THE BIOLOGICAL BULLETIN PUBLISHED BY * THE MARINE BIOLOGICAL LABORATORY Editorial Board DAVID W. BISHOP, Carnegie Institution of V. L. LOOSANOFF, U. S. Fish and Wildlife Washington Service HAROLD C. BOLD, University of Texas C. L. PROSSER, University of Illinois FRANK A. BROWN, JR., Northwestern University BERTA SCHARRER, Albert Einstein College of JOHN B. BUCK, National Institutes of Health Medicine LIBBIE H. HYMAN, American Museum of FRANZ SCHRADER, Duke University Natural History WM. RANDOLPH TAYLOR, University of Michigan J. LOGAN IRVIN, University of North Carolina CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 119 AUGUST TO DECEMBER, i960 Printed and Issued by LANCASTER PRESS, Inc. PRINCE LEMON STS. LANCASTER, PA. ii 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 No. 1. AUGUST, 1960 PAGE Annual Report of the Marine Biological Laboratory 1 ALDRICH, DAVID V., AND WILLIAM B. WILSON The effect of salinity on growth of Gymnodinium breve Davis 57 BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB A magnetic compass response of an organism 65 CAMPBELL, JAMES W. The occurrence of /3-alanine and j3-aminoisobutyric acid in flatworms. . 75 ( ".OODBODY, IVAN The feeding mechanism in the sand dollar Mellita sexiesperforata (Leske) 80 HARVEY, ETHEL BROWNE Cleavage with nucleus intact in sea urchin eggs 87 MUN, A. M., AND I. L. KOSIN Developmental stages in the broad breasted bronze turkey embryo. ... 90 NADAKAL, A. M. Carotenoid and chlorophyllic pigments in the marine snail, Cerithidea californica Haldeman, intermediate host for several avian trematodes. . 98 PRITCHARD, AUSTIN W., AND AUBREY GORBMAN Thyroid hormone treatment and oxygen consumption in embryos of the spiny dogfish 109 READ, C. P., J. E. SIMMONS, JR., J. W. CAMPBELL AND A. H. ROTHMAN Permeation and membrane transport in parasitism : studies on a tape- worm-elasmobranch symbiosis 120 RIZKI, M. T. M. Pigmented fat cells in a mutant of Drosophila melanogaster 134 TURNER, HARRY J., JR., AND JAMES E. HANKS Experimental stimulation of gametogenesis in Hydroides dianthus and Pecten irradians during the winter 145 WIGLEY, ROLAND L. A new species of Chiridotea (Crustacea : Isopoda) from New England waters 153 Xo. 2. OCTOBER, 1960 FRIZ, CARL T., ARNOLD LAZAROW AND S. J. COOPERSTEIN Studies on the isolated islet tissue of fish. III. The effect of substrates and inhibitors on the oxygen uptake of pancreatic islet slices of toadfish 161 7, iv CONTENTS HlLGARD, GALEN II()\V.\KI) A study of reproduction in the intertidal barnacle, Mitella polymerus, in Monterey Bay, California 169 JENNINGS, J. B. Observations on the nutrition of the rhynchocoelan Linens ruber ((). F. Miiller) . . 189 MKRKII.L, ARTHUR S., AND JOHN B. BURCH Hermaphroditism in the sea scallop, Placopecten magellanicus (Gmelin) 197 METZ, CHARLES B., AND KI:RT KOHLER Antigens of Arbacia sperm extracts 202 MOULTON, JAMES M. Swimming sounds and the schooling of fishes 210 PARMENTER, CHARLES L., MARVIN DEREZIN AND HAZELTENE S. PARMENTER Binucleate and trinucleate oocytes in post-ovulation ovaries of Raua pipens 224 PITKOW, RONALD B. Cold death in the guppy 231 SCOTT, ALLAN C. Furrowing in flattened sea urchin eggs 246 SCOTT, ALLAN C. Surface changes during cell division 260 TRIPP, M. R. Mechanisms of removal of injected microorganisms from the American oyster, Crassostrea virginica (Gmelin) 273 Abstracts of papers presented at the Marine Biological Laboratory 283 No. 3. DECEMBER, 1960 ABBOTT, WALTER, AND J. AXVAPARA Sulfur metabolism in the lugworm, Arenicola cristata Stimpson 357 ANDERSON, JOHN MAXWELL Histological studies on the digestive system of a starfish, Henricia, with notes on Tiedemann's pouches in starfishes 371 CONOVER, ROBERT J. The feeding behavior and respiration of some marine planktonic Crustacea 399 GOREAU, THOMAS F., AND NORA I. GOREAU The physiology of skeleton formation in corals. IV. On isotopic equilib- rium exchanges of calcium between corallum and environment in living and dead reef-building corals 416 GREGG, JOHN R. Respiratory regulation in amphibian development 428 GROSS, WARREN J., AND LEE ANN MARSHALL The influence of salinity on the magnesium and water fluxes of a crab. 440 GWILLIAM, G. F. Neuromuscular physiology of a sessile scyphozoan 454 PARKER, JOHNSON Seasonal changes in cold-hardiness of Fucus vesiculosus 474 CONTENTS v ROGICK, MARY D. Studies of marine Bryo/oa. XIII. Two new genera and new species from Antarctica 478 SCHNEIDERMAN, HOWARD A. Discontinuous respiration in insects: role of the spiracles. . 494 STUNKARD, HORACE \Y. Further studies on the trematode genus Himasthla with descriptions of H. mcintoshi n. sp., H. piscicola n. sp., and stages in the life-history of 1 1. compacta n. sp 529 WELLS, HARRY W., AND I. E. GRAY The seasonal occurrence of Mytilus edulis on the Carolina coast as a result of transport around Cape Hatteras 550 WILLIAMS, AUSTIN B. The influence of temperature on osmotic regulation in two species of estuarine shrimps (Penaeus) 560 Vol. 119, No. 1 August, 1960 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE MARINE BIOLOGICAL LABORATORY SIXTY-SECOND REPORT, FOR THE YEAR 1959 SEVENTY-SECOND YEAR I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 15, 1959) ... 1 STANDING COMMITTEES II. ACT OF INCORPORATION 4 III. BY-LA \vs OF THE CORPORATION 4 IV. REPORT OF THE DIRECTOR 6 Statement 7 Memorials 9 Addenda : 1. The Staff 18 2. Investigators, Lalor and Lillie Fellows, and Students 19 3. Fellowships and Scholarships 30 4. Tabular View of Attendance, 1955-1959 31 5. Institutions Represented 31 6. Evening Lectures 33 7. Shorter Scientific Papers ( Seminars ) 34 8. Members of the Corporation 35 V. REPORT OF THE LIBRARIAN 54 VI. GENERAL BIOLOGICAL SUPPLY HOUSE, INC 55 I. TRUSTEES EX OFFICIO 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 MARINE BIOLOGICAL LABORATORY EMERITI W. C. CURTIS, University of Missouri PAUL S. GALTSOFF, Woods Hole, Mass. E. B. HARVEY, Woods Hole, Mass. 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. LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y. TO SERVE UNTIL 1963 L. G. EARTH, Columhia 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 TO SERVE UNTIL 1960 H. F. BLUM, Princeton University K. S. COLE, National Institutes of Health S. W. KUFFLER, Harvard Medical School C B. METZ, Florida State University G. T. SCOTT, Oberlin College A. H. S/TURTEVANT, California Institute of Technology E. ZWILLING, Brandeis University TRUSTEES EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES GERARD SWOPE, JR., ex officio. Chairman EDGAR ZWILLING, 1960 JAMES H. \\'ICKERSHAM, ex officio \Y. I). MCELROY, 1961 ARTHUR K. PARPART, ex officio F. A. BROWN, JR., 1961 P. B. ARMSTRONG, ex officio JOHN BUCK, 1962 RUDOLF KEMPTON, 1960 ALBERT I. LANSING, 1962 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 WILLIAM I). MCELROY 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; Noiv, 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, Di- rector, 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. BY-LAWS OF THE CORPORATION 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 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 Emeritus 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 6 MARINE BIOLOGICAL LABORATORY and shall be organized and operated under the general supervision and authority of the Trustees. 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-second session of the Marine Biological Laboratory. During the past year the Laboratory made significant progress in rehabilitating some of its research space and facilities and also funds were obtained for a new research building and additional housing. 1. Policy During the past several years there have been about thirty people including investigators and their co-workers engaged in research on a year-round basis at the Laboratory. Investigators have made application through the Laboratory to various granting agencies for support for their various projects. In general, such investigators have been provided with laboratory space and services for a limited tenure, usually no more than five years. In addition, there have been other inves- tigators, either retired or on sabbatical leave, who have availed themselves of the opportunity to work at the Laboratory continuously for one or more years at the discretion of the Executive Committee. There has been a growing interest among the members of the Corporation, par- ticularly some of those engaged in invertebrate zoological research, in the possibility of developing year-round research programs at the Laboratory in marine systematics and ecology. It is felt by many that such programs will serve to strengthen the summer research programs and will represent the most profitable type of year- round research. The Board of Trustees concurred in a recommendation from the Executive Committee that such a combined systematics-ecology program be oper- ated by the Laboratory, the staff to be selected by the Executive Committee. Efforts are being made to receive support for this program. Also, a grant has been obtained from the Office of Naval Research w r ith which to explore the feasibility of developing a year-round research program in marine microbiology. The Laboratory will continue to make research space and facilities available to REPORT OF THE DIRECTOR 7 retired scientists and those on sabbatical leave on the approval of the Executive Committee. 2. Research Training Programs In 1959 the course in Physiology operated as a research training program under the direction of Dr. \Yilliam McElroy with support from the National Institutes of Health. The success of this operation strongly supported the desirability of devel- oping the other courses along similar lines. Research training program support has been obtained from the National Institutes of Health for Experimental Embryology starting in 1960 and for Marine Ecology starting in 1961. During this present winter the Old Lecture Hall has been completely remodeled to house the Experi- mental Embryology Training Program. Also in 1960 Invertebrate Zoology and Marine Botany will start operating as research training programs with support from the National Science Foundation. It is confidently anticipated that these programs will attract staff members of the same high caliber as have the courses in the past. At this writing the trainee applicants far exceed the numbers that can be accommo- dated in the various programs, which permits the selection of highly qualified individuals. All the training programs will run for the entire summer season. Each pro- gram will operate in the manner best calculated in the minds of its director and his staff to develop background knowledge, technical competence and research interest in the trainees. It is anticipated that these programs will stimulate in- creased interest in marine biology since it will introduce the beginning investigator to marine research material early in his career and emphasize the unique advan- tages that such material presents for a great variety of problems. 3. Xcw Laboratory Building The current progress on the new building assures completion for 1960 summer occupancy barring unforeseen delays in the construction. The schedule is a very tight one so any interruption of the work will be most serious. Mr. Homer P. Smith, General Manager of the Laboratory, merits the highest commendation for the energetic and effective way in which he has promoted the construction of the building. The original planning called for completion of the building for 1961 summer occupancy so that the present schedule represents the gain of a full year. 4. Grants, Contracts and Contributions The total income from these sources of support amounted to $181,726.00 in 1959. This represents 29.6% of the total income and is made up of the following accounts : American Cancer Soc. RC4A (+) Studies in Radiobiology $ 3,300.00 AEC--1343 Program of Research on the Physiology of Marine Or- ganisms L'sing Radioisotopes 16,165.00 NIH 1-359 Biological Research on the Morphology, Ecology, Physi- ology, Biochemistry and Biophysics of Marine Organisms 40,000.00 NIH 5143 Training Program in Nerve Muscle Physiology 21,654.00 MARINE BIOLOGICAL LABORATORY ONR 1497 Studies in Marine Biology 1 5,000.00 ONR 09701 Studies on Isolated Nerve Fibers 6,072.00 ONR 09702 Studies in Ecology 3,055.00 NSF 8295 Studies in Marine Biology 40,000.00 AEC-MBL-BM-78-59 Equipment . . '. 13,690.00 A1BL Associates 4,170.00 Abbott Laboratories 1 ,000.00 Carter Products 1,000.00 Ciba Pharmaceutical Products, Inc 1,000.00 Josephine B. Crane Foundation 2,000.00 Eli Lilly & Company 5,000.00 Merck Company Foundation 1 ,000.00 Sobering Foundation, Inc 1 ,000.00 Smith, Kline & French Foundation 3,000.00 The Upjohn Company 1 ,000.00 AYveth Laboratories, Inc 1,000.00 Olin Mathieson Chemical Corporation Charitable Trust 500.00 George Frederick Jewett Foundation 1 ,000.00 Miscellaneous Individual 120.00 $181,726.00 5. Future Plans During the summer of 1959 the Executive Committee in several meetings con- sidered the immediate future needs of the Laboratory and recommended that steps be taken to (1) develop plans and obtain funds for a dormitory-dining hall build- ing, (2) construct an additional 25 cottages on the Devil's Lane Property, and (3) prepare an application for funds to cover the detailed planning and construc- tion of a research instruction building. The concentration of both the research and dormitory buildings on our present campus creates a serious problem of congestion. The ten old wooden residences used as dormitories take up space out of proportion to the numbers they accommo- date. Also they do not adapt satisfactorily to dormitory use. The present dining hall, constructed for table service, has been modified to cafeteria service but it leaves much to be desired in fully meeting the needs of the Laboratory. A new dormitory-dining hall facility is urgently needed in a convenient location off of our present limited campus. There were sixty applications in 1959 for the twenty-five new cottages built in the spring in the Devil's Lane Tract. An additional twenty- five cottages are urgently needed particularly since the training programs will at- tract a larger number of advanced students. A research training building to replace the wooden laboratories will provide modern facilities for the training pro- gram staffs and the trainees. Such a building will permit the consolidation under one roof of the variety of services which must be provided the present training programs housed in three wooden buildings. It will also permit certain economies in operation and maintenance. Respectfully submitted, PHILIP B. ARMSTRONG, Director REPORT OF THE DIRECTOR 9 MEMORIAL EDMUND NEWTON HARVEY . by Aurin M. Chase The death of Edmund Newton Harvey from a heart attack on the morning of July 21st at his Penzance Point home was so unexpected as even now hardly to seem possible to his multitude of friends. Indeed, so difficult is it to realize that for a long time many of those who knew him will still expect to hear his voice in the corridors of the M.B.L., or to see him at their doors asking what's new, or reporting, with that characteristic enthusiasm, the most recent discovery in bioluminescence or in any one of a dozen other fields. It is rare indeed for a man to reach the age ordinarily associated with retirement and still remain so young. Newton Harvey was equally at home with people of all ages, and they with him. He will be greatly missed at Woods Hole and the many other places where he lived, worked and played. Having graduated from Germantown Academy and then received his Bachelor of Science at the University of Pennsylvania, Harvey went to Columbia for his graduate work and was awarded the Ph.D. in 1911. He started teaching immediately at Prince- ton where, virtually single-handed, he initiated courses in general physiology and bio- chemistry, subjects not often included in biology curricula at that time. In 1916 he mar- ried Ethel Nicholson Browne, herself a Columbia Ph.D. in zoology. They shared lab- oratories at Princeton and Woods Hole over the years, except for the period when it was necessary for her to devote full time to raising their two sons, Ned and Dick. The Har- veys had a host of friends and greatly enjoyed interesting company. Many will remem- ber, for example, the "Harvey Table" at the M.B.L. Mess as a center of good conversa- tion and congeniality. At Princeton, Newton Harvey was promoted to full professor in 1919 and, fourteen years later, became Henry Fairfield Osborn Professor of Biology, occupying that chair until his retirement in 1956. During his years of teaching he attracted many graduate students, most of whom based their Ph.D. theses on some aspect of bioluminescence. Nearly all his summers were spent at the M.B.L., where he had been elected a member of the Corporation in 1910, when barely out of college, and became a trustee in 1929. He served as Vice President of the Corporation from 1942 until 1952, and was always most active and influential in the affairs of the Laboratory. He was elected Trustee Emeritus in 1958. Although it was only natural that a man of his interests and energy should become involved in the direction and operation of many organizations he was, for example, a trustee of the Bermuda Biological Station the activities and welfare of the Marine Biological Laboratory were always closest to his heart. With as many interests as he had, it is not surprising that Harvey belonged to some twenty societies covering the fields of physiology, biochemistry and biophysics, as well as less specialized areas of biology. He was a member of The National Academy of Sciences and The American Philosophical Society, and had been vice president and president of The International Society of Cell Biology. In 1953 he was president of The American Society of Zoologists, as he was also of The American Society of Naturalists in 1956. He was an associate editor of several journals, and served as Managing Editor of The Journal of Cellular and Comparative Physiology during the first sixteen years of its existence. Among the formal awards made to him were the John Price Wetherill Medal of The Franklin Institute, in 1934, for his development of the centrifuge-microscope, and the Rumford Medal, by The American Academy of Arts and Sciences, in 1947, for his work 10 MAKIXK BIOLOGICAL LABORATORY in bioluminescence. He received the Certificate of Merit of the U. S. Armed Forces for his services, both experimental and advisory, during- the second world war. More re- cently, the Johns Hopkins University awarded him an honorary Doctor of Science de- gree, and Temple University the honorary degree of Doctor of Laws. HarveyV attitude toward research was alwavs that of the explorer and pioneer. He opened up new regions for others to develop. His tremendous curiosity and drive were not satisfied by the sort of routine experimentation required to wrap up completely all the loose ends of a problem. Without his kind, the discovery of new things would be slow if it occurred at all. So extensively did he explore in the field of bioluminescence, for example, that it is difficult to do any experiment involving a luminous organism with- out finding, sooner or later, that Harvey had had the same idea and tested it at least in a preliminary way years before. A person of such consuming curiosity could never be satisfied in a single line of re- search. His 250 or so published papers include such topics as cell permeability and oxidations, physical measurements at cell surfaces, brain potentials, effects of supersonic waves and of centrifugal forces on cells and during the war decompression sickness and wound ballistics. He loved instrumentation and was always eager to apply new apparatus and techniques to biological problems. His vast knowledge of organisms was most useful in this connection. But his greatest interest, and the one to which he re- turned again and again, and which was occupying him at the time of his death, was bio- luminescence. It is for this that he is best known and the acknowledged authority throughout the world. He wrote four books on the subject, and was working on a fifth. One who did not know Newton Harvey might get the mistaken impression from a re- view of his many scientific accomplishments that his time was spent entirely in the lab- oratory and the study. Nothing could be farther from the truth ! He enjoyed life to the full ! He was an excellent tennis player in his time and an experienced mountain climber, went on numerous scientific expeditions, and at one time was even reported to have been eaten by cannibals ! At Woods Hole he always found time for swimming and sailing, and the Harveys' sloop, the "Rip Tide," carried many a happy party across Vineyard Sound to Edgartown, or down the islands to Tarpaulin Cove or Cuttyhunk. He loved to surprise his friends in unusual and pleasant ways, such as pulling off the road into a secluded grove, opening the luggage compartment of his big Buick, and serv- ing cocktails from the portable bar which nobody had suspected was there. Then would follow dinner at some nearby restaurant which he had previously tested and found to be good. Yet in the midst of a social gathering his scientific interest might suddenly come to the fore. As when, once, he tossed a corked bottle to the ceiling so that all could observe that the large air bubble trapped inside did not rise in the bottle while it was in "free fall." All those who have come in contact with Edmund Newton Harvey, his friends, col- leagues, graduate students, and those fortunate undergraduates who did their senior theses under his direction, have absorbed some of his boundless enthusiasm and spirit. Cer- tainly he will lie sorely missed. But surely he would not have preferred to have gone otherwise than as he did; in full possession of all his faculties; vigorous, joyful and active until the last. MEMORIAL GEORGE H. A. CLOWES by Philip B. Armstrong Rarely do we encounter in one man such a variety of talents and interests as charac- terized George Henry Alexander Clowes. With unbounded energy, he used these tal- ents effectively, following up his interests with persistent determination. He was a REPORT OF THE DIRECTOR 11 scientist and investigator who sparked many practical applications of basic research. He was a man of business, a civic leader, and patron of the fine arts and of music. Dr. Clowes was born in Ipswich, England, in 1877, in a period of developing scien- tific interest. Through environment and natural bent, he early developed an interest in science. His family was in the business of producing intermediates for the brewing industry. He attended the Royal College of Science in London from 1893-1896 and completed his studies for a Ph.D. degree in chemistry in 1899 at the University of Gottingen. His thesis was concerned with the methyl derivatives of sugar. After additional work in some of the other leading chemical laboratories in France and Germany, he came to this country in 1901 as chemist at the Institute for the Study of Malignant Diseases at Buffalo. For the following period of fifteen years, Dr. Clowes published extensively, using a variety of approaches to the problem of cell division and growth, particularly as it ap- plied to cancer. An important contribution concerned the evidence for immunity against cancer in mice after spontaneous remissions. Other papers dealt with the mechanism of the action of mustard gas. enzyme action in fermentation, ion antagonisms, and the action of anesthetics in biological and physical systems. His outstanding scientific contributions were on the effects of calcium or sodium at oil-water interfaces. Surface phenomena remained a life-long interest resulting in many practical developments. Dr. Clowes left Buffalo in 1918 to serve in the Chemical Warfare Service where he continued his work on mustard gas, particularly its mechanism of action. In order to study its action under most favorable experimental conditions, he spent the summer of 1918 at the Marine Biological Laboratory, working with Robert Chambers and Ralph Lillie. Although Dr. Clowes had a highly practical turn of mind, he had a deep appre- ciation of the potential significance of basic research. The scientific intellectual atmos- phere of the Marine Biological Laboratory was so attractive to Dr. Clowes that he spent most of the following 40 summers at Woods Hole. At the Laboratory he found what he considered ideal biological materials for many of the basic problems in which he was interested. After the war he joined the research staff of Eli Lilly and Company and became Di- rector of Research in 1921. Here he served very effectively in the development of the commercial production of several products, starting with insulin. His earlier interest in salt antagonisms and the effects of pH on proteins served him in good stead in the precipitation and purification of insulin. He also played a prominent role in the develop- ment of liver extract, protamine insulin and penicillin. From the Marine Biological Laboratory with collaborative workers Homer Smith, Maurice Krahl, Anna Keltch and others, Dr. Clowes published a series of papers on the possible control of mitosis by chemical agents. In 1918 he was elected a member of the Corporation of the Marine Biological Lab- oratory, served three terms as a trustee, and was elected a Trustee Emeritus in 1948. He was active in seeking support for the Laboratory as well as contributing generously himself. In Indianapolis he played an active role in the development of the Indianapolis Symphony Orchestra and the John Huron Art Institute. Dr. Clowes enjoyed a felicitous marriage. He is survived by his wife, Mrs. Edith Whitehill Clowes, who shared with him many of his civic and philanthropic interests. Also surviving him are two sons, Dr. G. H. A. Clowes, Jr. and Allen W. Clowes. Dr. Clowes died in Woods Hole on August 25, 1958. He will be remembered by his Woods Hole friends for his outstanding generosity and kindness and his active participation in the scientific and physical development of the Marine Biological Laboratory. He had a keen interest in the work of others, participated actively in the scientific discussion at the Laboratory, and was always ready to give help in the development of science. 