1958. The Salton Sea. Physical and chemical characteristics. Limnol. Occanog., 3: 373-386. DAWSON, W. R., AND G. A. BARTHOLOMEW, 1956. Relation of oxygen consumption to body weight, temperature, and temperature acclimation in lizards Ufa stansburiana and Sccloporus occidentalis. Physiol. Zoo/., 29: 40-51. DEHNEL, P. A., 1955. Rates of growth of gastropods as a function of latitude. Phvsiol. Zoo!., 28: 115-144. GEORGE W. BARLOW DEHNEL, P. A., 1960. Effect of temperature and salinity on the oxygen consumption of two intertidal crabs. Biol. Bull., 118: 215-249. DOBZHANSKY, T. G., 1951. Genetics and the Origin of the Species. Columbia University Press, New York, 364 pp. FRY, F. E. J., 1957a. The lethal temperature as a tool in taxonomy. L'Ann. Biol., 3rd scr., 33: 205-219. FRY, F. E. J., 1957b. The aquatic respiration of fish. In: The Physiology of Fishes, M. E. Brown, ed., Acad. Press, New York, Vol. 1, pp. 1-63. FRY, F. E. J., AND J. S. HART, 1948. The relation of temperature to oxygen consumption in the goldfish. Biol. Bull.. 94: 66-77. GEYER, F., AXD H. MANN, 1939. Beitrage zur Atmung der Fische II. Weiteres zur Atmung des ungarischen Hundsfisches (I' mbm lacustris Grossinger). Zoo/. Anz., 127: 305-312. GIBSON, M. B., 1954. Upper lethal temperature relation of the guppy, Lcblstcs rcticulatus. Canad. J. Zoo!.. 32: 393-407. HART, J. S., 1952. Geographic variations of some physiological and morphological characters in certain freshwater fishes. Pitbl. Ontario Fish. Res. Lab., (72) : 1-79. HEUTS, M. J., 1947. Experimental studies on adaptive evolution in Gasterosteus acnlcatus L. Evolution, 1 : 89-102. HEUTS, M. J., 1956. Temperature adaptation in Gasterosteus aculeatus I>. Pufahl. Sta. Zool. Napoli. 28: 44-61. HOEL, P. G., 1954. Introduction to Mathematical Statistics. Wiley and Sons, New York, 331 pp., 2nd ed. JOB, S. V., 1955. The oxygen consumption of Salrclinus jontinalis. Toronto {'nk'. Biol. Scr., (61) ; Pitfal. Ontario Fish. Res. Lab.. ( 73 ) : 1-39. KIRPICHNIKOV, V. S., 1945. Viability, rate of growth and morphology of carps of different genotypes as affected by rearing conditions. C. R. Acad. Sci. U. R. S. S.. 47: 503-506. MANN, K. H., 1956. A study of the oxygen consumption of five species of leech. /. Exp. Biol., 33 : 615-626. MEUWIS, A. L., AND M. J. HKUTS, 1957. Temperature dependence of breathing rate in carp. Hiol. Bull.. 112: 97-107. MINAMORI, S., 1957. Physiological isolation in Cobitidae. VI. Temperature adaptation and hybrid inviability. /. Sci. Hiroshima L'nir.. Scr. B.. Dir. 1. 17: 66-119. MOORE, J. A., 1957. An embryologist's view of the species. In: The species problem, E. Mayer, ed., Pufal. Amcr. Ass. Adranc. Sci., (50) : 325-338. PRITCHARD, A., 1955. Oxygen requirements of some Hawaiian tuna baitfish. L' . S. Fish and ll'ildl. Sen>.. Spec. Sci. Reft.. Fish ( 145) : 1-30. PROSSER, C. L., 1955. Physiological variation in animals. Biol. Rci<., 30: 229-262. PROSSER, C. L., 1957. The species problem from the viewpoint of a physiologist. In: The species problem, E. Mayer, ed., Pufal. Anicr. Ass. Adranc. Sci., (50) : 339-369. PROSSER, C. L., 1958. General summary: The nature of physiological adaptation. In: Physio- logical Adaptation, C. L. Prosser, ed., American Physiological Society, Washington, D. C., pp. 167-180. RAO, K. P., 1953. Rate of water propulsion in Alvtilits californianus as a function of latitude. Biol. Bull.. 104: 171-181. RAO, K. P., AND T. H. BULLOCK, 1954. Qn, as a function of size and habitat temperature in poikilotherms. Amcr. Nat.. 88: 33-44. ROBERTS, J. L., 1957. Thermal acclimation of metabolism in the crab Pachygrapsus crassipcs Randall. II. The influence of body size, starvation, and molting. Physiol. Zoo/., 30: 232-242. ROSCOFF, STATION BIOLOGIQUE, 1957. Colloque International de Biologic Marine. L'Ann. Biol., 3rd scr.. 33. SCHOLANDER, P. F., W. FLAGG, V. WALTERS AND L. IRVING, 1953. Climatic adaptation in arctic and tropical poikilotherms. Physiol. Zool., 26: 67-92. SCHOTTLE, E., 1931. Morphologic und Physiologic der Atmung bei wasser-, schlamm- und landlebenden Gobiiformes. Zeitsclir. i(.'iss. Zoo!.. 140: 1-115. OXYGEN CONSUMPTION IN GILLICHTHYS 229 SPOOR, W. A., 1946. A quantitative study of the relationship between the activity and oxygen consumption of the goldfish, and its application to the measurement of respiratory metabolism in fishes. Biol. Bull., 91 : 312-325. SUMNEK, F. B., AND U. N. LANHAM, 1942. Studies on the respiratory metabolism of warm and cool spring fishes. Biol. Bull., 82: 313-327. SVARDSON, G., 1953. The coregonid problem. V. Sympatric vvhitefisli species of the lakes Idsjb'n, Storsjon, and Hornayan. hist. Fresh-water Res. Drottninyholin, (34) : 141-166. TASHIAN, R. E., 1956. Geographic variation in the respiratory metabolism and temperature coefficient in tropical and temperate forms of the fiddler crab, Uca pugna.r. Zoologica, 41 : 39-47. THORPE, W. H., 1940. Ecology and the future of systematics. /;;: The New Systematics, J. S. Huxley, ed., Clarendon Press, Oxford, pp. 341-364. TIMOFEEFF-RESSOVSKY, N. W., 1940. Mutations and geographical variation. In: The New Systematics, J. S. Huxley, ed., Clarendon Press, Oxford, pp. 73-136. VERNBERG, F. J., 1959a. Studies on the physiological variation between tropical and temperate zone fiddler crabs of the genus Uca. II. Oxygen consumption of whole organisms. Rial. Bull.. 117: 163-184. VERNBERG, F. J., 1959b. Studies on the physiological variation between tropical and temperate zone fiddler crabs of the genus Uca. III. The influence of temperature acclimation on oxygen consumption of whole organisms. Biol. Bull., 117: 582-593. VERNBERG, F. J., AND R. E. TASHIAN, 1959. Studies on the physiological variation between tropical and temperate zone fiddler crabs of the genus Uca. I. Thermal death limits. Ecology, 40: 589-593. WELLS, N. A., 1935a. Change in rate of respiratory metabolism in a teleost fish, induced by acclimatization to high and low temperature. Biol. Bull., 69: 361-367. WELLS, N. A., 1935b. The influence of temperature upon the respiratory metabolism of the Pacific killifish, Fiinditlus parvipin-nis. Physiol. Zoo!., 8: 196-227. WOHLSCHLAG, D. E., 1957. Differences in metabolic rates of migratory and resident freshwater forms of an arctic whitefish. Ecology, 38: 502-510. ZEUTHEN, E., 1953. Oxygen uptake as related to body size in organisms. Quart. Rev. Biol., 28: 1-12. ZEUTHEN, E., 1955. Comparative physiology (respiration). Ann. Re?'. Physiol. 17: 459-482. THE ABSENCE OF DAILY PHOTOSYNTHETIC RHYTHM IN SOME LITTORAL MARINE ALGAE L. R. BLINKS AND CURTIS V. GIVAN 1 Hopkins Marine Station of Stanford I'nii'crsity, Pacific (rrorc, California A photosynthetic periodicity in algae has been reported by several workers in recent years. Investigations with natural populations of planktonic forms have shown a marked variation in the ability of these organisms to fix carbon dioxide at different times of day. The rate of photosynthesis is reportedly highest in the middle and late morning, and lowest in the late afternoon and evening, a difference of several fold occurring (Doty and Oguri, 1957; Yentsch and Ryther, 1957; Shimada, 1958). In the coenocytic alga Hydrodictyon it has been found that a photosynthetic rhythm established with light-dark cycles persists for two or three periods in continuous light, the period of the induced rhythm depending upon that of the previous light-dark regime ; thus, it is in this case not an inherent but an im- posed or conditioned rhythm ( Pirson, 1957). In Chlorclla it has been found pos- sible to establish synchronous cultures by means of light-dark cycles (Tamiya, 1957; Sorokin, 1960). In this case the rate of photosynthesis varies with the age of the cells, younger cells in general showing a rate higher than that of older cells. In view of these observations it was thought desirable to determine whether some of the multicellular algae show a photosynthetic rhythm ; therefore, the following ex- periments were carried out at the Hopkins Marine Station during June and July of 1960. MATERIALS AND METHODS The algae utilized in this study were common littoral marine genera collected in the vicinity of Pacific Grove, California. The organisms were maintained in running sea water in a laboratory for a period of not greater than 24 hours prior to the start of the tests. The tissues were placed in one-liter jars completely filled with natural sea water and exposed for a period of one-half hour to incandescent light (2 bulbs) of an intensity approximating 800 foot candles. The temperature within the sampling jars was held constant at about 15 C. by submerging them in a battery jar through which sea water was circulated rapidly during the course of the light exposure. The sampling jars were turned around once during the exposure period to stir the contents. At the conclusion of the exposure, the oxygen content of the water within the sampling jars was determined by the Winkler technique, and the rate of oxygen production per gram fresh weight calculated. Values presented are for net oxygen production, uncorrected for respiration ; however, since respira- tion is low in relation to photosynthesis at the light intensity used (about 10% as shown by occasional dark-bottle checks), variations in respiration would be small 1 Predoctoral fellow, Division of General Medical Sciences, United States Public Health Service. 2. SO ABSENCE OF PHOTOSYNTHETIC RHYTHM 231 compared to total oxygen production. In these experiments the initial exposure was made in the middle of the morning between 9:00 and 10:30 A.M., the second exposure in the early afternoon between 12 :00 and 1 :30 P.M., and the third in the late afternoon between 4 :30 and 6 :30 P.M. The final exposure was made the next morning between 6 :30 and 9 :00 A.M., the same piece of tissue being used in all four exposure periods. Two of the genera, Uh'a and Porphyra, were subjected to cycles of 12 hours of white light followed by 12 hours dark for a period of four to ten days to see if a photosynthetic rhythm could be induced in the same manner as in Hydrodictyon and Chlorella. After the light-dark treatment, the rate of photosynthesis with con- tinuous light and with intermittent light (5 minutes light, 5 minutes dark) was measured by affixing the tissue to a platinum electrode as described previously (Haxo and Blinks. 1950). An electrical potential (0.5 volt) was applied to the electrode, the rate of oxygen production being reflected in the current flow. The latter was recorded on a Varian Graphic Recorder. A flow of sea water around the tissue was maintained throughout this experiment. The light intensity was approx- imately 80 foot candles. RESULTS The results of experiments with four of the genera tested for a naturally oc- curring diurnal rhythm are summarized in the accompanying table. It is clear that no rhythm such as that observed in the plankton was detected in the organisms tested here. In addition to the species listed in the table, the following organisms were tested and did not show any photosynthetic rhythm : Prionitis lanceolata, Endocladia muricata, Gigartina papillata, G. canaliculata, Porphyra pcrforata, Rhodoglossum offinc. Fucus jurcatus, Coilodesme calijornica, and Egregia wiensicsii. In some cases small variations of 5 to 10% were observed, but these are minor com- pared to the differences of several fold reported for the plankton and for Hydro- dictyon. In a few cases the late afternoon value was significantly lower than that of the previous morning, but in these cases an even lower value was observed the next morning, indicating poor survival of the tissue rather than a rhythm. The samples of Porphyra and Uha subjected to the regular light-dark cycles also showed no subsequent photosynthetic rhythm. In the case of Porphyra the ability to photosynthesize in either intermittent or continuous light did not change during the course of the day. In Uh'a the rate of photosynthesis was at first high on exposure to light after a dark period of several hours, gradually dropping to a lower steady level after about two hours of constant or intermittent illumination. The lower level was maintained as long as the illumination was continued, so that TABLE I Rate of oxygen production in cubic centimeters per hour per gram fresh weight Organism Morning Early afternoon Late afternoon Next morning Hesperophycus harveyanus .15 .19 .19 .19 Chaetomorpha aerea .35 .30 .38 .33 Ulva sp. 1.08 1.16 1.10 1.16 Iridaea flaccida .34 .38 .35 .35 232 L. R. BLINKS AND CURTIS V. GIVAN FIGURE 1. Recording of photosynthetic oxygen production by Ulva sp. in intermittent light (80 F.C.) ; 5 minutes light alternated with 5 minutes dark, a, b, and c constitute a continuous 40-hour sequence ; d and e another 28-hour sequence. No diurnal rhythm is apparent, only a slow falling away of the original photosynthetic rate. Tissues were previously exposed to 12 hours light, 12 hours dark for 10 days. Time marks one hour apart ; downward excursion of the recorder is proportional to oxygen production. no rhythm was occurring. The accompanying figure shows typical patterns dis- played by Ulva. Similar records were taken for Porphyra. DISCUSSION It is apparent from the results presented here that no diurnal rhythm occurs in the organisms tested, and that in Ulva and Porphyra no photosynthetic periodicity is ABSENCE OF PHOTOSYNTHETIC RHYTHM 233 induced by a 12 hour light- 12 hour dark regime. In the latter connection, no in- formation is available as to whether or not cell division is synchronized by the light-dark cycles, although the absence of any detectable photosynthetic rhythm tends to argue against such synchronization. No explanation appears to be at hand for the diurnal periodicity reported by Doty and Oguri and others. The rhythm in Hydrodictyon may be the result of physiological differences in the cells at different stages of development, as Tamiya (1957) has suggested. LITERATURE CITED DOTY, M. S., AND M. OGURI, 1957. Evidence for a photosynthetic daily periodicity. Limnol. Occanogr., 2: 37-40. HAXO, F. T., AND L. R. BLINKS, 1950. Photosynthetic action spectra of marine algae. /. Gen. Physio I.. 33 : 389-422. PIRSOX, A., 1957. Induced periodicity of photosynthesis and respiration in Hydrodictyon. In: Research in Photosynthesis. Edit, by H. Gaffron ct al, pp. 490-499. New York: Interscience Publishers, Inc. SHIMADA, B. M., 1958. Diurnal fluctuation in photosynthetic rate and chlorophyll "a" content of phytoplankton from Eastern Pacific waters. Limnol. Occanogr., 3 : 336-339. SOROKIN, C., 1960. Photosynthetic activity in synchronized cultures of algae and its dependence on light intensity. Arkiv f. MikrobioL, 37: 151-160. TAMIYA, H., 1957. Mas's culture of algae. Ann. Rev. Plant PhysioL, 8 : 309-334. YENTSCH, C. S., AND J. H. RYTHER, 1957. Short-term variations in phytoplankton chlorophyll and their significance. Limnol. Occanogr., 2: 140-142. CONTROL OF FLASHING IN FIREFLIES. I. THE LANTERN AS A NEUROEFFECTOR ORGAN JOHN BUCK AND JAMES F. CASE 1 Marine Biological Laboratory, Woods Hole, Mass.; Laboratory of Physical Biology, National Institutes of Health; Department of Zoology, State University of Iowa The ability of some fireflies to produce remarkably uniform and brief flashes of light is of much interest in relation to cellular control mechanisms. Neural involve- ment has been implicit or explicit in most theories of flash regulation, but there are two main ideas as to its ultimate mechanism. According to one view the lumines- cence of the photocyte is controlled by limiting the access of oxygen by supposed mechanical valves (tracheal end cells). According to the other view the terminal nerves stimulate the photocyte directly. The oxygen theory has been dealt with in previous papers (Buck, 1948; Hastings and Buck, 1956). The present series of papers explores responses to electrical stimulation. Work by Macartney (1810), Macaire (1821), Todd (1826), Joseph (1854), Kolliker (1858), Owsjannikow (1868), Bellesme (1880), Arnold (1881), Ver- worn (1892), Perkins (1931), Brown and King (1931), Snell (1932), Alexander (1943) and others showed that voluntary luminescence is abolished by decapitation and can be elicited by electrical stimulation. Dubois (1886), Heinemann (1886), Fuchs (1891), Lund (1911 ) and Gerretsen (1922) attempted to localize the stim- ulation to specific efferent nerves, though at a very crude level. Steinach (1908) reported evidence of summation of subliminal stimuli, and Chang (1956) recorded facilitated series of responses from both the larval and adult firefly and compared them with responses in muscle. Hanson (1961) showed that regional excitation corresponds to the gross innervation of the lantern, and Carlson (1961) found that even the "pseudoflash," caused by passive entry of oxygen into an hypoxic lantern, is affected by prior neural stimulation. Nonetheless, the type and degree of neural involvement in flash control remain uncertain because of ignorance of where the nerves in the lantern terminate. Thus, nerves might control either end cells (oxygen control) or photocytes directly, and the hypoxic effect might be a direct limitation or, as Briicke (1881) suggested long ago, an inactivation of control nerves. In view of this impasse it seemed possible that understanding of the role of nerve in triggering the firefly flash might be gained by analyzing the flash as an effector event and exploring its variation with changing stimulus parameters. Subsequent papers in this series will deal with central nervous aspects of excitation, peripheral nervous phenomena, and effector unit response. Preliminary summaries of some of the work have been given (Case and Buck. 1957. 1958, 1959a, 1959b). 1 Supported by PHS research grant B 1890 from the National Institute of Neurological Diseases and Blindness. 234 FLASH CONTROL IN FIREFLIES 235 MATERIALS AND METHODS The fireflies investigated were adults of Photinus f>y rails (Linne) from Mary- land and Iowa, Photinus inarginellus Le Conte and Photinus consanguineus Le Conte from Woods Hole. Photinits pitnctnlatns Le Conte from Iowa, and both larvae and adults of the most common photurid (tentatively identified as Photuris versicolor Barber) from each of the three localities. (Identifications were made via the papers of Barber and McDermott (1951 ) and Green (1956) . ) Adult speci- mens were netted in the field and used within a week, meanwhile being kept unfed in humidified jars at room temperature. Larvae were collected on lawns near streams and kept in petri dishes on moist paper, either at room temperature for immediate use or at 5-10 C. for use weeks or months later. Preparations included intact individuals, decapitated specimens, and isolated lanterns. To prepare the adult lantern, which consists of ventral plaques in ab- dominal segments 6 and 7, these segments were excised and all viscera overlying the photogenic organ, usually including the terminal ganglia of the ventral nerve cord, were removed. The larval light organs, a pair of small oval spots on the ventro- lateral surface of abdominal sternite 8, were excised with part of the surrounding sternite. Stimuli were delivered by Grass S-4 stimulators via radio-frequency isolation units and electrodes of unshielded 30-gauge silver or platinum-indium w r ire. Intact or decapitated specimens were de-legged and anchored on dental wax with wire staples spanning the body transversely. Isolated light organs were laid across a parallel electrode pair in a small moist chamber. Two methods were used in temperature experiments. In one, the specimen, with electrodes in position, was bound to the bulb of a thermometer reading to 0.01 suspended in air in a test tube immersed in a water bath ; in the other the specimen was attached to a thermistor thermometer readable to 0.5, in a small copper air chamber through the walls of which water was circulated from a large temperature- stabilized reservoir. Light emission was followed with an RCA 931- A photomultiplier leading directly to one channel of a dual beam oscilloscope and photographed simultaneously with a second trace carrying a stimulus marker. In all except the temperature experiments the preparation was mounted on the stage of a stereoscopic microscope magnifying up to 120 X. Unless otherwise speci- fied, all experiments were done at 25 3 C. RESULTS Both spontaneous and induced flashing exhibit a high degree of consistency, and most of the illustrations and data presented are typical of responses obtained in dozens or hundreds of measurements. Instances of refractoriness associated with apparent central nervous phenomena will be considered in the following paper. 1. The spontaneous flash Spontaneous flashing is seen only in individuals with brain still connected with cord. As a general rule it does not occur in either field or laboratory below 18- 236 JOHN BUCK AND JAMES F. CASE vAAJUUUUUUUUUU FLASH CONTROL IN FIREFLIES 237 20 C. Under the conditions of most of our experiments (animal immobilized; electrodes inserted ) , spontaneous flashes were generally infrequent, although single responses could sometimes be induced by various types of mechanical stimulation. Photuris was exceptional in its spontaneous activity, some specimens flashing with almost clock-like regularity. Figure 1 shows the first 10 and last 10 of 80 consecu- tive flashes given by a male of Photnris in 32 seconds. In this series the mean interflash interval was 405 3.2 milliseconds ( S.E. -- 0.8% of mean ) and mean flash intensity was 21.3 0.5 arbitrary light units (S.E. -- 2A/c of mean ). Insofar as flash kinetics are concerned, oscilloscopic observation confirms field impressions in showing characteristic differences between species. Thus, the dura- tions of flashes of comparable magnitude may vary from 200 mscs. or less in Photuris (Fig. 9 ) to a second or more in Photinns pyralis (Fig. 2). Single flashes may have nearly symmetrical accretion and decay phases (Figs. 3, 4, 5) or show marked skewing ( Figs. 2, 9 ) . Flashes may be either single or multiple ( Figs. 6, 7 ) . We have already emphasized the usual constancy of flash type within a species (Figs. 1, 13, 32 etc.) but it is also relevant to the control problem to point out differences that may occur between the flashes of the two sexes of one species (Figs. 6 Z's. 3 i's. 9), between different individuals of the same sex and species (Figs. 3 i f s. 9 ; 6 ) , and even between successive flashes of one individual ( Fig. 7 ) . Sometimes these variants have ready explanations, such as differences in degree of disturbance of the specimens, or asynchrony or variable involvements of different lantern re- gions ; but some are not presently understood. In most species the spontaneous flash under laboratory conditions appears com- parable to that emitted in natural flight. However, in Photuris, which is a variable and confusing genus even in field behavior (Barber and McDermott, 1951), the free-flying male of the Woods Hole variety typically gives a rapid four-peaked twinkle every second or two, whereas the female gives a long lingering glow at irregular intervals ; in the laboratory both sexes usually give single flashes, although FIGURES 1-15. Note that in photos of oscilloscope traces the time scale (S) is given as the width of the print. Some stimulus artifacts have been retouched. ( 1 ) Woods Hole Photuris, male. First and last 10 of a series of 80 spontaneous flashes. S = 8 sec. (2) Maryland Photinns pyralis, male. Spontaneous flash. Light did not regain base level for 1 second. S = 735 mscs. (3) Woods Hole Photuris, male. Spontaneous flash. S = 220 mscs. (4) Photinns marginellus, male. Spontaneous flash. S = 450 mscs. (5) Maryland Photuris, male. Spontaneous flash. S = 130 mscs. (6) Woods Hole Photuris, female. Two frames of spon- taneous flash of two individuals. S = 300 mscs. (7) Photinns consain/ui/icns, male. Five successive spontaneous flashes. S = 640 mscs. (8) Woods Hole Photuris, male. Responses of isolated lantern to 5, 7. 8 and 9 volts, all at 10 mscs. S = 200 mscs. (9) Woods Hole Photuris, male. Spontaneous flash. S = 240 mscs. (10) Woods Hole Photuris, female, decapitated, electrodes in light organ. Eight successive responses to 10 mscs./15 v. with the fourth, fifth and sixth occurring in a 4 v. B.C. field. S = 10.5 sees. (11) Photuris larva, decapitated, electrodes immediately anterior and posterior to light organ. Induced glow, 10 mscs./15 v. S = 3.6 sees. (12) Photinns pnnctulatns, female, isolated lantern. Three frames showing responses to 4, 8 and 10 v./4 mscs. S = 350, 500, 500 mscs. (13) Woods Hole Photuris, sex unknown, decapitated, electrodes in lantern. Response to stimulus train of 4 mscs./5 v. at 10 per second. S 2.2 sees. (14) Woods Hole Photurix, female, decapitated, electrodes in lantern. Responses to paired stimuli of 6 mscs./15 v. with intervals of 15, 20, 30, 40, 50, 60, 70, 90, 150 and 200 mscs. S = 360 mscs. except 430 mscs. for last two frames. Stimulus indicated by break in photomultiplier trace. (15) Maryland Photinus pyralis, male, decapitated, electrodes immediately anterior and posterior to lantern. Responses to two equal stimuli 1200 mscs. apart. S = 2 sees. 238 JOHN BUCK AND JAMES F. CASE at least the female occasionally produces compound flashes (Fig. 6) as does the female of the Maryland Photuris (Fig. 8 of Hastings and Buck). In larvae of most lampyrid fireflies the photogenic organ is much smaller and simpler than the adult lantern, and light is emitted as a long glow rather than a short flash. No spontaneous glows were recorded, because of their very irregular timing and low frequency, but the kinetic contrast between larval and adult lumines- cence is well illustrated by the induced glow (Fig. 11 ; note time scale). Actually, the duration of the larval glow is usually even longer in nature often several sec- onds but in view of our observations at different temperatures (see below) this is quite possibly accounted for by the temperature of the soil surface being considerably below 25, particularly on the damp evenings in early autumn when larvae are most easily observed. 2. Induced flashing : general Although, as will be shown, it is possible to alter flash duration, intensity and form by changing stimulus parameters, a single, moderate electric shock usually elicits a flash indistinguishable from the normal spontaneous flash characteristic of the species. Seemingly normal flashes can result with the electrode pair at any level of the body from head to lantern, but are usually most reliably produced with ab- 30 i 20 - C/) ~O 10 Duration 20 Mscs 30 FIGURE 16. Representative strength-duration curve for minimum visually detectable response induced by direct stimulation of lantern of a decapitated Woods Hole Photuris male. FLASH CONTROL IN FIREFLIES 239 dominal stimulation. In the observations described below, induced responses, un- less specifically excepted, were recorded with electrodes either in abdominal segment 6 at the level of the last ganglionic mass of the cord, or actually inserted in lantern tissue. In both instances excitation is presumably via peripheral nerve. In connection with changes in flash characteristics, it is important to avoid being misled by differences due simply to magnitude. In Figure 39, for example, the weakest and strongest flashes might be thought to be quite different in form, whereas in fact the relative rates of rise and fall in light intensity are the same in both, and the two flashes could be made to coincide by equalizing their peaks. 