12 MARINE BIOLOGICAL LABORATORY MEMORIAL EUGENE FLOYD Du Bois by Paul Reznikoff Dr. Eugene Floyd Du Bois died on February 12, 1959, at his home at the age of 76. His death is not only a great loss to medicine and physiology but to his many stu- dents, house officers and colleagues who were associated with him at Cornell University Medical College, Bellevue Hospital, the New York Hospital and the Marine Biological Laboratory, it is a personal tragedy. Doctor Du Bois was born on June 4. 1882 in West New Brighton, Staten Island, New York, the son of Eugene and Anna Brooks Du Bois. He attended the Staten Island Academy and Milton Academy in Massachusetts and received his A.B. degree from Har- vard in 1903 and his M.D. degree from Columbia in 1906. In 1948 he was awarded an honorary Doctor of Science degree by Rochester University. After interning at the Presbyterian Hospital (1907-08) and acting as assistant pathol- ogist (1909) at this institution he decided to secure a training in bacteriology. But just before he was about to leave for France he changed his plans at the suggestion of Dr. John Rowland and he went to Germany to study metabolism. Dr. Graham Lusk visited the laboratory in Berlin and met Dr. Du Bois there. This was the beginning of a life- long association of these two pioneers in the field of metabolism. When Dr. Du Bois returned to the United States he soon became the Medical Director of the Russell Sage Institute of Pathology, of which Dr. Lusk was the Scientific Director. Thus Dr. Du Bois became a scientific descendant of Lavoisier. Under Du Bois' guidance the Russell Sage Institute has had world-wide influence in advancing scientific knowledge in the field of metabolism and from his "Calorimeter Room" there have gone forth many of our most important scientists and medical educators. Doctor Du Bois was Director of the Second (Cornell ) Medical Division of Bellevue Hospital from 1919 to 1932, Professor of Medicine at the Cornell University Medical College from 1930 to 1941. Physician-in-Chief at the New York Hospital from 1932 to 1941, and Professor of Physiology at Cornell from 1941 to 1950 when he became Emeritus Professor. Some of Dr. Du Bois' most important contributions were concerned with his work for the United States Navy. Because of his modesty few of his associates knew that he was an outstanding authority in the fields of submarine warfare and aviation medicine. For heroism in the conduct of hazardous experiments during World War I he received the Navy Cross. During the second World War he was recognized by Commendation and Ribbon Bar. He retired from the Navy with the rank of Captain and continued to work with the military service until the very day of his death. He belonged to many societies the National Academy of Sciences, the Philosophical Society, the American Physiological Society, the Aero Medical Association, the Society for Experimental Biology and Medicine, and was president of the American Society for Clinical Investigation, of the Association of American Physicians, of the Harvey Society and of the Institute of Nutrition. He became a member of the Corporation of the Marine Biological Laboratory in 1929 and was elected a Trustee in 1942. He was re-elected in 1944 for an additional full term to 1948. In 1952 he was elected Trustee Emeritus which position he held until his death. He was as conscientious in his duties to the Laboratory as he was in all undertakings and even during the later years of his incapacity he attended the meetings of the Trustees despite his physical limitations. He planned to be present at the Annual Winter Meet- ing which took place just one day after his death. REPORT OF THE DIRECTOR 13 Many honors came to Dr. Du Bois, such as the Kober Medal of the Association of American Physicians in 1947 and the Academy Medal of the New York Academy of Medicine in 1956. He was to receive the John Phillips Memorial Award of the Ameri- can College of Physicians in April. Doctor Du Bois' accomplishments and honors are of minor importance compared to the influence he has had upon his students and associates by virtue of his personality and character. As his life-long- friend and successor as Professor of Medicine, Dr. David P. Barr. has said, "This extraordinary influence has been attributable only in part to his mastery of experimental procedure and the intrinsic value of his scientific contributions. Its essence derives from his own character and personality. Inspiration has come to others from his abiding faith in principles of scientific and personal conduct, from his integrity and tolerance, and from his sympathetic understanding of the problems of those about him. His character has influenced behavior of his colleagues. It has also influ- enced innumerable students who have learned from him lessons of critical evaluation, clear expression, unvarying courtesy, and true humility." Doctor Du Bois was a gentleman, gentle in all his dealings with his fellow men and with suffering, and a man in his uncompromising attitude toward injustice and dishonesty. A colleague once asked him why he did not delegate some of his difficult and unpleas- ant problems to his subordinates. His reply was that such tasks were the duties of the chief. The principles which guided him in educating the medical students were described in an article entitled "The Clinical Clerkship in Medicine," published in the Journal of the American Medical Association, August 21, 1926. and were these: "The purpose of instruc- tion is to teach the students to teach themselves ; the manner of instruction is by example and work ; the spirit of instruction is sympathy for and faith in the students." All his friends join his widow, his three children and his nine grandchildren in being proud of their association with a great and good man. His life may be summarized by the citation on the Academy Medal : "Eugene Floyd Du Bois, physiologist, physician, educator, patriot. His life and work have brought honor to the profession of medicine" and to science. MEMORIAL JACQUES LOEB by W. J. V. Osterhout This year marks the 100th anniversary of the birth of Jacques Loeb, who contributed so much to the study of marine biology, and it seems appropriate that in a marine bio- logical station where he worked, we should recall his activities. Thirty-five years have passed since his death and yet our memory of him is still fresh and vivid. He was above all an idealist. Protected by his devoted wife who knew how to help him, he lived in a world of ideals. Their inspiration dominated his life and set him apart from others. Yet he had also a tender heart, and his sympathy was always with those who were in need of help. His outstanding feature was his creative imagination, implying prophetic vision, the intuitive, and emotional urge of ideas. Fortunately his poetic imagination was associated with a keen critical sense. He would test his conceptions over and over again and repeat his experiments very carefully. He published only a small part of his experimental work. It is remarkable that his ob- servations remain valid without fundamental modifications. 14 MARIXH HIOI.OCICAL LABORATORY The questions lie put to nature were never dull and the answers he received were al- ways interesting and at times startlingly so. He was not content to pursue a special part of a problem without considering its rela- tion to all the rest. To achieve this, it was necessary both to simplify and to generalize, and these powers he possessed to an extraordinary degree. Courage played a great part in his success. He did not select problems because they were easy but because of their importance. His courage sprang largely from his faith in the mechanistic conception to which he consecrated his life. He had a truly lovable and sympathetic personality that drew men irresistibly to him. His teaching was inspiring and unforgettable, so that it was not strange that young men gladly followed him. One felt instinctively that he cared only for truth and that in its quest he would spare no labor and sacrifice. The breadth of his knowledge made it natural for him to utilize in his work recent advances in other fields of science. Thus he took the ideas of tropism and of hetero- morphosis from botany. He applied to biology theories of dissociation and osmotic pres- sure which resulted in the discovery of artificial parthenogenesis and antagonistic salt action. To the very end of his life he kept in touch with recent progress in physics and chemistry which he applied to his own studies. Death came while he was actively engaged in what he regarded as the most funda- mental investigation of his life. In the midst of this research on proteins he was stricken down. Here we may pause to ask ourselves, how are we to remember him ? He was an idealist, sympathizing with all suffering, consecrating his gifts to humanity; a scientist with an artist's soul, emotional, intuitive, creative; a thinker, strangely original, born to blaze fresh trails and teach new doctrines; a dreamer, regarding the world of life with poetic insight and seeking with creative imagination rarely equalled to sweep aside its mystery and set free the mind of men. His visions, that have made others see visions, cannot but continue to shed inspiration ; and in shaping the soul of the future he may serve humanity more than he dared to dream. MEMORIAL FRANK M. MACNAUGHT by Charles Packard Long and faithful was the service rendered to this Laboratory by Frank M. Mac- Naught who died in June of this year at the age of 83. Coming to Woods Hole as an- accountant in 1913 when the Laboratory began its rapid growth, he was soon made Registrar, and then, in 1916, the Business Manager, a position which he held for 34 years. For much of this time his only assistant was Miss Polly Crowell. Among his many responsibilities was the task of assigning laboratory rooms and tables, and the much more difficult work of apportioning space in the Apartment House and dormitories. Only those who worked closely with him can appreciate the care which he exercised in selecting places best adapted to the needs of each applicant. In addition to these duties, he was responsible for the Mess and its many employees. From the first he devoted himself to these various tasks, discharging them with great efficiency. Always in the Office, even on Sundays and holidays, he was quick to help newcomers unfamiliar with the operation of the Laboratory. His memory was extra- ordinary. He could at once call by name investigators and students returning after an REPORT OF THE DIRECTOR 15 absence of many seasons, even recalling the year of their last attendance and the rooms they occupied. His friendliness endeared him to all. He once remarked that if he should take a trip across the country he could spend each night at the home of investi- gators or students who had especially asked him to visit them. In his relations with all he worked with he showed patience, good judgment, and great tact. Many times he re- lieved a tense situation with an apt, humorous remark. He was active in Town affairs, serving on the Finance and other committees, and in the village, as Treasurer and Trustee of the Woods Hole Public Library, and Clerk of the Coonamesset Ranch. The Laboratory has lost an exceptional man, a devoted friend whose outstanding services and genial personality will long be remembered. MEMORIAL TO CHARLES R. CRANE ON HIS HUNDREDTH ANNIVERSARY by Lawrason Riggs On the eighth day of August 100 years ago was born the greatest benefactor and friend of the Marine Biological Laboratory, Charles R. Crane. Mr. Crane was most interested in education. I think this was because he had no formal education beyond grade school. His father, the founder of the Crane Company, did not believe in colleges, in fact he wrote a book against college education, enlisting in the writing of the book two employees of the Crane Company, both of whom turned out to be college graduates. He went to work at an early age for his father. In 1878 he happened to be in New York. He wandered down to Front Street where in those days the bowsprits of sailing vessels projected over the street. Young Crane went aboard one of these ships and on telegraphic consent from his father arranged to sail on her to Java as the sole passenger. The only additions he made to his baggage were a set of Herbert Spencer and 12 dozen bottles of Guiness Stout. On the voyage he read the Spencer, drank a bottle of stout every day and learned navigation, and on his 21st birthday he furled the main royal in a gale off the Cape of Good Hope. Toward the end of the voyage the first mate died and Mr. Crane was offered his position. He did not accept as he wanted to see as much of Asia as possible. This was fortunate as on the ship's return voyage the captain and most of the crew died, undoubtedly of beri-beri. Later his doctor informed him that his health had been preserved by those 144 bottles of Guiness Stout. So began his informal education, which he pursued with unrelenting vigor so that in time he became one of the best informed Americans about the Moslem world, Russia and the Far East. During the Peace Conference after World War I he was appointed on a Commission with President King of Oberlin to investigate and make recommendations on the future of Syria. This mission further deepened and extended his contacts with the Near East. Subsequently he was for a number of years American Ambassador to China. He had an extraordinary interest in exotic places and a real flair for people. He was as much at home in Paris, St. Petersburg, Cairo, Damascus, Constantinople, Samarkand and Pekin as he was in Chicago, and he numbered among his friends, presidents, espe- cially President Wilson, cabinet members, educators, judges, Moslem leaders, including the King of Hejaz and Sherif of Mecca and his son Feisal, later King of Iraq. He made some 32 visits to Russia, penetrated the most remote parts of Asia, includ- 16 MARINE BIOLOGICAL LABORATORY ing Bokhara and the Transoxnas, and went with one servant on horseback through Al- bania after being deserted by the Turkish bodyguard supplied by the Sultan. The M.B.L. was not the only beneficiary of his interest in education and research. He was interested in the Near East colleges, especially the American College for Girls on the Bosphorus and the Sofia-American Schools in Bulgaria and also a school in Al- bania and in many universities in this country. The main purpose of his Foundation, The Friendship Fund, was to assist individuals to get an education and the purpose of the Institute of Current World Affairs, which he also founded, was to train specialists in critical areas under conditions that would develop their talents and personality. His first important contact with the Marine Biological Laboratory was his joining in an offer of assistance with several other persons through President Harper of Chicago University. This happened in 1901. When this offer and that of the Carnegie Institution were finally rejected 1>y the Lab- oratory because the trustees and members did not wish to allow the Laboratory to lose its independence, Mr. Crane became more interested through his brother-in-law, Dr. Frank R. Lillie, and before long was contributing about $20,000 a year towards its expenses. He had a large part in purchasing real property for the Laboratory and in 1913 pro- vided the first brick building, the so-called Crane Building. He had a very important part in the expansion and endowment of the Laboratory between the years 1919 and 1925. He not only capitalized his annual contribution of $20,000 by a gift of $405,000 to endowment, but he guaranteed to pay any cost of the new Rockefeller Building in excess of $500,000. This guarantee cost him $221,000. He had a large part in inter- esting Mr. John D. Rockefeller, Jr. and the Rockefeller Foundation, as is clear from Mr. Rockefeller's letter found at page 75 of Dr. Lillie's book. While his gifts were important and always timely, his appreciation of the spirit of the Laboratory and of its democratic and self-governing organization a group of sci- entists running their own affairs was almost more important. He acted as President from 1904 to 1925. That the Laboratory came through its early and difficult years and survived to be- come the great institution that it now is is, I feel, largely due to his help and encourage- ment. ZOOLOGY I. CONSULTANTS F. A. BROWN, JR., Morrison 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, Assistant Professor of Zoology, University of Minnesota ; in charge of course. JOHN B. BUCK, Senior Biologist, National Institutes of Health RALPH I. SMITH, Associate Professor of Zoology, University of California, Berkeley BERNARD L. STREHLER, Chief, Cellular and Comparative Physiology, Division of Geron- tology, National Institutes of Health PAUL P. WEINSTEIN, Laboratory of Tropical Diseases. National Institutes of Health RICHARD C. SANBORN, Professor of Zoology, Department of Biological Sciences, Purdue University REPORT OF THE DIRECTOR 17 MORRIS ROCKSTEIN, Associate Professor of Physiology, New York University College of Medicine MILTON FINGERMAN, Assistant Professor of Zoology, Tulane University III. LABORATORY ASSISTANTS ROBERT ASHMAN, Wabash College DONALD HALL, University of Michigan EMBRYOLOGY I. INSTRUCTORS MAC V. EDDS, JR., Professor of Biology, Brown University; in charge of course PHILIP GRANT, Assistant Professor of Pathobiology, Johns Hopkins 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 Biological Sciences, Brandeis University LIONEL REBHUN, Assistant Professor of Biology, Princeton University II. LABORATORY ASSISTANTS CHANDLER M. FULTON, Rockefeller Institute for Medical Research DAVID S. LOVE, University of Colorado PHYSIOLOGY I. CONSULTANTS MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania ARTHUR K. PARPART, Professor of Biology, Princeton University ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Marine Biological Laboratory II. INSTRUCTORS W. D. McELROY, Professor of Biology, Johns Hopkins University ; in charge of course FRANCIS D. CARLSON, Associate Professor of Biophysics, Johns Hopkins University BERNARD D. DAVIS, Professor of Bacteriology, Harvard Medical School DONALD GRIFFIN, Professor of Zoology, Harvard University HOWARD SCHACHMAN, Virus Laboratory, University of California TIMOTHY GOLDSMITH, Fellow, Harvard University ROBERT LOFTFIELD, Massachusetts General Hospital III. LABORATORY ASSISTANT Louis OTERO, University of Puerto Rico, Rio Piedras BOTANY I. CONSULTANT WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan 18 MARINE BIOLOGICAL LABORATORY II. INSTRUCTORS RICHARD C. STARR, Associate Professor of Botany, Indiana University; in charge of course JOHN M. KINGSBURY, Assistant Professor of Botany, Cornell University WALTER R. HERNDON, Assistant Professor of Biology, University of Alabama III. COLLECTOR G. BENJAMIN BOUCK, Columbia University IV. LABORATORY ASSISTANTS LARRY HOFFMAN, University of Texas ROBERT W. KORN, Indiana University ECOLOGY I. CONSULTANTS PAUL GALTSOFF, U. S. Fish and Wildlife Service, Woods Hole ALFRED C. REDFIELD, Woods Hole Oceanographic Institution BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution EDWIN T. MOUL, Rutgers University CHARLES E. TENNER, University of North Carolina HOWARD L. SANDERS, Woods Hole Oceanographic Institution II. INSTRUCTORS EUGENE P. ODUM, Alumni Foundation Professor of Zoology, University of Georgia; in charge of course HOWARD T. ODUM, University of Texas HAROLD J. HUMM, Associate Professor of Botany, Duke University JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution III. LABORATORY ASSISTANT RICHARD B. WILLIAMS, Harvard University 1. THE LABORATORY STAFF, 1959 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, De- IRVINE L. BROADBENT, Office Manager partment 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. SHIRLEY A. ELDER MRS. LORETTA J. MCCARTNEY REPORT OF THE DIRECTOR 19 LIBRARY MRS. M. VERNA HANKS MRS. NAOMI BOTELHO MRS. GWENDOLYN S. BLOMBERG ALBERT K. NEAL MAINTENANCE OF BUILDINGS AND GROUNDS ROBERT ADAMS RALPH H. LEWIS ELDON P. ALLEN RUSSELL F. LEWIS ARTHUR D. CALLAHAN ALAN G. LUNN ROBERT GUNNING ALTON J. PIERCE WALTER J. JASKUN ROBERT H. WALKER, JR. DONALD B. LEHY JAMES S. THAYER DEPARTMENT OF RESEARCH SERVICE GAIL M. CAVANAUGH SEAVER R. HARLOW JOHN P. HARLOW MRS. ARLENE BROWN SUPPLY DEPARTMENT DONALD P. BURNHAM ROBERT M. PERRY MILTON B. GRAY BRUNO F. TRAPASSO GEOFFREY J. LEHY JOHN J. VALOIS ROBERT O. LEHY JARED L. VINCENT MRS. MILDRED H. MIXSON HALLETT S. WAGSTAFF 2. INVESTIGATORS, LALOR AND LILLIE FELLOWS, AND STUDENTS Independent Investigators, 1959 ADELMAN, WILLIAM J., Assistant Professor of Physiology, University of Buffalo ALLEN, ROBERT D., Assistant Professor of Biology, Princeton University AMBERSON, WILLIAM R., Investigator, Marine Biological Laboratory ARMSTRONG, PHILIP B., Professor and Chairman of Anatomy, State University of New York, College of Aledicine at Syracuse ARNOLD, WILLIAM, Principal Biologist, Oak Ridge National Laboratory BALTUS, ELYANE, Charge de Course, University of Brussels, Belgium BANG, FREDERIK B., Professor of Pathobiology, Johns Hopkins University School of Hygiene BARTH, L. G., Professor of Zoology, Columbia University BAYLOR, MARTHA B., Independent Investigator, Marine Biological Laboratory BELL, EUGENE, Assistant Professor of Biology, Massachusetts Institute of Technology BENESCH, REINHOLD, Investigator, Marine Biological Laboratory BENIGNA, SISTER MARIA, Professor of Biology, Saint Joseph College BENNETT, MICHAEL, Research Associate, Columbia University, College of Physicians and Surgeons BENNETT, MIRIAM F., Assistant Professor of Biology, Sweet Briar College BEXZER, SEYMOUR, Professor of Biophysics, Purdue University BERGMANN, FELIX, Research Fellow, College of Physicians and Surgeons, Columbia University BERMAN, MONES, Physicist, National Institutes of Health BERNARD, GEORGE R., Assistant Professor of Biology, University of Notre Dame BERNSTEIN, MAURICE H., Assistant Professor of Anatomy, Wayne State University BISHOP, DAVID W., Staff Member, Carnegie Institution of Washington BOSLER, ROBERT, Instructor of Physiological Optics, Johns Hopkins Hospital BRETT, WILLIAM J., Associate Professor of Biology, Indiana State Teachers College BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University 20 MARINE BIOLOGICAL LABORATORY BRYANT, SHIRLEY H., Assistant Professor of Pharmacology, University