3. Threshold The isolated light organ, 15 minutes after preparation, gives reproducible thresh- old values apparently not much affected by unavoidable variations in tissue-electrode contact. Using this preparation, the interrelations of stimulus voltage and duration for minimum detectible response were studied. The results (Fig. 16) show strength-duration relations similar to insect nerve-muscle. In seven males of Photuris with electrodes in the lantern the average rheobase was 2.1 volts and the chronaxie was 3.9 mscs. Minimum values were 0.7 volts and 1.7 mscs., respec- tively. Values for the female of Photuris and for males of Photinus pyralis and P. punctulatus were in the same range as for the male of Photuris. Threshold for direct current stimulation is relatively uniform. In Photinus pyralis, for example, the values in three individuals were 5.5-6.0, 3.5-4.0 and 7.5-8.0 volts. Threshold is much increased at lower temperatures. In typical measurements on Photuris a 10 mscs./6 v. shock elicited a flash of good intensity at 25, whereas, in the same individual, 50-60 volts at the same duration were required in the 9-12 range to obtain even a much weaker response. 4. Factors affecting flash intensity a. Stimulus strength and duration. Within limits, flash intensity varies with voltage of single shocks of given duration (Figs. 8, 17). The same effect is seen during stimulation imposed upon B.C. fields (Fig. 10). In certain species, partic- ularly if the lantern still has central connections, increasing strength of stimulus may induce multiple flashes (Fig. 12). These probably autoexcitatory responses will be considered in detail in the second paper of this series. As would be expected from the strength-duration findings, variation in stim- ulus duration alone also affects flash intensity. These effects are considered in the section on flash form. b. Effects of stimulation frequency on flash intensity: facilitation. With sub- liminal stimulation, the adult firefly lantern exhibits typical summation, response first occurring after several shocks and then growing (Figs. 13, 44). Additive effects occurring with supra-threshold stimuli are well displayed by paired shocks (Figs. 14, 18). The minimum delay which produced detectible summation (i.e., the absolute refractory period) was not investigated in detail, but is clearly less than 5 mscs. in Photuris and is apparently between 6 and 8 mscs. in Photinus pyralis, using near-threshold stimuli in both instances. Above these minima, facilitation 240 JOHN BUCK AND JAMES F. CASE 60l 40- (S) c. CD c 20 - CD to c O Q. CD 5 Stimulus Intensity - Volts FIGURE 17. Relationship between stimulus strength and flash intensity at constant 10 mscs. stimulus duration. Open circles represent ascending voltage series, closed represent descending series. Woods Hole Photuris male, isolated lantern. increases rapidly to a maximum that shows considerable variation both intra- and inter-specifically. In six specimens of Photuris maximum facilitation occurred with a stimulus interval of 10-20 mscs. (Fig. 18), but in two other individuals the most effective intershock interval seemed to be at least 30 mscs. In most specimens of Photinus pyralis the buildup and decay of facilitation was more gradual, the most effective delay lying between 50 and 100 mscs. After maximum facilitation is attained, flash intensity declines sharply with increasing delay. In Photuris the facilitating effect of the second shock is prac- tically dissipated by the time the delay reaches 60 mscs. Concurrently with changes in flash intensity, increasing intershock delay causes changes in flash form, manifested by a slowed rate of rise without change in latency i.e., a shift in peak position toward later time (Fig. 14, first vs. fifth frames), relegation of the original flash to a shoulder on the rise phase of the second response (Fig. 14, sixth and seventh frames), and finally by a splitting of the flash into two peaks representing the separate responses to the two shocks (Fig. 14, eighth and ninth frames). In Photuris the separation is complete at an inter-stimulus interval of approximately 85 mscs : in Photinus pyralis not until 300 mscs. At the interval for which separate responses to paired shocks are first detectible, the first flash is sometimes the more intense. However, as the peaks draw apart FLASH CONTROL IN FIREFLIES 241 the second usually becomes larger than the first (Fig. 14, frame seven et seq.*). In both Photuris and Photinus pyralis this apparent facilitation of one response by the preceding one persists, in some instances, for more than a second (Fig. 15). Facilitation is also shown by the Photuris larva, according to data collected by Dr. Albert Carlson (Fig. 19). Here the maximal summation effect of the second stimulus on the response to the first is reached only after 50-60 mscs., and persists at least 500 mscs., a much longer time than in the adult. The augmentation of the second response of a pair as a function of the first response was not investigated in larvae, but, if comparable to other adult/larval response rate ratios, would be ex- pected to persist at least 10 seconds. When repetitive stimulation is extended from paired shocks to trains, response varies widely depending on stimulus frequency and voltage, temperature, degree of neural integrity, degree of facilitation, and species. Figures 13 and 22 illustrate the rather stable sort of response seen typically with moderate stimulation, in which each flash is independent and the responses increase progressively in intensity and duration for several successive stimulation intervals, then reaching a plateau. As might be expected, the gain in flash intensity is usually more rapid the higher the rate of stimulation (Figs. 20, 21), and in species or individuals producing double flashes, both flash components augment (Fig. 22). The stimulation frequency limit for production of completely or partially inde- pendent responses of course varies with flash duration, among other factors. In DUl ' \ o / s I ' 6 -t / \ 15 / V \ *> 40- ' \ \ i i ' \ i i / o I . \ 1 o ^ o ^ T 30- / \ , \ \ CO / \ 1 \ c 1 CD / \ ]E 20- 1 1 \ 1 X 9 CD ; \ CO o \ 10- \ \ \ CL X \ CO cr-^^ CD cr - o o i i i i i i 20 40 60 Stimulus Interval - Mscs. FIGURE 18. Temporal facilitation of electrically induced flashes. Abscissa represents inter- val from first to second stimulus of a pair, in all instances 3 mscs./5 v. Woods Hole Photuris female, isolated lantern. Typical of five other specimens. 242 JOHN BUCK AND JAMES F. CASE Pliotiiris, completely separate responses to each stimulus of a train can usually be elicited at frequencies of at least 10/sec. (Fig. 13), whereas in Photinus pyralis any frequency higher than about one per second prevents the luminescence from falling to baseline before the next response. As stimulation frequency is still further in- creased the individual flashes necessarily begin to merge, the fusion giving a main- tained luminescence that changes more or less in proportion to flash intensity, and, in combination with facilitation, may produce responses quite similar to the familiar staircase and tetany of striated muscle (Figs. 23, 24). However, it is not uncom- mon, when close to the limit of 1 : 1 response to continued serial stimulation, to find the average response intensity falling off markedly after a brief rise (Figs. 25, 27, 30,33). Species differences are strikingly illustrated in ability to follow the stimulation frequency. The Woods Hole photurid, for example, is able to respond 1:1 up to about 20 shocks per second (Fig. 27). The Iowa photurid fails at between 10 and 5 H _Q O I I I .^ 3 H co C 0> oxic glow and pseudoflash Electrical stimulation of a firefly in air normally produces a facilitating series of flashes. When stimulation continues into the hypoxic period, flashes diminish and a high intensity hypoxic glow appears (Fig. 5). A pseudoflash can be induced i i FIGURE 6. Effect of electrical stimulation during hypoxia upon hypoxic glow in an adult male, Photinus pyralis. Top trace: up, 17.25% oxygen; down, .05% oxygen. The middle and bottom traces both indicate light intensity, the bottom trace at 10 times the amplification of the middle trace. Stimulating electrode pair in abdomen dorsal to light organ. Stimulation: 5 volts, 50-msec. duration, frequency 1 per second. Short parallel lines indicate a break in the record. In this figure and subsequent figures where used, arrows pointing up indicate stimulus onset and arrows pointing down indicate stimulus cessation. The figure shows first an anoxic sequence without electrical excitation followed by two stimulus trains during hypoxia. In last two episodes glow rose temporarily after stimulation ceased. NEURAL EFFECTS ON PSEUDOFLASH 271 as soon as electrically excited flashes undergo an increase in duration, which appears to be characteristic of the hypoxic state. Electrical stimulation during hypoxia increases the rate of hypoxic glow rise and intensity of glow attained (Fig. 6). It will also be noted that when stimulation ceases a rapid brightening usually occurs followed by a decline, an effect which is typically observed. The hypoxic glow was maintained in one animal for 20 minutes by periodic stimula- tion in 0.6 % oxygen ; without stimulation it might be expected to persist less than two minutes. Although prolonged stimulation during hypoxia favors a pseudoflash of in- creased duration, stimulation, when continued during readmission of air, super- imposes no discrete flashes on the pseudoflash rise. However, this may occur during pseudoflash decay as illustrated in Figure 7 ' . Electrical stimulation com- monly augments the hypoxic glow by recruiting new, discrete luminescing areas \ FIGURE 7. Electrical stimulation during readmission of 17.75% oxygen and consequent pseudoflash in an adult male, Photinus pyralis. Top trace: up, 17.75% oxygen; down, .05% oxygen. Middle trace : light intensity. Bottom trace : stimulus signal. Stimulating electrode pair placed in sides of abdomen dorsal to light organ. Stimulation : 50 volts, 40-msec. duration, frequency 1 per second. Tiny marks on middle trace most apparent during hypoxia are stimulus artifacts via ground circuit, not light emission. of the lantern in serial fashion, one or more areas per pulse. The effect is illus- trated in Figure 8 ; each pulse initiates luminescence which covers one additional area completely. 3. Effects of an anticholinesterase and of denervation on hypoxic glozv and pseudoflash In order to examine further the role of possible neural activity on the pseudo- flash, the effects of injection of an anticholinesterase and of nerve transection on the hypoxic glow and pseudoflash were studied. Eserine, 10~ 4 M, in Roeder's saline (Roeder, 1953), injected into the body cavity of the decapitated adult, after removing the tip of the abdomen to permit flow of the solution through the body, 272 ALBERT D. CARLSON FIGURE 8. Effects of electrical stimulation during rising phase of hypoxic glow in an adult male, Photinus pyralis. With each shock hypoxic glow augmented as new areas begin to glow and remain glowing. Same effect can be noted in Figure 6, after first arrow. Top trace: up, 17.25% oxygen; down, ,.05% oxygen. Middle and bottom traces: light intensity, amplification of bottom trace being 1000 times middle trace. Stimulating electrode pair on right and left sides of seventh abdominal segment dorsal to light organ. Stimulation : 10 volts, 50-msec. duration, frequency 1 per second. produces nearly continual high intensity scintillation and flashing in air (Case and Buck, 1959). Under these conditions the rate of rise and maximal intensity of hypoxic glow are increased greatly. Pseudoflashes of normal characteristics can be induced after as little as 5 seconds of hypoxia (Fig. 9). FIGURE 9. Effect of perfusion of 10~* .17 eserine through a decapitated adult male, Photinus pyralis. Note the continual scintillation and rapid onset of hypoxic glow. Top trace: up, 17.25% oxygen; down, .05% oxygen. Middle and bottom traces: light intensity, amplification of bottom trace being 200 times middle trace. NEURAL EFFECTS ON PSEUDOFLASH 273 I**" 1 I-H FIGURE 10. Effect of transaction of the ventral nerve cord between the sixth and seventh abdominal segments of a female, Pliotnris sp. Except where noted otherwise the following conditions were maintained. Top trace: up, 21% oxygen; down, .05% oxygen. Middle and bottom traces : light intensity except where otherwise noted, amplification of bottom trace being 500 times middle trace. (A) Normal pseudoflash, both sixth and seventh abdominal segments give a total flash. Spontaneous flashing is seen in this figure, as well as in B, during onset of hypoxia. (B) Normal pseudoflash, sixth segment masked. Note the reduction in intensity of the pseudoflash since only the seventh segment is being recorded. (C) Inability to induce pseudoflash in segment 7, 8 minutes after transection, sixth segment masked. Top trace: up, 21.4% oxygen; down, 0.4% oxygen. Light leak from sixth segment appears as small flashes on left, bottom trace. (D) Pseudoflash 20 minutes after transection, seventh segment masked, showing response in sixth segment still normal. Amplification of light signal in bottom trace, 1000 times middle trace. (E) Pseudoflash 45 minutes after transection with stimulation, sixth segment masked. Stimulating electrode pair inserted laterally into the seventh segment dorsal to the light organ. Middle trace : stimulus signal. Bottom trace : light intensity with same amplification as before. Stimulation : 15 volts, 20-msec. duration, frequency 20 per second. In some instances, transection of nerves supplying the light organ of abdominal segment 7, at the level of the intersegmental membrane between segments 6 and 7, abolishes spontaneous flashing in the seventh segment and leaves unaffected the light organ of segment 6. Gradual reduction of hypoxic glow and pseudoflash intensity occurs in segment 7 until both are completely abolished after two hours, while the response in segment 6 can be maintained by mechanical irritation, eliciting 274 ALBERT D. CARLSON spontaneous flashes. In other cases, particularly in Photinus, the pseudoflash is immediately abolished in segment 7, except in one or two tiny spots which gradu- ally lose the ability to generate pseudoflashes. Stimulation with electrodes placed dorsally in the seventh (denervated) segment partially restores the pseudoflash and hypoxic glow. The hypoxic glow can also be intensified by stimulation during hypoxia as in the normal light organ (Fig. 10). FIGURE 11. Recording of neural action potentials, spontaneous flashes, and pseudoflash in the lantern of an adult male, Plwtnris pcnusylranica. Photomultiplier output on upper trace (increasing intensity down). Action potentials on lower trace. Time base indicated by horizontal, dark line which represents 100 milliseconds. Time base reads from left to right, (a) Spontaneous flash and generating neural burst prior to hypoxia. (b) Extremely reduced spontaneous flash and preceding neural burst after hypoxia onset, (c) Pseudoflash induced with air. Neither neural nor muscle action potentials are seen, (d) Spontaneous flash and preceding neural burst following pseudoflash in air recovery period. NEURAL EFFECTS ON PSEUDOFLASH 275 4. Demonstration of lack of spontaneous neural activity during pseudoflash In order to further define the role of the nerves in pseudoflash generation, nerve action potentials were sought during the hypoxic period and pseudoflash by placing recording electrodes on the exposed ventral surface of the light organ. The results are illustrated in Figure 11. The animal was flashing spontaneously prior to the onset of hypoxia and before each flash a discrete burst of neural activity was noted (Fig. lla). Immediately after onset of hypoxia two flashes of reduced magnitude were observed, each associated with an undiminished train of action potentials (Fig. lib). Following these flashes no bursts or even isolated nerve impulses could be observed. Although no hypoxic glow was observed, a pseudoflash was produced without associated action potentials (Fig. lie). Spon- taneous flashes and associated neural bursts resumed immediately following the pseudoflash in air (Fig. lid). DISCUSSION These observations on active, quiescent, electrically stimulated and eserine- injected animals, and the results of nerve transection, strongly implicate the inner- vation in the pseudoflash. Our inability to detect nerve impulses during the hypoxic glow period, however, indicates that individual pseudoflashes in the intact animal can be induced independently of neural activity. One supposition seemingly warranted by the decline of successive pseudoflashes in quiescent animals is that previous neural activity serves as a priming mechanism. A priming activity with regard to spontaneous activity has been suggested by Case and Buck (1959). The priming mechanism of neural activity on hypoxic glow and pseudoflash can be explained on the basis of the scheme of firefly luminescence developed by McElroy and his associates. Hypoxia may allow substrate accumulation from precursors in the intact animal just as it does in vitro as shown by Hastings, McElroy and Coulombre (1953 ), who were able repeatedly to induce flashes similar to pseudoflashes by alternating hypoxic and aerobic conditions until surface denaturation of the enzyme occurred. The reduction of intensity in successive pseudoflashes in our experiments would represent, according to this scheme, accumulation of an inhibitory complex. When pseudoflashes can no longer be induced, presumably all the enzyme is inhibited. As McElroy and Hastings (1955) speculated, one result of neural stimulation might be release of inorganic pyro- phosphate which on the basis of in vitro observations would free the active enzyme and result in the ability to again induce high intensity pseudoflashes. If nerves operate in fact as a priming mechanism by mediating destruction of an inhibitor, a number of observations of previous investigators could be explained. This action might explain why it is difficult or impossible to elicit pseudoflashes in quiescent animals since in them the active enzyme would be com- pletely inhibited. This suggests that during the daily non-flashing periods the light organ would contain large amounts of this inhibitor, and neural activity would be low or absent. The great deal of areal variation observed in the pseudo- flash response may simply be the result of spotty neural priming. This is sup- ported by the observation that a total organ pseudoflash is usually obtained in actively flashing animals and by the observations that electrical stimulation during 276 ALBERT D. CARLSON hypoxia in quiescent animals commonly augments the hypoxic glow by recruiting new, discrete luminescing areas (Fig. 8). SUMMARY 1. Hypoxic glows and pseudoflashes were studied in adults of the lampyrid fireflies, Photinus pyralis Linn, and Photuris sp. 2. In spontaneously flashing Photinus adults a large number of successive pseudoflashes of high intensity can be induced. Induction of pseudoflashes in quiescent animals results in decline of successive pseudoflash and hypoxic glow intensities, and increase in the time required not only for hypoxic glow onset but also for development of maximal hypoxic glow intensity. 3. Electrical stimulation during hypoxia increases the rate of hypoxic glow rise, commonly by recruiting new luminescing areas within the lantern, and can maintain the hypoxic glow beyond the normal period. Electrical stimulation when continued during readmission of air superimposes flashes on the pseudoflash decay phase only. 4. Perfusion of the body cavity with 10~ 4 M eserine enhances the ability to induce high intensity pseudoflashes and hypoxic glows. 5. Transection of the nerves supplying the seventh abdominal segment reduces the ability of that segment to produce pseudoflashes, while pseudoflashes can be obtained from that portion of the lantern occupying the sixth segment. Electrical stimulation of the seventh segment after nerve transection can partially restore its pseudoflash capability. 6. No nerve impulses were observed during the hypoxic interval immediately prior to and during induction of the pseudoflash although neural bursts and their associated flashes were noted before and after hypoxia and during its early period. 7. The observations implicate the innervation in the pseudoflash. A possibility suggested is that previous neural activity serves as a priming mechanism by mediat- ing the removal of a chemical inhibitor. LITERATURE CITED ALEXANDER, ROBERT S., 1943. Factors controlling firefly luminescence. /. Cell. Comp. Physiol., 22: 51-71. CARLSON, ALBERT D., 1959. Neural involvement in the firefly pseudoflash. Biol. Bull., 117: 407. CASE, JAMES F., AND JOHN BUCK, 1959. Central nervous aspects of firefly excitation. Biol. Bull, 117: 393. DAHLGREN, ULRIC, 1917. The production of light by animals. /. Franklin Inst., 183 : 323-348. HASTINGS, J. WOODLAND, AND JOHN BUCK. 1956. The firefly pseudoflash in relation to photo- genic control. Biol. Bull, 111: 101-113. HASTINGS, J. WOODLAND, WILLIAM D. MCELROY AND JANE COULOMBRE, 1953. The effect of oxygen upon the immobilization reaction in firefly luminesence. /. Cell. Comp. Physiol., 42 : 137-150. MCELROY, W. D., AND J. W. HASTINGS, 1955. Biochemistry of firefly luminescence. Pp. 161- 198 in The Luminescence of Biological Systems (Ed. F. H. Johnson), Amer. Assoc. Adv. Sci., Washington. ROEDER, K. D., 1953. Insect Physiology. John Wiley and Sons, Inc., New York. SNELL, PETER A., 1943. The control of luminescence in the male lampyrid firefly, Photuris Pennsylvania?, with special reference to the effect of oxygen tension on flashing. /. Cell. Comp. Physiol., 1: 37-51. u, EUGLENA GRACILIS IN SYNCHRONOUS DIVISION. II. BIO- SYNTHETIC RATES OVER THE LIFE CYCLE JAMES R. COOK* Department of Zoology, University of California, Los Angeles 24, California - Mapping of the major metabolic and synthetic events occurring over the course of the life cycle of single cells forms one of the principal endeavors of current physiological efforts, and very refined (albeit tedious) techniques have permitted notable successes in this area. However, the small size of growing cells im- poses serious quantitative and qualitative limitations on the information to be gleaned from this approach, since many of the coarser techniques require masses several orders of magnitude above that provided by the single cell. Furthermore, natural variability of cells grown under identical conditions best exemplified perhaps in the scatter of individual generation times (Powell, 1955; Prescott, 1959) can often be extensive. The use of synchronously-dividing populations as a model of the single cell promises to surmount some of these difficulties, though new ones may well be introduced. That populations of cells can be induced to divide rhythmically has been known for some time (von Denffer, 1949), but a clear appreciation of the possibilities offered by such systems appeared only within the last decade (Hotchkiss, 1954; James, 1954; Scherbaum and Zeuthen, 1954). It has already been made the subject of several review articles (Scherbaum, 1960; Zeuthen, 1958). Increased interest in this field has resulted in the elaboration of certain obvious restrictions which should be satisfied by the synchronously-dividing population before it may fairly be viewed as a working model of the single cell. For example, the time consumed in the burst of division activity should be relatively short compared to the over-all generation time; and the increase in population number occurring in each burst should be commensurate with the normal division behavior of member cells, if the division burst is to be equated to division of the single cell. These are but two of the more obvious parameters which characterize synchronous cell divi- sion. More subtle restrictions include continued and repetitive division bursts, i.e., populations in which synchrony can be maintained indefinitely (as long as the environmental stimulus causing synchrony is continued) (Padilla and James, 1960). A first corollary of continued division synchrony is the "balanced growth" of all member cells, in the usage of Earner and Cohen (1956). A further restric- tion is always implied, namely that growth of member cells in the population be 1 This investigation was carried out during the tenure of a Predoctoral Fellowship from the National Cancer Institute, United States Public Health Service. It was also supported in part by California State Cancer Grant No. 536 and in part by the National Science Foundation Grant 5666, granted to Dr. T. W. James. 2 Present address : Biology Department, Tokyo Metropolitan University, Setagaya-Ku, Tokyo, Japan. 