of Cincinnati BUCK, JOHN B., Physiologist, National Institutes of Health, Laboratory of Physical Biology BURBANCK, W. D., Professor of Biology, Emory University BURK, REV. JOSEPH A., Assistant Professor, Loyola College CABRERA, GUILLERMO, Assistant Professor of Biochemistry, New York University College of Medicine CARLSON, FRANCIS D., Associate Professor of Biophysics, Johns Hopkins University CASCARANO, JOSEPH, Instructor of Pathology, New York University College of Medicine CASE, JAMES, Assistant Professor of Zoology, State University of Iowa CHAET, ALFRED B., Associate Professor of Biology, American University 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 COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health COLWIN, ARTHUR L., Professor of Biology, Queens College COLWIN, LAURA H., Lecturer, Queens College COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve University School of Medicine COSTELLO, DONALD P., Kenan Professor of Zoology, University of North Carolina CRANE, ROBERT K., Associate Professor of Biological Chemistry, Washington University Medi- cal School CROSTI, NICOLETTA, Foreign Italian Student, Bryn Mawr College CROWELL, SEARS, Associate Professor of Zoology, Indiana University CSAPO, ARPAD, Associate Professor, Rockefeller Institute for Medical Research DAVIS, BERNARD, Professor of Bacteriology, Harvard Medical School DOOLIN, PAUL F., Assistant Professor of Biology, Washington and Jefferson College DuBois, ARTHUR B., Associate Professor of Physiology, University of Pennsylvania School of Medicine ECHALIER, GUY P. R., Research Fellow in Biology, Harvard College Eons, MAC V., JR., Professor of Biology, Brown University EDMONDS, MARY, Research Associate, Montefiore Hospital Research Institute FAILLA, G., Professor of Radiology, Columbia University FEIGELSON, PHILIP, Assistant Professor of Biochemistry, College of Physicians and Surgeons FINGERMAN, MILTON, Assistant Professor of Zoology, Newcomb College of Tulane University FISHMAN, Louis, Research Associate, New York University College of Dentistry FORREST, HUGH S., Associate Professor of Zoology, University of Texas FRANK, ULRICH, Extraordinary Professor, Technical University, Darmstadt, Germany FRIZ, CARL T., Public Health Fellow, University of Minnesota FUJIMORI, EIJI, Investigator, Institute for Muscle Research, Marine Biological Laboratory FUORTES, M. G. F., Physiologist, National Institutes of Health FURSHPAN, EDWIN, Instructor in Ophthalmic Physiology, Johns Hopkins University FURUKAWA, TARO, Instructor in Ophthalmic Physiology, Johns Hopkins University GALTSOFF, PAUL S., Director, Shellfish Laboratory, U. S. Bureau of Commercial Fisheries GARDELLA, JOSEPH W., Assistant Dean, Harvard Medical School GLADE, RICHARD W., Assistant Professor of Zoology, University of Vermont GOLDSMITH, TIMOTHY H., Junior Fellow, Harvard University GONSE, PIERRE H., Research Fellow, University of Pennsylvania GOREAU, THOMAS F., Lecturer in Physiology, University College of the West Indies GORINI, LUIGI, Lecturer, Harvard Medical School GRANT, PHILIP, Assistant Professor of Pathobiology, Johns Hopkins University School of Hygiene GRAY, I. E., Professor of Zoology, Duke University GREEN, JAMES W., Associate Professor of Physiology, Rutgers, The State University GREIF, ROGER L., Associate Professor of Physiology, Cornell University Medical College GRIFFIN, DONALD R., Professor of Zoology, Harvard University GROSCH, DANIEL S., Professor of Genetics, North Carolina State College GROSS, PAUL RANDOLPH, Associate Professor of Biology, New York University GROSS, SAMSON RICHARD, Assistant Professor, Rockefeller Institute for Medical Research REPORT OF THE DIRECTOR 21 GRUNDFEST, HARRY, Associate Professor of Neurology, College of Physicians and Surgeons, Columbia University GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College HARVEY, ETHEL BROWNE, Investigator in Biology, Princeton University HARVEY, E. NEWTON, Professor of Physiology, Emeritus, Princeton University HAY, ELIZABETH D., Assistant Professor of Anatomy, Cornell University Medical College HAYASHI, TERU, Professor of Zoology, Columbia University HEGYELI, ANDREW, Investigator, Institute for Muscle Research, Marine Biological Laboratory HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania HENLEY, CATHERINE, Research Associate, University of North Carolina HERNDON, WALTER R., Assistant Professor of Biology, University of Alabama HERVEY, JOHN P., Senior Electronic Engineer, Rockefeller Institute HIATT, HOWARD H., Assistant Professor of Aledicine, Harvard Medical School HIBBARD, HOPE, Professor, Oberlin College HILL, ROBERT B., Instructor in Zoology, University of Maine HOLZ, GEORGE G., JR., Associate Professor of Zoology, Syracuse University HUMM, HAROLD J., Associate Professor of Botany, Duke University HURSH, JOHN B., Professor of Radiation Biology, University of Rochester HURWITZ, JERARD, Assistant Professor of Microbiology, New York University ISENBERG, IRVIN, Investigator, Institute for Muscle Research, Marine Biological Laboratory JACOBS, WILLIAM P., Associate Professor of Biology, Princeton University JENNER, CHARLES E., Associate Professor and Chairman of Zoology, University of North Carolina JONES, MARY ELLEN, Assistant Professor of Biochemistry, Brandeis University JONES, RAYMOND F., Visiting Research Associate, Marine Biological Laboratory KAMINER, BENJAMIN, Senior Lecturer in Physiology, University of Witwatersrand, Johannes- burg, South Africa KANE, ROBERT E., Assistant Professor of Biochemistry, Brandeis University KAPLAN, NATHAN O., Professor and Chairman of Biochemistry, Brandeis University KEMPTON, RUDOLF T., Chairman, Department of Zoology. Vassar College KINGSBURY, JOHN M., Assistant Professor of Botany, Cornell University KLEIN HOLZ, L. H., Professor of Biology, Reed College KOHLER, KURT, Research Associate, Florida State University KUFFLER, STEPHEN W., Investigator, Johns Hopkins Hospital KURIYAMA, HIROSI, Rockefeller Institute for Medical Research KURY, LIVIA REV, Investigator, Institute for Muscle Research, Marine Biological Laboratory LANSING, ALBERT L, Professor and Chairman of Anatomy, University of Pittsburgh LASH, JAMES W., Associate in Anatomy, University of Pennsylvania, School of Medicine LAZAROW, ARNOLD, Professor and Head, Dept. of Anatomy, University of Minnesota LEONE, VINCENZO, Professor, Istituto di Zoologia, Milano, Italy LEVY, MILTON, Professor and Chairman, Dept. of Biochemistry, New York University College of Dentistry LEWIN, RALPH ARNOLD, Investigator, Marine Biological Laboratory LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont LOFTFIELD, ROBERT B., Associate in Organic Chemistry, Harvard Medical School LORAND, L., Associate Professor of Chemistry, Northwestern University LOVE, W'ARNER E., Assistant Professor of Biophysics, Johns Hopkins University LOWENHAUPT, BENJAMIN, Research Associate, Rockefeller Institute for Medical Research MCELROY, WILLIAM D., Head, McCollum-Pratt Institute, Johns Hopkins University MARKS, PAUL A., Assistant Professor of Medicine, College of Physicians and Surgeons, Co- lumbia University MARSH, JULIAN B., Assistant Professor of Biochemistry, University of Pennsylvania MARSHALL, JEAN M., Assistant Professor of Physiology, Johns Hopkins University School of Medicine MARSLAND, DOUGLAS, Professor of Biology, New York University, Washington Square College MATEYKO, GLADYS M., Assistant Professor of Biology, New York University, Washington Square College METZ, CHARLES B., Professor, Florida State University MARINE BIOLOGICAL LABORATORY MIDDLEBROOK, W. ROBERT, Research Fellow, Institute for Muscle Research, Marine Biological Laboratory MONIER, ROGER, Post-doctoral Associate, University of Paris MOORE, JOHN W., Associate Chief, Laboratory of Biophysics, National Institutes of Health NACE, PAUL FOLEY, Associate Professor of Biology, McMaster University NELSON, LEONARD, Assistant Professor, University of Chicago NEWTON, JACK W., Research Associate, Brandeis University ODUM, EUGENE P., Professor of Zoology, University of Georgia OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research PALINCSAR, EDWARD E., Instructor of Biology, Loyola University PAPACONSTANTINOU, JOHN, Post-doctoral Research Fellow, Carnegie Institution of Washington PARPART, ARTHUR K., Chairman, Department of Biology, Princeton University PATERSON, MABEL C., Assistant Professor of Zoology, Vassar College PERLMANN, GERTRUDE E., Associate Professor, Rockefeller Institute for Medical Research PROSSER, C. LADD, Professor of Physiology, University of Illinois RANZI, SILVIO, Full Professor, Istituto di Zoologia, Milano, Italy RAPPORT, MAURICE M., Professor of Biochemistry, Albert Einstein College of Medicine RASMUSSEN, HOWARD, Graduate Fellow, Rockefeller Institute for Medical Research READ, CLARK P., Associate Professor of Parasitology, Johns Hopkins University REBHUN, LIONEL I., Assistant Professor of Biology, Princeton University RIESER, PETER, Research Associate, University of Pennsylvania ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of Medicine ROSE, S. MERYL, Professor of Zoology, University of Illinois ROSENBERG, EVELYN E., Associate Professor of Pathology, New York University-Bellevue Medical Center ROSLANSKY, JOHN D., Research Associate, Princeton University ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College ROTHMAN, ALVIN H., Research Fellow, Johns Hopkins University School of Hygiene RUDOMIN, P., Research Fellow, College of Physicians and Surgeons, Columbia University RUSHTON, W. A. H., Reader in Physiology, Trinity College, Cambridge, England RUSTAD, RONALD C., Instructor in Physiology, Florida State University RYTHER, JOHN H., Staff, Woods Hole Oceanographic Institution SANBORN, RICHARD C., Professor of Zoology, Purdue University SANDEEN, MURIEL I., Assistant Professor of Zoology, Duke University SANDERS, HOWARD L., Research Associate, Woods Hole Oceanographic Institution SAUNDERS, JOHN W., Professor of Zoology, Chairman Department of Biology, Marquette Uni- versity SCHACHMAN, HOWARD K., Associate Professor of Biochemistry, University of California, Berkeley SCHUH, REV. JOSEPH E., Associate Professor of Biology and Chairman of Department, Saint Peter's College SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College SCOTT, GEORGE T., Professor and Chairman, Department of Biology, Oberlin College SELIGER, HOWARD H., Guggenheim Fellow, Johns Hopkins University SKOGLUND, CARL RUDOLF, Associate Professor, Karolinska Institutet, Stockholm, Sweden SMITH, PAUL FERRIS, Electronics Engineer, Rockefeller Institute for Medical Research SMITH, RALPH I., Associate Professor of Zoology, University of California, Berkeley SPECTOR, ABRAHAM, Instructor, Harvard Medical School SPEIDEL, CARL C., Professor and Chairman of Anatomy, University of Virginia SPIEGEL, MELVIN, Assistant Professor of Biology, Colby College SPRATT, NELSON T., Chairman, Department of Zoology, University of Minnesota SPYROPOULOS, CONSTANTINE S., Neurophysiologist, National Institutes of Health STARR, RICHARD C., Associate Professor of Botany, Indiana University STEELE, RICHARD, Associate Professor of Biochemistry, Tulane University STEINBACH, H. BURR, Professor and Chairman, Department of Zoology, University of Chicago STEINHARDT, JACINTO, Director, Operations Evaluation Group, Massachusetts Institute of Tech- nology REPORT OF THE DIRECTOR STEPHENS, GROVER C, Associate Professor of Zoology, University of Minnesota STETTEN, DEWixx, Associate Director in Charge of Research, National Institutes of Health STETTEN, MARJORIE R., Biochemist, National Institutes of Health STEVENS, CHARLES F., Medical Student, Yale University School of Medicine STONE, WILLIAM, Director of Ophthalmic Plastics Laboratory, Massachusetts Eye and Ear Infirmary STREHLER, BERNARD L., Chief, Comparative Physiology Section, National Institutes of Health STRITTMATTER, PHILIPP, Assistant Professor of Biochemistry, Washington University STUNKARD, HORACE W., Research Scientist, U. S. Fish and Wildlife Service SUDAK, FREDERICK N., Instructor in 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, Investigator, Institute for Muscle Research, Marine Biological Lab- oratory TASAKI, ICHIJI, Chief, Special Senses Section, National Institutes of Health TAYLOR, ROBERT E., Neurophysiologist, National Institutes of Health TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan TEORELL, TORSTEN, Professor of Physiology, Uppsala University, Sweden TORCH, REUBEN, Assistant Professor of Zoology, University of Vermont TROLL, WALTER, Assistant Professor of Industrial Medicine, New York University-Bellevue Medical Center TSUBOI, KENNETH K., Assistant Professor of Biochemistry, Cornell University Medical College TWEEDELL, KENYON S., Assistant Professor of Biology, University of Notre Dame DEVILLAFRANCA, GEORGE W., Assistant 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, State Univer- sity of New York WAINIO, W T ALTER W., Associate Professor of Biochemistry, Rutgers University WATANABE, AKIRA, Research Fellow, College of Physicians and Surgeons, Columbia University WEBB, H. MARGUERITE, Assistant Professor of Biology, Goucher College WEINSTEIN, PAUL P., Senior Scientist, National Institutes of Health WEISS, LEON P., Assistant Professor of Anatomy, Harvard Medical School WELLS, G. P., Professor of Zoology, University College, London, England WERMAN, ROBERT, Research Associate, College of Physicians and Surgeons, Columbia University WHITING, ANNA R., Guest Investigator, University of Pennsylvania WICHTERMAN, RALPH, Professor of Biology, Temple University WIERCINSKI, FLOYD J., Research Associate, University of Pennsylvania WILLEY, C. H., Professor of Biology and Chairman of Department, New York University WILSON, WALTER L., Assistant Professor of Physiology, University of Vermont College of Medicine WITTENBERG, JONATHAN B., Assistant Professor of Physiology, Albert Einstein College of Medicine WRIGHT, PAUL A., Associate Professor of Zoology, University of New Hampshire ZWEIFACH, B. W., Professor of Pathology, New York University-Bellevue Medical Center ZWILLING, EDGAR, Associate Professor, University of Connecticut Lalor Fellows, 1959 BERNSTEIN, M. H., Wayne State University CROSTI, NICOLETTA, Bryn Mawr College ECHALIER, G. P. R., Harvard College GONSE, P. H., University of Pennsylvania LEONE, V., Istituto di Zoologia, Milano, Italy MARSH, J. B., University of Pennsylvania NELSON, LEONARD, University of Chicago PALINCSAR, E. E., Loyola University RANZI, SILVIO, Istituto di Zoologia, Milano, Italy ROSLANSKY, J. D., Princeton University 24 MARINE BIOLOGICAL LABORATORY RUSTAD, R. C, Florida State University SKOGLUND, CARL, Karolinska Instituted Stockholm, Sweden STRITTMATTER, P., Washington University Lillie Fellow RANZI, SILVIO, Istituto di Zoologia, Milano, Italy Grass Fellows LIPICKY, RAYMOND, University of Cincinnati STEVENS, CHARLES, Yale University, School of Medicine Beginning Investigators, 1959 BENSAM, BERTRAND J., State University of New York, Upstate Medical Center, Syracuse BROBERG, PATRICIA L., Brandeis University BURNSTOCK, G., University of Illinois BUTTERWORTH, FRANK M., University of Pennsylvania BYERS, THOMAS J., University of Pennsylvania CAMPBELL, JAMES WAYNE, Johns Hopkins University CARLSON, ALBERT D., State University of Iowa CHERNETSKI, KENT EUGENE, University of California CURTIS, BRIAN A., Rockefeller Institute for Medical Research DAVIDSON, MORTON, New York University Medical College DUBNAU, DAVID, Columbia University DUDEL, JOSEF, Johns Hopkins University FAUST, ROBERT GILBERT, Princeton University FILOSA, MICHAEL, Princeton University GRAHAM, CHARLES EDWARD, Johns Hopkins University School of Medicine GRIFFIN, DEAN H., American University GUMP, DIETER W., Johns Hopkins University GUTTMAN, BURTON S., Institute of Molecular Biology, University of Oregon HURWITZ, CHARLES, VA Hospital, Albany HUVER, CHARLES W., Yale University JACKSON, JAMES A., Western Reserve University KUPERMAN, ALBERT S., Cornell University Medical College LAURIE, JOHN S., Tulane University LIPICKY, RAYMOND JOHN, University of Cincinnati DE LORENZO, A. J., Johns Hopkins University Medical School NAGLER, ARNOLD L., New York University-Bellevue Medical Center NARBAITZ, ROBERTO, Carnegie Institution of Washington and Universidad de Buenos Aires ORKAND, RICHARD K., University of Utah PEPE, FRANK A., University of Pennsylvania POLGAR, GEORGE, University of Pennsylvania School of Medicine POTTER, DAVID, Johns Hopkins University REUBEN, JOHN P., College of Physicians and Surgeons, Columbia University RUBIN, ARNOLD D., New York University College of Medicine SCHAFER, DAVID G., New York University College of Medicine SHEPHARD, DAVID, University of Chicago SMITH, THOMAS G., JR., College of Physicians and Surgeons, Columbia University SMYTH, THOMAS, JR., Pennsylvania State University SUDDUTH, SOLON SCOTT, Johns Hopkins University School of Medicine SWAMI, KARUMURI S., University of Pennsylvania THEORELL, KLAS T. G., Karolinska Institute!, Stockholm, Sweden TOBIN, MICHAEL, New York University Medical Center, Downstate WARNER, ELDON D., University of Wisconsin REPORT OF THE DIRECTOR 25 WERTHEIM, GUTA, Hebrew University WHEELER, JAMES ENGLISH, Johns Hopkins University School of Medicine WINICK, PAUL, Columbia University Research Assistants, 1959 ALLEN, CONSTANCE, Massachusetts Eye and Ear Infirmary ASHMAN, ROBERT F., Wabash College ASHTON, FRANCIS T., University of Pennsylvania ASTERITA, HARVEY L., New York University BAIRD, SPENCER, Marine Biological Laboratory BARN WELL, FRANKLIN H., Northwestern University BARRON, EVELYN, Massachusetts Eye and Ear Infirmary BERMAN, LAWRENCE J., Princeton University BLUMSTEIN, JOYCE R., Albert Einstein College of Medicine BOLEYN, BRENDA J., University of Rhode Island BOUCK, G. BENJAMIN, Columbia University BRANHAM, JOSEPH, Florida State University BRAVERMAN, MAXWELL H., University of Illinois BROOKS, KENNETH H., Indiana University BUNIM, LESLEY S., Barnard College CICAK, ANNA, Albert Einstein College of Medicine CLARK, ELOISE E., University of California CLARK, LYNNE G., Queens College CONWAY, DOROTHY M., Rockefeller Institute for Medical Research CORLETTE, SALLY L., Institute for Cancer Research COUSINEAU, GILLES, University of New York DELSON, ROZANNE, Massachusetts Institute of Technology DINGLE, AL D., University of Illinois DOOLITTLE, RUSSELL F., Harvard University Doss, DICKY E., American University DUNSKY, MILTON H., Rockefeller Institute for Medical Research EIGNER, ELIZABETH ANN, Massachusetts General Hospital EIN, DANIEL, New York University-Bellevue Medical Center ERSKINE, LOUISE, Institute for Muscle Research, Marine Biological Laboratory ESPER, HILDEGARD, Columbia University FELDHERR, CARL M., University of Pennsylvania FIELDEN, ANN, University of Illinois FINKEL, ARNOLD, New York University College of Medicine FIORENTINO, EILEEN, Hahnemann Medical College FISHER, FRANK M., JR., Purdue University FORAN, ELIZABETH H., Smith College FRIEDLER, GLADYS, Tufts Medical School FRIEDMAN, LEONARD, Rutgers University FULTON, CHANDLER M., Rockefeller Institute for Medical Research GASSELING, MARY T., Marquette University GEBHART, JOHN H., National Institutes of Health GOLDFARB, DAVID, Johns Hopkins University GOUDSMIT, ESTHER M., University of Michigan GRIFFIN, JOE L., Princeton University HAAS, FLORENCE ANNE, Western University Medical School HALEY, BARBARA, Brandeis University HALL, DONALD J., University of Michigan HAMPSON, GEORGE RICHARD, Northeastern University HANSON, FRANK E., JR., State University of Iowa HASKELL, JUDITH ANN, Purdue University HATHAWAY, RALPH R., Florida State University 26 MARINE BIOLOGICAL LABORATORY HIKE, SALLY JAYNE, Mount Holyoke College HILLMAN, CELIA A., Harvard University HIMMELFARB, SYLVIA, University of Maryland School of Medicine HOFFMAN, LARRY R., University of Texas HOLSTEN, GEORGE H., Ill, Rutgers University HOLT, CHARLES E., Ill, Massachusetts Institute of Technology JACKSON, THOMAS JOHN, Lehigh University JOHNSON, CHRISTINE A., Wheaton College KAIGHN, MORRIS E., Massachusetts Institute of Technology KORN, ROBERT WILLIAM, Indiana University LAMONT, HAYES C, Columbia University LEIGHTON, CHARLES, Colby College LIBBIN, DICK, Bard College LIPPERT, BYRON E., Indiana University LONIGRO, NORMA, Seton Hill College LORING, JANET, Harvard Medical School LOVE, DAVID S., University of Colorado McCoNNAUGHY, R. A., American University McGowAN, BERNARD L., Johns Hopkins University MCLAUGHLIN, JANE, Institute for Muscle Research, Marine Biological Laboratory MANGUM, CHARLOTTE PRESTON, Vassar College MAKINEN, PAULA, University of Minnesota MALKOFF, DONALD B., University of Pittsburgh Medical School MAVRIDIS, PARASKEVI J., Purdue University MERRILL, CHARLOTTE F., Massachusetts Institute of Technology MERSON, GERALD, New York University MINGIOLI, ELIZABETH S., Harvard University MOBBERLY, WILLIAM C., Tulane University MORRISON, ROBERTA ANNE, Smith College MOULE, JOHN WILLIAM, McMaster University MOULE, MARGARET, McMaster University MUELLER, HELMUT, Institute for Muscle Research, Marine Biological Laboratory OTERO-VILARDEBO, Luis, University of Puerto Rico PALUBINKAS, BERTHA, College of Physicians and Surgeons, Columbia University PERRY, BARBARA, Institute for Muscle Research, Marine Biological Laboratory PHILPOTT, CHARLES W., Tulane University REICH, MELVIN, Rutgers University REUBEN, JOHN PHILLIP, University of Florida ROBERTS, MARY Lou, Washington University Medical School ROGERS, ANNETTE, North Carolina State College ROSE, JEANNETTE, Bates College ROSENBLUTH, RAJA, Columbia University ROTHSTEIN, HOWARD, University of Pennsylvania SATUREN, JANICE, State University of New York Upstate Medical Center at Syracuse SCHROEDER, PAUL C., St. Peter's College SCHUEL, HERBERT, University of Pennsylvania SELLERS, RICHARD LEE, American University SIEGEL, PAULA, University of Cincinnati SIGER, ALVIN, Johns Hopkins University SILKOVSKIS, IZOLDE, McMaster University SIMMONS, JOHN E., Johns Hopkins University SPENCER, JOYCE M., Harvard Medical School STAUB, HERBERT W., Rutgers University STOLL, LOUISE, Johns Hopkins University School of Hygiene SWOPE, JULIA, Massachusetts General Hospital SZENT-GYORGYI, EVA, Institute for Muscle Research, Marine Biological Laboratory SZENT-GYORGYI, MARTA, Institute for Muscle Research, Marine Biological Laboratory REPORT OF THE DIRECTOR 27 TEYAN, FRED, Albert Einstein College of Medicine THOMAS, CYNTHIA, Massachusetts Eye and Ear Infirmary VANLIEW, HUGH D., U. S. Navy, Bethesda VANNORMAN, EARL, Princeton University WAHBE, VERA, Kansas University WATKINS, DUDLEY T., Oberlin College YATES, LLOYD AUSTIN, University of Minnesota YIP, CECIL C, McMaster University Library Readers 1959 BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School BAYLOR, MARTHA B., Investigator, Marine Biological Laboratory BEIDLER, LLOYD M., Professor of Physiology, Florida State University BODANSKY, OSCAR, Chief, Division of Metabolism and Enzyme Studies, Sloan-Kettering Institute BROWN, DUGALD, Professor of Zoology, University of Michigan BUTLER, ELMER G., Professor of Biology, Princeton University CHASE, AURIN M., Associate Professor of Biology, Princeton University CLARK, ELIOT R., University of Pennsylvania COHEN, SEYMOUR S., Professor of Biochemistry, University of Pennsylvania School of Medicine COLLIER, JACK R., Marine Biological Laboratory FOERSTER, THEODOR, Professor of Physical Chemistry, Technische Hochschule, Stuttgart, W. Germany FRIES, E. F. B., Associate Professor, City College of New York GABRIEL, MORDECAI L., Associate Professor of Biology, Brooklyn College GAFFRON, HANS, Professor of Biochemistry, University of Chicago GINSBERG, HAROLD S., Associate Professor of Preventive Medicine, Western Reserve University GOLDTHWAIT, DAVID A., Assistant Professor of Biochemistry, Western Reserve University HUNTER, F. R., Professor and Head, Dept. of Biology, Univ. de los Andes, Bogota, Colombia JACOBS, M. H., Professor Emeritus, University of Pennsylvania KARUSH, FRED, Professor of Immunochemistry, University of Pennsylvania KASHA, A!ICHAEL, Professor of Chemistry, State University of Florida KLEIN, MORTON, Professor of Microbiology, Temple University School of Medicine KOZLOFF, LLOYD M., Associate Professor of Biochemistry, University of Chicago LEIGHTON, JOSEPH, Associate Professor of Pathology, University of Pittsburgh School of Medicine LUBIN, MARTIN, Assistant Professor of Pharmacology, Harvard Medical School LUDWIG, GEORGE