277 278 JAMES R. COOK at least as synchronous as their division. Unfortunately, none of the cell types thus far synchronized can completely satisfy all these restrictions ; as noted by Zeuthen (1958), however, even a modest degree of synchrony can be useful. It is possible to synchronize division activity of free-living photosynthetic cells, at a constant temperature, merely by growing the cells autotrophically on light- dark cycles (Sweeney and Hastings, 1958: Tamiya ct al., 1953). If the light and dark periods are of appropriate lengths, the average cell grows uninterruptedly over one whole generation, and stores sufficient energy in the light period to satisfy demands for those processes culminating in cell division (Bernstein, 1960; Cook and James, 1960). In all photosynthetic systems so grown, the "burst" of division activity is confined for the most part to the dark period, so that growth and division are truly separated. In populations of Euglcna gracilis, the percentage of cells completing division in successive bursts gradually decreases with increased population density, pre- sumably because mutual shading limits effective utilization of the photosynthetic apparatus. At population densities below about 5000 cells per ml., 90-95% of the cells complete division at each burst ; if the culture is diluted once in each generation with fresh salt medium (at the temperature of the culture), so that the population density never approaches levels of the stationary phase, division syn- chrony can be maintained without abatement for long periods of time, perhaps indefinitely (see Figure 1). It can be seen from Figure 1 that divisions commence o o 38 36 35 E_ qra cilis Division Synchrony with Periodic Dilution 10 20 20 15 S 10 > 5 30 40 50 60 70 Time (Mrs) 80 90 100 no 120 FIGURE 1. Light-induced division synchrony of Euglcna gracilis. The upper curve is a plot of the logarithm (to the base 10) of the population density. The 8-hour dark periods, to which nearly all of the cell divisions are confined, are represented by the shaded areas. The light periods are of 16 hours duration. The broken lines indicate dilution with fresh salt medium at the temperature of the culture (20 C). The lower curve shows the proportion of cells in recognizable fission. BIOSYNTHESIS IN SYNCHRONOUS EUGLENA 279 at about the third hour of each dark period, and are terminated some two or three hours after the onset of the subsequent light period. Except for this very early portion, no cell divisions occur in the light period, which has a total duration of 16 hours. Of those cells completing division, 95% do so within a 6-hour span of time (James, 1960). Thus, about 75%, of the total generation time is devoid of cell divisions, and most of this fraction is spent in the light, where major biosynthetic activities of Euglena should occur. Since the division synchrony of Euglena gracilis is well-defined and repetitive, the metabolic regulation of syn- thetic rates must be in fine adjustment at all ages, especially since the energy supplied (as light) is just that needed for growth and division of the average cell over one life cycle. Characterization of growth patterns in synchronized Euglena is therefore profitably made, and cultivation on a near-basal medium, where the photosynthetic apparatus itself must be duplicated, perhaps renders such a study all the more meaningful. Moreover, synchronous growth at a constant temperature obviates difficulties associated with the interpretation of temperature-sensitive rate processes, and thus circumvents some of the criticisms which have been leveled against other methods of synchronizing cell division (Abbo and Pardee, 1960; Mitchison and Walker. 1959; Prescott, 1960). Mass increase and the distribution of volumes in member cells of synchronized Euglena populations have been reviewed in a previous paper, and the analogy of the population as a single cell discussed in some greater detail (Cook, 1961). While a wide range of cell volumes exists at any time, the (weighted mean) volume and dry mass exhibit a linear increase, to a doubling, over the whole of the 16-hour light period. The average Euglena in synchronous populations, then, is to a first approximation a successful working model of the single cell. The present report will describe growth patterns of other important cell parameters over the life cycle. METHODS AND MATERIALS Culture methods have been described in detail previously (Cook and James, 1960). A constant-temperature water bath (American Instrument Co.) circulated water continuously through a thin transparent plastic jacket surrounding the cylin- drical culture vessel, so that the culture was maintained at 20 C. An in-line reservoir of fresh medium, also kept at 20, was used to dilute the culture once in each generation (see Figure 1), so that the population density at harvest always lay between 3000 and 5000 cells per ml., well below- levels of the stationary phase. While this approach is only quasi-chemostatic, it is not likely that the medium ever became limiting for growth of Euglena; and in addition, changing characteristics of the average cell as a function of population density were avoided (Summers, I960). The medium was that of Cramer and Myers (1952), slightly modified (Padilla and James, 1960) : (NH 4 ).,HPO 4 , 1.0 gm./L. ; KH,PO 4 , 1.0 gm./L. ; MgSO 4 -x- H 2 O, 0.2 gm./L.; CaCL, 6.02 gm./L.; Fe,(SO 4 ),-xH.,O. 3.0 mg./L. ; MnCl,-4 H 2 O, 1.8 mg./L.; CoSO 4 -7H 2 O, 1.5 mg./L.; ZnSO 4 , 0.4 mg./L.; Na,MCv2H 2 O, 0.2 mg./L. ; CuSO 4 -5H 2 O, 0.02 mg./L. These salts are chelated by sodium citrate (0.8 gm./L.), which is not used as a carbon source by Euglena. The vitamins B x (0.1 mg./L.) and B 12 (0.0005 mg./L.) are required by Euglena, and were added aseptically to the autoclaved salt-citrate solution after sterilization by filtration. The phosphates buffer the medium to a pH of 6.8. In this medium, (NH 4 ) 2 HPO 4 280 JAMES R. COOK is the sole nitrogen source. However, small amounts of the sulfhydryl-bearing amino acids methionine (10~ 5 M) and cysteine hydrochloride (6.4 X 10~* M) were routinely added to the medium (after sterilization by filteration). While these concentrations do not support growth, they can effectively improve division char- acteristics of Euglcna in synchronous culture (Cook and James, 1960; James, 1960). Incandescent lamps (General Electric Co.), with an incident intensity of 130 foot- candles, were the sole energy source, and nutrition, save for catalytic amounts of the vitamins and two amino acids, was strictly autotrophic. In continuous light at 20 C, this intensity is saturating for growth of Euglena. Cultures were continuously stirred, by means of a teflon-covered magnetic impeller bar, to ensure even illumination and rapid exchange of gases. Harvests were made by means of a gravity-flow siphon, and stirring further served to randomize the distribution of cells within the culture. After the syphon was flushed, all aliquots were representative of the population as a whole. Cultures were axenic, and were kept on the light-dark cycle from inoculation. Dilution of the medium was initiated when the appropriate population density was reached, and repeated once in each generation ; harvests of any given culture were deferred until at least five light-dark cycles had elapsed after the first dilution. Generally, two light-dark cycles suffice for the induction of division synchrony, and initial harvests were delayed only as a precaution against the possible tardy stabiliza- tion of other cellular parameters. All cell counts were made with the Coulter cell counter (Coulter Co.). Details of this procedure can be found elsewhere (Mattern ct a/., 1957) ; the mean values of replicate counts made with this instrument are within 2% of the true value when no correction for coincidence is necessary. For the present case, aliquots were always diluted to cell densities yielding a linear counting rate, so that coincidence was negligible. At harvest, cells were first packed by gentle centrifugation, and then re- suspended to densities appropriate to the analytical procedure being followed. Representative aliquots of this concentrated suspension were then diluted with the salt medium for cell counts. Total protein content of the average cell was estimated by the Folin-Ciocalteau phenol test as modified by Lowry et al. (1951), after prior extraction of pigments and other soluble fractions with 90% aqueous acetone. Bovine serum albumin in known concentrations was simultaneously run as a standard, and protein levels in Euglena are expressed in these equivalents. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were extracted according to the method of Schmidt and Thannhauser (1945). Optical densities of the hydrolyzed DNA and RNA (in 5% perchloric acid) were measured at 260 and 315 m^t with the Beckman DU spectrophotometer. Regression curves obtained with serial concentrations of commercial DNA and RNA permitted conversion of the observed difference reading (O.D. 260 O.D. 315 ) into absolute amounts per cell. Pigments of the photosynthetic apparatus were estimated according to the method of Richards with Thompson (1952). After overnight extraction of a known number of cells in 90% aqueous acetone at 15 C., the initial extract was made up to volume (10 ml.) with two washes. Optical densities were determined in the Beckman DU spectrophotometer at wave-lengths of 750, 665, 645, 630, 510, BIOSYNTHESIS IN SYNCHRONOUS EUGLENA 281 and 480 m/t, with 90% acetone as a blank. Pigments in Euglena include the chlorophylls a and b, and several carotenoids, of which beta-carotene is the most important in terms of absolute amounts (Fogg, 1953). None of these absorb appreciably at 750 m/A, and readings at this wave-length (usually negligible) were routinely subtracted from those at the other wave-lengths as a correction for tur- bidity. Simultaneous equations developed by Richards with Thompson (1952) permit calculation of pigment concentrations from the observed optical densities. For the chlorophylls, concentrations in absolute amounts are obtained. The method serves as a satisfactory measure of chlorophyll a in Euglena. Chlorophyll b is present in much smaller amounts, however (about 10% that of chlorophyll a). This pigment is therefore largely masked by the other pigments, and accurate meas- urements are not feasible by this method. Since the several carotenoids are esti- mated as a mixture, concentrations must be expressed in arbitrary units. Richards with Thompson have proposed the Standard Pigment Unit (SPU) for this measure, and this unit is retained here. Their equations are satisfactory for estimates of carotenoid content in Euglena (Cook, 1960). The method of cultivating cells which was adopted for these studies limited the working volume of the culture to about one liter per day, i.e., a yield of no more than 5 X 10 6 cells in each generation. Since some of the procedures required almost this many cells for a single determination, a complete picture of synthetic rates could 1100- ,1000 = 900 o 800 700 600 500 . Qf OClllS Dry moss increase during the light period 8 10 12 14 16 Time (Mrs. Since Light On) 20 22 FIGURE 2. Dry mass of the average cell at different times during any given light-dark cycle. The abscissa is the time scale for one complete cycle, beginning arbitrarily with the onset of the 16-hour light period. The shaded area at the left represents the end of the preceding dark period, and that at the right the whole 8-hour dark period for the cycle shown. Limits of one standard deviation are indicated. JAMES R. COOK be compiled only by pooling data from many different light-dark cycles. Continued synchronous culture by periodic dilution, as illustrated in Figure 1, thus served practical as well as theoretical reasons. In the following, statistical estimates of experimental error are shown as the standard deviation about the mean, and repre- sent both errors of analysis and of cell counts. Unfortunately, the cell used in these studies was previously referred to as Euglena gracilis var. bacillaris (Cook and James, 1960). Certain characteristics, not discovered until after that paper had been accepted for publication, made it clear that the cell was not in fact E. gracilis var. bacillaris, but another strain of the species. Pending positive identification, the cell is called merely Euglena gracilis. RESULTS Figure 2 shows the dry weight of the average Euglena, measured at different times during the light-dark cycle. These data have been reviewed before (Cook, 1961), and are shown here for reference. The dark period is represented by the shaded portion, and the abscissa is the time-scale for one complete cycle 24 hours- beginning arbitrarily with the onset of the light period. The rate of dry mass increase is clearly linear over the whole of the light period, and is terminated when the cells enter the dark period. Since cell divisions are still occurring in the first two hours of the light period, meaningful mass measurements on a per cell basis are not easily made at this time. It seems fair to estimate mass changes in the early hours of the light period by extrapolation from the rate observed after cell divisions are complete. The extrapolated portion of the rate curve in Figure 2 is indicated by the broken line. (This treatment ignores the possibility of a brief lag in mass accumulation very early in the light period.) Since Euglena essentially doubles in number at each burst of division activity, continued balanced growth demands a duplication of major cell constituents over the life cycle. Figure 2 indicates that this is indeed the case for the dry weight of Euglena; the average filial cell has a dry mass of about 500 /x/^grams, and the average parent cell 1000 /x/xgrams. Mass loss in the 8-hour dark period is negligible. Synthetic rates for total protein are not so straightforward. As can be seen from Figure 3, protein synthesis is terminated (after a doubling) at the tenth or eleventh hour of the light period, at a level which remains constant thereafter. The filial cells contain about 200 /u^grams protein, and the parent cells 400. The protein : dry mass ratio is 0.4 both at the beginning and end of the light period, but goes through a slight maximum (0.5 ) in the interim. Nucleic acid levels in the average cell were determined less frequently. Figure 4 shows the RNA content at four different ages. If the rate curve is again extra- polated to the beginning of the light period, it can be seen that a doubling occurs linearly during the entire light period. The filial cell contains 23 ^grams, and the parent cell 46 /x/zgrams. Less confidence can be placed in the DNA data, and this study will bear repetition with culture conditions yielding larger numbers of cells for this analysis. However, Figure 5 indicates that DNA is probably doubled in a linear fashion throughout the light period. Duplication of chlorophyll a is completed by the fourteenth hour of the light period, after which no further increase occurs (Fig. 6). Similarly, carotenoid synthesis (in Euglena, mostly beta-carotene) is terminated about two hours before BIOSYNTHESIS IN SYNCHRONOUS EUGLENA 283 500 400 n" 300 20O- grocilis Totol protein increase during the light period ft 10 12 14 16 Time (Mrs Since Light On) 20 22 FIGURE 3. Total protein of the average Euglena at different times during any given light-dark cycle. the onset of the dark period (Fig. 7). The ratio carotenoids : chlorophyll a is thus constant throughout the life-cycle of the average cell. Chlorophyll a contributes about 3% to the total dry weight of Euglena, although this value goes through a slight maximum at the fourteenth hour of the light period. From data of Goodwin and Jamikorn (1954), it can be calculated that the carotenoids in synchronized SO 45 40' a. a. 35- Z a. 30 t'5 20 E_ grocilis RNA increase during the light period _0_ 6 8 10 12 14 Time (Mrs Since Light On) 18 20 22 24 FIGURE 4. RNA synthesis by the average cell during the light period. 284 JAMES R. COOK < z o E grocilis DNA increase during the light period 24 1 5 Z 15 E" Time (Mrs Since Light On) 22 FIGURE 5. DNA content of the average Euglcna during the light period. Euglena constitute one to two per cent of the total dry weight (Cook, 1960), and this, too, goes through a slight maximum in the light period. Table I lists the concentrations of chlorophyll b calculated for different ages. While there is considerable error involved in estimates of this pigment by the method used here, the expected two-fold difference in chlorophyll b levels should at least E. grocilis Chlorophyll a. increose during the light period 4 6 8 10 12 14 16 18 2O 22 24 Time (Mrs Since Light On) FIGURE 6. Chlorophyll a content of the average cell during any given light-dark cycle. BIOSYNTHESIS IN SYNCHRONOUS EUGLENA 285 10- a. a. E gracilis Carolenoid increase during the light period 24 e 10 12 14 Time (Mrs Since Light On) 16 20 24 FIGURE 7. Carotenoid synthesis by the average Euglena. be indicated ; that is to say, even rough approximations should reveal some tendency toward increasing amounts if chlorophyll b is indeed duplicated in the light period. No such differences are seen over the major portion of the light period, however; at the sixth hour, the average cell has 2.60 0.38 ^grams chlorophyll b, and at the onset of the dark period, 2.67 0.24 /^grains. It is not at all unlikely that chlorophyll b is duplicated in the very early portion of the light period. The uncertain role of chlorophyll b in the family of photosynthetic pigments need not after all demand a constant ratio of chlorophyll a : chlorophyll b. Thus, Smith (1949) and Koski (1950) have shown that the biosynthesis of chlorophylls a and b in etiolated seedlings proceeds at different and apparently unrelated rates. For Euglena, a more sensitive analysis than that used here must be adopted before accurate rate measurements of chlorophyll b synthesis can be known. The possi- bility that a continuously changing ratio of photosynthetic pigments occurs over the life cycle of Euglena implies a regulation of metabolism which could be of first importance to the over-all balanced growth of the cell. TABLE I Chlorophyll b content of Euglena gracilis in synchronous culture, calculated according to the equations of Richards with Thompson (1952). The numbers following "Age" represent the time in hours following onset of the 16-hour light period. Age 2 6 8 10 12 14 16 17.5 19 Chlorophyll b (jujugm./cell) 1.87 2.60 2.53 2.44 2.79 3.21 2.67 2.86 3.11 .42 .38 .08 .06 .22 .21 .24 .14 .45 286 JAMES R. COOK DISCUSSION The energy supply supporting cell division, an event which comes in the dark period in synchronous populations of Euglcna gracilis, as well as maintenance of the cell in this period, must ultimately be derived from photosynthetic products elab- orated in excess of immediate needs for incremental growth, and stored up during the light period against future requirements of the dark period. In point of fact, an estimate of the quantity of this stored energy was the basis leading to the partic- ular light-dark cycle on which Euglcna is grown (Cook and James, 1960). One might therefore have expected the total dry mass of the average cell to somewhat more than double during active synthesis in the light period. Figure 2 indicates that this is not the case, however. Each parent cell of 1000 juyxgrams dry weight yields two filial cells of 500 /x/Agrams (assuming equal division), and each of these enters the subsequent light period with just this weight. Thus, parent and filial cells traverse the entire 8-hour dark period with no significant reduction of mass. Cramer and Myers (1952) have estimated that Euglena gracilis consumes twice its own volume of oxygen every hour. While respiratory rates of Euglena in synchronous culture have not yet been determined, it is probable that similar rates will be found. With this assumption, and with the further assumption that an RQ of 1 obtains, it can be calculated that the total carbon lost (as CO 2 ) in the dark period is only 35 ju/tgrams for the average parent and generated filial cells. This is less than 4% of the total dry weight, not likely to be detected by a technique which has a probable error of about 5%. In most euglenoid flagellates, protein constitutes about 30-40% of the total dry weight (Cook, 1960). In autotrophic strains, photosynthetic pigments contribute about 5%, and the nucleic acids 4 or 5%. Most of the remaining dry mass, some 50%, will be made up of storage products lipids and paramylum, the characteristic polysaccharide of these cells (Kudo, 1954). Although these storage products were not measured over the life cycle of Euglcna, it is inferred that their accumulation must complement protein synthesis in such a way that the over-all increase in dry mass, of which these fractions make up the bulk, follows the linear rate shown in Figure 2. The ratio of storage products to dry mass therefore probably passes through a minimum at the same time of the light period as the protein : dry weight ratio exhibits a maximum. Major synthetic patterns of the average cell in synchronized populations of Euglcna gracilis, growing autotrophically on a simple, near-basal salt medium at 20 C, are summarized in Figure 8. The ordinate is in relative units, from 1 to 2, the range of increase in these parameters expected for the single cell over its life cycle. The time scale on the abscissa is shifted somewhat from that seen in the earlier graphs, with respect to the light-dark cycle, to more nearly accommodate the actual age of the average cell. This cell has its inception, by division of the parent, some two hours before the onset of the light period, and 24 hours later is itself the parent of two filial cells, an event which occurs at the sixth hour of the subsequent dark period, being preceded by a mitosis which is probably of three or four hours duration (Leedale, 1958). Most of the interphase is spent in the light, where the cell completes its physical growth and duplicates most (if not all) of its principal biochemical constituents. Increase in dry mass is limited to the light period, and follows a linear rate over the entire 16 hours, as does the cell volume (Cook, 1961). BIOSYNTHESIS IN SYNCHRONOUS EUGLENA 287 E. grocilis Growth and Division of the Average Cell in Synchronous Culture _?i>L ._CJU associated with a substance showing polarographic reversibility (as appears within gut content) is more likely attributable to a variable pH than to varying ionic strength in an unaltered medium with regard to buffer. No direct evidence for the correlation between E i/o and the initial gut content pH for use as a reference point was sought. It was anticipated that delay, in the event sufficient sample remained for polarographic study after pH measurement of a volume as small as 0.1 ml., would be accompanied by sample deterioration. Cell activity gradually changes the chemical nature of hind-gut content as confined metabolites accumulate. The pH of freshly obtained gut content samples ranges between 6.6 and 7.2. Attempted sample pH control during polarography by oxygen-free buffer addi- tion probably resulted in failure for one or more of the following reasons: (1) sudden medium alteration due to contact with lysed cell content, (2) severe sample dilution, and (3) ionic strength change leading to nonspecificity of E !/o identical to that caused by variable pH for which control was sought ionically. Slight variation, therefore, in E l / 2 between polarograms of different freshly obtained gut content samples is attributed to pH variation between samples. Initial standardization of pH, and, for that matter, pH control during a series of recorded polarograms tracing changes produced by aging of any one sample as reported later, cannot be achieved successfully with buffer. Regardless, a low oxidation-reduction potential is indicated, and specificity range is sufficiently limited to allow positive identity of the reducing agent. 2. Tests used in identification of the reducing agent A. Polarography Separate polarograms for solutions of varied substances in 0.1 M KC1 failed to produce waves even remotely resembling the shape of Curve A, Figure 2, much less a half-wave potential falling within the desired range. Among compounds studied 334 HOPE RITTER, JR. -0.4 volts FIGURE 1. Polarograms of reduced glutathione: (A) 1.4 X 10~ 3 M GSH in 0.1 M KC1, (B) 7.2 X 10~ 4 M GSH in a complex salt medium also containing yeast, blood serum and methyl- cellulose (sensitivity X 5; pH 6.6). were L-cysteine, adrenochrome semicarbazone, ascorbic acid, sodium anthraquinone sulfonate, acetylcholine chloride, riboflavin, and sodium thioglycollate. A 1.4 X 10~ 3 M solution of reduced glutathione (GSH) in a supporting electro- lyte of 0.1 M KC1 at pH 6.6 produced a polarogram (Curve A, Fig. 1) closely resembling that of Curve A, Figure 2. Addition of 0.005 gm. GSH to a gut content sample increased the wave height without significant change in the previously re- corded half-wave potential. The comparative method of wave height-concentration plots indicated 7.2 X 10~ 4 M GSH in an isotonic medium of mixed salts, nutrients, and methyl cellulose results in a polarogram (Curve B, Fig. 1) almost identical to that recorded from the natural gut content (Curve A, Fig. 2). The Ilkovic equation, mentioned earlier, was not employed in the calculation of GSH concentration. Application of this equation would require a viscosity reading for hind-gut content, and this has not yet been determined. B. Chemical GSH produces a violet color in the presence of sodium nitroprusside. A posi- tive reaction occurs when a thin layer of dark brown Cryptocercus gut content is tested on a white porcelain surface. Although ascorbic acid does not react with nitroprusside, cysteine and possibly other sulfhydryl compounds effect the positive reaction (Patterson and Lazarow, 1953). GLUTATHIONE AND ANAEROBIOSIS 335 -0.4 FIGURE 2. Polarograms of the same hind-gut sample from Cryptocercus: (A) untreated, (B) following air passage for one hour, and (C) following nitrogen gas flow, 20 minutes (sensitivity X 5; pH approximately 6.8 at start). Patterson, Lazarow and Levey (1949) report alloxan in excess reacts with the sulhydryl of GSH forming an unknown substance with an absorption spectrum maximum at 305 m/x. By this method 3 X 10~ 4 M GSH has been identified in hind-gut content cleared of cellulose and protozoa. The authors mentioned also report equivalent molecular amounts of cysteine, cysteinylglycine, glutamylcysteine, and ascorbic acid in the presence of GSH lower the value of GSH determination. C. Bioassay Loomis (1955a, 1955b) showed that a concentration of 1C)- 5 M GSH elicits a characteristic feeding response in starved Hydra littoralis. A 1 : 3000 dilution of Cryptocercus gut content induced a strong response, indicating a concentration of GSH somewhere in the order of 10~ 3 M. This bioassay, however, is no longer specific for GSH. Lenhoff and Bovaird (1960) obtained the same response with trypsin, and Cliffe and Waley (1958) obtained it with ophthalmic acid. 3. The apparent source of GSH Since variables involved with polarograph operation were held constant, slight variations in GSH concentration, observed in fresh unaltered samples, must be inherent in sample fluids. The ratio between number of cells and surrounding fluid volume probably varies between hosts. If GSH is protozoan and/or bacterial in 336 HOPE RITTER, JR. origin, it could fluctuate with the rise and fall of these populations. If GSH is a secretion of the host tissues, its concentration could vary in direct proportion with the mass of the insect. The question of primary concern, however, was the source of GSH. After squeezing, sufficient protozoa remain within the roach to repopulate all species back to normal level within four to six weeks. During this period, contrary to conditions in unsqueezed roaches, many cells appear in various stages of mitosis. Protozoan repopulation is a relatively slow process, but the fluid content is rapidly reestablished. An adequate volume of fluid can be removed from the same roach 24 hours after squeezing for polarographic study. Polarograms were made at 24 hours, and at the end of the first, third, and fourth weeks during the repopulation period with two groups of squeezed roaches. Results indicate GSH concentration increases directly proportional to the increasing proto- zoan population. For the present, the relatively crude method used in estimating repopulation rate during the four-six-week period appears adequate. Three stages in the process were easily recognized. (1) That time immediately following squeezing when the gut fluid contains a few random, scattered, residual cells. (2) A mid-stage follows when fluid contains a large number of cells, but also cell-free areas. (3) Maximum population is reached when a mass of cells appears, each in contact at many points with other cells. An estimate of one week for total bacterial repopulation, following squeezing, exceeds the mean reproductive potential of such organisms. If host tissue was responsible for GSH secretion, a maximum concentration would be expected (polarographic inspection) within one week after squeezing. Usually three weeks were required before GSH concentration reached 50% of normal. This corre- sponds to a protozoan population judged to be at the approximate refaunation half-way mark. Several protozoan species are intimately associated with one or more species of bacteria. The cell membrane of Barbnlanympha shows uniform cellular arrange- ment for one species of bacterial rod. The association is permanent. This example of subordinate symbiosis, and other associations of a similar nature, prevent final decision regarding the exact source of GSH the protozoa per se, intimate bacterial associates, or both. It is, however, significant to know GSH is not host elicited. 