D., Assistant Professor of Medicine, University of Pennsylvania MCDONALD, SISTER ELIZABETH SETOX, Professor of Biology, College of Mt. St. Joseph on the Ohio MINARD, FREDERICK, Research Biochemist, Abbott Laboratories MOUL, EDWIN T., Associate Professor of Botany, Rutgers University MUSACCHIA, X. J., Associate Professor in Biology, St. Louis University NOVIKOFF, ALEX B., Research Professor, Albert Einstein College of Medicine PULLMAN, BERNARD, Professor of Theoretical Chemistry, University of Paris, France RHULAND, LIONEL E., Research Section Head, The Upjohn Company ROCHOVANSKY, OLGA M., Research Assistant, Public Health Research Institute of New York ., City ROOT, WALTER S., Professor of Physiology, College of Physicians and Surgeons, Columbia University ROTH, FR. OWEN H., Associate Professor of Zoology, St. Vincent College SCHLAMOWITZ, MAX, Associate Cancer Research Scientist, Roswell Park Memorial Institute SERBER, BARBARA Jo, Assistant Professor of Anatomy, New York University-Bellevue Medical ' Center SONNENBLICK, B. P., Professor of Biology, Rutgers University SULKIN, S. EDWARD, Professor and Chairman, Dept. of Microbiology, University of Texas, Southwestern Medical School MARINE BIOLOGICAL LABORATORY TRURNIT, HANS J., Senior Scientist, Research Institute for Advanced Study WARNER, ROBERT C, Associate Professor of Biochemistry, New York University College of Medicine WEIGLE, WILLIAM O., Assistant Research Professor, University of Pittsburgh School of Medicine WHEELER, GEORGE E., Instructor in Biology, Brooklyn College YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate Medical Center ZINN, DONALD J., Associate Professor of Zoology, University of Rhode Island Students 1959 BOTANY BROWN, MALCOLM, University of Texas CHURCHILL, ALGERNON C., Harvard University CORRELL, DAVID L., Michigan State University EDWARDS, JACKIE L., University of Alabama EHRLICH, DIANA LEE, College of the City of New York FINDLEY, DAVIS L., University of Alabama FLACH, MARY E., Vassar College FOLDATS, ERNESTO, Universidad Central de Venezuela FREDERICKS, WALTER W., Johns Hopkins University GOLAS, MARY, Marquette University KALIL, MILDRED, Wellesley College KOOB, DERRY DELOS, Cornell University MASON, CHARLES P., Cornell University MILES, MARJORIE L., Acadia University MORRIS, RUTH CAROL, Cornell University NOLAN, RICHARD A., University of Nebraska SHOR, BERNICE C., Rollins College WAGNER, KENNETH A., College of William and Mary WILLIAMS, RICHARD B., Harvard University ZACHARIA, KURUVILA, Princeton University EMBRYOLOGY ASHMAN, ROBERT F., Wabash College BAKER, JOHN R., University of Minnesota BERGMANN, FRED H., Brandeis University BIRKY, C. WILLIAM, JR., Indiana University CORDES, EUGENE H., Brandeis University CURTIS, JOSEPH C., Brown University GIBLEY, CHARLES W., JR., Iowa State College GRAND, THEODORE L, Brown University GRINNELL, ALAN D., Harvard University ,HARRIS, THOMAS M., University of North Carolina HENNEN, SALLY H., Indiana University HOLT, CHARLES E., Ill, Massachusetts Institute of Technology KESSLER, DIETRICH, University of Wisconsin LAWRENCE, IRVIN E., Kansas University LESSUPS, ROLAND J., S. J., Johns Hopkins University "LEVINE, STEPHEN, Brandeis University MERSON, GERALD, New York University Medical School PIERCE, GORDON B., University of Pittsburgh ROSE, IRWIN A., Yale University SCHULER, MARGERY E., Wesleyan University REPORT OF THE DIRECTOR 29 STEINBERG, SONIA NAOMI, Northwestern University WHITTAKER, J. RICHARD, Yale University VATES, ROBERT D., University of Alabama Medical Center PHYSIOLOGY ALVAREDO, FRANCISCO, New York University, College of Medicine ANGELES, LETICIA, Tulane University BENJAMIN, THOMAS, Amherst College EISEN, JAMES, Emory University GARRICK, MICHAEL, Johns Hopkins University GILLESPIE, BARBARA, Radcliffe University GOTTLIEB, ABRAHAM, New York University-Bellevue Medical Center GREEN, MORRIS, University of Rochester HAMILTON, MARY, Sloan-Kettering Institute HANDLER, JOSEPH, University of Pennsylvania HOMER, Louis, Medical College of Virginia KALEY, GABOR, New York University KEAN, EDWARD, University of Pennsylvania KINSOLVING, CLYDE, Vanderbilt University LIEBMAN, PAUL, Barnes Hospital LUCHI, ROBERT, University of Pennsylvania PLOTZ, PAUL, Harvard Medical School PURPLE, RICHARD, Rockefeller Institute ROSENBAUM, JOEL, Syracuse University RYSER, HUGUES, Massachusetts General Hospital SLAYMAN, CLIFFORD, Rockefeller Institute THEORELL, HENNING, Karolinska Inst., Stockholm TODARO, GEORGE, New York University College of Medicine TOWNSEND, EDITH, McGill University WALCH, CAROLYN, Johns Hopkins University WEISS, CHARLES, Harvard University WRITTEN BURY, GUILLERMO, Harvard Medical School INVERTEBRATE ZOOLOGY ANDREW, OLIVER T., Franklin and Marshall College BERCHMANS, SISTER ANN, St. Mary of the Woods College BREBBIA, DANTE R., Fordham University Graduate School BRENOWITZ, HARRY, Adelphi College BUCKLEY, BROTHER WILLIAM, Fordham University CHURCHILL, ALGERNON, Harvard University COXROW, MARY M., Wilson College CORRELL, DAVID, Michigan State University DELONG, KARL T., Oberlin College EDDY, JANE, Tufts University EDWARD, BROTHER C, Fordham University ELLISON, ESTHER, University of Minnesota ENGLUND, PAUL, Hamilton College EPEL, DAVID, University of California, Berkeley FEIR, DOROTHY J., University of Wisconsin FERGUSON, JOHN, Cornell University GAGE, ELIZABETH M., Gushing Academy GATES, DAVID A., Clark University GOLDMAN, LAWRENCE, University of California, Los Angeles GREENE, LAUREL E., Goucher College GUTKNECHT, JOHN, University of North Carolina 30 MARINE BIOLOGICAL LABORATORY HAYES, WILLIAM, University of Michigan HENDERSON, OLIVER, JR., The Citadel HUBER, SALLY A., Mt. Holyoke College IZOWER, JACK, City College of New York JONES, LYNNE A., Connecticut College KRAUSE, HELEN, University of Massachusetts LAFAUCI, GRACE, Wilson College MANGUM, CHARLOTTE, Vassar College MARZOLF, GEORGE, University of Michigan MCDOWELL, SISTER MARGARET ANN, College of St. Mary of the Springs AIcWniNNiE, DOLORES J., DePaul University MESCHER, SISTER ALMA L., University of Notre Dame AIoFFEY, ELIZABETH S., University of Michigan MOULTON, JOHN, Hastings College and Clark University NORBECK, BETTY, University of Minnesota NORDLIE, FRANK, University of Minnesota PROSSER, JANE ELLEN, Earlham College RAPPAPORT, LUCINDA, Brandeis University SEECK, MARGARET A., Oberlin College SHAW, WILLIAM N., Bureau of Commercial Fisheries SHOR, BERNICE, Rollins College SIMPSON, MARGARET, Catholic University of America STERNS, CAROL W., Peekskill, New York STONG, CYNTHIA C, Wellesley College THOMAS, CAROLINE, University of Vermont VERRUSIO, A. CARL, Drew University WILLIAMS, JUNARDEN, Northwestern University ZIMMERMAN, WILLIAM, Princeton University ZOTTOLI, ROBERT, Bowdoin College ECOLOGY ABBIATE, LORRAINE M., Douglass College BACHMAN, ROGER W., University of Michigan BIANCHI, CARLA F., Chatham College BURKHOLDER, K. M., Emory University DAVEY, TESSA, Mount Holyoke College HAYWARD, GEORGE E., Drew University PALMER, JOHN D., Northwestern University PINCHOT, GIFFORD B., Johns Hopkins University MCLAUGHLIN, ELLEN, University of North Carolina SWEENEY, EDWARD F., Boston University SWIFT, ELIJAH, Swarthmore College TAYLOR, WALTER R., Johns Hopkins University WATT, WALTON D., Dalhousie University WHITELEY, GEORGE Co., The Hill School WILLIAMS, ELSIE LOUISE, Goucher College 3. FELLOWSHIPS AND SCHOLARSHIPS, 1959 Lucretia Crocker Scholarships : CHARLES P. MASON, Botany Course JOHN D. PALMER, Ecology Course Conklin Scholarship : STEPHEN LEVINE, Embryology Course Bio Club Scholarships : DIANA LEE EHRLICH, Botany Course JACK IZOWER, Invertebrate Zoology Course REPORT OF THE DIRECTOR 31 4. TABULAR VIEW OF ATTENDANCE, 1955-1959 1955 1956 1957 1958 1959 INVESTIGATORS TOTAL 250 304 326 410 427 Independent 162 184 186 203 215 Under Instruction 9 20 23 39 45 Library Readers 54 50 42 54 51 Research Assistants 25 50 75 114 116 STUDENTS TOTAL 148 140 139 138 134 Invertebrate Zoology 56 55 55 55 49 Embryology 30 28 27 22 23 Physiology 30 30 30 27 27 Botany 19 18 18 18 20 Ecology 13 9 9 16 15 TOTAL ATTENDANCE 398 444 465 548 561 Less persons represented as both investigators and students 2 3 4 398 442 462 543 557 INSTITUTIONS REPRESENTED TOTAI 129 130 129 142 143 By Investigators 95 97 94 110 98 By Students 34 33 35 74 73 SCHOOLS AND ACADEMIES REPRESENTED By Investigators 3 3 5 12 By Students 2 1 1 2 8 FOREIGN INSTITUTIONS REPRESENTED By Investigators 8 9 11 20 29 By Students 6 6 5 6 9 5. INSTITUTIONS REPRESENTED, 1959 Abbott Laboratories A & M College of Texas Adelphi College Agricultural Research Center Alabama, University of Albert Einstein College of Medicine American Heart Association American University Amherst College Barnes Hospital Bowdoin College Brandeis University Brooklyn College Brown University Bryn Mawr College Buffalo, University of California, University of Carnegie Institution of Washington Catholic University Chatham College Chicago, University of Cincinnati, University of City College of New York Colby College College of St. Mary of the Springs College of William and Mary Columbia University Columbia University, College of Physicians and Surgeons Connecticut College Connecticut, University of Cornell University Cornell University Medical School Cushing Academy Department of the Interior DePaul University Drew University Duke University Earlham College Emory University Florida State University Fordham University Franklin and Marshall College Georgia, University of Goucher College Hahnemann Medical School Hamilton College Harvard University Harvard University Medical School Hastings College Illinois, University of Indiana State Teachers College Indiana University Institute for Muscle Research 32 MARINE BIOLOGICAL LABORATORY Iowa State University Johns Hopkins University Kansas University Louisiana State University Loyola College Maine, University of Manhattan College Marquette University Maryland, University of Massachusetts Eye and Ear Infirmary Massachusetts General Hospital Massachusetts Institute of Technology Medical College of Virginia Michigan State University Michigan, University of Minnesota, University of Montefiore Hospital Research Institute Mount Holyoke College Mt. St. Joseph, College of National Institutes of Health Nebraska, University of New Hampshire, University of New York State University College of Medi- cine at Syracuse New York University New York University, Bellevue Medical Center New York University School of Dentistry New York University, Washington Square College North Carolina State College North Carolina, University of Northwestern University Notre Dame University Oak Ridge National Laboratory Oberlin College Ohio Wesleyan University Oklahoma, University of Oregon, University of Orleans High School Pennsylvania, University of Pennsylvania Medical School, University of Pittsburgh, University of Princeton University Purdue University Queens College Radcliffe College Reed College Research Institute for Advanced Studies Rhode Island, University of Rochester, University of Rockefeller Institute for Medical Research Rollins College Roswell Park Memorial Institute Rutgers University St. Joseph's College St. Louis University St. Mary of the Woods College. St. Peter's College St. Vincent College Seton Hill College Single Cell Research Foundation Sloan-Kettering Institute Smith College Swarthmore College Sweet Briar College Syracuse University Temple University Texas, University of Texas, University of, Southwestern Medical School The Hill School Tufts University Tulane University Upjohn Company Utah, University of U. S. Fish and Wildlife Service U. S. Public Health Service Vanderbilt University Vassar College Vermont, University of Veterans Administration Hospital Virginia, University of Washington University Washington University Medical School Washington and Jefferson College Wayne State University Wellesley College Wesleyan University Western Reserve University Wilson College Wisconsin, University of Woods Hole Oceanographic Institution Yale University FOREIGN INSTITUTIONS REPRESENTED, 1959 Institute de Anatomica y Embriologia, Uni- versidad de Buenos Aires, Argentina University of Brussels, Belgium Arcadia University, Canada Dalhousie University, Canada McGill University, Canada McMaster University, Canada University de los Andes, Bogota, Colombia King's College, England Trinity College, England University College, England Sorbonne, Paris, France University of Paris, France Max-Plank Institiit fur Virusforschung, Ger- many Technical University, Darmtstadt, Germany REPORT OF THE DIRECTOR Technische Hochschule, Germany Karolinska Institutet, Stockholm, Sweden Madras Christian College, Madras, India Uppsala University, Sweden Hebrew University, Israel Clinique Medicale Universitaire, Switzerland University of Milan, Milan, Italy University of Witwatersrand, Johannesburg, University of Tokyo, Japan South Africa University of Puerto Rico, Puerto Rico Universidad Central de Venezuela, Venezuela University of the Philippines, Philippines University College of the West Indies, Ja- Centro Investigaciones Biologicas, Madrid, maica, West Indies Spain SUPPORTING INSTITUTIONS AND AGENCIES, 1959 American Cancer Society Olin Mathieson Chem. Corporation, Charitable Associates of the Marine Biological Labora- Trust tory National Institutes of Health Atomic Energy Commission National Science Foundation Josephine B. Crane Foundation Office of Naval Research The Grass Foundation The Rockefeller Foundation The Lalor Foundation Smith, Kline and French Foundation F. R. Lillie Fellowship CORPORATE ASSOCIATES Abbott Laboratories Merck Company Foundation Ciba Pharmaceutical Products, Inc. Schering Corporation Carter Products, Inc. The Upjohn Company Eli Lilly and Company Wyeth Laboratories 6. EVENING LECTURES, 1959 June 26 R. E. BILLINGHAM "Studies on the Y chromosome antigen in rodents" July 3 ALEXANDER FORBES "The growth of physiology" July 6 ALEXANDER FORBES "Electrophysiology of color vision" July 10 I. M. KLOTZ "Protein hydration and behavior" July 17 C. LADD PROSSER ' 'The origin' after a century; prospects for the future" July 24 SILVIO RANZI "Protein differentiation during embryonic and larval development" July 31 V. G. DETHIER "Chemical sense of the blowfly and hunger'' August 7 CLAUDE A. VILLEE "Interrelations of hormones and enzymes" August 14 HUGO THEORELL "Mode of action of enzyme-coenzyme com- plexes" 34 MARINE BIOLOGICAL LABORATORY August 21 W. A. H. RUSHTON "The retina is the net of a fisherman who catches quanta and barters them for infor- mation" August 28 EUGENE P. ODUM "The energy flow to the study of populations in nature" 7. TUESDAY EVENING SEMINARS, 1959 July 7 J. L. GRIFFIN "Isolation and chemical identification of the crystalline cytoplasmic inclusions in the large, free-living amebae" MILTON FINGERMAN "Physicochemical characterization of chro- matophorotropins in the crayfish, Cam- barellus shujeldti" WILLIAM H. JOHNSON AND ANDREW G. SZENT GYORGYI "The molecular basis for the 'catch mech- anism' in molluscan muscles" July 14 JAMES D. EISEN "A study on the physiology of the predator- prey relationship existing between Para- mecium aurelia and Didinium nasututn" WOLFGANG WIESER "Growth, metabolism and coexistence in ma- rine nematodes" RALPH A. LEWIN "Uptake of strontium by Syracosphaera" July 21 A. B. NOVIKOFF "Lysosomes in the physiology and pathology of cells" J. R. COLLIER "Localization and synthesis of ribonucleic acid in the development of Ilyanassa ob- soleta" G. G. HOLZ, JR. AND C. C. SPEIDEL "Mating behavior of x-rayed Tetrahymena pyriformis." Motion pictures C. FULTON "Polarized tissue movement in hydroid re- generation." Motion pictures July 28 PHILIP PERSON, JAY W. LASH AND ALBERT FINE "Myoglobin and cytochrome oxidase in odon- tophore cartilage of Busycon" W. TROLL, S. BELMAN AND N. NELSON . ."Aromatic amine metabolism and bladder cancer" PAUL S. GALTSOF'F AND D. E. PHILPOTT "Ultra structure of the spermatozoon of the oyster" August 4 VINCENZO LEONE "Some structures found in electron micro- scopic pictures of an amphibian tumour" ALFRED W. SENFT "Ultrastructure of the human parasite, Schis- tosoina mansoni" REPORT OF THE DIRECTOR 35 GEORGE W. DE VILLAFRANCA AND DELBERT E. PIIILPOTT "A study of the fine structure of skeletal muscle from Limuhis polyphemus" August 11 MAURICE M. RAPPORT "Present status of the problem of plasmalo- gen structure" ERIC G. BALL "On the mode of action of insulin" WALTER S. VINCENT AND ELYANE BALTIS "Incorporation of isotopic label into RNA : synthesis or terminal addition?" August 18 L. V. HEILBRUNX "The action of glycerol on protoplasm" WALTER L. WILSON AND K. S. SWAMI "Electrophoretic studies on protoplasm" FRANCIS T. ASIITON "Germinal vesicle breakdown in the eggs of Spisula and Hydroides" R. D. ALLEN "Polarized optical studies on Ameba" F. CHILD "Isolation and analysis of cilia" 8. MEMBERS OF THE CORPORATION, 1959 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 DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut 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 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 YOUNG, DR. B. P., Cornell University, Ithaca, New York 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 36 MARINE BIOLOGICAL LABORATORY ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York ADOLPH, DR. EDWARD F., University of Rochester School of Medicine and Dentistry, Rochester, New York ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota ALLEN, DR. M. JEAN, Department of Biology, Wilson College, Chambersburg, Pennsylvania ALLEN, DR. ROBERT D., Department of Biology, Princeton University, Princeton, New Jersey ALSCHER, DR. RUTH, Department of Physiology, Manhattanville College, Purchase, New York AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 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, Pennsylvania ARMSTRONG, DR. PHILIP B., 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 Pediatrics, University of Chicago, Chi- cago, 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 BAKER, DR. H. B., Department of Zoology, University of Pennsylvania, Philadel- phia 4, Pennsylvania BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medical School, Boston 15, Massachusetts BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School of Hygiene, Baltimore 5, Maryland BARD, DR. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland EARTH, DR. L. G., Department of Zoology, Columbia University, 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 Pitts- burgh School of Medicine, Pittsburgh 13, Pennsylvania BEERS, DR. C. D., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina REPORT OF THE DIRECTOR 37 BEHRE, DR. ELINOR H., Black Mountain, North Carolina BENESCH, DR. REINHOLD, Marine Biological Laboratory, Woods Hole, Massa- chusetts BENESCH, DR. RUTH, Marine Biological Laboratory, Woods Hole, Massachusetts BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet Briar, Virginia BERG, DR. WILLIAM E., Department of Zoology, University of California, Berke- ley 4, California BERMAN, DR. MONES, Institute for Arthritis and Metabolic Diseases, National Insti- tutes 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 University College of Medicine, Detroit 7, Michigan BERTHOLF, DR. LLOYD, Illinois Wesleyan University, Bloomington, Illinois BEVELANDER, DR. GERRIT, New York University School of Medicine, 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, Apt. C 7, Philadelphia 4, Pennsylvania 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, 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 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. GIULIO, 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, D. C. CHAET, DR. ALFRED B., Department of Biology, American University, Washing- ton 16, D. C. CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical School, Coral Gables, Florida CHANG, DR. JOSEPH J., Akademiestr. 3, Physiologiscb.es Inst., Postfach 201, Heidelberg, Germany CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton, New Jersey CHENEY, DR. RALPH H., Biology Department, Brooklyn College, Brooklyn 10, New York CLAFF, DR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts CLARK, DR. A. M., Department of Biology, University of Delaware, Newark, Delaware CLARK, DR. E. R., The Wistar Institute, Woodland Avenue and 36th Street, Phila- delphia 4, Pennsylvania CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, New York CLARKE, DR. GEORGE L., Harvard University, Biological Laboratories, Cambridge 38, Massachusetts REPORT OF THE DIRECTOR 39 CLELAND, DR. RALPH E., 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 Pennsyl- vania School of Medicine. Philadelphia 4, Pennsylvania COLE, DR. KENNETH S., National Institutes of Health (NINDB), Bethesda 14, Maryland COLLETT, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts COLLIER, DR. JACK R., Department of Zoology, Louisiana State University, Baton Rouge, Louisiana 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 Zoology, University of Florida, Gaines- ville, Florida COOPERSTEIN, DR. SHERWIN J., Department of Anatomy, Western Reserve Uni- versity Medical School, Cleveland, Ohio COPELAND, DR. D. E., 8705 Susanna Lane, Chevy Chase 15, Maryland COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine COPLEY, DR. A. L., Medical Research Laboratories, Charing Cross Hospital, 8 Ex- change Ct., Strand, London, W. C. 2 CORN MAN, 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., Goucher College, Towson, Baltimore 4, Maryland CROWELL, DR. P. S., JR., Department of Zoology, Indiana University, Blooming- ton, Indiana CSAPO, DR. ARPAD I., Rockefeller Institute for Medical Research, 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 DAWSON, DR. A. B., Biological Laboratories, Harvard University, Cambridge 38, Massachusetts 40 MARINE BIOLOGICAL LABORATORY DAWSON, DR. A. J., College of the City of New York, New York City, 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, 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, Ashland, Virginia DONALDSON, DR. JOHN C., University of Pittsburgh School of Medicine, Pitts- burgh, Pennsylvania 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, D. C. EDDS, DR. MAC V., JR., Department of Biology, Brown University, Providence 12, Rhode Island EDWARDS, DR. CHARLES, University of Utah, Salt Lake City, Utah EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis, Missouri ELLIOT, 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, Iowa City, Iowa FAILLA, DR. G., Columbia University, College of Physicians and Surgeons, New York 32, New York FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France FERGUSON, DR. F. P., Department of Physiology, University of Maryland Medical School, Baltimore 1, 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 University, New Orleans 18, Louisiana FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Richmond 19, 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 FORBES, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge 38, Massachusetts REPORT OF THE DIRECTOR 41 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 FURTH, DR. JACOB, 18 Springdale Road, Wellesley Farms, Massachusetts FYE, DR. PAUL M., Director, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn 10, New York GAFFRON, DR. HANS, Research Institutes, University of Chicago, 5650 Ellis Avenue, Chicago 37, Illinois GALL, DR. JOSEPH G., Department of Zoology, University of Minnesota, Minneapolis 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., Western Reserve University School of Medicine, Cleve- land, Ohio GOLDSTEIN, DR. LESTER, Department of Zoology, University of Pennsylvania, Phila- delphia, Pennsylvania GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, Atlanta 22, Georgia GOODRICH, DR. H. B., 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., Dade County Cancer Institute, 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 Brunswick, New Jersey GREEN, DR. MAURICE, Microbiology Department, 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 42 MARINE BIOLOGICAL LABORATORY GRIFFIN, DR. DONALD R., Biological Laboratories, Harvard University, Cam- bridge 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 City, New York GUDERNATSCH, DR. FREDERICK, 41 Fifth Avenue, New York 3, New York GUTHRIE, DR. MARY J., Detroit Institute for Cancer Research, 4811 John R. Street, Detroit, Michigan GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn 10, New York HAJDU, DR. STEPHEN, U. S. Public Health Institute, 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 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 HARRISON, DR. Ross G., Osborn Zoological Laboratories, Yale University, New Haven, Connecticut HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research, 66th Street and York Avenue, New York 21, New York HARTMAN, DR. FRANK A., Hamilton Hall, Ohio State University, Columbus, Ohio HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo 3, New York HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institute 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, Wellesly 81, Massachusetts HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, Philadelphia 4, Pennsylvania 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 HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts HESS, DR. WALTER N.. Hamilton College, Clinton, New York HTATT, DR. HOWARD H., Department of Medicine, Harvard Medical School, Boston 15. Massachusetts REPORT OF THE DIRECTOR 43 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, Pennsylvania HOLLAENDER, DR. ALEXANDER, Biology Division, O.R.N.L., Oak Ridge, Tennessee HOLZ, DR. GEORGE G., JR., Department of Zoology, Syracuse University, Syracuse, New York HOPKINS, DR. HOYT S., New York University College of Dentistry, New York City, New York HUNTER, DR. FRANCIS R., University of the Andes, Calle 18-a Carreral-E, Bogota, Colombia, South America HUTCHENS, DR. JOHN O., Department of Physiology, University of Chicago, Chicago 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 ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts JACOBS, DR. M. H., University of Pennsylvania School of Medicine, Philadelphia 4, Pennsvlvania ^ 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., Biology Department, Princeton University, Princeton, New Jersey JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of Florida, Gainesville, Florida KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachu- setts RABAT, DR. E. A., Neurological Institute, College of Physicians and Surgeons, New- York City, 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 44 MARINE BIOLOGICAL LABORATORY 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 Chemistry, 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 KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York KLEIN, DR. MORTON, Department of Microbiology, Temple University, Philadel- phia, Pennsylvania KLEIN HOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland. 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 5, Maryland KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois KUFFLER, DR. STEPHEN, Department of Ophthalmology, Johns Hopkins Univer- sity, Baltimore 5, Maryland KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York LACKEY, DR. JAMES B., Box 497, Melrose, Florida LANCEFIELD, DR. D. E., Queens College, Flushing, New York 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 L, Department of Anatomy, University of Pittsburgh Medical School, Pittsburgh 13, Pennsylvania LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts- burgh, Pennsylvania LAVIN, DR. GEORGE L, 3714 Springdale Avenue, Baltimore, 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., Brookhaven Apartments, Upton. Long Island, New York REPORT OF THE DIRECTOR 45 LEHMANN, DR. FRITZ, Zoologische Institut, University of Berne, Berne, Switzerland 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., Marine Biological Laboratory, Woods Hole, Massachusetts LEWIS, DR. IVEY F., 1110 Rugby Road, Charlottesville, Virginia LING, DR. GILBERT, 307 Berkely 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. LEO, 40 Crestwood Drive, St. Louis 5, Missouri LOEB, DR. R. F., 950 Park Avenue, New York 28, New York LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans- ton, Illinois 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. RUTH STOCKING, Department of Botany, University of California, Los Angeles 24, California LYNCH, DR. WILLIAM, Department of Biology, St. Ambrose College, Davenport, Iowa LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America, Washington, D. C. McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School, Philadelphia, 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 MACDOUGALL, DR. MARY STUART, Mt. Vernon Apartments, 423 Clairmont Avenue, Decatur, Georgia MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 136 Harrison Avenue, Boston, Massachusetts MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York MARSHAK, DR. ALFRED, Department of Biology, University of Notre Dame, Notre Dame, Indiana MARSLAND, DR. DOUGLAS A., New York University, Washington Square College, New York 3, New York 46 MARINE BIOLOGICAL LABORATORY 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, Lankenau Research Institute, Philadelphia, Pennsylvania MEINKOTH, DR. Norman A., Department of Biology, Swarthmore College, Swarth- more, Pennsylvania MENKIN, DR. VALY, Agnes Barr Chase Foundation for Cancer Research, Temple University Medical School, Philadelphia, Pennsylvania 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 Laboratory, Woods Hole, Massachusetts MILLER, DR. J. A., JR., Department of Anatomy, Emory University, Atlanta 22, Georgia 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 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 MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick, New Jersey MOUNTAIN, MRS. J. D., 8 Coolidge Avenue, White Plains, New York MULLER, DR. H. J., Department of Zoology, Indiana University, Bloomington, Indiana MULLINS, DR. LORIN J., Biophysical Laboratory, Purdue University, Lafayette, Indiana MUSACCHIA, DR. XAVIER J., 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, Canada NACHMANSOHN, DR. DAVID, Columbia University, College of Physicians and Sur- geons, New York City, New York NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts REPORT OF THE DIRECTOR 47 NELSON, DR. LEONARD, Department of Anatomy, University of Chicago, Chicago, Illinois NEURATH, DR. H., Department of Biochemistry, University of Washington, Seattle 5, Washington NICOLL, DR. PAUL A., Indiana Contract, Box K, A.P.O. 474, San Francisco, California Niu, DR. MAN-CHIANG, Rockefeller Institute for Medical Research, 66th Street and York Avenue, New York 21, New York OCHOA, DR. SEVERO, New York University College of Medicine, New York 16, 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 OSTER, DR. ROBERT H., University of Maryland School of Medicine, Baltimore 1, Maryland OSTERHOUT, MRS. MARION IRWIN, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York OSTERHOUT, DR. W. J. V., 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 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, Veterans Adminis- tration 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 Medical Center, New York City, New York PIERCE, DR. MADELENE E., 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 PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti- more 12, Maryland PROSSER, DR. C. LADD, 401 Natural History Building, University of Illinois, Urbana, Illinois 48 MARINE BIOLOGICAL LABORATORY PROVASOLI, DR. LUIGI, Raskins Laboratories, 305 E. 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 East 15th Street, New York 9, New York RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta 22, Georgia READ, DR. CLARK P., Johns Hopkins University, Baltimore, Maryland 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 REINER, DR. J. M., V. A. Hospital, Albany, New York RENN, DR. CHARLES E., 509 Ames Hall, Johns Hopkins University, Baltimore 18, Maryland REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue, New York City, New York RICE, DR. E. L., 2241 Seneca Avenue, Alliance. Ohio RICHARDS, DR. A., 2950E 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., Harvard University, Museum of Comparative Zoology, Cambridge, 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 City, New York ROSE, DR. S. MERYL, Department of Zoology, University of Illinois, Champaign, Illinois 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 REPORT OF THE DIRECTOR 49 ROTH, DR. JAY S., Department of Biochemistry, Hahnemann Medical College Philadelphia 2, Pennsylvania 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 Bldg., 215 S. 34th Street, Philadel- phia 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, R. D. 7, Bryn Mawr, Pennsylvania SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of Cali- fornia, Berkeley 4, California SCHARRER, DR. ERNST A., 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 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, Minneapolis 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 SCHRADER, DR. FRANZ, Department of Zoology, Duke University, Durham, North Carolina SCHRADER, DR. SALLY HUGHES, Department of Zoology, Duke University, Dur- ham, North Carolina 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, 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, Massachu- setts SENFT, DR. ALFRED W., Woods Hole, Massachusetts SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and Surgeons, New York City, New York 50 MARINE BIOLOGICAL LABORATORY SHANES, DR. ABRAHAM M., Experimental Biology 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., 35 Henderson Terrace, Burlington, Vermont SILVA, DR. PAUL, Department of Botany, University of Illinois, Urbana, Illinois 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., General Manager, Marine Biological Laboratory, Woods Hole, Massachusetts SMITH, MR. PAUL FERRIS, Marine Biological Laboratory, Woods Hole, Massachu- setts 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 SPEIDEL, DR. CARL C., Department of Anatomy, University of Virginia, University, Virginia SPIEGEL, DR. MELVIN, Old Waterville, Maine SPRATT, DR. NELSON T., JR., Department of Zoology, University of Minnesota, Minneapolis 14, Minnesota SPYROPOULOS, DR. C. S., Department of Neurophysiology, 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 15, Illinois STEINBERG, DR. MALCOLM S., Department of Biology, Johns Hopkins University, Baltimore 18, Maryland STEIN HARDT, DR. JACINTO, Director of Operations Evaluation Group, Massachu- setts Institute of Technology, Cambridge, Massachusetts STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota, Minneapolis 14, Minnesota STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois STOREY, 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 Medical School, Baltimore 5, Marvland REPORT OF THE DIRECTOR 51 STUNKARD, DR. HORACE W., American Museum of Natural History, 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, New York SZENT-GYORGYI, DR. ALBERT, Marine Biological Laboratory, Woods Hole, Massa- chusetts SZENT-GYORGYI, DR. ANDREW G., Marine Biological Laboratory, Woods Hole, Massachusetts TASAKI, DR. ICHIJI, Laboratory of Neurophysiology, National Institute of Neurological Diseases and Blindness, Bethesda 14, Maryland TASHIRO, DR. SHIRO, University of Cincinnati, Medical College, Cincinnati, Ohio TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology, National Institute of Neurological Diseases and Blindness, Bethesda 14, Maryland 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, DR. J. PHILIP, Osborn Zoological Laboratories, Yale University, New Haven, Connecticut TROLL, DR. WALTER, Department of Industrial Medicine, New York University College of Medicine, New York City, New York TWEEDELL, DR. KENYON S., Department of Biology, University of Notre Dame, Notre Dame, Indiana TYLER, DR. ALBERT, Division of Biology, California Institute of Technology, Pasadena 4, California UHLENHUTH, DR. EDWARD, University of Maryland School of Medicine, Baltimore, Maryland URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago, Chicago, Illinois DEViLLAFRANCA, DR. GEORGE M., Department of Zoology, Smith College, North- ampton, 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 Reserch, Rutgers University, New Brunswick, New Jersey 52 MARINE BIOLOGICAL LABORATORY 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., Osborn Zoological Laboratories, 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., University of Pennsylvania, Philadelphia 4, Pennsylvania 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 WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania WHITING, DR. ANNA R., University of Pennsylvania, Philadelphia 4, Pennsylvania WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania, Philadelphia 4, Pennsylvania WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, Pennsylvania WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts WIERCINSKI, DR. FLOYD J., Department of Biological Sciences, Drexel Institute of Technology, 32nd and Chestnut Streets, Philadelphia 4, Pennsylvania 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 College of Medicine, Burlington, Vermont WITSCHI, DR. EMIL, Department of Zoology, State University of Iowa, Iowa City, Iow r a WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry, Albert Einstein College of Medicine, New York 61, New York WOLF, DR. ERNST, Pendleton Hall, Wellesley College, Wellesley, Massachusetts WOODWARD, DR. ARTHUR A., Army Chemical Center, Maryland (Applied Physi- ology 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 College of Medicine, Syracuse 10, New York YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings- ton, Rhode Island REPORT OF THE DIRECTOR 53 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 City, New York ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham 54, Massachusetts 3. ASSOCIATE MEMBERS ALDRICH, Miss AMY 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 CRANE, MR. AND MRS. BRUCE CRANE, MR. JOHN CRANE, Miss LOUISE CRANE, MRS. MURRAY CRANE, MR. STEPHEN CRANE, MRS. W. CAREY COWDRY, DR. AND MRS. E. V. 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, MR. AND MRS. FREDERICK EWING, MR. AND MRS. FREDERIC 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. GlLCHRIST, MR. AND MRS. JOHN M. GlLDEA, DR. AND MRS. E. F. GREEN, Miss GLADYS M. HAIG, MRS. R. H. HAMLEN, MR. AND MRS. J. MONROE HARRELL, MR. AND MRS. JOEL E. HARRINGTON, MR. AND MRS. ROBERT HERRINGTON, MRS. A. W. S. HERVEY, DR. AND MRS. JOHN P. HlRSCHFELD, MRS. NATHAN B. HOUSTON, MR. AND MRS. HOWARD JEWETT. MRS. G. F. KEITH. MR. AND MRS. HAROLD C. KING, MR. AND MRS. FRANKLIN KOLLER, MR. AND MRS. LEWIS LEMANN, MRS. BENJAMIN 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 54 MARINE BIOLOGICAL LABORATORY MITCHELL, MRS. JAMES McC. MIXTER, MRS. W. JASON MOSSER, MRS. BENJAMIN D. MOTLEY, MRS. THOMAS NEWTON, Miss HELEN NICHOLS, MRS. GEORGE NICHOLSON, REV. ROBERT W. NIMS, MRS. E. D. NORMAN FUND, INC., AARON E. PACKARD, DR. AND MRS. CHARLES 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. LAWRASON RIVINUS, MRS. F. M., JR. ROOT, MRS. WALTER S. ROZENDAAL, DR. H. M. 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 STOCKARD, MRS. CHARLES R. STONE, MR. AND MRS. LEO STONE, MR. AND MRS. S. M. STRAUS, DR. AND MRS. DONALD B. 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, Miss EMILY WILSON, MRS. EDMUND B. WOLFINSOHN, MRS. WOLFE V. REPORT OF THE LIBRARIAN During 1959, forty-eight new journals were acquired making a total of 1665 currently-received titles. Of these, there were 484 (12 new) Marine Biological Laboratory subscriptions, 638 (9 new) exchanges and 184 (5 new) gifts; 100 (7 new) were Woods Hole Oceanographic Institution subscriptions; 199 (12 new) were exchanges and 60 (3 new) were gifts. Between the years 1950 and 1959, 439 new journals were obtained with initial date of publication, in each case, falling within that period. The Laboratory purchased 92 books (15 of these from the Montgomery Memo- rial Fund), received 119 complimentary copies (7 from authors and 112 from publishers) and accepted 43 miscellaneous gifts. The Institution purchased 50 books and received 6 as gifts. The total number of books accessioned amounted to 310. Through purchase, exchange and gift the Laboratory completed 10 journal sets and partially completed 15. The Institution completed 6 sets and partially com- pleted 6. There were 5,629 reprints added to the collection, of which 1772 were of current issue. At the close of the year there were 76,073 bound volumes and 212,627 reprints. The Library mailed out on inter-library loan 384 volumes and borrowed 72. About 900 volumes were bound, as well as 85 pamphlets. Dr. E. V. Cowdry presented his large collection of reprints to the Library, of which 2000 were added to the shelves. Among his collection there were several journal numbers which filled in gaps of long standing. Dr. F. A. Hartman pre- sented a collection which will be processed in 1960. Also, gifts of reprints and books were received from Dr. H. W. Kaan, Dr. P. W. Whiting and Mrs. A. R. REPORT OF THE LIBRARIAN 55 Memhard. Dr. W. R. Amberson donated a long series of the serial entitled "Onderzoekingen gedaan in het Physiologisch Laboratorium der Rijksuniversiteit te Utrecht," as well as several books. To each of these generous friends the Lab- oratory wishes to extend grateful thanks for the valuable literature acquired by the Library. Two foreign institutions benefited from the collection of duplicates, namely, the National Institution of Oceanography in England and the Marine Biological Lab- oratory at Helsingor, Denmark. During 1959, the Staff noticed a considerable increase in the use of the Library during the winter months. This is very gratifying, as it indicates further year- round use of the Library facilities, as Woods Hole becomes more and more a scientific research center. Respectfully submitted, DEBORAH L. HARLOW, Librarian VI. GENERAL BIOLOGICAL SUPPLY HOUSE, INC. It would seem that a short resume of the history of "Turtox" would be of interest to the members of the Corporation at this time. In 1913, Morris Wells was a graduate student in the Department of Zoology of the University of Chicago. Dr. Frank R. Lillie was Chairman of the Depart- ment, and also President of the Marine Biological Laboratory. Prior to this, Mr. Wells had taught biology for one year in a high school in Kansas. This experience made him realize that biology teachers needed aid in obtaining material for instruc- tion. In 1914, he and his wife prepared a one-page mimeographed sheet, listing slides and other material, which was mailed to a list of biology teachers. Several orders for $1.00 each were received, which were processed in the cellar of Mrs. Wells' parents' home. Mr. Wells received his Doctor's degree in 1915, and accepted a position as Instructor under Dr. Frank R. Lillie, at the University of Chicago. He was promoted to the rank of Assistant Professor three years later. By 1918, Dr. Wells realized he could no longer do justice to his teaching, and take care of his growing business. He discussed this with Dr. Frank Lillie. Dr. Lillie suggested that if Dr. Wells wished to devote all his time to furnishing biological supplies to teachers, he might interest his brother-in-law, Mr. Charles Crane, in the business. A corporation was formed, under the laws of the State of New York ; Dr. Frank Lillie was Chairman of its Board of Directors for many years. Mr. Crane purchased 51% of the stock of the new company, and turned it over to the Marine Biological Laboratory as a gift. The amount involved was $10,000. In 1920, due to the growth of the business, the General Biological Supply House increased their stock by $5000, and the M. B. L. purchased one-half of the new issue for $2500. In 1921, the Treasurer's report of the M. B. L. shows the entire holdings of General Biological Supply House stock held by M. B. L. listed at $12,700. This figure includes 51% of the voting stock. The purpose of this arrangement was to keep the business in the control of a scientific institution, which would prevent any 56 MARINE BIOLOGICAL LABORATORY possible future hazards of the business being run just for the profit of the private owners of the stock. Dr. Wells insisted from the start that the most important functions of the new company were to help biology teachers get material, and pass on to them information concerning new techniques. The new company was called "The General Biological Supply House Inc." and Dr. Wells coined the word "Turtox," with a design of a turtle holding up the world. Information about the new company during the first five years is not available, except that 8% of the par value of the stock was paid in dividends during that period. From 1923 to the present date, a period of 36 years, the M. B. L. has received $476,144 in dividends and its stock interest in the company at this time is worth well over $500,000. In 1919 Dr. Wells had a student in one of his courses who seemed to have potentialities. Dr. Wells suggested that he continue his courses in fundamental biology, and take courses in commerce. This student followed Dr. Wells' sugges- tion, and took his degree in Business Administration. He worked part time, as a student, for "Turtox," and became a full time employee when he graduated. Dr. Wells was ill for long periods, and this man was elected Vice President. When Dr. Wells died, he became President, a position he still holds. He is, of course. Mr. C. Blair Coursen. Part of Blair Coursen's work until recently was to edit the magazine, "Turtox News" ; Mrs. Shepherd, who worked with Mr. Coursen for several years, is the present editor. "Turtox News" devotes more than half its pages to non-advertising material. Secondary school biology teachers find it "good reading." In 1955, General Biological Supply House moved to a new building, especially designed for their operation. This building has proved to have been a wise investment. In 1957, the Board of Directors established the "Turtox Scholarship." Any American citizen who is currently, or has been, enrolled in a graduate school of biology is eligible. The award is based upon evidence bearing upon the promise of the applicant as a prospective teacher and research scholar. The stipend is $5000 per year one of the largest scholarships available. The Scholarship Com- mittee consists of Dr. Frank A. Brown, Jr., Chairman, Dr. Philip B. Armstrong, Dr. C. E. Olmstead, Dr. D. P. Rogers and Dr. S. Meryl Rose. In preparing this report, I wish to acknowledge help from Mr. C. Blair Coursen, Mrs. Edith Wells, Dr. Winterton C. Curtis, Mr. Homer P. Smith and the Librarian, Mrs. Deborah L. Harlow\ The successful operation of the General Biological Supply House Inc. reflects the management of its President, C. Blair Coursen, ably assisted by the Vice Presi- dent, Arold Blaufuss, the Export Manager, Charles Coursen, Jr., and the As- sistant to the President and Editor of "Turtox News," Mrs. Ruth L. Shepherd. They, together with a group of well trained and loyal employees, have rendered a unique service to teachers of biology throughout the world. Thus, the original purpose of its founder. Dr. Morris Wells to give aid and assistance to biology teachers is being carried out. Respectfully submitted, C. LLOYD CLAFF THE EFFECT OF SALINITY ON GROWTH OF GYMNODINIUM BREVE DAVIS DAVID V. ALDRICH AND WILLIAM B. WILSON Biological Laboratory, U. S. Bureau of Commercial Fisheries, Galveston, Texas Field observations have established the close physical association of mass mortalities of marine animals in the Gulf of Mexico with high concentrations of the non-thecate dinoflagellate Gymnodiniuw breve Davis (Davis, 1948; Galtsoff," 1948, 1949; Gunter ct al, 1948"; Wilson and Ray, 1956, among others). With the development of satisfactory culture media (Wilson and Collier, 1955) and successful methods for growing the organism in the absence of bacteria (Ray and Wilson, unpublished results), more definitive study of this association became possible. Subsequently Ray and Wilson (1957), and Starr (1958) conclusively demonstrated the toxicity of G. breve to fishes. The catastrophic manifestations of naturally-occurring G. breve blooms have attracted considerable attention. The sporadic nature of the outbreaks has stimu- lated particular interest in possible relationships between environmental factors and these "red tides." In this regard, various investigators, drawing from rela- tively sparse field data, have postulated the importance of salinity, dissolved nutrients, and meteorological conditions (see Ryther, 1955, for review). This report deals with the effect of salinity on the growth in vitro of G. breve and compares these findings with the field observations of other workers. The technical assistance of Mrs. Alice Kitchel is gratefully acknowledged. MATERIALS AND METHODS Bacteria-free cultures (10-ml. aliquots in 16 X 125 mm. screw-capped Pyrex tubes) were employed throughout this work. These tubes, together with the flasks and pipettes used in medium preparation or inoculation, were rigorously cleaned before each use. The cleaning routine, found by Ray and Wilson (un- published results) to be an important factor in the successful culturing of this organism, included the use of a detergent, hot lO^o nitric acid, and repeated rinses in tap water and distilled water. In an additional step, culture tubes were filled with, and inoculation micropipettes immersed in, triple-distilled water and autoclaved for 15 minutes at 15 p.s.i. All control tubes contained a completely synthetic medium (Table I), com- pounded by one of us (W.B.W.) which supported good growth of G. breve. The medium in experimental tubes differed from control medium only in major salt content (NaCl, MgSO 4 , MgCl,, CaCL and KC1). In varying salinity these major constituents were varied proportionally, thus producing no change in the ion ratios. 