4. The effect of oxygen and nitrogen on GSH The relationship betw r een absence of a polarographic oxygen wave, following exposure of gut content to air or pure oxygen, and GSH concentration must be considered. A sample of hind-gut content bubbled with air at a slow rate for as long as one hour in the H-cell solution chamber failed to produce an oxygen wave. GSH con- centration, however, decreased approximately one-half from a reading of approx- imately 5.4 //.a to 2.5 //.a shown in a comparison of Curves A and B, Figure 2. The reduction potential also was shifted 0.04 volt more positive. Roughly 90% of the initial GSH concentration (presumably oxidized) was reduced in the gut content during nitrogen passage for one hour. Recovery was indicated by an increase in the height of the wave (Curve C, Fig. 2) and a reduction GLUTATHIONE AND ANAEROBIOSIS 337 -0 4 volts FIGURE 3. Polarograms of the same hind-gut sample from Cryptoccrcus: (A) untreated, (B) following pure oxygen passage ten minutes, (C) following a 20-minute additional period of pure oxygen passage, and (D) nitrogen gas passage, 40 minutes (sensitivity X 5; pH approx- imately 6.8 at start). to a more nega- potential shifted back to a more negative reading. Shift of --E tive position was first viewed as a result of alkalinity increase following a CO blozv-off. But, upon further consideration, oxygen passage would also be expected to achieve the same effect, and does not. A change in pH, nevertheless, first in one direction and then the other, appears responsible for corresponding shifts in reduction potential. Curves B and C, Figure 3, show a progressive reduction in GSH concentration for one sample following contact with pure oxygen. Curve A represents the sample's GSH level prior to oxygen exposure. Curve A differs from the previous experiment's unexposed sample (Curve A, Fig. 2), registering approximately 0.7 //a more GSH and a slightly higher pH, judging from the more negative E ^ position. Curve B, Figure 3, was recorded following oxygen passage for ten minutes. From a comparison of microampere readings between Curve A (original concentra- tion) and Curve B, estimated GSH drop was 50%. Curve C, Figure 3, was recorded following oxygen passage lasting 20 additional minutes, in which the GSH level was further reduced. Even under these extreme conditions, in a sample less than 0.5 ml., it was not possible to demonstrate the presence of dissolved oxygen. Continuing with the same sample, nitrogen passage following oxygen exposure of the extreme intensity just described failed unaccountably to demonstrate the 338 HOPE RITTER, JR. reversibility achieved with nitrogen after air exposure in the earlier experiment (Curve C, Fig. 2). Even forty minutes' contact with nitrogen (Curve D, Fig. 3) did not bring about a curve height increase beyond that recorded in Curve C, Figure 3. The height of Curve D, Figure 3, was depressed even further. In addition, again contrary to results obtained when the air-treated sample was bubbled with nitrogen, a comparison of half-wave potentials in Figure 3 illustrates nitrogen passage was accompanied by a reduction potential shift to a more positive reading. The phenomena just described are based on repeated observations. Polaro- grams illustrated trace events related to two treated gut samples ; one sample using air passage followed by nitrogen gas (results verified by fourteen experiments), and one sample using "prepurified" oxygen followed by nitrogen gas (results verified by seven experiments). Although it has not been determined how much oxygen a small sample of gut fluid can absorb or bind without losing its potential with respect to GSH reduction during nitrogen passage, experiments suggest oxygen can overwhelm the glutathione recovery system. A. Evidence suggesting enzymatic reduction of GSH That partial GSH reduction can occur during nitrogen passage through a gut sample under prolonged exposure to air suggests strongly the presence of an appro- -4 -04 FIGURE 4. Polarograms of the same hind-gut sample from Cryptocercus: (A) heated ten minutes, and (B) following pure oxygen passage five minutes (sensitivity X 2). GLUTATHIONE AND ANAEROBIOSIS 339 priate enzyme system. Further evidence favoring the enzyme hypothesis appears when partial glutathione recovery, to the reduced state following air exposure, cannot be achieved in a sample altered by heating over a water bath for 10 minutes at 80 C. Even negligible atmosphere exposure encountered in transferring a heat- treated sample to the electrolysis vessel is sufficient to oxidize GSH, as indicated by disappearance of the reduction wave (Curve A, Fig. 4). Five minutes of oxygen passage then results in such marked saturation of the sample that a prominent oxygen wave (Curve B, Fig. 4) can be recorded. Glutathione reductase and dehydrogenase assays have not yet been undertaken. Such studies will be resumed following application of present findings to protozoan culture. It is entirely possible that more than one enzyme will be found present in the hind-gut content, accounting for rapid glutathione auto-reduction demonstrated prior to heat alteration. 5. Evidence suggesting a chemical relationship betiveen glutathione and carbon dioxide Polarographic experiments and related observations repeatedly point out an- other phenomenon in the gut content which might support M. Calvin's (1953) hypothesis for addition of mercaptans across C = O in CO 2 in slightly alkaline media to form S-alkyl monothiocarbonate. He predicted an acid medium could reverse the reaction to release CO 2 and restore GSH : O O II OH- || GSH + C = O + OH- ;= = R - - S - - C - O~ + H 2 O H 2 A dense resident population of microorganisms in the hind-gut of Cryptocercus ferments ingested cellulose. The fermentation products would be expected to be similar to those reported by Hungate (1939) for Zootermopsis CO 2 , hydrogen, and predominantly acetic acid. Complete acid analysis has not been made, but total CO., in freshly removed gut content measures 0.12 volume per cent by Scholander's (1947) method. Fermentation probably continues briefly, following sample introduction into a polarographic vessel (due to continued cell activity), which partially accounts for increase in acidity. Twenty to 30 minutes later the pH drops from the 6.6-7.2 range to 6.4. Within five hours the pH drops to 4.8-5.1, and at 18 hours 4.4. This could explain the shift of half- wave potential in polarograms (Curves A, B, and C, Fig. 5) from 0.196 volt vs. S.C.E. made immediately to a more positive potential approximating 0.133 volt after 17 hours. The protozoa are dead, most are deformed, and some are lysed when the pH reaches 6.4. Motile bacteria are no longer active. The remaining bacterial species constitute a very small number of cells in what was an overwhelmingly populated milieu. These bacterial species can be cultured in a cellulose-nutrient medium which supports growth and reproduction of five protozoan genera normally asso- ciated with the bacteria (Ritter, 1959). Bacterial metabolites produced in this culture medium in the absence of protozoa cause the pH to increase, not decrease. 340 HOPE RITTER, JR. Bacterial influence in this phenomenon of acidification seems to be completely ruled out following observation of toluene-treated gut content samples. Toluene addition to the extent of 20% final dilution failed to alter the pattern of pH decrease to the 4.0-5.0 range within 18 hours. The cause of pH drop from 6.4 to 4.4 in 18 hours must be accounted for if, as seems to be the case, protozoa and bacteria are not responsible. It appears that bound CO 2 is released in sufficient quantity to cause this pH drop. CO 2 was identified as a major gas constituent, accumulating after several hours as a bubble mound at the interface of a large gut fluid sample layered with heavy mineral oil. Correlated with this rapid accumulation of CO 2 in the absence of fermentation, polarograms (Curves A and C, Fig. 5) lend additional support to Calvin's hy- pothesis. As the pH registers progressively lower, GSH concentration increases. From comparative wave height measurement of polarograms A and C, GSH (which almost doubles in quantity within five hours) increases nearly four times the original amount within 17 hours. This marked wave height increase could indicate mercaptide formation. It is generally accepted that a typical mercaptan characteristic is reaction with heavy metals such as mercury, and with organic compounds, to form mercaptides. And, apparently, there is every opportunity for this reaction to occur in a sample which rests on mercury in the electrolysis vessel. The mercaptide phenomenon, if it occurs in this gut fluid, does not appear polarographically. The wave height increase represents GSH concentration in- -o 4 volts FIGURE 5. Polarograms of the same hind-gut sample from Cryptocercus, untreated, and recorded at intervals: (A) immediately, (B) at 20 minutes, and (C) at 17 hours (sensitivity X 5 Curves A and B ; X 10 Curve C ; pH approximately 6.8 at start ; 4.4 at end) . GLUTATHIONE AND ANAEROBIOSIS 341 -0.4 volts FIGURE 6. Polarograms of two different hind-gut samples from Cryptocercus: (A) aged 5 hours in the electrolysis vessel and in contact with the mercury pool, pH 5.1, and (B) aged 4% hours in a vessel without mercury contact until transfer to the electrolysis vessel, pH 4.9 (sensitivity X 5). crease, and the shift in -E y 2 to a more positive reduction potential correlates with pH drop. Evidence for this conclusion rests on : 1. A fluid sample aged between four and five hours in a mercury-free container, after removal from the roach and deposit inside the electrolysis vessel, registers a wave (Curve B, Fig. 6) coresponding closely with a fluid sample aged the same period in solution chamber contact with mercury (Curve A, Fig. 6). 2. According to Benesch and Benesch (1952), more energy is required for mercaptide complex reduction than for the free compound that gave rise to the complex. Therefore, the resulting reduction wave would register at a more nega- tive E y% for the mercaptide not more positive as recorded in this study to reflect the low dissociation of the Hg-S bond. 3. Also in the event of mercaptide formation, two separate waves would be expected to appear polarographically. The difference between the --E i/> of the gradually forming mercaptide and the more negative E !/o of disappearing GSH would likely be great enough to initiate two separate reduction waves separated by a shifting plateau. DISCUSSION The appreciable presence of atmospheric oxygen coupled with diverse, unique, and efficient animal systems that transport oxygen suggest unspecified claims of anaerobiosis (as to degree) should be questioned. Living membranes present an 342 HOPE RITTER, JR. ineffectual barrier to oxygen. For that matter, full appreciation of oxygen per- meability through synthetic membranes cannot be realized until experience is acquired with a working chamber from which all oxygen is meant to be excluded. In other words, at least a minute amount of oxygen probably reaches an organism's most remote anatomical niche. If symbionts dwelling in such a site require an absolute oxygen-free environment, either they or the surrounding host cells must chemically maintain it. 1. Absence of molecular oxygen in Cryptocercus hind-gut content A. Direct evidence The entire problem of determining environmental p(X at lower limits is ham- pered by inadequate indicators and mechanical devices for measurement. Polar- ography, in spite of extreme sensitivity for detecting oxygen in solution, is in- capable of registering ultra-trace amounts. Failure to obtain typical reduction waves for oxygen in Cryptocercus hind-gut fluid polarograms is of great analytical significance. However, the question of degree in anaerobiosis still remains un- answered. To report that this environment is free of oxygen to the extent of 10' 6 M is informative, though admittedly inadequate, for culture investigation. Technically, therefore, the term anaerobic should not be used merely on the basis of this information. B. Indirect evidence The polarograph also indicated concentration decrease of an unknown substance when gut content was exposed to pure oxygen. Prolonged oxygen exposure failed to leave enough molecular oxygen in solution for polarograph detection. In addi- tion to indicating a substance is exploited in proportion to the amount of oxygen passing into gut fluid, the observation strongly suggests chemical affinity between oxygen and this unknown substance. Oxygen most certainly diffuses into the lumen from the gut epithelium under natural conditions. Conceivably the oxygen is instantly bound molecularly. Inter- pretation of these phenomena, based on direct and indirect evidence obtained in polarograms, suggests Cryptocercus protozoa live in an anaerobic environment- anaerobic to the extent of maximum physical and chemical maintenance. 2. Reduced glutathione in Cryptocercus hind-gut content A. Methodology in identification Identification of the substance, which registers so prominently on polarograms and appears to function as a strong reducing agent, was difficult. Analyses offering unequivocal interpretation are rare when attempting to single out one organic constituent in the complexity of a biological medium. Investigative methods included comparative polarography, the classic sodium nitroprusside test, the alloxan "305" method, and the Loomis bioassay technique. Comparative polarography is a highly accurate procedure in which closely controlled physical conditions are imposed to measure known and unknown solu- tions. A more extensive survey of potential reducing agents appeared unnecessary because of the method's relative specificity. A known GSH solution produces GLUTATHIONE AND ANAEROBIOSIS 343 a polarogram of nearly identical structure to gut content polarograms. The sup- porting electrolyte of mixed salts used in the GSH medium was determined by flame photometry of the gut fluid lacking major suspended elements. Viscosity was approximated with methyl cellulose, and the pH of 6.8 required no adjustment. Variation in any one of these especially pH caused an expected fluctuation in the E 1/2, but the polarographic curve remained essentially unchanged. As mentioned earlier, the standard addition method of polarography confirmed the presence of glutathione. Addition of GSH to the unknown sample increased the wave height (concentration) without changing other aspects of the curve. Following preliminary evaluation of other available methods of GSH analysis for application to this study, one-dimensional paper chromatography of use after blocking the sulfhydryl group with N-ethylmaleimide (Hanes, Hird and Isher- wood, 1950) indicated greatest specificity. Inadequate sensitivity of the chroma- tographic method, using GSH concentration determined polarographically as reference, eliminated chromatographic application. The alloxan "305" method functioned as a qualitative test only. GSH concen- tration determined in the supernatant portion of centrifuged gut content was less than half the amount demonstrated polarographically. Although substances already mentioned compete with GSH in alloxan reaction, it is possible only two need be considered. Cysteine and ascorbic acid, though potentially reactive at the dropping mercury electrode, do not appear polarographically. Glutamylcysteine and/or cysteinylglycine might be involved in competitive absorption. Awaiting further investigation, one might guess that y glutamylcysteine, an intermediate in GSH synthesis probably to the exclusion of cysteinylglycine (Snoke and Bloch, 1952), is of more vital concern in evaluating potential alloxan competition with GSH. The nitroprusside method and the bioassay technique using Hydra lack specificity in GSH identification. Nevertheless, nitroprusside identified the substance as a sulfhydryl compound, and Hydra gives a feeding response only in the presence of reduced glutathione. Trypsin, as yet unidentified in Cryptocercus, has not been ruled out as an alternative stimulant for Hydra. This represents by no means an exhaustive methodology for assaying biologi- cally-occurring glutathione. Each chemical test available is nonspecific or limited to some degree. General disagreement usually exists regarding the number of chemical tests necessary before identification is scientifically acceptable. In this polarographic study, however, more than mere substantial evidence accumulated to support glutathione assessment. Results derived from both nitroprusside and Hvdra assays were conclusive yet predicated upon far less specific analysis. On the strength of collective information afforded by these tests, along with confirming opinions of Professors K. E. Bloch, J. T. Edsall, and A. M. Pappen- heimer, GSH is a constituent of Cryptocercus hind-gut content. GSH is responsible for maintenance of the oxidation-reduction potential observed polarographically. B. The site of synthesis Since more than 30 species of protozoa inhabit Cryptocercus, it must still be determined which of these are involved in GSH synthesis. The problem is further complicated by intimate, and, in some cases, permanent, bacterial association with the cell membrane of certain protozoan species. Evidence suggests protozoa are the site for this svnthesis, but not necessarily the sole metabolic source of GSH. 344 HOPE RITTER, JR. C. Oxidation-reduction activity It was impossible to assess fully results obtained in experiments in which gas was directed through freshly obtained gut content samples. It was clear that oxygen decreased the concentration of reduced glutathione. This was interpreted as an oxidative phenomenon in which GSH was converted to the disulfide form (GSSG). Although nitrogen passage was capable of partially restoring the gluta- thione wave height in air-exposed samples, the precise mechanism was not dis- cernible. It has been assumed temporarily that this wave extension represents a reduction of GSSG to GSH the nitrogen gas being most indirectly involved. Wieland (1953) stated reduction of GSSG can be accomplished with various reducing agents, other mercaptans in excess, nascent hydrogen, Na or Li in NH 3 , and by other substances. He also pointed out enzymatic reduction is frequently responsible. Whatever the cause, a point exists polarographically demonstrated at which nitrogen can no longer carry out its indirect role. This occurs following gut content exposure to pure oxygen in excess of ten minutes. Glutathione oxidized to this extent could not be reactivated as could samples exposed to lesser amounts of oxygen. Opinions sought are totally inadequate in accounting for contradictory evidence concerning the effect of nitrogen following varied aerations of gut content air versus pure oxygen. Interpretation of present phenomena obtained polaro- graphically must await correlation with results from additional analyses now contemplated. Since the oxidation-reduction potential of gut content can also be rendered inactive by heat, the tentative conclusion for the presence of at least one enzyme system seems justified. Glutathione reductase, according to Vennesland and Conn (1953). is the thermolabile enzyme responsible for the reduction of GSSG by tissues. GSSG is reactive with TPN-reducing systems. Accordingly : TPNH + GSSG + H + -* TPN + + 2GSH D. A proposed role in pCO 2 and pH regulation Culture studies in progress demonstrate glutathione can now be assigned an extracellular role in anaerobic maintenance. The importance of this mercaptan in growth and other physiological activities is well substantiated, but whether it can be assigned an additional function in the gut of Cryptocercus is still problematical. Speculation regarding GSH capacity to combine with CO 2 to form an alkyl compound, as proposed by Calvin, is a tempting hypothesis. Culture studies of the protozoa definitely show a very narrow range of pCO 2 tolerance. Experi- mentally, membrane selectivity can be altered dramatically when cells are exposed to an excess of this gas, but protozoa move directly to a higher pCO 2 gradient when placed in a sub-optimal region of the culture medium. Hind-gut protozoa, therefore, engaged in active fermentation yet highly sensi- tive to an altered pCO., threshold, would have marked survival potential if a mechanism were available for chemical regulation of this metabolite. Theoretically GSH could function as a pH-regulated pCO 2 buffer, supplementary to more direct pCOo control by the roach's external respiratory apparatus. Conceivably, even though pH might trigger this mechanism to reestablish an equilibrium between S-alkyl monothiocarbonate and GSH, pH, in turn, could be regulated, in part at least, by the mechanism. Certainly the host roach and its protozoa are confronted GLUTATHIONE AND ANAEROBIOSIS 345 by environmental factors that challenge optimal metabolism, such as conditions imposed during molting and winter. 1 am very much indebted to Dr. L. R. Cleveland for invitation to work at the Biological Laboratories, for his advice, and for his criticism of the manuscript. I am grateful for the assistance of Dr. W. F. Loomis, and for advice and sugges- tions regarding polarography and the interpretation of polarograms from Professor J. J. Lingane and Dr. Allen J. Bard. Professors Bloch, Edsall, Pappenheimer, and Wald were consulted during various phases of this study. Their interest and counsel is greatly appreciated. SUMMARY 1. Polarography, employing a mercury-dropping electrode and an electrolysis vessel for analysis of sample slightly less than 0.1 ml., was applied in this study. 2. Polarograms of Cryptocercus hind-gut content lack an oxygen wave and indicate absence of dissolved oxygen to the extent of apparatus sensitivity (10~ 6 M). 3. A single polarographic wave registers a half -wave potential approximating 0.2 volt vs. S.C.E. within a 0- to 0.4- volt span. Specificity of this method enabled identification of reduced glutathione (7.2 X 10~ 4 M) following compara- tive inspection of numerous reducing agents. Supplementary assays using sodium nitroprusside, alloxan monohydrate, and Hydra confirmed the presence of reduced glutathione in hind-gut content. 4. Flagellate protozoa, symbionts in the hind-gut fluid of Cryptocercus, repre- sent the site of glutathione synthesis. 5. Prolonged oxygen passage through hind-gut samples (0.1-0.5 ml.) fails to alter polarograms with respect to negative pO 2 registration, but reduced glutathione concentration is decreased in direct proportion to oxygen exposure intensity. 6. Partial restoration of reduced glutathione concentration following nitrogen gas passage through samples moderately exposed to oxygen cannot be explained. 7. Brief atmosphere contact with heat-altered hind-gut samples results in polaro- grams showing pO 2 contamination and an absence of reduced glutathione. Until enzyme analyses are completed, sample alteration by heat merely suggests presence of a mechanism for glutathione maintenance in the reduced state. 8. Cryptocercus hind-gut content is anaerobic to the extent of maximal physical and chemical maintenance as are the microorganisms normally living within it. Reduced glutathione is considered of major importance in this role. 9. Some evidence exists which may support Calvin's hypothesis for addition of mercaptans across carbonyls in a biological sample. The value of such a mechanism to Cryptocercus and its symbionts is theorized. LITERATURE CITED BEECHER. H. K., R. FOLLANSBEE, A. J. MURPHY AND F. N. CRAIG, 1942. Determination of the oxygen content of small quantities of body fluids by polarographic analysis. J. Bio!. Chcm.', 146: 197-206. BENESCH, R., AND R. E. BENESCH, 1952. Reactions of thiols with organic mercury com- pounds. Arch. Biochcm. and Biophys., 38 : 425-441. BENESCH. R., R. E. BENESCH AND W. I. ROGERS, 1953. The reactivity of the sulfhydryl group in glutathione and related peptides. In: Glutathione, 1954 (S. Colowick ct al., editors), Academic Press, Inc., New York: 31-42. 346 HOPE RITTER, JR. CALVIN, M., 1953. Mercaptans and disulfides : some physics, chemistry, and speculation. In: Glutathione, 1954 (S. Colowick et a!., editors), Academic Press, Inc., New York: 3-30. CLEVELAND, L. R., 1925a. The effects of oxygenation and starvation on the symbiosis between the termite Tcrmopsis, and its intestinal flagellates. Biol. Bull., 48: 309-326. CLEVELAND, L. R., 1952b. Toxicity of oxygen for protozoa in vivo and in vitro: animals de- faunated without injury. Biol. Bull, 48: 455-468. CLEVELAND, L. R., S. R. HALL, E. P. SANDERS AND J. COLLIER, 1934. The wood-feeding roach Cryptoccrcus, its protozoa, and the symbiosis between protozoa and roach. Mem. Am. Acad. Arts and Set., 17: 185-342. CLEVELAND, L. R., AND A. W. BURKE, 1956. Effects of temperature and tension on oxygen toxicity for the protozoa of Cryptocercus. J. Protosool., 3 : 7477. CLIFFE, E. E., AND S. G. WALEY, 1958. Effect of analogues of glutathione on the feeding re- action of Hydra. Nature, 182: 804-805. GILMOUR, D., 1940. The anaerobic gaseous metabolism of the termite, Zootermopsis nevadensis Hagen. /. Cell. Comp. Physiol, 15 : 331-342. GRUNERT, R. R. AND P. H. PHILLIPS, 1951. A modification of the nitroprusside method of analysis for glutathione. Arch. Biochem. and Biophys., 30: 217-225. HANES, C. S., F. R. HIRD AND F. A. ISHERWOOD, 1950. Synthesis of peptides in enzymic re- actions involving glutathione. Nature, 166: 288-292. HUNGATE, R. E., 1939. Experiments on the nutrition of Zootermopsis. III. The anaerobic carbohydrate dissimilation by the intestinal protozoa. Ecology, 20: 230-245. KOLTHOFF, I. M., AND J. J. LiNGANE, 1952. Polarography. Second ed. Interscience Publishers, Inc., New York. LENHOFF, H. M., AND J. BOVAIRD, 1960. Enzymatic activation of a hormone-like response in Hydra by proteases. Nature, 187 : 671-673. LOOMIS, W. F., 1955a. Specific qualitative microbioassay for reduced glutathione. Fed. Proc., 14: 247. LOOMIS, W. F., 1955b. Glutathione control of the specific feeding reactions of Hydra. Ann. N. Y. Acad. Set., 62: 211-227. LOWRY, O. H., AND O. A. BESSEY, 1946. The adaptation of the Beckman spectrophotometer to measurements on minute quantities of biological materials. /. Biol. Chcm., 163: 633-639. MEITES, L., 1955. Polarographic Techniques. Interscience Publishers, Inc., New York. PATTERSON, J. W., A. LAZAROW AND S. LEVEY, 1949. Reactions of alloxan and dialuric acid with the sulfhydryl group. /. Biol. Chcm., 177: 197-204. PATTERSON, J. W., AND A. LAZAROW, 1953. Methods of glutathione assay : a review of classical methods. In: Glutathione, 1954 (S. Colowick ct a!., editors), Academic Press, Inc., New York : 63-78. RITTER, H. T. M., 1959. Factors essential for in vitro reproduction of Cryptocercus protozoa. /. Protozool., 6 (August supplement) : 21. SCHOLANDER, P. F., 1947. Analyzer for accurate estimation of respiratory gases in one-half cubic centimeter samples. /. Biol. Chcm., 167 : 235-250. SNOKE, J. E., AND K. BLOCK, 1952. Formation and utilization of 7-glutamylcysteine in gluta- thione synthesis. /. Biol. Chcm., 199: 407-414. TRACER, W., 1933. The cultivation of some intestinal flagellates of termites and the nature of the symbiosis between these protozoa and their insect host. Thesis, Harvard University. VENNESLAND, B. AND E. E. CONN, 1953. The enzymatic oxidation and reduction of glutathione. In: Glutathione, 1954 (S. Colowick et al., editors), Acadameic Press, Inc., New York: 105-127. WIELAND, T., 1953. Chemistry and properties of glutathione. In: Glutathione, 1954 (S. Colo- wick et al., editors), Academic Press, Inc., New York: 45-57. YAMASAKI, M., 1931. Studies on the intestinal protozoa of termites II. Oxygenation experi- ments under the influence of temperature . Mem. Col. Sci., Kyoto Imp. Univ., Series B, 7: 179-188. COBALT AND GLUTATHIONE IN THE PRESERVATION OF FERTILITY AND LIFE OF SAND DOLLAR EGGS 1 OLIN RULON Department of Biological Sciences, N orthzvestern University, Evanston, Illinois, and The Hopkins Marine Station, Pacific Grove, California The loss of fertility of the unfertilized egg of Dcndraster cxcentricus within a few hours after removal from the ovary is of interest. During the breeding season the eggs may be 95-100% fertilizable for 24-48 hours when allowed to stand uncrowded in finger bowls of sea water at 18 C. After this interval, the capacity for fertilization decreases and the eggs soon disintegrate. When conditions are not optimum the eggs show poor fertility from the beginning. This is commonly the case with over-ripe eggs. The decay of fertility seems to be correlated with the onset and progression of the death processes which lead to disintegration. Fertility may be prolonged or regained under certain conditions. In experi- ments involving the treatment of Dcndraster eggs with lithium before fertilization (Rulon. 1946) it was found that a \% ethyl alcohol solution would insure the fertility of the egg sufficiently to permit an adequate period of exposure to the test agent. In other experiments (Rulon, 1948) it was found that certain lots of Arbacia eggs, which were only 25-50% fertilizable, could be stimulated to 90-100% fertilization by treatment with Ca-free sea water for eight hours before the addition of a few drops of sperm suspension. For a number of years the writer has been interested in modifying and con- trolling developmental patterns in echinoderm embryos with various enzyme inhibi- tors and other agents. In several cases interesting modifications have been caused by treatment of the unfertilized egg (Rulon, 1941, 1946). Cobaltous chloride was chosen for the present study because previous work (Rulon, 1956) had shown it to alter development in a definite manner when applied to the newly fertilized egg and the mid-blastula. In the work reported here it was found that cobalt had little effect on the pattern of the developing larva when only the unferti- lized egg was treated. It was found, however, that cobalt possessed remarkable properties for maintaining fertility long beyond the normal period. MATERIALS AND METHODS The sand dollars (D. c.vccntricus} were dredged from Monterey Bay during the summer of 1959 and maintained in healthy condition in tanks in the aquarium room of the Hopkins Marine Station, Pacific Grove, California. Ova were taken in large numbers from ripe females by cutting away the oral surfaces of the tests and allowing the exposed gonads to shed. Droplets of ova were drawn into medi- 1 This investigation was supported by the Graduate School of Northwestern University. The writer is also much indebted to Dr. L. R. Blinks, Director of the Hopkins Marine Station, Pacific Grove, California. 347 348 QLIN RULON cine droppers and released in fresh sea water. After washing, the ova were placed in the various concentrations of cobaltous chloride and cobaltous-glutathione solu- tions for the duration of the experiments. At 24-hour intervals a number (50-1 00 ) of the eggs were removed to fresh sea water and washed. Fresh sperm suspension was added to test fertility. The chief criterion for fertilization was nuclear division. Throughout the work the eggs were kept uncrowded in covered fingerbowls out of direct sunlight and at 18 1 C. Smoking was not permitted in the laboratory since previous work by Child and others has shown that in sufficient density, tobacco smoke may cause modifications in developing embryos (Child. 1941, p. 222 ) . EXPERIMENTAL I. The effects of cobaltous chloride on fertility and cleavage Numerous preliminary experiments indicated unfertilized eggs, in sea-water solutions of cobaltous chloride, retain their fertility long beyond those in sea-water controls. They also showed that on fertilization the cobalt-treated eggs behaved somewhat differently from the controls and that these differences were related to concentration of the cobalt and length of exposure period. One of the most conspicuous effects of exposure to cobaltous chloride was the presence of tight fertilization membranes on fertilized eggs from the more concen- trated solutions (compare controls, Figures 1-3, with tests, Figures 4-15). Eggs dividing in such membranes gave elongated and often incompletely separated blastomeres. If the exposure was of sufficient duration cytoplasmic division was partially or totally inhibited but nuclear division still occurred (as in Figures 1115). If the concentration of cobalt was somewhat less but the exposure time was of long duration, the presence of a fertilization membrane could not be detected at all and cytoplasmic division graded from complete to partial (Figs. 16-24). When the blastomeres were fully cleaved they tended to become spherical and loosely attached. With long treatment many inequalities in size of cells and irregu- larities in cleavage patterns were to be noted. It was soon evident that cobalt was exerting its effects largely on the cell cortex and that cytoplasmic division was more subject to inhibition than nuclear division. In general, it was found that most of the eggs that could be fertilized showed recovery in sea water and developed into plutei that were not far from normal. In some cases, however, plutei were somewhat stunted and granular but none developed in a manner comparable to those caused by post-fertilization treatment (see Rulon, 1956). The effects of various concentrations of cobalt on the preservation of fertility are shown in Table I. Here are tabulated the results of a single series from eggs taken from one female. In this series, approximately 45% of the control eggs were fertilizable at the end of 24 hours (not in table) but none could be fertilized at 48 hours. Approximately 50% responded after treatment with M/400-M/800 for 96 hours while approximately 25% retained fertilizability for as long as 168 hours with M/1600-M/3200. Even in a weak solution of cobaltous chloride (M/6400) 50% of the eggs retained fertility for 96 hours. These experiments definitely showed that certain concentrations of cobalt prolong the fertile life and even life itself of the egg of Dendraster e.rcentricus. FERTILITY OF SAND DOLLAR EGGS 349 23 FIGURES 1-24. Figure 1, newly fertilized egg of Dendraster with elevated fertilization membrane. Figures 2-3, normal early cleavage within fertilization membrane. Figures 4-10, eggs cleaving within tight fertilization membrane following exposure to strong (M/400- Af/1600) solutions of cobalt. Figures 11-15, eggs with nuclear division but inhibited cyto- plasmic division following long exposure to cobalt. Figures 16-24, eggs with aberrant cyto- plasmic division and undetectable fertilization membranes following exposure to less concentrated solutions of cobalt. 350 OLIN RULON TABLE I The effects of different concentrations of cobaltous chloride for various intervals of time on fertilization of the eggs of Dendraster excentricus ( Values given in percentage) Solution used 48 hours 96 hours 144 hours 168 hours 192 hours Sea water (control) M/200 cobaltous chloride 20 10 M/4)00 cobaltous chloride 50 60 1 M/800 cobaltous chloride 60 40 1- M/1600 cobaltous chloride 80 50 40 30 j M/3200 cobaltous chloride 80 50 25 25 M/6400 cobaltous chloride 80 50 II. The effects of cobaltous glutathione on fertility and cleavage It has been thought that cobalt causes much of its effect by the fixation of thiol ( SH) groups (see Marston, 1952). Zinc, which has much the same action as cobalt on differentiation but at much lower concentration, appears, also, to unite with sulfhydryl (Rulon, 1955). Since the effect of zinc may be prevented with glutathione, it was suggested that this agent may well inhibit the effect of cobalt on the preservation of fertility. Accordingly, tests were run in which eggs were placed in solutions of cobaltous-glutathione in which the concentration of cobalt varied but that of glutathione was kept constant at 0.025% (a concentration which had no detectable effect by itself on the unfertilized egg). It seemed apparent that there was actual chemical union of these two substances in that clear pink solution of cobalt becomes a clear brown when the colorless glutathione was added. TABLE II The effects of cobaltous chloride and glutathione for various intervals of time on fertilization of the eggs of Dendraster excentricus ( Values given in percentage) Solution used 48 hours 96 hours 168 hours 216 hours 312 hours Sea water (control) 0.025% glutathione 50 cc. M/100 cobaltous chloride plus 50 cc. 95 95 95 90 0.05% glutathione 50 cc. M/200 cobaltous chloride plus 50 cc. 95 95 90 70 50* 0.05% glutathione 50 cc. M/400 cobaltous chloride plus 50 cc. 95 90 75 75 0.05% glutathione 50 cc. M/800 cobaltous chloride plus 50 cc. 90 0.05% glutathione 50 cc. M/1600 cobaltous chloride plus 50 cc. 0.05% glutathione 50 cc. M/3200 cobaltous chloride plus 50 cc. 0.05% glutathione * Different series of experiments. FERTILITY OF SAND DOLLAR EGGS 351 The results of this series of experiments are tabulated in Table II where it is shown that glutathione prevented the fertility-preserving effects of cobalt only at the lower concentrations. In the stronger solutions (50 cc. M/100-M/400 cobaltous chloride plus 50 cc. of 0.05% glutathione) cobaltous-glutathione was a far more effective agent in prolonging the fertile life of the eggs of Dendraster than was cobalt alone. It will be noted that the eggs are still highly fertile after 216 hours in these solutions and that in another experiment there was 50% fertility after 13 days. It was of interest to note (data not shown in table) that while with the most concentrated solution (50 cc. of M/100 cobalt plus 50 cc. of 0.05% glutathione) 90% of the eggs were fertile, only nuclear division resulted. In treatment with next strongest solution, where 70% of the eggs were fertile, 30% showed only nuclear division while the remaining 40% showed some cytoplasmic cleavage as well. The 75% fertile eggs from the next solution (50 cc. M/400 cobalt plus 50 cc. of 0.05% glutathione) showed cytoplasmic along with nuclear division. DISCUSSION It does not seem likely that the effects of cobalt in the concentrations used in these experiments were due to osmotic factors. Although measurements of egg size were not made, the test eggs, before fertilization, appeared little different from the controls. In previous work by the author and many others, agents far more con- centrated (i.e., lithium) have been used with only slight osmotic effects. Minor changes (of the order of the concentrations used in these experiments) in salt content of the sea water produce little or no effects. It is known, however, that as echinoderm eggs age there is an increase in permeability and viscosity (see Goldforb, Landowne and Schechter, 1937, for references). It is not known that such physical changes destroy fertility in the egg but there seems to be a correlation. It should be remembered, however, that permeability to water and ions is quite different from permeability to (or penetration by) spermatozoa. The failure of the fertilization membrane to become elevated after treatment with cobalt is difficult to explain. Tight membranes occur under a variety of conditions in these forms (i.e., aging itself will cause them). It may be that long exposure to cobalt partially denatures the vitelline membrane or it may be that cobalt prevents the breakdown of the cortical granules. Certainly the subject de- serves further study. Any explanation of the mechanisms for the increase or preservation of fertility is not easy. To this worker it long has been a question as to why an ovum, con- taining so much in the way of nutrients, should lose its capacity to be fertilized so early. Other investigators have dealt with the problem in other eggs. The fertile life of Arbacia eggs may be prolonged in low-calcium sea water (Schechter, 1937). Perhaps such procedure prevents the release of calcium from its protein binding in the cortex for internal clotting (Heilbrunn, 1943, pp. 88-89) and thereby pre- vents or slows down processes concerned with loss of fertility and death. The fertilizable life of Urechis eggs may be extended with 1% alcohol or dextrose (Whitaker, 1937). It was suggested that these agents are effective by furnishing nourishment to the egg or by decreasing permeability of the egg surface. Runn- 352 OLIN RULON strom (1952) reports the use of various amino acids and proteins to improve fertilization (but not to preserve fertility) in echinoderm eggs. Of the several hypotheses given in the past it seems that the present work with cobalt may lend itself to an interpretation that is similar, in part, to one proposed by Loeb (1912, p. 155) who found that the fertilizable life of the starfish egg could be extended by cyanide or oxygen lack. He concluded that the egg becomes unfertilizable, in the normal course of events, after a damaging amount of aerobic oxidation takes place and that such agents as potassium cyanide preserved fertility by slowing down this action. The writer has come to a tentative explanation as to the action of cobalt. This substance is well known because of its action in inducing polycythemia, possibly as the result of physiological compensation to partial anoxia, caused by the fixation of SH groups (see Marston, 1952). It is suggested that the cortex and surface of the newly shed egg are abundant in such groupings. It is well known that essential -SH groups on an enzyme may be rapidly oxidized by atmospheric oxygen (see Singer, 1945). It seems not unreasonable to assume that under normal aerobic conditions in sea water, labile or soluble sulfhydryl compounds may be expected to unite with each other by oxidation to disulfide. In other words, simple proteins are united into large stable, insoluble protein chains in which the union of the individual members is through S S bonding (see Jensen, 1959). Such a change at the egg surface may well account for the loss in fertility in aging eggs. ^ It is believed that cobalt prevents this chain reaction by uniting with -SH and in this manner serves in preventing the decay of fertilizability. This, indeed, seems to be the case as is shown in the experiments in which glutathione is added to the cobaltous chloride solutions. Concentrations of cobalt that were highly effective in prolonging fertility (71//1600-M/3200) were rendered partly or entirely ineffective by the addition of 0.025% glutathione. This shows the affinity of the two substances and supports the suggestion that cobalt unites with thiol groupings at the egg surface in the preservation of its integrity. But with more concentrated solutions of cobaltous-glutathione the preservation of fertility zvas greatly enhanced! It is apparent that a different situation has now developed. The combination gives more protection to the egg surface and cortex than does the cobaltous ion alone. In the adsorption of cobaltous-glutathione, changes (oxidation of SH) with the loss of fertility are considerably less than with cobalt alone. Such an explanation seems highly speculative until one considers the more recent work with Co 60 Vit. B ]2 in which the combining power of this important substance with the serum proteins (alpha and beta globulins) and the protein of cerebro-spinal fluid has been so aptly demonstrated (see Meyer ct a/., 1959). It seems quite reasonable to assume that the remarkable preserving action of cobaltous-glutathione is brought about by its union with the substances which would otherwise gradually change as fertilizability is lost. SUMMARY 1. A range (1//200-.M/6400) of cobaltous chloride in sea water increases the fertile life of Dendraster eggs. The most effective concentrations (M/1600- FERTILITY OF SAND DOLLAR EGGS 353 JW/3200) prolong fertilizability for over 168 hours in 25-30% of the eggs tested. None of the control eggs is fertile at 48 hours. 2. The exposure of unfertilized eggs for long periods or to high concentrations leads to tight fertilization membranes and gradual suppression of cytoplasmic divi- sion when the eggs are finally fertilized. 3. Glutathione ( 0.025 %') prevented the fertility-preserving action of cobaltous chloride in lower concentrations but greatly enhanced it in higher concentrations. One mixture (50 cc. of 0.05% glutathione plus 50 cc. M/200 cobaltous chloride) preserved fertility in 50% of the eggs for 312 hours. 4. The work suggests that cobalt and cobaltous-glutathione unite with R-SH at the egg surface (each in a somewhat different manner) preventing oxidation and a deterioration of fertilizability. LITERATURE CITED CHILD, C. M., 1941. Patterns and Problems of Development. Chicago: University of Chicago Press. GOLDFORB, A. J., M. LANDOWNE AND V. SCHECHTER, 1937. Effects of age on Arbacia eggs. VI. Increased stretching and bursting of egg membrane in hypotonic sea water. Physiol. Zoo!.. 10: 59-70. HEILBRUNN, L. V., 1943. An Outline of General Physiology. Second Edition. Saunders, Philadelphia. JENSEN, E. V., 1959. Sulfhydryl-disulphide interchange. Science, 130: 1319-1323. LOEB, J., 1912. The Mechanistic Conception of Life. University of Chicago Press. MARSTON, H. R., 1952. Cobalt, copper and molybdenum in the nutrition of animals and plants. Physiol. Rev., 32: 66-121. MEYER, L. M., R. W. BERTCHER AND C. MULZAC, 1959. Co. 60 Vit. Bis binding capacity of normal human cerebrospinal fluid. Proc. Soc. Exp. Biol. Mcd., 100 : 607-608. RULON, O., 1941. Modification of development in the sand dollar by NaCNS and Ca-free sea water. Physiol. Zoo}., 14: 305-315. RULON, O., 1946. Modification of sand dollar development by exposure to lithium chloride and sodium thiocyanate before and after fertilization. Physiol. Zoo!., 19 : 58-66. RULON, O., 1948. The experimental modification of developmental patterns in Arbacia with malonic acid. Physiol. Zool., 21 : 100-105. RULON, O., 1955. Developmental modifications in the sand dollar caused by zinc chloride and prevented by glutathione. Biol. Bull., 109: 316-327. RULON, O., 1956. Effects of cobaltous chloride on development in the sand dollar. Physiol. Zoo}., 29 : 51-63. RUNNSTROM, J., 1952. The cell surface and its relation to fertilization. Symposia of the Society for Experimental Biology, VI : 39-88. University Press, Cambridge. SCHECHTER, V., 1937. Calcium reduction and the prolongation of life in the egg cells of Arbacia punctulata. Biol. Bull, 72 : 366-376. SINGER, T. P., 1945. Enzyme inhibitors and the active groups of proteins. Breivcrs' Digest, 20: 43-46 (August), 42-44 (September). WHITAKER, D. M., 1937. Extension of the fertilizable life of unfertilized Urcchis eggs by alcohol and by dextrose. /. E.vp. Zool., 75: 155-167. LEARNING IN THE SPINY LOBSTER PANULIRUS ARGUS HERMANN SCHONE Bermuda Biological Station^ St. George's West, Bermuda, and Max-Planck-lnstiiut fiir Verhaltensphysiologie, Scezviescn, Obb., Germany Although Bethe (1898) believed crustaceans in general could not learn by experience, subsequent research by many investigators has proved that cladocerans, isopods, crayfishes, hermit crabs and several kinds of brachyuran crabs can variously show habituation, discrimination learning and trial-and-error learning (reviews : Warden, Jenkins and Warner, 1940; Thorpe, 1956; Schone, 1961; recent research paper: Datta, Milstein and Bitterman, 1960). In these different cases visual, chemoreceptor, kinesthetic or tactile elements were involved in the afferent com- ponents of the behavior patterns studied. As is typical for learning, these data (plotted as error scores) usually fall on curves resembling exponential decay func- tions with a large initial number of errors which, with training, are rapidly reduced at first and then later decrease more and more slowly (Hull, 1952; Hilgard, 1956; Bush and Estes, 1959). In attempting to demonstrate polarized light vision in decapod crustaceans, training experiments were undertaken with Panulirus to determine whether dif- ferent planes of polarization could be learned as cues for the solution of a two-choice spatial problem. While the lobsters failed to use the polarization pattern in this way, they were able to make their spatial choice on the basis of brightness discrim- ination or to learn always to choose the same side for attaining a goal. The present report describes these experiments and briefly considers their comparative be- havioral implications. This research was supported by NSF Grant 7387, for which Talbot H. Water- man, Dept. of Zoology, Yale University, was Principal Investigator, and by grants from the American Academy of Arts and Sciences as well as the Penrose Fund of the American Philosophical Society. Thanks are due to Dr. Waterman for his encouragement and stimulating discussions ; also to Dr. William H. Sutcliffe, Jr., Director of the Bermuda Biological Station where this work was done, for his keen interest and unfailing help. METHODS The experimental animals were juvenile specimens of Panulirus argus (Lat- reille) 8-12 cm. in body length. They were tested in an experimental discrimina- tion box in which they were trained to walk forward (in air) to one of two com- partments at the far end ( Fig. 1 ) . Each compartment had a large rectangular exit hole in its floor; one was open and led down to the aquarium below in which the lobster was normally kept ; the other was closed by a wire screen which prevented 1 Contribution number 290. 354 LEARNING IN THE SPINY LOBSTER 355 10 cm FIGURE 1. Two-choice spatial discrimination box used to train Panulirus. A, aquarium containing sea water ; D, partition dividing front of box into two compartments ; Ei, open exit from one compartment to aquarium below ; E 2 , blocked exit in other compartment ; L, light source illuminating conditioning stimuli (Pi, P 2 ) ; O, slit for observing the lobster's behavior during tests ; Pi, Pa, circular illuminated areas used in experiments training to brightness and those designed to train to plane of polarization ; S, starting place for runs. the animal's return home. The lobsters were observed through a peep hole and their course recorded on a floor plan of the box. Two conditioning stimuli were presented when desired at a circular opening in the far wall of each compartment. These apertures were covered with translucent paper and illuminated from behind. The intensity of these stimuli could be altered by using additional layers of the translucent paper, and polarizers (Polaroid HN38 neutral polarizing filters) could be placed in the light paths to effect nearly 100% linear polarization of the illuminated areas. Experiments were carried out as follows. The Panulirus were tested one at a time by placing them at the entrance of the experimental box facing the two goal compartments which were at the opposite end (Figs. 1 and 2). For animals which walked to the compartment with the open exit hole without entering the closed com- partment a score of "correct" was kept; an error was recorded for those which walked into the closed compartment (score of "incorrect") and for those which stopped and remained anywhere in the back part of the box for five minutes ("non- spontaneous runs"), after which the lobsters were prodded into action and led in the correct direction with a small stick. When spontaneous locomotion was partic- ularly circuitous or slow, this stick was also used to prod the lobster into action. In addition to the error scores (in per cent per three trials) two other kinds of data were taken: (1) percentage of nonspontaneous runs and (2) percentage of runs in which tail-flexing reactions occurred. 356 HERMANN SCHONE In various experiments groups of individuals varying in number from 6 to 11 were given 2 to 6 tests per day, and the observations were repeated for 6 to 22 days. In certain cases the performances of trained and inexperienced individuals were compared. The three main series of training experiments all followed the general procedure outlined above but differed in certain important details. 1. Training to brightness. Ten individual inexperienced lobsters which had never been in the experimental box before were used in these tests. The condition- ing stimuli differed in intensity, and the more intense one was always on the side of the compartment with the exit open to the aquarium below (positive side). During the first 9 days / pos was three times as great as / neg ; during the remaining 13 days 7 pos was five times as great as 7 neg . The first day each animal was tested three times, for the next 9 days five times per day, and for the last 12 days six times per day (about 119 runs per individual or 1188 altogether). To control against a) [ V 9 W b) Lobster No. 20 FIGURE 2. First ten runs of (a) a lobster (No. 17) which learned relatively quickly to discriminate brightness in choosing the right course to the open exit, and (b) a lobster (No. 20) which was slow in learning this task. The different parts of the animal's track are marked with the following symbol : full line = running forward ; zig-zag line = running backward ; small open circle = stop ; cross hatching next to line guiding by a stick ; crosses = tail flexing movement. the lobsters merely learning "side" the positive side was changed alternately from right to left for the first 12 days; then sides were shifted in random order for the next 10 days. 2. Training to polarised light. One series of measurements (A) was made on 11 inexperienced lobsters; a second series (B) was run with 10 experienced animals. 