57 DAVID V. ALDRICH AND WILLIAM B. WILSON Pasteur capillary pipettes were used to inoculate each of a series of tubes of medium with 100-200 cells from well-established cultures of G. breve. Two or three cultures were used to inoculate each experiment. Equal numbers of replicates from each salinity group were inoculated with a given culture so that physiological differences between inocula would not bias results. After inoculation, the new cultures were maintained at a temperature of 26-27 C., and illuminated by two 30-watt "standard cool white" fluorescent lights two to three inches from the culture tubes. Glassware and media were sterilized by autoclaving at 15 p.s.i. for 15 minutes. After inoculations were completed, two bacterial sterility tests were conducted for each culture used as inoculum. These tests involved pipetting 1 ml. of the inoculum culture into each of two culture tubes, one containing 10 ml. TABLE I Gymnodinium breve culture medium NaCl* 29.0 gm. KNO 2 1.0 mg. MgSO 4 -7H 2 O 6.0 gm. KNO 3 1.0 mg. MgCl 2 -6H 2 O 4.5 gm. Thiaminef 1.0 mg. CaCl 2 0.7 gm. Vitamin B J2 t 1.0 Mg- KC1 0.6 gm. Biotinf 0.5 /ig. Tris(hydroxymethyl)- 20.0 mg. Sulfide solution! 5.0 ml. aminomethane** Na 2 S-9H 2 O 3.0 mg. Metals solution 20.0ml. K 2 HPO 4 1.0 mg. Triple-distilled water 1000 ml. * A. R. grade, recrystallized from triple-distilled water by the addition of C. P. HC1. All other inorganic compounds were C. P. or A. R. grade. ** Fisher Scientific Co. Added as 50 ml. of a stock solution adjusted to pH 8.2 by the addition of HC1. t Nutritional Biochemicals Corp. J Five ml. of this solution (derived from van Niel, 1931) contains: NH 4 C1, 1.0 mg. ; NaHCOs, 1.0 mg.; Na 2 S-9H 2 O, 0.8 mg. ; KH 2 PO 4 , 0.5 mg. ; MgCl 2 -6H 2 O, 0.2 mg. Twenty ml. of this solution contains: (Ethylenedinitrilo)tetraacetic acid disodium salt (Eastman Kodak Co.), 3.0 mg.; Mn as MnCl 2 -4H 2 O, 0.2 mg. ; Rb as RbCl, 0.2 mg. ; Alas A1C1 3 - 6H 2 O, 0. 1 mg. ; Co as CoCl 2 6H 2 O, 0. 1 mg. ; Cs as CsCl, 0. 1 mg. ; B as H 3 BO 3 , 0. 1 mg. ; Se as H 2 SeO 3 , 0.1 mg.; Cr as K 2 CrO 7 , 0.1 mg. ; Mo as Na 2 MoO 4 -2H 2 O, 0.1 mg. ; Sr as SrCl 2 -6H 2 O, 0.1 mg. ; Ti as TiO 2 , 0.1 mg. ; Zn as ZnCl 2 , 0.1 mg.; Zr as ZrOCl 2 -8H 2 O, 0.1 mg.; Ba as BaCl 2 , 0.02 mg. ; Cd as CdCl 2 -2iH 2 O, 0.02 mg.; Cu as CuCl 2 , 0.02 mg.; Fe as FeCl 2 -4H 2 O, 0.02 mg. ; Ce as (NH 4 ) 2 Ce(NO 3 ) 6 , 0.02 mg. ; V as NH 4 VO 3 , 0.02 mg. ; Ni as NiCl 2 -6H 2 O, 0.02 mg. ; Rh as RhCl 3 , 0.02 mg. ; Ru as RuCl 3 , 0.02 mg. ; Sn as SnCl 2 -2H 2 O, 0.02 mg. of peptone sea water broth, the other a 10 ml. peptone sea water agar slant (Spencer, 1952). Growth of the dinoflagellate was estimated by visual examination of the tubed cultures with the aid of a stereoscopic miscroscope, using 9x magnification for most cultures, and 18 X or 27 X when populations were low. Eleven graded population categories were adopted and "peak population" arbitrarily defined to include the top three. A rough calibration of this method was carried out by making estimates and actual cell counts from the same cultures, and comparing results. This check was conducted on four occasions, and, in all, 99 test cultures were examined by both methods. Cultures showing peak population by visual SALINITY AND GYMNODINIUM BREVE 59 100 50 2 o < 100 i| Q. o Q. UJ Q. 50 t too CO UJ oe { s 1 1 *l 1 1 1 1 1 1 35 DAYS 1 i s 1 t , M I 1 1 1 1 1 1 * 25 DAYS i !i i i 50 o u. o 18 DAYS [. I I |-L 50 10 DAYS 22 24 26 28 30 32 34 36 38 40 42 44 46 SALINITY %o FIGURE 1. Rate of peak population development of Gymnodinium breve at various salinities. Each point is based on 10 replicate cultures. 60 DAVID V. ALDRICH AND WILLIAM B. WILSON estimate never proved to have fewer than 750 cells per ml., and usually contained from one thousand to several thousand cells per ml. Although sacrificing a degree of quantitative accuracy, the visual estimate method was selected because of the ease, speed, and lack of bacterial contamination with which it could be performed. All cultures were examined by this method at 4, 10, 18, 25, and 35 days after inoculation. The five-week period was considered adequate, since cultures seldom grow after five weeks post-inoculation. Five experiments were conducted, each having nine salinity levels and ten replicate tubes at each level. The first experiment had the widest salinity range (6.3, 8.4, 11.9, 13.7, 17.8, 18.3, 25.8, 31.7, and 41.1% ). The four later'experi- ments were designed for a salinity range of 22.5 to 4l.l%o with intervals of ap- proximately 2.3%c. However, due to technical error, the last two experiments had ranges of 25.4 to 36.3%c and 23.8 to 46.0% and somewhat irregular intervals. RESULTS Cultures of G. breve grew well throughout a salinity range of 27 to 37%c (Fig. 1). Within this range, at least 80% of replicate cultures reached popula- tions of 750 cells or more per ml. during the five-week experimental period. More variable results and generally poorer growth occurred at salinity levels immediately adjacent to the optimal range. No instances of optimum growth occurred at less than 24% c or more than 44%c. Some indication of comparative growth rates may also be obtained from Figure 1. It is apparent that cultures reach high populations more rapidly within the optimal range (27 to 37%o). Some organisms survived throughout a salinity range of 22.5 to 46.0%o. From 24.8 to 46.0%c, 91% of cultures contained living cells at the end of the five-week observation period. There was no indication of reduced survival at the extremes of this range. Below this range the incidence of survival was lower; at 23.8%o the organism survived in only one of 10 replicate cultures, and at 22.5%c 10 of 20 replicates contained surviving cells. No instances of five-week survival were noted at any of the tested salinity levels below 22.5% c . At 18.3 and 17.8% three and two, respectively, of the 10 tubes in each group contained a few live organisms 10 days after inoculation, but no survivors were found eight days later. Media with salt concentrations of 13.7/ansa, Thysanosoma actinioidcs, and Cittotaenia per pie xa. Exp. Parasit., 9: 1-8. CAMPBELL, J. W., 1960b. Pyrimidine metabolism in parasitic flatworms. Biochem. J., in press. CANELLAKIS, E. S., J. J. JAFFE, J. R. MANTSAVINOS AND J. S. KRAKOW, 1959. Pyrimidine me- tabolism. IV. A comparison of normal and regenerating rat liver. /. Biol. Chan., 234: 2096-2099. CRUMPLER, H. R., AND C. E. DENT, 1949. Distinctive test for a-amino acids in paper chroma- tography. Nature, 164 : 441-442. CRUMPLER, H. R., C. E. DENT, H. HARRIS AND R. G. WESTALL, 1951. /3-Aminoisobutyric acid (a-methyl-jtf-alanine) : A new amino acid obtained from human urine. Nature, 167: 307-308. FINK, R. M., C. MCGAUGHEV, R. E. CLINE AND KAY FINK, 1956. Metabolism of intermediate pyrimidine reduction products in vitro. J. Biol. Chcm., 218 : 1-7. FOWDEN, L., 1951. The quantitative recovery and color imetric estimation of amino acids separated by paper chromatography. Biochem. J., 48 : 327-333. HAUSMANN, W., 1952. Amino acid composition of crystalline inorganic pyrophosphatase isolated from bakers yeast. /. Amer. Chcm. Soc., 74: 3181-3182. HULME, A. C., AND W. ARTHINGTON, 1950. 7-Amino-butyric acid and /3-alanine in plant tissues. Nature, 165: 716-717. LANG, C. A., 1958. Simple microdetermination of Kjeldahl nitrogen in biological materials. Analyt. Chem., 30: 1692-1694. LEVY, A. L., AND D. CHUNG, 1953. Two-dimensional chromatography of amino acids on buffered papers. Analyt. Chcm., 25 : 396-399. PoCHEDLEY, D. S., 1956. II. The chromatographic separation of amino acids from insect blood. Trans. N. Y. Acad. Sci., 19: 19-22. p-AMINO ACIDS IN FLATWORMS 79 ROBERTS, E., S. FRANKEL AND J. H. PINCKNEY, 1950. Amino acids of nervous tissue. Proc. Soc. Exp. Biol Med., 74: 383-387. SIMPSON, J. W., K. ALLEN AND J. AWAPARA, 1959. Free amino acids in some aquatic inverte- brates. Biol. Bull.. 117: 371-381. SYNGK. R. L. M., 1951. Methods for isolating omega-ammo acids: gamma-aminobutyric acid from rye grass. Bioclicin. J., 48 : 429-435. TALLEN, H. H., S. MOORE AND W. H. STEIN, 1954. Studies on the free amino acids and re- lated compounds in the tissues of the cat. /. Biol. Chcm., 211: 927-939. VIRTANEN, A. L, AND T. LAINE, 1937. The decarboxylations of d-lysine and 1-aspartic acid Enzymologia, 3 : 266-270. THE FEEDING MECHANISM IN THE SAND DOLLAR MELLITA SEXIESPERFORATA (LESKE) IVAN GOODBODY Department of Zoology, University College of the West Indies, Mono, Jamaica MacGinitie and MacGinitie (1949) give the following account of feeding in the scutellid echinoderm Dendraster excentricus (Eschscholtz) (p. 237) : "The spines on the upper side of the sand dollar are club shaped and are covered by cilia. These cilia create currents that flow from the direction in which the animal is moving toward what could be called the posterior edge. ... As the currents flow through these spines, little eddies are created at the posterior sides of the spines. These eddies allow tiny particles and organisms to become trapped in mucus that is secreted onto the surface of the spines. This mucus goes downward and is led into tiny tracts to unite with others. These in turn unite again, passing around the edge to the underside, until near the mouth five tracts or strings of mucus feed directly into the mouth of the sand dollar." This appears to be the only available account of feeding in the Clypeasteroida. Hyman (1958) studied the five-lunuled sand dollar Mellita quinquiesperforata (Leske), but was unable to elucidate the feeding mechanism. The present paper describes observations made on another of the lunuled sand dollars, Mellita (Lcodia) sexiesperjorata (Leske), and shows that it is a ciliary mucus feeder collecting particles on the aboral surface and transporting them through the lunules and around the margin of the test to food tracts on the oral surface. M. sexiesperjorata is common in certain shallow water sandy areas around Jamaica and normally lives either in the surface layer of the sand, so that its outline is discernible from above, or else buried in the sand very close to the surface. The animals used for these observations were collected on the Port Royal Cays and observations were made both in the aquarium and in dishes under a microscope. MORPHOLOGY Figure 1 shows an individual in both oral and aboral views. The size of the animals varies considerably, fully grown animals being about 70 to 80 mm. in diameter. In surface view the animal has a roughly pentagonal outline and its surface is pierced by six slit-like lunules : five of these are in the ambulacral areas, the sixth or anal lunule is interambulacral and marks the posterior side of the animal. The anterior side of the test is markedly pointed. Both aboral and oral surfaces are densely clothed with short spines which are described below. On the aboral side of the only other structures visible are lunules, petaloicls, and gono- pores. On the oral surface the mouth is central and the anus lies posterior to it, just on the edge of the anal lunule. Leading away from the oral margins of the five ambulacral lunules are a number of broad food tracts: one of these, the radial tract, leads straight from the inner tip of the lunule to the mouth, the remainder 80 FEEDING IN MELLITA 81 run laterally from the lunule and terminate in the ambulacra! or food grooves. These are deep hut narrow grooves, a pair to each ambulacral area running into the mouth; the two members of a pair form a petaloid outline and unite just before reaching the mouth. The food tracts of the anal lunule are less well developed and discharge laterally to the ambulacral grooves of the two neighboring lunules. Figure 2 shows the profile of a sagittal section through the test and shows that it is thin around the margin and dome-shaped in the center ; the anterior margin is thicker than the posterior margin. The mean measurements for the ten indi- viduals of 60 to 65 mm. diameter are : Anterior margin : 1.8mm. 0.04 Central dome: 5.9mm. 0.49 Posterior margin : 1.0mm. 0.01 It is significant that the animal progresses through the sand with the anterior and thicker margin foremost. Podia On the oral surface there are dense concentrations of podia around the periphery of the test, around the margins of the food tracts and in the lunules. They are less dense on the remainder of the ambulacral areas and are absent from areas FIGURE 1. a. The aboral surface of a living Mcllita sexiesperforata. The anterior end is towards the top of the picture. The club-shaped spines appear as minute spots all over the surface of the animal. Notice that the peripheral ( ambulatory ) spines are more dense anteriorly than posteriorly. The ring of protective spines can be seen clearly around the margin of each lunule. 82 IVAN GOODBODY FIGURE 1, b. The oral surface of a living McUita sexiesperforata stained for two minutes in toluidine blue. Orientation as in Figure 1, a. The stain is taken up by podia and other structures, and was used to improve contrast. The mouth is central and the anal papilla appears as a black spot between it and the anal lunule. The anal spines between anus and mouth are just visible. A Ambulatory spine; F = Food tracts; G = Food (ambulacra!) grooves. with ambulatory spines. On the aboral surface there are only a few scattered podia. When extended the podia are very long and thin with a slightly swollen tip and poorly developed sucking disc. They function exclusively as an accessory food collecting device (see below) and do not appear to play any part in locomotion. Spines There are four principal types of spine. On the oral surface ambulatory and non-ambulatory spines can be distinguished. Ambulatory spines are confined to five locomotory areas in the interambulacra of the oral surface. In the anal inter- ambulacrum they form a transverse group behind the anal lunule, in the other four interambulacra they form a radially disposed wedge-shaped group (Fig. Ib). These spines, which are long and thick (1300//, X 85 /A) normally have a rounded tip but in many spines the tip is abraded into a roughened end (Fig. 3, A) ; their FIGURE 2. Profile of McUita sexiesperforata. The anterior end is toward the right of the picture. FEEDING IN MELLITA 83 function appears to be entirely that of locomotion. Similar spines are found all around the margin of the test hut they are almost twice as thick (1300 /A X 160 /A) ; they are densely arranged anteriorly and more scattered posteriorly. Their extra thickness is probably correlated with stresses set up during movement through the sand in a horizontal direction. Non-ambulatory or protective spines of the oral surface are shorter and thinner (430 /AX 30 /A) than ambulatory ones and are often bent near the middle of their length (Fig. 3, B) ; they cover all the re- maining areas of the oral surface. Similar spines are found in the lunule and on the aboral margin of the lunule they form a protective ring projecting up higher than the other spines of the aboral surface ; here they prevent very large sand particles from entering the lunule and blocking it. On the aboral surface there are two types of spine distributed together all over the animal. The larger of these are club-shaped spines which move the A/ FIGURE 3. The principal types of spine found in Mcllita sexiesj>erforata. A = Ambulatory spine with abraded tip. B = protective spine of oral surface. C : - Two views of club-shaped spines of aboral surface. D = Aboral miliary spine. sand grains posteriorly over the aboral surface (Fig. 3, C). The club-shaped head of these spines is set at a slight angle to the stem and its tip is oriented toward the margin of the test. The others or miliary spines (Fig. 3, D) are shorter than the club-shaped spines and are characterized by having a sac-like swelling on the tip ; this sac contains yellowish granules which stain darkly with toluidine blue. The epithelium at the base of the cilia on these spines stains purple with the same stain and it seems plausible therefore to suggest that these spines are the principal site of mucus secretion. Miliary spines also occur on the inner walls of the lunules and along the food tracts of the oral surface ; the latter have a smaller sac than those of the aboral surface or lunule. As well as these four main types of spine there is a group of very large spines forming a circle between the mouth and anus and with their tips overarching the anus. They play an important part in preventing feces from entering the mouth. 84 IVAN GOODBODY These spines are in three groups: seven or eight very long ones (up to 5 nun. in length) form a group nearest to the anus, outside these there are ten to twelve of intermediate size, then a group of many smaller ones forming an outer ring next to the mouth. (Fig. Ib). Cilia Cilia are confined to the epithelia of the spines and are found on only one side of the stem. This side of the stem is here referred to as its back and the cilia beat in such a way as to drive a horizontal current from it in a backward direction. On the aboral surface the club-shaped spines have dense ciliation at the base while the miliary spines have dense ciliation along the whole length of the stem. The club-shaped and miliary spines are oriented in such a way as to produce centrifugal currents across the aboral surface leading away from the center and towards the margins and lunules. On the oral surface the ambulatory spines have a thin ciliation at the base while the non-ambulatory spines have dense ciliation at the base producing a strong current. The ciliation of the miliary spines of the food tracts is similar to aboral miliary spines. At the margins of the oral surface the spines are oriented so as to drive currents in towards the center. In the food tracts they produce currents towards the mouth in the radial tracts and laterally towards the ambulacral grooves in the remainder. Over the rest of the ambulacral areas the currents drive towards the adjacent ambulacral grooves. FEEDING BEHAVIOR M. sexiesperjorata is a microphagous feeder. Removal and microscopal exami- nation of the food cord passing along the ambulacral groove shows that most particles are less than 20 p. in diameter and in a high proportion of them are of the order of 1 /A in diameter. Within these size limits there appears to lie no selection of different types of food algal cells, detrital particles and sand grains are all collected. Larger particles are sometimes collected and sand grains which only just fit in the groove have been seen passing along it and entering the mouth. An active animal ploughs slowly through the surface sand always keeping the anterior end (Loven's Ray III) forward. As it moves it builds up a small wall of sand in front of itself, the sand grains of which constantly fall down on the aboral surface where they are carried slowly backwards on the tips of the club-shaped spines. This movement of sand appears to be oriented posteriorly across the aboral surface without special reference to the lunules, but small sand grains which reach the margins of the lunules are carried down through them ; larger particles are carried off the posterier margin of the test. Particles entering the lunule usually do not drop through it, but are lowered slowly down by means of the spines and then deposited back in the substratum ; during this time they may be actively probed by podia in the lunule. suggesting that the latter may remove from it minute absorbed particles. If carmine particles instead of sand are placed on the aboral surface of the animal a further process of selection may be observed. Larger particles are treated exactly as sand and it can also be seen that the very small particles (visible only under the microscope) drop down between the club-shaped spines and are carried away by the ciliary currents around the spines. At this lower level some particles FEEDING IN MELLITA 85 travel to the margin of the test and then around it to the oral surface, other particles are carried to the lunules and pass down through them and onto the food tracts on the oral surface. By placing carmine particles in the food tracts of an inverted animal it can he shown that the ciliary currents lead to the ambulacral grooves, except in the radial tract where they lead directly to the mouth. Carmine particles placed on the aboral surface of an animal appear in the ambulacral grooves in a few moments. In the ambulacral groove food can be seen to be loosely aggregated in mucus but the precise point at which the mucus is secreted is still in doubt. As pointed out above the miliary spines of the aboral surface are the most probable site of mucus secretion. In the ambulacral groove the food is carried along by podia and not by cilia. In summary, then, an animal ploughing through the sand pushes sand onto the aboral surface where it is crudely sorted, large particles being carried along on the tips of the club-shaped spines and ultimately deposited back in the sand either through the lunules or off the posterior edge of the test. Fine particles fall down between the bases of the spines where they are carried away by ciliary currents through the lunules to food tracts on the oral surface. From the food tracts cilia carry them to the ambulacral groove thence to the mouth in a mucus aggregation carried along by podia. There remains the question of food collection by the podia. Because they are confined to the oral surface it is difficult to see them in normal function, but following the method of Nichols (1959) I have examined them in a perspex box with the aid of a prism and a binocular microscope. Under these circum- stances the podia at the margin are seen to be constantly extending and contracting and probing the surroundings of the animal. If carmine particles or yeast stained in congo red are pipetted around the margin, particles may sometimes be seen to be picked up by the podia and drawn in to the margin of the animal where they are released and carried inwards by the ciliary currents. At the same time man}- podia are seen to extend and contract with no visible particles attached. However, only relatively small magnifications ( X 30) can be used successfully in examining with the prism and 1 believe that the principal function of these podia is to probe the surrounding sand for very small particles of food, i.e., particles of about 1 p. diameter. Mention has already been made of the manner in which sand grains are probed by podia as they pass down through the lunules. In the perspex box only a few scattered grains of sand can be included with the animal ; otherwise they obscure it from view. The animal thus rests directly on the bottom of the box and only the marginal podia, which extend laterally, can be extended effectively. It seems certain, however, that under normal conditions the podia of the oral surface all probe the sand in the same way, collecting small food particles. DEFECATION In an animal such as M. sexiesperjorata in which the anus is in close proximity to the mouth, special precautions are required to ensure that feces do not re-enter the mouth. Although many animals have been examined from time to time defecation has only been observed on a very few r occasions and it appears that it must be an intermittent and not a continuous process. Defecation commences with a pumping action of the anal papilla followed by 86 IVAN GOODBODY cessation of feeding activity; passage of the mucus cord in the ambulacra! groove stops completely. The spines between the mouth and the anus beat gently away from the mouth and over the anus, and all around this area pedicellariae become intensely active. The tip of the anus is directed towards the anal lunule and feces are ejected in intermittent puffs of loose particles and not in a mucus string. Examination of a defecating animal from below shows that feces are not, as might 1)e expected, carried up through the anal lunule and so removed in water currents. They fall down from the animal and must in normal circumstances be deposited back in the sand in which the sand dollar community is feeding. Grateful acknowledgment is made to the Nuffield Foundation from whom the author was in receipt of a research grant when these observations were made. SUMMARY 1. A brief description of Mellita sc.riespcrforata is given and the process of food collection and defecation are described. 2. Sand pushed onto the aboral surface is sorted by club-shaped spines. Fine particles drop down between the spines and are carried round to the oral surface by ciliary currents, thence to the ambulacra! grooves. Mucus is probably secreted by the miliary spines on the aboral surface ; podia play an accessory role in food gathering. 3. Defecation is an intermittent process and feeding stops while it is in progress. Spines and pedicellariae prevent feces from reaching the mouth. LITERATURE CITED HYMAN, L. H., 1958. Notes on the biology of the five-lunuled sand dollar. Biol. Bull., 114: 54-56. MAcGiNixiE, G. E., AND N. MAcGiNixiE, 1949. Natural History of Marine Animals. New York. McGraw-Hill Book Co. NICHOLS, D., 1959. Changes in the chalk heart urchin Micrastcr interpreted in relation to living forms. Phil. Trans. Roy. Soc. Scr. B. 242 : 347-437. CLEAVAGE WITH NUCLEUS INTACT IN SEA URCHIN EGGS ETHEL BROWNE HARVEY Marine Biological Laboratory, Woods Hole, Massachusetts, and the Biological Laboratory, Princeton University, Princeton, Nezv Jersey Though, in general, nuclear changes associated with mitosis precede the cleavage of a cell, there are some rare cases in which this is not true. It has been found that in some cases in developing sea urchin eggs, the nucleus may remain as it is in a resting cell, hut nevertheless the cell may cleave as it does after mitosis, and produce two quite normal "resting" cells. This has been found to occur in centrifuged eggs which have been stimulated to develop parthenogenetically by treatment with hypertonic sea water (30 grams of NaCl per liter of sea water) for 5 to 15 minutes. After one half to one hour, the egg nucleus remains un- changed but a cleavage plane may come in between the nucleate part of the cell and the non-nucleate part, as it does in such eggs when fertilized (see E. B. Harvey, 1932), resulting in two cells. This has been found to occur in Sphae- rcchinus granularis, Psaininccltiniis (Parccliinus) inicrotuberculatus and more recently in Arbacia punctulata and A. pustulasa (photographs of living eggs are reproduced in Figs. 1-8). These occasional cases have been observed over a period of twenty years. No further development or change has been observed. It has not been possible to produce such a cleavage lacking nuclear change, with any of many chemical substances tried. There are in the literature a few references indicating that the nucleus may be removed experimentally. Mazia and Dan (1952) succeeded in removing the mitotic apparatus in an isolated condition from the "fixed" Strongylocentrotus franciscanns egg, and later Dan and Nakajima (1956) removed it "fixed" from other sea urchins, Pscudoccntrotits depressus and Hcmicentrotus pulcherrimus, with observations also on Arbacia punctulata. According to Swann and Mitchison (1953), the eggs of the heart urchin, Clypeaster japonicus, may be treated with concentrated colchicine at mid-anaphase, completely abolishing the asters and spindle, and still the egg will divide. There is, of course, the possibility that some part of these structures still remains. To make the experiment more decisive, Hiramoto (1956) sucked out the spindle and asters with a micropipette inserted into the egg, and he found that cleavage still took place. Some years ago (1938), I made a reference in one of my papers to "cleavage planes coming in independently of any nuclear changes" (p. 182) in sea urchin eggs, and Holtfreter called attention to this in his 1948 paper (p. 723). My paper was accompanied by photographs (44 and 57, 58). SUMMARY There now seems no doubt that cleavage can take place without any visible change in the nucleus. 87 88 ETHEL BROWNE HARVEY j ' FIGURES 1-8. Living eggs. CLEAVAGE WITH NUCLEUS INTACT 89 LITERATURE CITED DAN, K., AND T. NAKAJIMA, 1956. On the morphology of the mitotic apparatus isolated from echinoderm eggs. Embryologia, 3 : 187-200. HARVEY, E. B., 1932. The development of half and quarter eggs of Arbacia pnnctulata and of strongly centrifuged whole eggs. Biol. Bull.. 62 : 155-167. HARVEY, E. B., 1938. Parthenogenetic merogony or development without nuclei of the eggs of sea urchins from Naples. Biol. Bull. 75: 170-188. HIRAMOTO, Y., 1956. Cell division without mitotic apparatus in sea urchin eggs. .r/>. Cell Res.. 11: 630-636. HOLTFRETER, J., 1948. Significance of the cell membrane in embryonic processes. Ann. N. ) . A cad. Sci.. 49: 709-760. MAZIA, D., AND K. DAN, 1952. The isolation and biochemical characterization of the mitotic apparatus of dividing cells. Proc. Nat. Acad. Sci., 38 : 826-838. SWANN, M. M., AND J. M. MITCHISON, 1953. Cleavage of sea-urchin eggs in colchicine. /. Exp. Biol., 30: 506-514. FIGURES 1-3. Stratification of unfertilized eggs of Arhacia piinctulnta with centrifugal force. FIGURES 4-6. Cleavage of centrifuged, parthenogenetic eggs of Arbacia pnnctulata. without the nucleus taking part. Notice the nucleus in Figure 4, slightly enlarged. It becomes some- times, not always, slightly enlarged. FIGURE 7. Cleavage of parthenogenetic, centrifuged eggs of Sphacrcchimts grannlaris, without the nucleus taking part. FIGURE 8. Cleavage of parthenogenetic, centrifuged eggs of Psammechinus (Parcchinus) microtuberculatus without the nucleus taking part. DEVELOPMENTAL STAGES OF THE BROAD BREASTED BRONZE TURKEY EMBRYO ' - A. M. MUN * AND I. L. KOSIN * Department of Poultry Science, Washington State University, Pullman, Washington In studying early mortality in the turkey embryo, it became necessary to determine with considerable accuracy the extent of development of the embryo. Phillips and Williams (1944) described the Black and the Beltsville Small White turkey embryos after different durations of incubation. However, chrono- logical age, Lc., incubation time, per sc, is not a reliable expression of the extent of morphological differentiation of the embryo. Such factors as temperature and humidity during incubation, genetic composition, and size of the egg. have been shown to affect the rate of growth of the avian embryo (for review, see Landauer, 1951). It has previously been shown in this laboratory (Kosin and St. Pierre, 1956) that storage of Broad Breasted Bronze hatching eggs for 8 to 14 days results in a lowered mean somite count after 60 hours of incubation, as compared with eggs held for 1 to 7 days. Hamburger and Hamilton (1951) established a series of normal stages of development for the chick embryo, based on various morphological criteria. These criteria were found to be useful in this laboratory in estimating the extent of development of the turkey embryo, although the turkey embryo takes approximately 28 days to hatch as compared with 21 days in the chicken. Thus, the major objective of the study reported in this paper was to determine for turkey embryos the period of incubation necessary to obtain the different stages of embryonic development described for the chicken by Hamburger and Hamilton (1951 ). Similar studies on "staging" of embryonic development in Aves have previously been reported by Rempel and Eastlick (1957) and Koecke (1958) for the White Silkie bantam chicken and the Khaki Campbell and White Indian Runner ducks, respectively. MATERIALS AND METHODS All embryos used in this study were obtained from eggs produced by a flock of Broad Breasted Bronze (BBB) turkeys maintained at the Station. The birds 1 Scientific Paper No. 1958, Washington Agricultural Experiment Stations, Pullman. Project No. 717. 2 Supported, in part, by Federal Funds for Regional Research (W-7) under the Hatch Amended Act. 3 Present address : Department of Embryology, Carnegie Institution of Washington, Balti- more 5, Maryland. 4 We wish to acknowledge the advice and help of Dr. Thomas J. Russell, Washington Agri- cultural Experiment Stations Statistician, in the analysis of the data on which this study is based. We are also indebted to Mrs. Lynne Frutiger, Mrs. Jewell Keeney, Mrs. Mary Ellen Schy, and Mrs. Jeannette Wright for their technical assistance in the collection of these data. 90 EMBRYONIC STAGES IN TURKKVS 91 were trapnested and the eggs were collected three times a day, after which they were placed in the holding room at 50 F. and 85 c /c relative humidity for not more than three to four days. The eggs were incubated for a desired length of time in a forced draft incubator at 99.5 F. The earlier embryos (1 to 7 days) were removed from the yolk, placed in chick Ringer's solution and then measured and staged. Each embryo was staged separately according to (1) the development of the mesodermal derivatives, e.g., somites; (2) the development of the ectodermal derivatives, e.g.. optic vesicles, neuromeres ; and (3) the development of the heart. The "average" stage of development of the embryo was then obtained from these three separate stagings. Although there were individual differences, no striking and consistent differences between the turkey and the chicken were observed in terms of rate of development in these three groups of morphological criteria. Older embryos ( 17 to 28 days) were fixed in Benin's fluid or Baker's calcium formol before staging. In the later stages of development (Hamburger-Hamilton, stage 36 to stage 40), the turkey embryo has a distinct structure, the "snood" or "leader," which was included among the criteria used for describing the stage of development of the embryo. Measurements of the beak and toe, which are the main criteria for identifying chick embryos from stage 40 to 44. were obtained for the turkey embryo. How- ever, owing to the relatively small increments of increase in length in these structures, measurements of the foot, i.e., from the outer edge of the tarsal joint to the tip of the claw of the third toe, were used to characterize the development from seventeenth to the twenty-seventh day of incubation. This study is based on the observation of more than 4000 embryos collected over a period of three years. FIGURE 1. Stage (Hamburger and Hamilton, 1951) of development of the turkey embryo after various periods of incubation. The figures in boxes indicate the number of specimens for each point on the coordinate. \. M. MUN AND 1. L. KOSIN TABLE I Anteroposterior (AP) lengths of the area pellucida and of the BRB turkey embryo at different stages of development Stage No. cases AP length (mm.) Sd Embryo length (mm.) Sd 4 11 3.3 .375 . _ 5 132 3.9 .423 6 233 4.3 .400 1.4* .390 7 101 4.7 .458 2.3* .242 8 52 5.3 .454 3.1** .341 9 2 5.6 3.8 10 5 6.3 4.6 . 11 9 7.0 .508 5.6 .225 12 12 7.0 .673 5.8 .310 * From the head fold to Hensen's node. ** From the tip of the head to Hensen's node. RESULTS AND DISCUSSION The approximate periods of incubation to obtain stages 1 to 39 can be obtained from Figure 1. There is a wide range of variation in the stage of development in the turkey embryo after a definite period of incubation. This becomes particu- larly apparent in the early stages. In the earlier stages (stages 4 to 12) the stage of the embryo can also be estimated from measurements of the anteroposterior lengths of the area pellucida or of the embryo, i.e., from the tip of the head, or in stage 6, from the head fold to Henson's node (Table I). These measurements are highly correlated with the stage and somite number ( Mun and Kosin, 1958). The development of the snood ("leader") is summarized in the following tabulation : Day of incubation 13th day 12 to 14 15 16 to 17 17 18 19th day Hamburger & Hamilton Stage 36 37 38 39 40 Snood characteristics Snood appears (Fig. 2). Snood is as high as it is wide at the base (Fig. 3). Snood is higher than wide and distinctly columnar in appear- ance (Fig. 4). Snood is columnar and almost twice as tall as it is wide. Papil- lae may be seen at the base of the snood (Fig. 5). The snood is covered with papillae (Fig. 6). The snood is larger and conical feather germs at base of snood are colored (Fig. 7). The feather germs are as long as the snood and may cover it completely (Fig. 8). The growth of the beak, toe, and foot from the seventeenth to the twenty-seventh day of incubation is presented, graphically, in Figure 9. Each point represents the average measurements of 10 to 21 embryos by three different individuals on FIGURE 2. Snood at 13th day of incubation. FIGURE 3. Snood at 14th day of incubation. FIGURE 4. Snood at 15th day of incubation. 93 FIGURE 5. Snood at 16th day of incubation. FIGURE 6. Snood at 17th day of incubation. FIGURE 7. Snood at 18th day of incubation. 94 EMBRYONIC STAGES IN TURKEYS 95 FIGURE 8. Snood at 19th day of incubation. bU o A Q FOOT TOE BEAK > { oc /i r\ i i : M r J )^^^ i > \ METERS 'fU c c j ^^--*" I < ^^ i -~ i _i d < 2 -2. ? ^" ""* ^ "*i p j i i , < X O z 1 /-\ < k < ! ' ^ ! : 1 ! ^ ! i 1 3 1 LU t _l [ 5 \ U J C XD | | ' I ' r ' r ' 3D I P f 1 1 7 1 8 1 9 2 2 1 2 2 2 3 2 4 2 5 2 6 2 PERIOD OF INCUBATION IN DAYS FIGURE 9. Length of beak, toe, and foot after different periods of incubation. 96 A. M. MUN AND I. L. KOSIN different groups of embryos at different times. The measurements were made with a pair of vernier calipers. As may be seen from Figure 9, the rate of growth of the foot is greater than the rate of growth of the beak and toe. A straight line obtained from the regression of the length of the foot on time was then constructed. The equation of this line (solid line in Figure 9) is as follows: Y -- - 25.3 + 2.9.v where Y - length of the foot in mm. and .v = period of incubation in days. The standard deviation of the regression line is 3.3 and the standard error of the slope is 0.051. The 95% confidence interval in age of the embryo, for any particular length of the foot, can be calculated from the following equation : = 36.6 + 0.35 To 0.7 o.00024(K - 40.0) 2 + 8.342 where Lx = upper limit of age of the embryo in days, Lx = lower limit of age of the embryo in days and Fo observed length of the foot in millimeters. Similarly, the 95% confidence interval for the length of the foot following a definite interval of incubation can be calculated from the following equation : = - 25.3 + 2.9J\r 6.47 l.002 + 0.00024(X - 22. 6) 2 Ly\ where Ly = upper limit of the length in mm., Ly = lower limit of the length in mm. and *o = observed period of incubation in days. This information has been used in our laboratory to approximate the time of death of the embryo, whether it was accidental, e.g., due to incubation failures, or due to causes associated with the problem of hatchability, and to compare the growth rates of embryos from different lines of BBB turkeys cultivated in vitro. SUMMARY 1. The period of incubation of Broad Breasted Bronze turkey eggs necessary to obtain the various normal stages of development established by Hamburger and Hamilton for the chick embryo is presented. 2. Data have been submitted on the development of the snood ("leader") in the turkey embryo. 3. Measurements of the beak, toe, and foot were obtained from the seventeenth to the twenty-seventh day of incubation. From these measurements, a straight line obtained from the regression of the length of the foot on time was constructed. The equation of this line is presented, as well as equations for determining the approximate age of the embryo from measurements of the foot, or the approximate length of the foot. LITERATURE CITED HAMBURGER, V., AND H. L. HAMILTON, 1951. A series of normal stages in the development of the chick embryo. /. Morphul., 88: 49-92. EMBRYONIC STAGES IX TURKEYS 97 KOECKE, H., 1958. Normalstadien der Embryonalentwicklung bei der Hausentee (Anas Boschas domestica). Embryologia, 4 : 55-78. KOSIN, I. L., AND E. ST. PIERRE, 1956. Studies on pre-incubation warming of chicken and turkey eggs. Poultry Sci., 35 : 1384-1392. LANDAUER, W., 1951. The hatchability of chicken eggs as influenced by environment and heredity. Bull. 262, Storrs Agric. Exper. Sta. ; 223 pp. MUN, A. M., AND I. L. KOSIN, 1958. The early turkey embryo, on media prepared from eggs of "high" and "low" hatchability hens. Growth, 22: 9-15. PHILLIPS, R. E., AND C. S. WILLIAMS, 1944. External morphology of the turkey during the incubation period. Poultry Sci., 23 : 270-277. REMPEL, A. G., AND H. L. EASTLICK, 1957. Developmental stages of normal White Silkie fowl embryos. Northzvest Science, 31 : 1-13. CAROTENOIDS AND CHLOROPHYLLIC PIGMENTS IN THE MARINE SNAIL, CERITHIDEA CALIFORNICA HALDEMAN, INTERMEDIATE HOST FOR SEVERAL AVIAN TREMATODES * 2 A. M. NADAKALs Department of Biology, University of Southern California, Los Angeles, California The marine snail, Cerithidea californica Haldeman, is a favorable host for more than twenty species of larval trematodes (Martin, 1955; Hunter, 1942). These larvae occupy different regions of the body of the snail such as the digestive gland, mantle, gills and part of the digestive tract. The digestive gland of the snail, which is the main organ of infection, presents a variety of coloration in different specimens. It may be green, brown, yellow, orange or creamy white. The visceral part including the digestive tract is frequently dark blue. The mantle and the integument are usually greenish-blue or blue and yellow intermingled. A striking similarity exists between the coloration of the snail tissues and that of the parasitic larvae harbored by them. A considerable amount of information is available concerning the occurrence and distribution of pigments, particularly carotenoids in various species of gastropod molluscs. Earlier work has been reviewed by Fox (1953) and Goodwin (1954). Although several species of snails are known to be hosts for pigmented larval trematodes, no critical study so far has been made concerning their pigments with a view to understanding the host-parasite relationship of pigmentation. In the snail, Littorina littorea, pigmented foot has been reported to be a means of recog- nizing infection with larval trematodes (Willey and Gross, 1957). Spectro- photometric absorption studies of L. littorea extracts indicated the presence of carotenoids ; however, chromatographic methods were not used for the separation of various pigments. The author was interested to study the chemical nature and origin of pigments found in certain species of larval trematodes harbored by the snail. Cerithidea (Nadakal, 1960a, 1960b). In order to trace the host-parasite relationship of pigments, it was necessary to analyze the pigments of the snail. The present paper describes the pigments found in Cerithidea with special reference to carotenoids. MATERIALS AND METHODS Specimens of Cerithidea and four species of algae, including three green and one red algae which serve as food for the snails, were collected from the mud flats of Newport Bay, California. The green algae were identified as Ulva sp., Chacto- 1 From a thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy, University of Southern California. - The author wishes to express his deep appreciation to Prof. W. E. Martin for the guidance, interest, and criticisms throughout the course of this work. 3 Present address : Department of Biology, Immaculate Heart College, Los Angeles 27, California. 98 PIGMENTS IN A MARINE SNAIL 99 morpha torta, and Enteronwrpha clathrata ; the red alga as Hypnca johnstonii. The algal pigments were studied by the methods of the following workers : Strain (1942) for chlorophyll a and b; Manning and Strain (1943) for chlorophyll d; and Haxo et al. (1955) for phycobilins. After sorting out the various snail tissues such as the digestive gland, mantle and foot, and visceral mass, they were lyophilized separately and ground in an ordinary mortar. The methods outlined by Fox and Pantin (1941) were followed with necessary modifications for the extraction and analysis of pigments from the snail tissues. Chromatographic separation of pigments was carried out according to the directions given by Karrer and Jucker (1950). A cylindrical glass tube measuring 20 cm. X 12 mm. was used for preparing the adsorption column. Among several adsorbents tried for the separation of various pigments, such as calcium hydroxide for epiphasic carotenoids, calcium carbonate and zinc carbonate for hypophasic carotenoids, and powdered sugar (C & H Confectioner's) for chlorophyll derivatives, activated alumina was found most satisfactory. Various pigment fractions obtained by chromatographic separation were eluted in appropriate solvents like petroleum- ether, methanol, etc. for determining the absorption spectra, with a Beckman Spectrophotometer. Efforts were made to identify the pigments by spectro- photometric absorption analyses, partition tests, color reactions, solubility, fluorescence, and chromatographic behavior. Figures 1-3 include spectral curves of the pigments A-E in petroleum-ether (b.p. 50-70 C.) and F and G in methanol. 0-4 03 LJ o LJ DQ gO-2 CO 00 --A 400 560 440 48O 520 WAVELENGTH (M|J) FIGURE 1. A. Light orange pigment HIII (Table II). B. Yellow pigment HII (Table II). 100 A. M. NADAKAL 0-4 400 560 440 480 52O WAVELENGTH (M|J) FIGURE 2. C. Orange pigment EIV (Table I). D. Orange pigment EIII (Table III), E. Violet pigment EIII (Table I). o 0-3 z UJ 00 CC 02 CD < o - -F G 400 800 500 GOO 700 WAVELENGTH (Mp) FIGURE 3. F. Yellowish-brown pigment EII (Table I). G. Pale green pigment Ella (Table I). PIGMENTS IN A MARINE SNAIL 101 RESULTS The pigment fractions obtained by chromatographic separation of the epiphasic and hypophasic portions of the pigment extracts of the various snail tissues and their characteristics are listed in Tables I-V. The pigment fractions in the case of epiphasic portions of the extracts are numbered in order of decreasing adsorption and hypophasic portions in order of increasing adsorption on the columns. Absorption maxima and the forms of the spectral curve (Fig. 3, G) of the pigments El and Ella (Table I) indicate that these pigments resemble chlorophyll or pigments derived from chlorophyll. However, the absorption maxima in the violet region of the spectrum are different from that of the chlorophyll reported from plant sources. The Gmelin reaction (Pearse. 1953) was negative for these pigments, suggesting that they are not open-ring tetrapyrrole compounds. The brown pigment Ellb (Table I) showed a maximum at 450 m^. This fraction could not be made hypophasic even after prolonged saponification. This may be a carotenoid acid. TABLE I Ep! 