2 For Series A the box's wooden walls were unpainted. For Series B the walls of the experimental box were painted with aluminum paint in order to minimize reflection-refraction artifacts arising from differential effects of the walls on the total light pattern visible to the lobsters. The two conditioning stimuli had the same intensity and degree of linear polarization, but in one (the positive side) the c-vector was horizontal, while in the other it was vertical. Five tests per day per animal were made in each series. The horizontal polarization was regularly alternated from left (1) to right (r) in successive tests of Series B, but there were some irregularities in Series A which ran : rrllrlrlrlrrlrlrlrlrlrlrlrlrlr. Series A 2 These had previously been used in the experiments on training to brightness. LEARNING IN THE SPINY LOBSTER 357 lasted 6 days (about 30 tests per animal, total 327), Series B, 11 days (55 tests per animal, total 550). 3. Training to side. Six inexperienced lobsters were run in the experimental box for 9 days with the left compartment's exit open to the aquarium throughout. The intensities of the two light windows were equal and they were not polarized. Two tests were made per animal on the first day and three during the succeeding 8 days, making 26 per animal (total 156). RESULTS A. General behavior In the present experimental situation Panulirus' behavior was made up of ac- tivity sequences containing one or more of four locomotor components : ( 1 ) no loco- motion, (2) forward walking, (3) backward walking, and (4) tail-flexing move- ments. 3 The actual paths followed were, of course, characteristically affected by the amount and rate of turning. Examples of the resulting trails are given as two series of 10 trials each for individual lobsters (Fig. 2). Evidently these activity sequences differ in three major ways which are inter- related : (1) degree of spontaneity, (2) directness from starting point to end point, (3) particular locomotor components involved. Thus the most direct trails (Fig. 2a, Runs 7 and 8; Fig. 2b, Run 8) were started spontaneously, and consisted only of forward walking, at first straight ahead, then gradually turning towards the side with the open exit. In slightly less direct paths the animal also started spontaneously and walked forward all the way but it turned more strongly towards the positive side, struck the side wall and followed this for a while until it turned in the opposite direction towards the exit (Fig. 2a, Runs 6 and 10 ; Fig. 2b, Run 9) . As the paths became less direct than this, more turning, stopping and backward walking appeared (Fig. 2a, Runs 5 and 9; Fig. 2b, Runs 4 and 10). Spontaneity was reduced and tail-flipping escape reflexes were evident. Finally, in the least direct runs the animals often did not leave the back part of the box spontaneously. In such cases they frequently had to be guided to the open exit with the stick. Backward walking, stopping and escape reactions occurred often (Fig. 2a, Runs 1, 2, 3 and 4 ; Fig. 2b, Runs 2, 5 and 7 ) . Some of the variation in these behavior patterns of Panulirus is due to individual peculiarities since the number and kind of certain loops, turns or backtracks tend to be similar in successive trials for the same lobster but to differ from one animal to another. The correlation of alterations in response with learning and the three types of training employed is considered in detail in the next section. B. Training experiments In running the two-choice maze of the present setup the short spontaneous paths over which the animals have walked directly forward to the open exit represent the best behavior if prompt return to sea water in their aquarium is considered the goal. 3 These arise as strong flexions of the abdomen (Lochhead, 1961; Wiersma, 1961) which may result in the animal's projecting itself backward several centimeters in a response often termed an escape reflex. 358 HERMANN SCHONE 90 80 70 60 50 : .T , './..f. . . . . x* ' Lobster No.17 Q- .. y ? -Z-X x^Sx- ,. 75 2/ 75 73 79 20 24 12 1-28 29-54 55-89 90-779 TRIALS 1-6 7-11 12-17 18-22 DAYS FIGURE 3. Learning curves for 10 individual lobsters training to brightness. Points indicate percentages of runs for the periods in trials (as well as days) specified on the abscissa. Conversely, meandering runs with stops, walking backward, escape reactions and failure to reach the goal without guidance represent the worst performances. Under appropriate conditions of training and with test animals having a tendency to asso- ciate a conditioning stimulus with the goal, the poor responses shown initially by inexperienced animals improve progressively to a better performance level. However, the amount of improvement and its rate during different parts of the training period were quite different in individual animals (Fig. 3). The factors involved in such variations remain largely unstudied. Nevertheless in certain ex- periments poor or insignificant improvement can be attributed to the development of certain preferences largely unaffected by the kind of associations tested. Thus in Series B of the training to polarized light, 5 of the 10 animals tested preferred one side of the box regardless of changes in the side of the open exit. This habit may be so rigid as to persist even when the positive side was never made the one preferred. An example of this type of behavior, quite resistant to conditioning in the present setup, is shown in Figure 4. Here the alternation of positive side (a, 1 ; b, 1) from left to right was succeeded by the open exit being maintained only on the left (a, 2 ; b, 2). Despite this, Animal 12 continued to turn right, and even after a few correct responses, returned to its persistent incorrect right turn. Animal 15 showed a com- parable predominance of right-hand turns for the greater part of the series but toward the end seemed to learn the left turn required to reach the positive side. A summary of the numbers and types of runs made in each of the three kinds of training experiments is presented in Table I. LEARNING IN THE SPINY LOBSTER 359 a) Lobster No ; 2 Lobster No. 75 A - FIGURE 4. Serial representation of training runs of two experienced lobsters to polarized light (sequence from left to right). In series al, bl the open exit (positive side) was alternated regularly ; in series a2, b2 the positive side was always to the left. Correct runs are represented by open circles, incorrect runs by filled circles. Development of a tendency to run towards the right side is shown by lobster No. 12 which kept this tendency even when the positive side remained left; lobster No. 15 did not, having finally changed to the positive side. 1. Training to brightness. Initially the average error score of the 10 animals tested was 93%. This had fallen to 35-40% after 30-40 trials and continued to diminish (but at a reduced rate) to about 20% by the end of the experiment (119 trials) (Fig. 5, error curve). The fraction of nonspontaneous runs was also high during the first trials (57%) but fell to about 10% after 10 trials and became zero for the last 40 trials (Fig. 5, curve of nonspontaneous runs). Similarly, the per- centage of runs showing tail-flexing movement was maximum in the first trials (30%), declined irregularly during the next 30 trials and settled down to 0-7% in TABLE I Occurrence of tail-flexing movements during different kinds of runs in four series of training experiments Training to polarized light Training to brightness Training to side Series A Series B With tail flexes With tail flexes With tail flexes With tail flexes D Tntal Tnt-al Total No. % No. % No. % No. % Correct 745 6 / 157 5 3 251 23 9 88 Incorrect* 394 82 21 156 66 42 271 86 32 33 5 16 Nonspon- 49 15 31 14 12 86 28 13 46 35 10 29 taneous Total 1188 103 9 327 83 26 550 122 22 156 15 10 * Incorrect runs = error runs minus nonspontaneous runs. 360 HERMANN SCHONE 100 60 60 40 U. o ?? 20 Errors U n - \ A/'J |/spon/on\ / VN H V P. a. M :\Tailflexing \ : \4 movement \. : \ \\! \A A o o \ * \AA \ 72 2/ JO 39 48 57 66 75 84 93 102 111 119 TRIALS 1 11 13 15 17 19 21 DAYS FIGURE 5. Mean learning curve for 10 lobsters training to brightness. Points represent percentages of the runs in three trials. 50 60 00 Q: U. 10 o; Errors ,'\ Tailflexing ! '. < movement 80 60 LO 5^0 ^ 20 J 72 27 JO TRIALS '"DAYS 1 Errors j> 9 /'. Tailflexing m. i > < i i < i / \ Nonspontaneous \> A A \ / \ / \ 90 ^ 12 21 30 39 48 55 TRIALS 7 11 DAYS FIGURE 6. Mean learning curves for lobsters where training was attempted to plane of polarization, (a) Series A, 11 previously untrained animals, (b) Series B, 10 previously trained animals. Points represent percentages of the runs in three trials. LEARNING IN THE SPINY LOBSTER 361 the last 30 trials (Fig. 5, curve of tail-flexing movement). The error curve and curve of tail-flexing movements at least show signs of flattening out, or even of a temporary upturn, starting with trial 61 when the regular right-left alternation of the positive side of the box was changed to a randomized sequence. These results demonstrate that Panulirus can discriminate the light intensities used in the two compartments and utilize their position as a conditioning stimulus in goal seeking. 2. Training to polarized liglit. In both Series A (inexperienced lobsters) and Series B (experienced lobsters) the error scores were no better than random even at the end of the 6- and 11 -day training periods (Fig. 6a, b). The inexperienced animals made a large number of errors (76%) in the first trials, but after 9 trials they made about 50^ (Fig. 6a, error curve). Rapid decreases in the proportion 80 60 u. o ^ 20 Errors Non- \ spon/on. 75 27 1 26 TRIALS ~9~ DAYS FIGURE 7. Mean learning curves for 6 lobsters training to side. Points indicate percentages of the runs in three trials. of nonspontaneous runs and runs with tail-flexing movements also occurred in the inexperienced group (Fig. 6a, curve of nonspontaneous and tail-flexing movement runs). In contrast, with the previously trained animals all three of these scores were about at the same level in the first as in the last trials (Fig. 6b). Although these two series showed that spontaneity and tail-flexing movements reached levels com- parable to those attained in the brightness training (Fig. 5 ), they give no indication that the two tested orientations of the r-vector of polarized light could be used as a conditioning stimulus to discriminate the spatial choice required. 3. Training to side. The general behavior of test animals in the above experi- ments suggested that they readily could learn the side with a reward since they often went toward that compartment which had been the positive one in the previous test. 362 HERMANN SCHONE This was confirmed by the results of experiments carried out to study this type of training. The error score was over 78% at first but decreased precipitously and quite steadily thereafter. It was 50% after 9 trials and about 10% after 26 trials at the end (Fig. 7, error curve). A similar course was run by the proportion of nonspontaneous runs which started at 50% and decreased after 15 trials to 11%. The fraction of runs with tail-flexing movements showed a general trend to decrease from about 20% initially to zero in the last trials. DISCUSSION Experience indicates that rate and extent of learning in various animals depend strongly on the particular problem tested and the sensory modalities involved in the afferent components of the process. Also the strength and type of drive and re- inforcement involved obviously will markedly affect learning (Hull, 1952). Never- theless, it seems clear in the present experimental situation that Panulirus did not learn even at best to make two-choice spatial discrimination either very quickly or very accurately. Mean error scores for a day never were substantially less than 20% and even these took more than 20 trials to achieve. Thus in the training-to- side experiment the error score dropped markedly below 20% only after 26 trials (Fig. 7) and in the training-to-brightness series an error score markedly below 20% first appeared after 108 trials (Fig. 5). The differences in learning shown in these two sets of training experiments may reflect the relative importance of different sensory modalities for the type of condi- tioning involved. The more rapid achievement of < 20% errors in choosing one side than in discriminating between the two circular areas of different brightness suggests that some tactile or kinesthetic cues are more important than visual ones in finding the open exit hole. Such an interpretation is supported by the cases cited above (Fig. 4) where persistent preference for one side was manifest in certain individual Panulirus and by previous work on the fiddler crab Uca (Schwartz and Safir, 1915) and the crayfish Pacifastacus (Gilhousen, 1927) which demonstrated turning predominantly in one direction or following of one wall. Such a pre- ponderant influence of mechanoreceptor afferent information for Panulirus is con- sonant with the animal's normal rocky surroundings in submarine holes and crevices, coupled with its essentially nocturnal habit. The lack of evidence for the lobsters learning to discriminate between vertically and horizontally polarized light sources is consonant with the conclusion that visual cues are not as significant as certain others in the present spatial choice problem. However, it does not seem to reflect the finding in the crab, Ocypode, that vertically polarized light has a significantly greater effect on eyestalk displacement than hori- zontal polarized light of the same photometric intensity (Schone and Schone, 1961). Previous attempts with other animals to demonstrate discrimination of different planes of polarization by training techniques have yielded both positive and negative results. In the pigeon, food-training discrimination tests gave no evidence for polarized light perception but indicated good light intensity discrimination (Mont- gomery and Heinemann, 1952), thus resembling some of the present results with Panulirus. On the other hand, in the honeybee a significant ability to discriminate vertically from horizontally polarized light was reported on the basis of food-seeking LEARNING IN THE SPINY LOBSTER 363 training (Stockhammer, 1956). * Reward and punishment training in Octopus has similarly demonstrated that animal's ability to discriminate vertical from horizontal polarized light as well as between oblique planes of polarization at 45 and 135 to the vertical (Moody and Parriss, 1960, 1961). However, the present failure to get discrimination learning to polarized light cannot be taken as evidence that the animal does not perceive the plane of polariza- tion, as a great variety of arthropods, including decapod crustaceans, must do (Stockhammer, 1959; von Frisch, Lindauer and Daumer, 1960; Jander and Water- man, 1960; Waterman, Jander and Daumer, unpublished; Schone, unpublished data). Negative evidence of the sort obtained here merely demonstrates an absence of training effect in the current experimental situation without decisive prejudice to the more general question. In the learning curves for the error scores of inexperienced lobsters (Figs. 5, 6a, and 7). the occurrence of initial levels significantly above the random choice value of 50% results directly from the way errors were counted. The inclusion of non- spontaneous runs as errors contributed substantially to the initial excess over what would be expected, since inexperienced animals frequently required prodding to initiate and maintain walking. On the other hand with experienced animals the initial error scores were near 50% (Fig. 6b) and the nonspontaneous runs were much less frequent during the first trials. This raises two points of interest. (1) Retention of training must occur for the period between experiments (3 days). (2) Learning, as evidenced by the reduction in nonspontaneous runs, took place even when the Panulirus were not learning to find the open exit. Such data suggest that the test situation consists of at least two stages : (1 ) learning to walk forward (probably related to the general factor of drive or appetitive behavior (Tinbergen, 1951), and (2) learning to dis- criminate the positive compartment. Another point of interest is the correlation of tail-flexing movements with in- experience and with progress in learning. The tail-flexing movements appeared to be characteristic of inexperienced individuals (Figure 2 and the first trials of the appropriate curves of Figures 5 and 7) and of those showing little tendency to learn (Fig. 6). In both of these cases tail-flexing is correlated with error runs. The detailed correlations of tail-flexing movements with correct, incorrect and non- spontaneous runs are shown in Table I which indicates that tail-flexing movements were most prominent in nonspontaneous runs, less so in incorrect runs, and at a minimum in positive runs where the open exit was reached spontaneously. The protocols show that tail-flexing movements were particularly associated with "indecisive" behavior. Thus, if the lobster (1) was inactive unless prodded with the stick, (2) chose the negative compartment and found the exit blocked, (3) made turns in more than one direction, stopped or walked backwards, then tail-flexing movements were likely to appear suddenly. In cases (1) or (2) above the tail-flexion response could be interpreted as an escape reaction from a threaten- ing situation as it often appears to be for lobsters in the sea when strong external stimulation occurs (such as the nearby appearance of a large fish) . However, when the same reaction appeared as a component of "indecisive" behavior patterns lacking * In these experiments no control is mentioned against the possibility that intensity artifacts due to differential reflection of the polarized light were involved in the training process. 364 HERMANN SCHONE any obvious external threat, the tail-flexing movement would seem to be a displace- ment activity in the ethological sense (Eibl-Eibesfeldt and Kramer, 1958; Schone, 1961). Thus, its diminution with experience could be considered as habituation to initially startling or conflicting situations. SUMMARY 1. Juvenile spiny lobsters (Panulirus argus) could be trained in air in a two- choice discrimination box to use two stimulating lights which differed in intensity by a factor of 3-5 times in learning to reach an open exit hole leading to an aquarium containing sea water. 2. Comparable training procedures yielded no better than chance selection of the two compartments (one with the open exit, the other with its exit blocked) when the conditioning lights were of equal intensity but were both linearly polarized, one horizontally, the other vertically. Demonstration that lobsters did not use polarization pattern in this spatial discrimination situation does not prove that the plane of polarization was not perceived by Panulirus. 3. Learning was more rapid than it was to intensity differences when the animals were trained always to go to one side of the discrimination box to reach the open exit. For intensity discrimination initial high percentage errors were reduced to less than 20% only on the twentieth day after 104 trials per animal, whereas for training to side less than 20% errors were achieved after 20 trials per animal. 4. Learning indicated by the curve of mean error scores per day resembles an exponential decay function and is accompanied by decreases in percentage of non- spontaneous runs and runs showing tail-flexing movements. Tail-flexing move- ments were correlated positively with error runs and occurred as a result of "indecisive" behavior. 5. Learning indicated by reduction in percentages of nonspontaneous runs showed retention of training between experiments and continued in cases where exit discrimination was not being learned. Hence over-all training involved : (a) learning to walk directly forward, and (b) learning to discriminate the side with the open exit. LITERATURE CITED BETHE, A., 1898. Das Centralnervensystem von Carcinus maenas. II. Teil. Arckiv. mikro- skop. Atiat. u. Entwickhtngsges., 51 : 382-452. BUSH, R. R., AND W. K. ESTES (Eds.), 1959. Studies in Mathematical Learning Theory. 432 pp., Stanford University Press, Stanford, California. DATTA, L-E., S. MILSTEIN AND M. E. BITTERMAN, 1960. Habit reversal in the crab. /. Comp. Physiol. Psycho!., 53: 275-278. EIBL-EIBESFELDT, I., AND S. KRAMER, 1958. Ethology, the comparative study of animal be- havior. Quart. Rev. Biol, 33: 181-211. VON FRISCH, K., M. LINDAUER AND K. DAUMER, 1960. Uber die Wahrnehmung polarisierten Lichtes durch das Bienenauge. E.rpericntia, 16: 289-301. GILHOXJSEN, H. C., 1927. The use of vision and of the antennae in the learning of crayfish. Univ. Calif. (Berkeley) Publs. Physiol., 7 : 73-89. HILGARD, E. R., 1956. Theories of Learning. 2nd Edition, 563 pp., Appleton Century Crofts Inc., New York. HULL, C. L., 1952. A Behavior System. 372 pp., Yale University Press, New Haven, Con- necticut. LEARNING IN THE SPINY LOBSTER 365 JANDER, R., AND T. H. WATERMAN, 1960. Sensory discrimination between polarized light and light intensity patterns by arthropods. /. Cell. Comfy. Physio!., 56: 137-159. LOCH HEAD, J. H., 1961. Locomotion. In: The Physiology of Crustacea (edited by T. H. Waterman). Vol. II, pp. 313-364, Academic Press, New York. MONTGOMERY, K. C, AND E. G. HEINEMANN, 1952. Concerning the ability of homing pigeons to discriminate patterns of polarized light. Science, 116: 454-456. MOODY, M. F., AND J. R. PARRISS, 1960. Discrimination of polarized light by Octopus. Nature, 186: 839-840. MOODY, M. F., AND J. R. PARRISS, 1961. The discrimination of polarized light by Octopus: a behavioural and morphological study. Zcitschr. verc/l. Physiol., 44 : 268-291. SCHONE, H., 1961. Complex behavior. In: The Physiology of Crustacea (edited by T. H. Waterman). Vol. II, pp. 465-520, Academic Press, New York. SCHONE, H., AND H. SCHONE, 1961. Eyestalk movements induced by polarized light in the ghost crab, Ocypode. Science (in press). SCHWARTZ, B., AND S. R. SAFIR, 1915. Habit formation in the fiddler crab. /. Animal Be- havior, 5 : 226-239. STOCKHAMMER, K., 1956. Zur Wahrnehmung der Schwingungsrichtung linear polarisierten Lichtes bei Insekten. Zcitschr. vcrgl. Physiol., 38: 30-83. STOCKHAMMER, K., 1959. Die Orientierung nach der Schwingungsrichtung linear polarisierten Lichtes und ihre sinnesphysiologischen Grundlagen. Ergeb. Biol., 21 : 23-56. THORPE, W. H., 1956. Learning and Instinct in Animals. 493 pp., Methuen, London. TINBERGEN, N., 1951. The Study of Instinct. 228 pp., Oxford University Press, London and New York. WARDEN, C. J., T. N. JENKINS AND L. H. WARNER, 1940. Comparative Psychology. Vol. II, 1070 pp., Ronald Press Company, New York. WIERSMA, C. A. G., 1961. Reflexes and the central nervous system. In: The Physiology of Crustacea (edited by T. H. Waterman). Vol. II, pp. 241-279. Academic Press, New York. ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY 1961 ABSTRACTS OF SEMINAR PAPERS JUNE 26, 1961 Varieties of ephaptic transmission in crayfish. HARRY GRUNDFEST AND AKIRA WATANABE. The septal membranes of the abutting segments of the lateral giant axons of the crayfish nerve cord form bidirectional ephaptic junctions. The properties of the junctional membranes are described accurately in terms of a simple (non-reactive) resistance which links the reactive, electrically excitable and spike generating membranes of the two segments. Thus, the septal membranes show no rectification, at least for a range of polarization of 25 mv., nor do they respond with electrogenesis to depolarization by 70 mv. The electrical inexcitability of these ephaptic membranes accounts for the bidirectional transmission of the junctions. In contrast, the membranes of the unidirectional ephaptic junctions between the cord and root giant axons have an electrically excitable component which is responsible for the high degree of rectification of these membranes. The available data are insufficient to indicate whether the conductance change is due to K-activation, as in frog slow muscle fibers (Belton and Grundfest, this issue) or to Cl-activation, as in Raia electroplaques. Bidirectional ephaptic junctions are also formed by the commissural connections between the branches of corresponding segments of the two lateral giant axons. Since these connections form a ladder of cross-linkages at the different segmental levels, circus propagation of impulses from one axon to the other is possible. These ephaptic pathways can give rise to reverberating activity of both lateral giant axons at rates as high as 400 impulses per second. This activity presumably can initiate corresponding activity in other neurons of the nerve cord. JULY 3, 1961 The advance of electrical models for cells and axons. KENNETH S. COLE. The electrical models of cells and axons have advanced rather steadily from the model for the red blood cell given by Fricke in 1925, which demonstrated the molecular dimensions of living membranes, to the detailed model for the squid giant axon given by Hodgkin and Huxley in 1952 in which much of classical electrophysiology is explained in terms of membrane ionic conductances obtained from voltage clamp experiments. JULY 10, 1961 The components of the resting potential in crayfish and lobster muscle fibers. LUCIEN GlRARDIER, JOHN P. REUBEN AND HARRY GRUNDFEST. In short range experiments involving changes of external potassium, lobster muscle fibers depolarize with a slope of 58 mv./decade increase of K, according to the thermodynamic (Nernst) relation. Under similar experimental conditions, crayfish muscle fibers do not manifest the theoretical E m log K relation. The latter is attained, however, when thermo- dynamic conditions are established, e.g. by keeping the product K X Cl constant. Furthermore, 366 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 367 the crayfish fibers exhibit a long-lasting electrochemical disequilibrium after modification of the bathing solution. Thus, remarkably slow transient potential changes are induced by altering the Cl-content of the medium. These properties are consonant with the high membrane re- sistance of crayfish muscle fibers and with their high volume-to-surface ratio, and the prepara- tion is thus particularly suitable for the study of the pharmacological effects on the ionic fluxes which can be deduced from the kinetics of the membrane potential. The synaptic agents, GABA and picrotoxin, which respectively initiate and block activity of the inhibitory synaptic membrane in these fibers, respectively increase and decrease the membrane resistance and the net Cl-flux in the resting membrane. Although the inhibitory synapses of the fibers operate through in- creased Cl-conductance, the effects of the drugs on net Cl-flux are due to complementary actions on the non-synaptic membrane component. They are produced only by high concentrations of the agents, well after the maximal effects on the synaptic membrane had been attained. Barium, which converts the normally graded responses of the fibers to spikes and also increases the membrane resistance, decreases the transport number for K. Procaine, which also converts the graded responses to spikes, but has little or no effect on the resting resistance, exerts no clear- cut action on any one ion flux. In lobster muscle fibers, by comparison, the synaptic drugs and Ba have similar actions on the membrane resistance. However, only Ba modifies the E m log K relation of lobster muscle fibers, as may be expected, since the relation in these muscle fibers is determined primarily by the K. JULY 11, 1961 Temperature adaptation in Drosophila melanogaster pupae. ROGER MILKMAN. Day-old (at 23 C.) D. melanogaster pupae respond to treatment at 39.5-41.5 by showing quantitative defects in the posterior crossvein. Longer treatments lead to death. Both re- sponses have a Q\ of 2.3, and both depend upon the temperature at which the first day of pupal life is spent. The higher the temperature, the greater the resistance, both to crossvein defect production and to death. Temperatures at 1 intervals between 20 and 30 have been tested. The rate of increase in resistance per degree rises greatly with higher temperatures in the range. The temperature coefficient measurements, previously published in detail, imply that these crossvein defects result from thermal protein denaturation. Moreover, duration-response curves for crossvein defects at 40.5, using pupae aged at 23 and at 28, are now shown to be parallel, the duration threshold being greater for the 28 pupae. This implies similar protein denaturation rates, and that the 28 pupae had more of the relevant protein to begin with. Since resistance to morphological effects and death is similarly temperature-dependent at various pupal stages in D. melanogaster and other Drosophila species, it is suggested that one mechanism of temperature adaptation may be the change in concentration of certain proteins, or perhaps proteins in general. Short prior exposures (5-60 minutes) to 32-38 obliterate the crossvein response and defer death. Production of crossvein defects and both forms of temperature adaptation have been shown in D. virilis, D. zvillistoni, D. pseiidoobscura, and D. busckii. This work supported by grants from NSF and NIH. Endoplasmic reticulum in the asters of invertebrate eggs. LIONEL I. REBHUN. Electron microscopic evidence was presented showing an orientation of the endoplasmic reticulum in the asters of the egg of Spisula solidissima. Such an orientation can be seen during formation of asters of the first polar body spindle after germinal vesicle breakdown and during aster formation, just prior to first cleavage. In eggs which have been centrifugally stratified during periods in which asters are present, the endoplasmic reticulum can be seen as a layer 4 to 5 microns wide, tightly pressed beneath the lipid layer. This corresponds to a layer distinguishable in the stratified egg with the light microscope and in the same sublipid position. During redistribution after stratification, asters reappear usually in the form of "one-sided" radiate bodies at first, and later, as full asters. In the electron microscope the endoplasmic reticulum shows a similar one-sided radial orientation, later changing to a 368 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY fully radiate body, again suggesting that an important component of the aster is the endoplasmic reticulum. The orientation of centrioles in dividing cells and its significance. DONALD P. COSTELLO. The centrioles of the resting first cleavage metaphase of the eggs of the acoel, Polychoerus carmelensis, are curved rods about 5 microns long, oriented at right angles to each other and to the spindle axis (Costello, 1961). By light microscopy a smaller rod, projecting at right angles from the middle of each centriole, but at an oblique angle to the spindle axis, can be discerned. At the end of this accessory rod is a small, clear vacuole, containing a stainable (with iron haematoxylin) granule. Extending into the centrosomal area, away from this vacuole, are other similar vacuoles, each with a granule. As Heidenhain (1894) pointed out, and Schreiner and Schreiner (1905) demonstrated, centrioles in many species reproduce by budding, with the daughter centriole produced at right angles to the mother centriole. The small rods are therefore centriolar buds (daughter centrioles) which will grow and separate, presumably at the angle indicated by their mode of origin, from the mother centriole. Comparing this centriolar orientation and behavior (including daughter centriole origin) in Polychoerus with that in the spermatocyte divisions of Gryllidae (Johnson, 1931) and Hemiptera (Payne, 1927) in relation to the arrangement of daughter cells, leads to a new hypothesis, which may account, in part, for cleavage patterns : (a) The orientation of the centrioles and their mode of reproduction determines the path of separation of daughter centrioles from mother centrioles (or of daughter centrioles from each other). (b) This, in turn, determines the position of the main axis of the spindle for the next division, and hence, the relative position of the daughter cells with respect to each other. (c) This arrangement of daughter cells is maintained, for a time, by the primary cell connective, of which the spindle remnant is the significant portion. (d) These relations obtain in the absence of secondary intervening factors. Aided by a grant from the National Institutes of Health, RG-5328. JULY 18, 1961 The nature of lamprey eel fibrinopeptide material. RUSSELL F. DOOLITTLE. When various components of the mammalian blood coagulation scheme are reacted with components from lower vertebrates, the ensuing clotting times are invariably longer than when all the components come from the same or closely related animals. An investigation of the chemical nature of this "species" specificity was undertaken, using purified preparations of lamprey eel thrombin and fibrinogen and their mammalian counterparts. Human thrombin will completely clot lamprey eel fibrinogen, but the reaction proceeds at only 50% of the rate of human thrombin clotting bovine fibrinogen. Lamprey eel thrombin will clot bovine fibrinogen at less than 10% the rate at which it clots lamprey fibrinogen. The clotting of mammalian fibrinogen by mammalian thrombin is effected by the enzymatic release of two different negatively charged peptides. It has been postulated that the negative charge clusters keep the fibrinogen molecules mutually repelled and that their loss permits aggregation of the monomeric units. There has been some dispute as to whether or not it is necessary to cleave both peptides in order for the fibrinogen to completely clot. When lamprey eel thrombin clots bovine fibrinogen, only peptide A is released, in spite of the fact that over 90% of the original protein is incorporated into the clot. Human thrombin and lamprey thrombin yielded identical products when exposed to lamprey fibrinogen. One electro- negative peptide and one peptide bearing a positive charge at neutral pH were detected. The amino acid composition of the two lamprey peptides was determined and compared with bovine fibrinopeptides. The nature of restricted and permissive mutations in the evolution of the thrombin-fibrinogen interaction was discussed. The details and methods of this work will be published elsewhere. PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 369 Equal and unequal effects of unit dosages of ultraviolet and x-ray irradiation of Arbacia gametes as recorded b\< cine photomicrography. RALPH HOLT CHENEY AND CARL CASKEY SPEIDEL. Eggs and sperm were subjected separately to either 2537 A ultraviolet or x-ray irradiation. UV-rayed gametes were exposed for periods ranging from one-half second to 4 minutes, the rate of delivery being 103 ergs per sq. mm. per second. Gametes exposed to x-rays received from 4 to 120 kr. Fertilization combinations were then made and ensuing developmental stages recorded by cinephotomicrography as follows: (1) elevation of fertilization membrane, (2) first cleavage, (3) 5-hour cleavage, (4) initiation of blastular motion at about 8 hours, (5) 24-hour development. Motion picture scenes of stages 3, 4, and 5 were especially useful in demonstrating that there was a striking difference in the relative effects induced by UV treatments as compared with x-ray treatments. A unit UV dose to eggs caused jar less developmental delay and injury than an equal UV dose to sperm. Even a 4- unit dose to eggs caused less damage than a 1-unit dose to sperm. An 8-unit dose to eggs, however, induced delay and damage approximately equivalent to a 1-unit dose to sperm. With x-ray irradiation, on the other hand, although a unit dose to eggs was followed usually by less damage than an equal dose to sperm, the difference was much less marked. A 2-unit dose to eggs, or even less, was sufficient to induce delay and damage equivalent to a 1-unit dose to sperm. Thus, there '^vas a sharp contrast between the ttvo types of irradiation. A ratio of 8 : 1 describes the relation between the dosages of UV irradiation to eggs and sperm, respectively, to induce equivalent effects ; a ratio of not more than 2 : 1 describes this relationship for x-ray. Time-lapse motion pictures of stages 1 and 2 illustrate varieties of fertilization membrane elevation (concentric, eccentric, and partial), also movements of intracellular structures. Supported by grant RG-4326(C4) to C.C.S. from the U.S.P.H.S. and by grant 144 to R.H.C. from the National Academy of Sciences. JULY 25, 1961 Fine structure of an unusual sperm in the flat-worm Plagiostomum. A. KENT CHRISTENSEN. (See Lalor Fellowship Reports}. Observations on the Golgi apparatus and related lysosomes. ALEX B. NOVIKOFF. Nucleosidediphosphatase has been localized in Golgi lamellae and a staining method developed that permits delineation of the Golgi apparatus from sites of high acid phosphatase activity. In epididymis and many other cells the acid phosphatase-rich lysosomes show a close topographical relation to the Golgi lamellae. Essner's observations in our laboratory on dif- ferentiated hepatoma cells suggest that there may be a "membrane flow" to and from the Golgi apparatus: elements of the endoplasmic reticulum (ER) transform into Golgi cisternae (in one case, this apparently involves flattening of individual smooth-surfaced sacs derived from the rough ER ; in the other, Golgi cisternae appear to arise in situ by transformation and reorientation of the smooth ER) ; the cisternae enlarge and separate into vesicles (lysosomes) containing condensed secretory products. Light microscopy and electron microscopy show that the ER (and nuclear membrane) of these cells, and another interesting group, possess nucleo- sidediphosphatase activity, and that the acid phosphatase reaction product is localized in the lysosomes of these tumor cells. The lysosomal hydrolases may be involved in the biochemistry of secretory granule condensation. How the enlarged Golgi vesicles acquire hydrolytic enzymes is unkown, but there is some suggestion that this may occur via small lysosomes (pinocytosis vacuoles?). In the zymogenic cells of parotid, pancreas and jejunum, acid phosphatase-rich granules are restricted to the Golgi area, and thus only the newly formed secretory granules appear to be lysosomes. Of many cells tested, only the spermatogonia and their derivatives always show high acid phosphatase activity in the Golgi apparatus. Acid phosphatase is present in the acrosome (frog sperm) which may be considered a specialized lysosome with a decisive role in fertilization. 370 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY Functional analysis of the corpus allatum of the insect, Leucophaea maderae, with the electron microscope. BERTA SCHARRER. In adult females of Leucophaea, periodic changes in corpus allatum activity are paralleled by structural variations whose details become particularly evident in electron micrographs. In the inactive gland, which due to inhibitory action of the brain does not furnish gonadotropin, the nuclei are densely packed and surrounded by scarce cytoplasm ; their chromatin appears clumped. Neighboring cells interdigitate by elaborate foldings of the cell boundaries. Further- more, the cells interlock by means of long, gradually thinning processes. Following cessation of inhibition, intermediate stages lead to the fully activated corpus allatum and the concomitant maturation of eggs. A considerable increase in cytoplasmic content of the corpus allatum cells is accompanied by gradual straightening of their cell membranes. The number of mito- chondria rises, and endoplasmic reticulum appears in the form of juxtanuclear spherical units which may be paired. In addition, small clusters of unattached ribosomes become more numer- ous, while Golgi elements remain inconspicuous. Electron-dense cytoplasmic granules are especially noticeable at the surface of the gland. With increasing nuclear diameter the chromatin becomes more evenly distributed. One, or not rarely two large nucleoli show a characteristic morphology. The folding and straightening of the cell membrane, as its content shrinks and expands with each reproductive cycle, demonstrates its structural identity. The stellate shape of the corpus allatum cells, which is observed also in fresh tissue dissociated by means of trypsin, seems to be maintained throughout the changes of volume. Branches of the basement membrane which ensheaths the corpus allatum and the corpus cardiacum penetrate between the corpus allatum cells. Some of these processes enclose neurosecretory elements. This morphological intimacy between the two organs reflects their close functional relationship. JULY 31, 1961 Biological transducer mechanism. WERNER R. LOEWENSTEIN. The data available presently on charge transfer through the receptor membrane of the nerve ending of Pacinian corpuscles can be fitted to the following excitation scheme : the receptor membrane separates two media of different ionic concentration. Mechanical stimulation causes the conductance of the membrane to increase in a relatively non-selective manner, and ions to flow through along their electro-chemical gradients. An activation energy barrier of about 16,000 cal./mole limits this process. The membrane reaction that leads to the conductance change is highly localized. It is localized at and confined to the membrane spot that has been stimulated mechanically, and does not spread to adjacent spots within the same receptor membrane by local circuit excitation. This stands in striking contrast to the well-known propagated nature of excitation of the adjacent axon membrane that gives rise to the action potential. The peculiarity of the receptor membrane is that excitation is produced by the mechanical stimulus alone, but not by the resulting local flow of current. As a result, the amount of charge transferred through the membrane, namely the generator current, increases with area of membrane excited. The area-generator current relationship is non-linear ; the current increases progressively at a diminishing rate with excited area. The area of membrane excited (that is, the lumped mem- brane conductance) is the main factor, if not the only one, in determining the intensity of the generator current. Repeated excitation leads to a transient inactivation of the receptor membrane. The effect manifests itself as a reversible depression in generator current which has low energy require- ments and is independent of resting membrane potential. Like excitation, inactivation is a localized process. AUGUST 7, 1961 Current voltage relations in Nitella. UICHIRO KISHIMOTO. The measuring cell and the voltage clamp setup (Cole and Moore, 1960) for squid giant axon was used for the Nitella experiment, because the size and shape of Nitella internode are just PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 371 about same as those of squid axon. Two microtips were inserted into vacuole of Nitella internode instead of the axial wire-microtip system used in the squid giant axon, since pene- tration of the 50- to lOO-^i axial wire caused a sudden rapid loss of vacuolar sap due to high osmotic concentration gradient between inside and outside of Nitella. One microtip is used for monitoring the potential between vacuole and the reference electrode just outside of Nitella membrane. Another is used for current supplying through the membrane. Space clamp condition is achieved with this arrangement, since the output of the feedback amplifier appears to be a perfect square wave, which is probably due to a large space constant of Nitella (2-3 cm.). The membrane current during step potential change is very similar to that of squid giant axon. The membrane current consists of three components. The first one is the sudden rise or fall of current which corresponds to leakage current, the second is the transient inward current and the third is the steady outward current which increases slowly with time and finally approaches to a constant level. Ions which contribute to the leakage current are not identified as yet. However, there is a good reason to suppose that the second one corresponds to the marked increase of chloride efflux and the third one to that of potassium efflux. Increase of outside chloride concentration to 10 mM causes a reduction of the transient inward current to about half. Increase of outside calcium concentration to 10 mM, maintaining the outside chloride concentration constant, brings about a longer duration for action potential without changing the height of the latter. The transient inward current is not influenced by high calcium. Threshold for excitation was increased and the leakage current decreased markedly. Decreasing the temperature to 7 C. promptly increased the leakage of the Nitella membrane and the threshold for excitation was increased greatly. The recovery from the effect of lowered temperature takes about an hour. Application of prepulse (subthreshold intensity) of 1-3 seconds in duration has no influence on the inward and outward currents, but the leakage current tends to decrease for hyperpolarization prepulse and to increase for depolarization prepulse. AUGUST 8, 1961 The physiological basis of acclimation of Tetrahymena to high NaCl medium. PHILIP B. DUNHAM. It has been known for some time that fresh-water ciliates can acclimate to concentrated media. However, little was known of the physiological basis of acclimation. Tetrahymena pyrifonnis strain W was acclimated to a !%NaCl-2% proteose-peptone medium, and compared to a normal (unacclimated) culture of the same animals. An investigation of ion regulation in normal Tetrahymena has been reported in this journal, in which it was shown that these animals have a sodium extrusion mechanism with a saturation level at 20 mM. medium Na and an apparent free sodium space in the cells of 48%. The acclimated culture was started with- out intermediate steps. Only 5% of the normal animals survive to divide in the high NaCl medium. The acclimated animals have a much lower sodium content (39 mM./l. of cells) than do normal animals equilibrated in high NaCl medium (103 mM./l. of cells). The potas- sium and chloride contents of the two cultures of animals are not significantly different. A comparison of the relationships between cell sodium and medium sodium in acclimated and normal animals suggested two kinds of mechanisms operative in the acclimation: (1) a decrease in apparent free sodium space in the cells from 48% to 20% ; (2) an increased saturation level of the sodium extrusion mechanism from 20 mM. Na to 45 mM. The acclimated animals have a slower growth rate, are smaller, and are differently shaped. Excretion rate of radio-isotopes as indices of metabolic rates in nature: biological half-life of zinc-65 in relation to temperature, food consumption, grozvth and reproduction in arthropods. EUGENE P. ODUM. Laboratory experiments and a preliminary field experiment with two species of terrestrial insects (Tenebrio molitor and Oncopcltus fasciatns) and a marine isopod (Idothca baltica) indicate that the excretion rate of tracer amounts of Zn 65 is proportional to certain activity rates, especially food consumption and egg production rates. Individuals were allowed to 372 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY ingest contaminated food or water in a "tagging chamber," then transferred to a clean environ- ment where the loss of radioactivity was followed by making periodic whole body counts in a well scintillation detector. Semi-log plots of radioactivity against time strongly indicated two pools of isotope : ( 1 ) a non-assimilated pool which is lost rapidly at a rate dependent on the level of the original "tag" and (in aquatic environments) on the physio-chemical nature of the environment, and (2) an assimilated pool which is distributed throughout the tissues of the body with some concentration in digestive organs and gonads and which is excreted much more slowly at a rate more directly related to metabolic processes. Biological half-life (Tbi/ 2 ) of the assimilated pool varied from as long as 250 days in inactive adults kept at low temperatures to 20 days in active egg-laying Oncopeltus females or rapidly growing Tenebrio larvae. Young adult Tenebrio kept for 30 days at 10, 20, and 30 C. averaged 163. 40, 23 days Tbi/r;, respectively, which is roughly inversely proportional to expected metabolic rate at these temperatures. Tbi/ 2 of 5 adult Oncopeltus which had been confined to small bottles in the laboratory decreased 5-fold (125-25 days) when released for 10 days in an outdoor habitat, indicating much greater activity in the field. It is concluded that the rate of loss from the assimilated pool provides a "metabolic clock" for comparing laboratory and field rates of energy flow in arthropod populations. Supported by AEC contract AT (07-2) -10 and NIH 2G-535 (MBL Ecology Training Program). AUGUST 22, 1961 In vitro culture of human tumors on ex plants of chick embryo organs. EMILIENNE WOLFF AND ETIENNE WOLFF. Previous researches of our laboratory have shown that it is possible to culture malignant cells from mammals on chick embryonic organs explanted in vitro. Mesonephros is one of the most favorable organs for tumor proliferation. Up to now, cells originated from fresh mouse tumors and from standard human strains have been cultured by this method. Recent improvements of our technique made it possible to obtain new developments of this investigation. 1. A thin membrane, the vitelline membrane of the hen's egg yolk, is placed between mesonephric explants and cancer cells, originated from the human strains HeLa, KB, Fogh, J III, Detroit 6 and Detroit 116. Malignant cells multiply intensely at the expense of substances which pass through this dialysing membrane. They form aggregates, the histological structure of which is similar to the primary tumor, from which they were originated. The same results were obtained with crude extracts of mesonephros or with "conditioned" media, i.e. media in which mesonephros was cultured during several days and then carefully removed. 2. Pieces of fresh human surgical tumors were wrapped up in a vitelline membrane together with explants of chick mesonephros. In many cases, such as epithelial carcinomas of stomach and uterus, an abundant proliferation of the cancerous cells takes place at the expense of the mesonephros which becomes more or less completely invaded. The growing tumorous masses retain the same histological structures as in the in situ tumors. The question arises whether the substances provided by the mesonephros are banal nutrients or specific growth promoting substances. GENERAL SCIENTIFIC MEETINGS AUGUST 28-31, 1961 Abstracts in this section (including those of Lalor Fellowship reports) are arranged alphabetically by authors under the headings "Papers Read," "Papers Read by Title," and "Lalor Fellowship Reports." Author and subject references will also be found in the regular volume index. PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 373 PAPERS READ Molybdenum uptake of marine plankton algae. ROBERT J. BARSDATE AND ROBERT R. L. GUILLARD. Molybdenum uptake by three species of marine planktonic algae was investigated using Mo-99 as a tracer. Coccolithns lut.vlcyi (BT-6) and Cyclotclla nana (clone 13-1) were iso- lated from the Sargasso Sea; Synechococcus sp. and Cyclotella nana (clone 3-H) are estuarine forms. All were grown in pure culture at 22 C. and 2000 lux in enriched sea water containing both nitrate and ammonia, and buffered to pH 8 with "Tris." The total molybdenum content of the medium was varied from 15 to 40 /*g./l. (1.5 to 4 times the Mo concentration of Woods Hole Harbor water). C. nana was grown in Mo-99-tagged media for about 7 cell divisions. Cell counts and molybdenum assay were then made, from which a concentration factor of ca. 6.5 was estab- lished. Labelled algae in darkness maintained their molybdenum concentration without change for 24 hours. There was no significant difference between the oceanic and estuarine clones. When C. nana cells were exposed in darkness to an increase in molybdenum concentration of the medium, they equilibrated completely (to a concentration factor of 6.5) in four hours, after which there was no change. However, in illuminated flasks the addition of molybdenum resulted in an initial uptake exceeding that corresponding to a concentration factor of 6.5. After 12 hours the concentration factor was again 6.5. The molybdenum uptake by chloroform- killed C. nana cells was 70% that of living organisms (at 40 Mg./l. of molybdenum). The coccolithophoride, C. huxleyl, with a cell volume 2.5 times that of the diatoms, had a concentration factor of 4.8. Synechococcus sp., with a cell volume ca. 10~ 3 that of the diatoms described, had a concentration factor of about 70. This study constituted one of of the staff-student research projects of the Marine Ecology Course (NIH Training Grant 2G-535) and was supported in part by a NSF grant. Formation and possible function of the "secretory packets" of the starfish tube foot. ALFRED B. CHAET AND DELBERT E. PHILPOTT. Further studies were carried out on the secretory structures found near the walking surface of the tube foot of Astcrias jorbcsi. The "secretory packets" were seen with the electron microscope to be formed intracellularly in an area proximal to the bottom surface of the tube foot where the "packets" were released to the outside. In most cases the cytoplasm of the cell consisted mainly of endoplasmic reticulum, a Golgi apparatus, and associated mitochondria. Within these cells, several "secretory packets" generally were being formed. It appeared that a clear area first formed in the cytoplasm, which then contained a "secretory packet." This newly formed elliptical "packet" had darkly staining (osmophilic) ends between which were attached 50 or so fibers (200 A diameter). During formation the osmophilic ends of the "packet" were attached to the rest of the cell by cytoplasmic strands, thus holding the "packet" in the center of the small clear area. Attempts to follow the individual paths of the "secretory packets" from their intracellular origin to the secretory surface at the base of the tube foot have been unsuccessful, due to the tortuous nature of the long narrow channels. In an effort to further the hypothesis that these "packets" functioned as a "glueing" mechanism, sections of the tube foot were stained by the P.A.S. technique. Observation with the phase contrast microscope demonstrated the "secretory packet" to be P.A.S.-positive, further suggesting their function as an adhering mechanism. These structures were not observable in the light or phase microscope until stained by the P.A.S. method. Supported by grants from the National Science Foundation (G-8718) and the National Institutes of Health (A-3362). Blood coagulation and fibrinolysis in the smooth dogfish. RUSSELL F. DOOLITTLE. The hemorrhagic nature of elasmobranch blood can be remedied in vitro by the presence of unphysiologically large concentrations of calcium ions. In a milieu containing 50-75 mM.fL. 374 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY calcium, dogfish blood plasma generates thrombin activity equivalent to mammalian systems maintained at physiological calcium levels. Under such conditions, other phases of the event become greatly exaggerated. Fibrinolysis occurs at a rate proportional to thrombin generation, and complete clot dissolution can be effected within 60 minutes of clot formation. Concomitantly, an intense serum inhibition develops which will prevent clotting when added to similar dog- fish plasma systems. The inhibition is thermo-labile, partially dialyzable, and not reversed by toluidine blue. The site of inhibition is the thrombin-fibrinogen interaction or the subsequent polymerization of fibrin monomer units. If serum is separated from the fibrin clot before fibrinolysis occurs (e.g., 30 minutes after clot formation), the inhibition does not develop, indicating that the fibrinolytic breakdown products are instrumental in the serum inhibition. Thrombin activity in such serum is neutralized as usual, however, suggesting two separate control devices. Comparisons of the ability of the generated thrombin to clot dogfish fibrinogen with its power to split the artificial substrates, tosyl-L-arginine methyl ester (TAMe) and L-lysine ethyl ester (LEE), showed that fibrinogen clotting power is neutralized much more rapidly than is the esterase activity. Other studies with TAMe and LEE suggest other esteratic or proteolytic events before and after clot formation which are distinct from the ability to clot fibrinogen. These include an initial sharp peak for TAMe esterase activity before the generation of thrombin and a slight increase in LEE esterase activity after clot formation. This study was aided in part by a grant from the ONR Research Contract No. 1497. In vivo and in vitro recovery of irradiated gametes of Arbacia punctulata. PATRICIA MCCLEMENT FAILLA. It is known that exposure to ionizing radiation of either sperm or eggs of Arbacia punctulata delays the time of first cleavage of the fertilized egg. The magnitude of cleavage delay decreases exponentially with the time the irradiated eggs remain in sea water before fertilization ("in vitro recovery"). No such recovery occurs in irradiated sperm. Use of the electrical stimulation method to procure gametes has permitted investigation of the recovery process in gametes remaining inside irradiated urchins for periods of time up to two weeks ("in vivo recovery"). It was found that some recovery occurs in these eggs but at a rate about 100 times less than in the in vitro case. After about eight days no further recovery is detectable, but a slight cleavage delay persists. As in the in vitro case, no significant recovery was found for sperm in vivo. Also the ability of irradiated eggs to recover in vitro is no longer present when the eggs have remained in the animal more than four hours after irradiation. It was thought that the slower recovery rate in vivo might be due to a lower metabolic activity of the eggs while in the ovary. The in vitro recovery process was followed, therefore, with the metabolic activity of the eggs reduced by keeping them in sea water in a nitrogen atmosphere for an hour after irradiation. Preliminary tests indicate that this recovery process is not affected by marked decreases in the oxygen tension. On the other hand, irradiation of the eggs in a nitrogen atmosphere caused a considerable reduction in the cleavage delay. The rate of subsequent in vitro recovery in air was reduced as well. Absorption, accumulation and loss of radioactive scandium by marine macroalgae. JOHN GUTKNECHT. Uniform discs of freshly collected Ulva lactnca and Porphyra umbilicalis were exposed to 20 uc./L. concentrations of high specific activity Sc 46 in initially sterile and strongly buffered pH 8.0 sea water solutions in the light and the dark. Radioactivity of the algae was meas- ured directly in a well scintillation counter. Uptake-time curves were characterized by a roughly hyperbolic rapid absorption phase, followed by a more linear accumulation phase. In 48 hours the light Ulva absorbed 35% more Sc 46 per unit weight than the dark, while the light Porphyra took up 15% more than the dark. High wet weight concentration factors were observed for both species, 2600 and 1500 times the external concentration in Ulva and Porphyra, respectively. Adsorption accounted for 40-60% of the total uptake as estimated by the intercept of the line of regression of the linear PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 375 part of the time curve. Thalli transferred after 48 hours from light to dark radioactive media showed a definite retardation of the Sc 46 uptake rate. Algae killed with ethylamine absorbed more scandium than live material. Loss of Sc* 6 was measured in buffered sea water in the light and the dark. Both species displayed a slow linear loss of scandium, less than \% per hour. The light and dark algae showed no difference in loss rate. Research supported by NIH Grant 2G-535 (MBL Ecology Training Program). Properties of a neu'lv isolated bacteriophage of luminescent bacteria. J. W. HASTINGS, A. KEYNAN AND K. McCLOSKEY. Phage wa-s isolated from a squid incubated with inoculations of marine mud and four strains of luminous bacteria. Phage containing samples were obtained by filtration and by treating with chloroform. Typical bacteriophage plaques were produced when tested with either A. fischeri or strain 60L (isolated from the Pacific Ocean by C. B. Van Niel) as host. The phage showed clear host specificity ; with five locally isolated bacteria strains and one from Jamaica no plaques were observed. However, various "dark" mutants of the sensitive strain were susceptible to infection. Plaques developed well in soft agar at temperatures between 20 and 30 C. The plaques always showed a turbid center, suggesting that the phage is a temperate one. In older plaques the phage-resistant bacteria completely overgrew the cleared areas, to an extent such that it was difficult to visualize the plaques. Observed in the dark, however, the formerly phage- cleared areas could be seen as non-luminescent spots on the background of the luminescent host strain. From this it was concluded that the phage-resistant cells growing in the center of the plaques were non-luminescent. "Dark" bacteria isolated from the center of the plaques exhibited the following properties which remained stable through several single colony iso- lations: (1) Ten of the fifteen isolates emitted light not visible to the eye but photometrically detectable; the others were intermediate in brightness. (2) All isolates were resistant to phage infection. (3) After being re-isolated four times from single colonies, all produced phage. These observations support the view that these are lysogenic bacteria produced by phage infection, and that lysogenicity is in some way inversely related to light production. The isolation and characterization of dark mutants of luminescent bacteria. A. KEYNAN AND J. W. HASTINGS. Numerous authors (Beijerinck, Van Niel, McElroy) have noted that dark mutants of luminescent bacteria accumulate in cultures. Biochemical studies of different dark mutants could yield information concerning the genetic and physiological control of bioluminescence. The present study is concerned with conditions under which dark mutants are found, their frequency and selection, and their physiological properties. Three strains were used in this work; (1) Achromobactcr fischeri, (2) a strain isolated by Van Niel (L-60), and (3) a strain with a low temperature optimum isolated locally (W-14). From cultures started from a single bright colony, maintained on solid media, no dark colonies (out of several thousand) were seen ; from similar cultures maintained in either minimal or complete liquid culture for extended periods (three days to three weeks), many dark colonies were observed upon plating out. From cultures maintained at high temperatures (up to 37) a high proportion (30%) of dark colonies were found. Fifty-one dark mutants have been isolated and studied. Although several isolates may be derived from the same parental mutant, many are clearly different in origin and in physiological characteristics. All of the mutants which are dark to the eye emit some small amount of light which may be detected photometrically ; the different mutants vary enormously with respect to their ability to emit, the intensity of the dimmest mutant being 10~ 5 of the wild type. The quantity of dark cells found in cultures suggested that the conditions gave selective advantage to dark mutants. This possibility was tested and sup- ported in experiments where different proportions of wild type and dark mutant were mixed. It will be of interest to see if conditions where the wild type has a selective advantage can be found. This might be an experimental approach for studying the possible competitive advantage of bioluminescence. 376 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY Recent observations on Botryllus schlosseri. ROGER MILKMAN AND SYLVIA BYRNE. We have confirmed Bancroft's observation of tremendous morphological diversity in the offspring of individual colonies of Botryllus schlosseri and his finding that the more closely related two colonies are, the more likely they are to fuse vascular systems when contiguous. Oka and Watanabe's demonstration of vascular budding in B. primigcnus has been extended to B. schlosseri. Sabbadin's demonstration of selfing has been repeated. Outcrossing, shown experimentally by Sabbadin, must be common in nature, too, for the great genetic heterogeneity of individual colonies would not otherwise be present. Fertiliza- tion by sperms arriving over a period of time probably accounts for the continuous variation in developmental stages we find in individual colonies of this viviparous organism, in contrast to the well-known synchrony of budding. Embryos may be raised outside the colony from before gastrulation. Attempts at external fertilization have failed. Numerous traits, stable in asexual reproduction, have been characterized. Most of these depend upon the nature, quantity, and distribution of pigments ; they are components of the colonies' over-all color patterns, of which there must be thousands. Inbreeding should permit development of homozygous strains to extend the recent observations of others on the genetic control of colony fusion and of pigmentation. B. schlosseri is notable for the unusual degree to which its genetic diversity is visibly obvious. This work supported by a grant from NIH (Milkman) and an NSF Cooperative Summer Fellowship (Byrne). Cortical granule extrusion during maturation of the egg of Astcrias forbesi. ARTHUR K. PARPART. The cortical granules of immature and mature eggs of Astcrias forbesi were observed by television microscopy under conditions where individual cells were exposed to 1 M solutions of glycerol, erythritol, d-xylose, glucose or sucrose. In no case did these egg cells alter in volume, therefore these compounds do not enter the egg in the time period of observation, one hour. The cortical granules of these eggs, however, reacted quite differently, dependent on the stage of maturation. The cortical granules of immature eggs showed no change, when exposed to the above solutions. The mature eggs exhibited a definite and violent explosion of their cortical granules in 1 M glycerol, erythritol or d-xylose ; while no such explosion occurred in either 1 M glucose or sucrose. Prior to first polar body formation no cortical granules exploded under these conditions. Within a half hour after polar body formation all of the mature eggs had cortical granules which exploded, and showed membrane elevation. These experimental observations suggest that the cortical granules of immature eggs are in the egg cytoplasm and that during the maturation process these cortical granules are extruded through the egg's plasma membrane and come to lie between it and the vitelline membrane. This is indicated by the fact that the polyhydric alcohols and sugars used cannot penetrate the plasma membrane. On the other hand a penetrating alcohol, ethylene glycol, caused the explo- sion of the cortical granules in both immature and mature eggs, and subsequently cytolysis. The fact that the above 1 M solutions caused an explosion of the cortical granules indicates that the granule's membrane must be permeable to the polyhydric alcohols and d-xylose but not to glucose and sucrose. The a, ft-unsaturated ether (plasnialogen) content of the tissues of several mollusks. MAURICE M. RAPPORT. It was recently shown that the lipids of a number of marine invertebrates contain high concentrations (10 to 15%) of plasmalogens having the a, /3-unsaturated ether structure (Rapport and Alonzo, J. Biol. Chem. 235: 1953, 1960). Comparative studies have now been made of the lipids of individual tissues from 8 species of mollusk including 6 pelecypods (Mya, Mytilus, Ostrea, Pecten, Spisula, Venus), one gastropod (Busycon), and one cephalopod (Loligo). The a, /3-unsaturated ether content of 5 or more tissues from each of these species was determined with respect to two reference bases : tissue dry weight and total lipid weight. PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 377 Among the pelecypods it was found that gill tissue contains the highest plasmalogen concen- tration, attaining values as high as 50 yumoles per gram dry weight and over 400 ^moles per gram of lipid (about 30% by weight). These values compare with those found in mammalian (rat) brain, the mammalian organ in which plasmalogens are most concentrated, namely, 60 /imoles per gram dry weight and 140 /".moles per gram of lipid. High plasmalogen concentra- tions are found in most pelecypod tissues, the lower values usually being associated with tissues of low lipid content (adductor muscle). Only crystalline style (Venus, Pccten) was found to be devoid of plasmalogen. In Lolii/n, a species in which plasmalogen concentrations are rather low, highest values were also observed in gills. In Eriochcir, the posterior gills, a salt- concentrating organ, were found by Dumont (Arch. Internat. Physiol. 66: 373, 1958) to contain a high concentration of plasmalogen phosphatide, and this led him to suggest the possibility of a role for this lipid in ion transport. The findings in this study of mollusk tissues indicate that elevated plasmalogen concentrations may be a general characteristic of gills of all species, and are consistent with the suggestion that plasmalogens are involved in some type of trans- membrane transport process. Supported by N.I.N.D.B. research grant B-1570, U. S. Public Health Service. Studies on the binding site of pinocytosis inducers. RONALD C. RUSTAD AND LYNNE C. RUSTAD. Pinocytosis is a method of transport in which the formation of membrane-limited vacuoles is dependent on temperature and aerobic respiration. The first step in this process involves the attachment of inducers to the cell surface. In Amoeba proteus basic dyes act as inducers. These dyes compete with proteins for the same surface sites. The molecular orientation of the binding sites can be demonstrated by the birefringence colors of the attached dye. The dye colors obscure the natural surface birefringence ; hence, the attachment is in immediate proximity to the major oriented component in the surface. Since proteins, which would not cross the permeability barrier, act as inducers, the binding region must be located outside the cell membrane. The Alcian blue staining of sections of amoebae indicates that the extracellular coat is an acid mucopolysaccharide. This conclusion is further substantiated by the demonstration in vivo of alpha metachromasia when toluidine blue is used to induce pinocytosis. This work suggests that pinocytosis inducers become attached to highly oriented sites in the extracellular acid mucopolysaccharide coat of the amoeba. Culture of gonads and neural gland of Molgula tnanhattensis on various natural and synthetic media. PHILIPPE SENGEL AND MADELEINE KIENY. The gonads and the neural gland were obtained from immature and mature animals. The gonads, including testes and ovaries, were explanted, together with the lateral wall of the body to which they are attached. The neural gland was cultured together with the brain and the vibratile organ. Several media were tested as to their ability to provide adequate conditions for the survival of the organs in vitro. Some media contained 7 volumes of a 1% agar saline solution, 5 volumes of sea water and one volume of either sea water (medium M), chicken plasma, horse serum (medium HS), chick embryo extract, or Parker's solution no. 199. Other media were made of 7 volumes of the agar saline solution and either 6 volumes of horse-shoe crab serum or three volumes of sea water and three volumes of horse-shoe crab serum. Immature gonads were associated on media M and HS with neural glands, including brain and vibratile organ, obtained from both juvenile and adult animals. In some cultures, however, the gonad was associated with the neural gland alone. The explants were cultured for seven days and survived throughout this period. In most cases, the vibratile activity of the ovarian cavity, the spermiducts and the vibratile organ con- tinued in vitro until the end of the cultivation period. The explants mostly maintained their organotypic organization, and very little outgrowth of cells occurred. The histology of the cultured gonads and the possible action of the neural gland on their differentiation will be studied and described ulteriorly. 378 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY Effect of temperature^ salinity, and food intake on the excretion of Zn G5 in small marine fish. JUDITH SHULMAN, I. LEHR BRISBIN AND WALTER KNOX. Fish of three species were tagged by feeding Venus meat soaked in Zn^CU solution of approximately 1 /nc./ml. concentration, and biological half-life (Tb^) of assimilated isotope determined hy making periodic whole-body counts in a well counter over a 15-20-day period. Fish were kept in clean tanks with running water or frequent changes of water. Oxygen consumption measurements were also made on two species, using the Winkler method. At 20 C. and 35% c salinity, Tbj was 13 days for Menidia mcnidia as compared with 58 days for Fitndulus heteroclitus; the metabolic rate of the former is about two times that of the latter. For Funduhis, Tbj was 75 days when kept at 10 C. and 35 days at 30 C., sug- gesting an inverse relationship of environmental temperature and excretion rate. A preliminary experiment showed that Fundiilus's Tbj was not different at 8% and at 35%c salinity, indicating that gill salt excretion at high salinity does not materially increase the exchange of zinc. No effect of varying food intake could be demonstrated. Zn 6;; -tagged specimens of Tauto- yolabrus adspcrsus at 20 C. and 35%c salinity averaged a Tb; of 44.5 days, at food levels varying from 153.7 to 58.1 cal./g. fish/day, over a 17-day period. Tbj of one specimen of T. adspcrsus, which was starved for the 17-day period, was 59 days. Research supported by N.I.H. grant 2G-535 (M.B.L. Ecology training program). Aminopterin effects on Arbacia punctulata development. RICHARD G. SKALKO. Aminopterin, a potent inhibitor of folic acid metabolism, produces a variety of develop- mental abnormalities in mammalian embryonic systems. This study was undertaken to deter- mine whether this compound also interfered with the normal development of the sea urchin, Arbacia. Eggs and sperm were obtained from mature Arbacia by mild electrical shock. Aliquots (100-300) of eggs were added to stender dishes containing the anti-metabolite dissolved in filtered sea water, either 30 minutes after fertilization or at the blastula stage. They were allowed to develop in a constant-temperature incubator (19 C.) and observations were made during cleavage, at hatching, at pre-pluteus and mature pluteus stages. Exposure at fertilization over a range of doses (200-50 /"g./10 ml. filtered sea water) produced no visible effect during cleavage. Beyond this stage, however, the effects were dosage-dependent. At 200 /xg./10 ml., the eggs rarely became free-swimming blastulae and were killed at that stage. Eggs treated with 100 ,u.g./10 ml. hatched normally but never developed beyond the blastula stage. They exhibited a characteristic circular swimming pattern and survived 10 to 14 hours beyond the 200 /"g. -treated eggs. Those treated with 50 ng./lQ ml. were completely inhibited as pre-plutei. When the eggs were exposed to the same doses of aminopterin at the blastula stage, a different range of effects was observed. Those exposed to 200 A'g./lO ml. developed into pre- plutei and rarely into immature plutei. Those treated with 100 /tg./10 ml. developed into pre-plutei and immature plutei, while those treated with 50 Mg-/10 ml. developed into mature, immature and pre-plutei. The results indicate that the effects produced by aminopterin are both dosage-dependent and time-dependent, the embryos being most sensitive during cleavage stages. It appears that, during cleavage, the anti-metabolite is interfering with a metabolic process that is essential for further development beyond the blastula stage. Difference in chlorophyll a content of Cyclotella caspia cells grown on nitrate and uric acid. MARGARET J. WALDREP AND R. R. L. GUILLARD. We determined the chlorophyll a content per cell and per unit of cell volume of Cyclotella caspia (clone 10-5) grown in pure culture in enriched sea water having either nitrate (100- 400 /J.M) or uric acid (25-100 fj.M) as nitrogen source. At the higher concentrations of either nitrate or uric acid the chlorophyll remained rela- tively high, varying from 5.1-8.0 X 10" 7 /ig./cell, or from 3.8-6.9 X 10~ 9 Mg-/M 3 of cell volume. PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 379 At the lower concentration of nitrate the chlorophyll a was lower, varying from 1.1-3.3 X 10- 7 /xg./cell or from 1.1-2.8 X 10~ 9 /ug.//u 3 of cell volume. At the lower concentration of uric acid the chlorophyll remained high (4.5 X 1(H fig. /cell, or 4.2 X 10- 9 /ug./M 3 ) during early growth of the culture but then fell in one day to 2.6 X 1Q- 7 /xg./cell or 2.2 X 10- 9 Mg-/M 3 of cell volume. All observations are consistent with the hypothesis that availability of nitrogen rather than the specific nature of the nitrogen source is responsible for the variation in chlorophyll a content. However, the availability of nitrogen is influenced both by the nature of the source and its concentration. C. caspia can utilize all the nitrogen atoms in the uric acid molecule (at 25 /*M). Photo- synthesis per cell, determined by C 14 uptake, was about 30% greater when cells were supplied uric acid (100 /^17) than nitrate (400 ^M ) . Active transport of o.vygen into the eye of fish. JONATHAN B. WITTENBERG AND BEATRICE A. WITTENBERG. The transport of oxygen against a concentration gradient has heretofore been described in only a single organ, the swimbladder of teleost fishes. M. Biot, in 1806, established that oxygen could be brought into the swimbladder against a pressure gradient of over 100 atmos- pheres. A century later W. N. F. Woodland, and J. S. Haldane showed that gas secretion was accomplished by a complex of two organs a gas gland and a rete mirabile. The rete consists of a regular and intimate intercalation of venous and arterial capillaries, which follow parallel courses and carry blood to and from the gas gland. An entirely similar rete mirabile supplies blood to the choriocapillaris underlying the retina of teleost fishes. The pigment cell layer of the retina may be regarded as analogous to the gas gland. It occurred to us that the rete mirabile-pigment epithelium complex might build up a large tension of oxygen behind the retina, which would provide a pressure head for diffusion to supply the vigorous oxygen demand of the relatively avascular retina. \Ye have used a polyethylene-covered polarographic oxygen electrode to measure the oxygen tension in the vitreous humor immediately in front of the retina. Eight species of teleost with well developed retia exhibited oxygen tensions greatly in excess of that in air (160 mm. Hg) ; tensions ranging from 400 to over 1000 mm. were commonly encountered. Two species of teleosts with poorly developed retia exhibited tensions not too different from air ; three elasmobranchs and two species of teleosts, which have no rete, displayed oxygen tensions at the retina substantially less than that of air. Our finding establishes a second instance of oxygen secretion against a large concentration difference. The parallel between the degree of development of the rete and the oxygen tensions measured at the retina constitutes strong evidence that the choroid rete is an important member of the oxygen-secreting complex. PAPERS READ BY TITLE Cytochemical observations of o.vidative activity in the developmental stages of Chaetopterus pcrganicntaceus, using succinatc and DPNH as substrates. M. JEAN ALLEN. Activity of DPN diaphorase and succinic dehydrogenase was demonstrated in whole mounts and serial sections following incubation with DPNH-Nitro-BT and succinate-Nitro-BT. At the germinal vesicle stage, using DPNH as substrate, purple to blue granules of diformazan ( indicating DPN diaphorase activity) are concentrated in a pe