'phasic portion of the digestive gland extract. Adsorbent: activated alumina. Developing solvent: petroleum ether (50-70 C.) with 1-5% methanol Band No. Color of band Percentage of solvent required for elution Absorption maxima mju Solvent El EII EIII Greenish-yellow Yellowish-brown Violet 2-3 methanol 2-3 methanol 3-4 methanol 410, 667 410, 450, 667 454 Ethanol Ethanol Petroleum ether EIV Orange 452, 482 Petroleum ether Band EII was chromatographed again on alumina column and the two fractions obtained are given below: Ella Pale green 2-3 methanol 416, 665 Methanol Ellb Brown 3-4 methanol with a few 450 Petroleum ether drops of glacial acetic acid The violet pigment EIII (Table I) showed a maximum at 454 m/x. The form of the spectral curve (Fig. 2, E) and the single absorption maximum are suggestive of a keto-carotenoid (Vevers and Millott, 1957). The orange pigment EIV (Table I) has been found to possess properties similar to those of /^-carotene (Fig. 2, C). The absorption maxima are in good agreement with the figures given by Karrer and Jucker (1950), and Lederer (1938). Besides, solubility, behavior on partition test, fluorescence (bluish-green in ultra violet light), color reactions (pigment in chloroform solution turned bluish-green on addition of concentrated sulfuric acid), color in solutions, and chromatographic behavior also indicate that this is /^-carotene. The absorption maxima of the pink pigment HI (Table II) in petroleum ether and benzene are in good agreement with the figures given by Karrer and Jucker (1950), and Goodwin (1953) for zeaxanthin. Its hypophasic behavior on parti- tion test and chromatographic behavior also lend support to the conclusion that 102 A. M. NADAKAL TABLE 11 Hypo phasic portion of the digestive gland extract. Adsorbent: activated alumina. Developing solvent: petroleum ether with 1-5% methanol Band No. Color of band Percentage of solvent required for elution Absorption maxima m/i Solvent HI Pink 2-3 ethanol 420, 450, 482 Petroleum ether 462, 490 Benzene HII Yellow 1-2 methanol 426, 448, 478 Petroleum ether 429, 457, 487 Chloroform Hill Light orange 1-2 methanol with a few 424, 454 Petroleum ether drops of glacial acetic acid HIV Blue-green 23 ethanol 416, 670 Methanol this is zeaxanthin. The fact that this pigment fraction stayed hypophasic even after continued saponification and could not he made epiphasic with acid treatment, showed that this pigment occurs in the snail tissues in the free state. The yellow pigment HII (Tahle II) showed maxima in petroleum ether and chloroform which are in good agreement with the figures quoted by Karrer and Jucker (1950) for the xanthophyllic pigment lutein (Fig. 1, B). This pigment turned bluish-green with concentrated sulfuric acid. These properties, coupled with the chromatographic behavior, hypophasic nature on partition test, and color in solutions, prove that this is lutein. A yellow pigment fraction, separated from the epiphasic portion of the un- saponified pigment extract, showed maxima in petroleum ether at 424, 445, and 475 niju. This pigment became hypophasic on saponification and could be made epiphasic again with acetic acid treatment. This indicated that some of the lutein in the snail's tissues is esterified. After saponification the pigment showed the maxima at 446 and 478 m/x in petroleum ether. The light orange pigment HIII (Table II) could be removed from the column with a few drops of acetic acid in the eluting solvent. It is difficult to identify this pigment conclusively ; it may be a carotenoid acid or some pigment derived from hypophasic carotenoids (Fig. 1, A). The blue-green pigment HIV (Table II) is characterized by absorption maxima and spectral curve suggestive of chlorophyll or a pigment derived from it. The brown pigment El (Table III) showed only one absorption maximum. TABLE III Pigment extracts from the mantle, branchial, and pedal tissues. No hypophasic fraction was obtained on partition of the original extracts between 90% methanol and petroleum ether systems. Pigments chromatographed as the epiphasic portion of the digestive gland extract Band No. Color of band Percentage of solvent required for elution Absorption maxima m/* Solvent El Brown 23 methanol 450 Petroleum ether EII Pink 1-2 methanol with a few 452 Petroleum ether EIII Orange drops of glacial acetic acid 424, 452, 480 Petroleum ether PIGMENTS IN A MARINE SNAIL 103 This fraction could not be made hypophasic even after prolonged saponification in 90% methanol-petroleum ether systems. Identification of this pigment fraction was difficult. The pink pigment El I (Table III) could be eluted with a few drops of glacial acetic acid. It showed a maximum at 452 m/x. On saponification this became hypophasic and could be made epiphasic again by treating with acetic acid. This behavior indicated that this pigment could be an esterified carotenoid acid. The orange pigment EIII (Table III) showed all characteristics of /3-carotene. However, a small shoulder at 424 m/x in the spectral curve of this pigment is remarkable (Fig. 2, D). The khaki pigment El (Table IV) showed an absorption maximum only in the violet region of the visible spectrum. This may be some breakdown product TABLE IV Pigment extract from the visceral mass. EpipJiasic portion chromatographed on activated alumina. Developing solvent: petroleum ether with 1-5% methanol Band N T o. Color of band Percentage of solvent required for elution Absorption maxima m/i Solvent El Khaki 1-2 methanol 425 Methanol EII Greenish-yellow 2-5 methanol 416, 667 Methanol EIII Yellowish-brown 2-4 methanol 420, 452, 667 Methanol EIV Violet 2-3 methanol 452 Petroleum ether EV Orange 450, 482 Petroleum ether Band EIII was chromatographed again on alumina column and the two fractions obtained are given below: EHIa Pale green 1-2 methanol 416, 665 Methanol EHIb Pale orange 1-2 methanol with a few 450 Petroleum ether drops of glacial acetic acid of chlorophyll or carotenoid pigments. Such breakdown products are known to be adsorbed at the top of the columns (Fox, 1953). The spectral properties of the pigments EII and Ellla (Table IV) indicated that they are chlorophyll derivatives. The pale orange pigment EHIb (Table IV) is considered to be a carotenoid acid because of its single absorption band and acidic properties. The violet pigment EIV (Table IV) was more or less similar to the violet pigment EIII (Table I) extracted from the digestive gland and may be a keto- carotenoid. The orange pigment EV (Table IV) was identical with the pigments EIV (Table I) and EIII (Table III) extracted from the digestive gland and mantle, respectively. It is therefore concluded to be /^-carotene. The pink pigment HI (Table V) was similar in properties to the one HI (Table II) recovered from the hypophasic portion of the digestive gland extract and is concluded to be zeaxanthin. 104 A. M. NADAKAL The yellow pigment HI I (Table V) has been identified as lutein, as its properties resemble those of the yellow pigment HII (Table II) extracted from the hypophasic portion of the digestive gland extract. The light orange pigment HIII (Table V) with its acidic properties and two absorption maxima in the blue-violet region of the visible spectrum may be considered as a carotenoid acid. No trace of chlorophyll derivatives could be detected in the hypophasic portion of the visceral extracts. A blue-green residue was left in the methanol-water fraction of the original extracts of the digestive gland and visceral tissues. Part of the colored substance could be taken up in ether after addition of a few drops of glacial acetic acid. It was then washed with water, evaporated to dryness under vacuum and finally taken up in methanol. The absorption maxima of this pigment at 416 and 667 m/* and the form of the spectral curve was characteristic of chlorophyll a (Atkins and Jenkins, 1953; Green, 1957). Even after extraction of the chlorophyllic pigments by acidified ether, a bluish residue was left behind in the aqueous methanolic solution. This was not extractable by any of the solvents tried. Attempts to TABLE V Hypophasic portion of the visceral extract. Chromatographed as the hypophasic portion of the digestive gland extract Band No. Color of band Percentage of solvent required for elution Absorption maxima m^ Solvent HI Pink 2-3 methanol 420, 450, 482 Petroleum ether HII Yellow 1-2 methanol 422, 448, 478 Petroleum ether HIII Light orange 1-3 methanol with a few drops of glacial acetic acid 424, 454 Petroleum ether separate the pigment on adsorption columns also failed. This bluish residue probably contained haemocyanin which is common in molluscan body fluids. PIGMENTS FOUND IN THE ALGAE The three species of green algae, Ulva sp.. Enter omorpha, clathrata, and Chaetomorpha torta, have been found to contain chlorophyll a and b, /3-carotene, and the xanthophyllic pigment lutein. No trace of a-carotene or any other pig- ments related to carotenoids could be detected. The red alga, Hypnea johnstonii, contained chlorophyll a and d, /3-carotene, lutein, phycoerythrin, and phycocyanin. The principal carotenoid pigment extracted from the algae was /2-carotene. DISCUSSION The occurrence of carotenoids and chlorophyll derivatives in Ccrithidca is in agreement with the previous findings in various species of molluscs. /3-Carotene and lutein have been reported from several gastropods (Fox, 1953; Goodwin, 1954). Zeaxanthin is known to occur in Patella rulgata and P. dcpressa (Goodwin, 1954; Goodwin and Taha, 1950), and Mytilus californianus (Scheer, 1940). Carotenoid acids with single absorption bands in the visible spectrum have also been reported PIGMENTS IN A MARINE SNAIL 105 from many invertebrates such as sponges and molluscs (Fox, 1953). Demonstra- tion of chlorophyll derivatives in molluscs has been made by MacMunn (1886a, 1886b), Dhere and Vegezzi (1916), and many others. A survey of the occurrence and distribution of carotenoid pigments in in- vertebrates reveals the fact that the digestive gland plays an important role in the storage of these pigments. Crane (1949) found that in the cephalopods, Octopus bimaculatus and Loligo opalescens, the liver-pancreas accumulated relatively large amounts of carotenoid pigments. In Cerithidea, undoubtedly the digestive gland functions as the chief organ for the storage of carotenoids. The preferential accumulation of carotenoids in the lipid-rich digestive gland is not surprising since these pigments have a tendency to be associated with lipids. The visceral extracts of Cerithidea have also been found rich in carotenoid and other pigments. This might be due to the presence in the gut of plant materials ingested as food. The mantle and branchial tissues contained relatively small amounts of carotenoids and no traces of chlorophyll derivatives could be detected in these tissues. A similar situation is described by Brooks and Paulais (1939) in the lamellibranchs, Ostrca edulis and Gryphaea angulata. In Cerithidea, the principal pigment found in various tissues is ^-carotene. Next in importance, on a quantitative basis, are the chlorophyll derivatives ; third comes the lutein and fourth only the zeaxanthin. Carotenoid acids and keto- carotenoids occur in traces only. The snail apparently shows a preference for storing /2-carotene in its tissues. This is perhaps due to the preponderance of /^-carotene in the algae which serve as food for the snail. Since Cerithidea assimi- lates and stores both hydrocarbons and xanthophylls as well as chlorophyll deriva- tives, it may be regarded as non-selective in its chromatic storage. However, the preferential accumulation of carotenes in the mantle and the branchial tissues is remarkable indeed. There is ample evidence for the elementary origin of carotenoids and chlorophyll derivatives in animals. The crustacean, Daphnia, builds up its carotenoid supply from the algae upon which it feeds (Green, 1957). The sea mussel, Mytilus calijornianus, is known to absorb and store carotenoid pigments "from a very plentiful and widely varied diet" (Fox and Coe, 1943). Dhere and Vegazzi (1916) concluded from experimental evidence that the greenish and grayish hepatic pigments of Helix pomatia were derived from the chlorophyll of its diet. The dark green pigment "chaetopterin" found in the intestinal epithelium of the polychaete worm, Chaetopterus, is derived from chlorophyll by the elimination of magnesium and the phytol chain (Lederer, 1940). Similarly, Lederer and Huttrer (1942) and Winkler (1957) showed that the sea slug, Aplysia, accumulates the pigment "aplysioviolin" in its ink-gland, which is derived from red algae consumed as part of its diet. As regards Cerithidea, sources of pigments could also be attributed to nutritional factors. Examination of four species of algae which serve as food for the snail indicated the presence of chlorophylls and carotenoids, in addition to the phycobilins and chlorophyll d in the red alga. The snail probably builds up its pigment supply from these algal sources. /3-Carotene seems to have been accumulated in the snail tissues without any metabolic alteration. However, the bluish-green color of mantle tissues is an indication that carotenoids may exist in them as a carotenoid- 106 A. M. NADAKAL protein complex. The fact that no chlorophyll derivatives could be detected from these tissues also lends support to this conclusion. Zeaxanthin, carotenoid acids, and keto-carotenoids were not observed in the algae studied ; they may be con- sidered as products of metabolic activities of the snail. Moreover, some of the lutein and carotenoid acids were found esterified in the tissues of the snail. The spectral properties of chlorophyll derivatives indicate that these pigments must have also undergone some kind of metabolic change, possibly oxidation. The absorption maxima of the chlorophyll derivatives in the red region of the spectrum suggest that these pigments are derived from chlorophyll a of the algae. There was no indication of the presence of chlorophyll d or phycobilins in the tissues of the snail, negating the possibility that the snail absorbs these pigments from the red alga, Hypnea. Several examples can be cited to prove that the accumulation of pigments in the body tissues of animals frequently results from catabolic activities. Such accumulation of pigments may or may not be significant in the functional economy of these organisms. It has been reported that the large pigment cells found in the deeper layer of connective tissues adjoining the intestinal caeca of the leech, Glossiphonia complanata, represent a kidney for the storage of waste products derived from haemoglobin metabolism (Bradbury, 1957). Wigglesworth (1943) found that in the blood-sucking bug, Rhodnius proli.vus, some of the ingested blood is denatured to form biliverdin which is subsequently either excreted through the gut or stored in the pericardial cells. Stephenson (1947) noticed that the pigment in the gut epithelium of Fasciola hepatica is derived from the haemolysis of the ingested blood. The occurrence of chlorophyll derivatives in Cerithidca may not have any functional significance ; they simply happen to be deposited in the tissues as metabolic wastes resulting from the digestion of algal food. Never- theless, the storage of carotenoids in various tissues, particularly in the digestive gland of the snail, may be beneficial since there are indications that certain carotenoids may serve to prevent autoxidation of lipids in animal tissues (Verne, 1936a, 1936b). It is yet to be found out whether the snail needs vitamin A for its metabolic activities, and, in case it does, it might make use of /3-carotene as a potential source. Although in certain invertebrates carotenoids are known to be utilized in sexual reproduction (Scheer, 1940) and in maintenance of mucous surfaces, nothing is known about their roles in similar processes in Cerithidea. SUMMARY 1. Evidences obtained from chromatography, spectrophotometric absorption analyses, partition tests, etc. suggested the occurrence of the following pigments in the marine snail, Cerithidea californica : /3-carotene, carotenoid acids, keto- carotenoids, lutein, and chlorophyll derivatives. 2. In an attempt to understand the dietary relationship of pigmentation in the snail, four species of algae were studied for their pigment contents. The three green algae were found to contain chlorophyll a and b, /3-carotene, and lutein ; the red alga, chlorophyll a and d, /3-carotene, lutein, and phycobilins. 3. The spectral properties of the chlorophyll derivatives recovered from the snail suggested that they are derived from chlorophyll a of the algae and that the molecular structure is still intact with the magnesium atom atached to it. However, PIGMENTS IN A MARINE SNAIL 107 absorption maxima in the violet region of the spectrum are shifted toward shorter, and in the red toward longer wave lengths, indicating some metabolic change in these pigments, possibly oxidation. 4. Part of the lutein and carotenoid acids were found to be esterified in the digestive gland and mantle tissues. No metabolic alteration has been noticed in the case of /3-carotene. All available evidence suggests that zeaxanthin, carotenoid acids, and keto-carotenoids are products of the snail's metabolic activities. 5. Apparently the snails do not absorb phycobilins or chlorophyll d from the red alga. 6. The snail has been found to be non-selective in its chromatic storage. 7. The nutritional relationship and biological significance of pigments in the snail have been discussed. LITERATURE CITED ATKINS, W. R. G., AND P. G. JENKINS, 1953. Seasonal changes in the phytoplankton during the year 1951-52 as indicated by spectrophotometric chlorophyll estimations. 7. Mar. Biol. Assoc., 31 : 495-507. BRADBURY, S., 1957. A histochemical study of the pigment cells of the leech, Glossiphonia complanata. Quart. J. Micr. Sci., 98: 301-314. BROOKS, G., AND R. PAULAIS, 1939. Repartition et localisation des carotenoides, des flavin et de l'acide-L-ascorbique chez les mollusques lamellibranches : cas des huitres et des gryphees vertes et blanches. C. R. Acad. Sci., Paris, 208 : 833-835. CRANE, S. C., 1949. Studies of hepatopancreatic function and carotenoid metabolism of the octopus, Octopus biiuaculatus. Dissertation, University of California. DHERE, C., AND G. VEGEZZI, 1916. Sur la composition pigmentaire de I'hepato-chlorophylle. C. R. Acad. Sci. Paris, 163: 399-401. Fox, D. L., 1953. Animal Biochromes and Structural Colors. Cambridge Univ. Press. Fox, D. L., AND W. R. COE, 1943. Biology of the California sea mussel (Mytilus calif ornianus). II. Nutrition, metabolism, growth, and calcium deposition. /. E.vp. Zoo!., 93: 205-249. Fox, D. L., AND C. F. A. PANTIN, 1941. The colors of the plumose anemone, Metridium senile. Philos. Trans. Roy. Soc.. Ser. B, 230: 415-450. GOODWIN, T. W., 1954. Carotenoids, Their Comparative Biochemistry. Chemical Publishing Company, New York. GOODWIN, T. W., AND M. M. TAHA, 1950. The carotenoids of the gonads of the limpets. Patella vulgata and P. depressa. Biochem. J., 47 : 249-251. GREEN, J., 1957. Carotenoids in Daphnia. Proc. Roy. Soc. London. Scr. B, 147 : 392-401. HAXO, F., COLM O'nEocHA AND P. NORRIS, 1955. Comparative studies of chromatographically separated phycoerythrins and phycocyanins. Arch. Biochem. Biophys., 54: 162-173. HUNTER, W. S., 1942. Studies on cercariae of the common mud-flat snail, Cerithidea californica. Ph.D. Thesis. University of California. KARRER, P., AND E. JUCKER, 1950. Carotenoids. Elsevier Publishing Co., Inc., New York. LEDERER, E., 1938. Recherches sur les carotenoides des Invertebres. Bull. Soc. On;;;. Biol.. Paris. 20: 567-610. LEDERER, E., 1940. Les pigments des Invertebres. Biol. Rei 1 .. 15: 273-306. LEDERER, E., AND C. HUTTRER, 1942. Pigments from the secretion of Aplvsia (sea hare or sea slug). Trans. Mem. Soc. Chim. Biol., 1055-1061. MACMUNN, C. A., 1886a. Further observations on enterochlorophyll and allied pigments. Philos. Trans. Roy. Soc., 177: 267-298. MACMUNN, C. A., 1886b. Further observations on some of the applications of the spectro- scope in biology, with special reference to the presence of chlorophyll in animals. Proc. Birmingham Nat. Hist. Soc., 5: 177-218. MANNING, W. M., AND H. H. STRAIN, 1943. Chlorophyll d, a green pigment of red algae. /. Bwl. Chetii., 151 : 1-19. 108 A. M. NADAKAL MARTIN, W. E., 1955. Seasonal infections of the snail, Ccrithidca californica Haldeman with larval trematodes. Hancock Commemoration Volume, pp. 203-210. NADAKAL, A. M., 1960a. Chemical nature of cercarial eye-spot and other tissue pigments. /. Parasitol. (in press). NADAKAL, A. M., 1960b. Types and sources of pigments in certain species of larval trematodes. /. Parasitol. (in press). PEARSE, A. G. E., 1953. Histo-chemistry, Theoretical and Applied. London. Churchill. SCHEER, B. T., 1940. Some features of the metabolism of the carotenoid pigments of the Cali- fornia sea mussel (Mytilus calif orniamts) . J. Biol. Chem., 136: 275-299. STEPHENSON, W., 1947. Physiological and histo-chemical observations on the adult liver fluke, Fasciola hcpatica L. II. Feeding. Parasitol., 38 : 123-127. STRAIN, H. H., 1942. Chromatographic Adsorption Analysis. Interscience Publishers, New York. VERNE, J., 1936a. Carotenoides et oxydation des lipides. C. R. Soc. Biol., Paris, 121 : 609-610. VERNE, J., 1936b. Observations histochemiques sur 1'oxydation des lipides et ses rapports avec les carotenoides. Bull. Histol. Tech. Micr., 13: 433-440. VEVERS, H. G., AND N. MILLOTT, 1957. Carotenoid pigments in the integument of the starfish, Marthasterias glacialis (L.) Proc. Mar. Biol. Assoc., 30: 569-574. WIGGLES WORTH, V. B., 1943. The fate of haemoglobin in Rhodnins prolixus (Hemiptera) and other blood-sucking arthropods. Proc. Roy. Soc. London, Scr. B, 131 : 313-339. WILLEY, C. H., AND P. R. GROSS, 1957. Pigmentation in the foot of Littorina littorca as a means of recognition of infection with trematode larvae. /. Parasitol., 43 : 324327. WINKLER, L. R., 1957. The biology of California sea hares of the genus Aplysia. Doctoral thesis. University of Southern California. THYROID HORMONE TREATMENT AND OXYGEN CONSUMPTION IN EMBRYOS OF THE SPINY DOGFISH 1 AUSTIN W. PRITCHARD AND AUBREY GORBMAN Department of Zoology, Oregon State College^ Corvallis, Oregon, and the Department of Zoology, Columbia University, New York 27 , N. Y. The inability of adult cold-blooded vertebrates to respond to thyroxine treatment with an increased oxygen consumption rate is now a well documented finding (Hoar, 1957; Gorbman, 1959). The two often cited exceptions to this general experience are the thyroxine-induced increases in oxygen consumption in adult goldfish observed by Miiller (1953), and in parrot fish of certain sizes, as described by Smith and Matthews (1948). Both of these claims have been denied by opposite results in the same species (Etkin, Root and Mofshin, 1940; Chavin and Rossmore, 1956; Matty, 1957). Measurements of metabolic rate in fishes are subject to numerous variables which are not as easily controlled as they are in mammals (responses to handling, previous temperature history, illumination, endogenous activity cycles) (Fry, 1957), so that it is not surprising that con- flicting claims may exist for some species. Of the factors which may contribute misleading information in measurements of oxygen consumption in fishes, among the most significant is muscular activity. Hoar (1958) has shown clearly that treatment of fishes with thyroid hormone induces behavioral changes, expressed primarily by an increased spontaneous motor activity. If this is so, then any valid test for basal metabolic stimulation by thyroxine must exclude the variable of locomotor muscular work. Although testing systems are available which make this possible (Fry, 1957), neither of the two exceptional claims mentioned above utilized them. While working with near-term embryos of the spiny dogfish, Sqitalus sucklevi, removed from the uterus and kept in flowing sea water, we noticed a behavioral feature which makes this animal useful in respiratory studies. When kept in subdued light they remain still, even after treatment with thyroid hormone. Since under these circumstances, spontaneous muscular movements are rare, then respira- tory measurements can be taken to reflect "basal" requirements (or at least "standard" metabolism as defined by Fry, 1957), not a thyroxine-induced increase in swimming. In these experiments oxygen consumption of such exteriorized dogfish pups was measured after treatment with thyroxine, or two of its analogues, or propyl thiouracil. To our knowledge the only other studies of the metabolic responsiveness to thyroid hormone in larval vertebrates have concerned anuran tadpoles. In this regard, too, the published literature is in disagreement (Etkin, 1955; Lewis and Frieden, 1959). Accordingly, it was hoped that the experiments 1 Supported by grants from the National Science Foundation. We would like to express our appreciation to the Friday Harbor Laboratories of the University of Washington for generous assistance rendered during this investigation. We also thank Dr. Frederick L. Hisaw, Jr., who contributed time, experience, and materials towards collecting the dogfish, and Mr. Robert Lasher, who aided in capturing the dogfish and in running oxygen analyses. 109 110 AUSTIN W. PRITCHARD AND AUBREY GORBMAN with dogfish embryos would prove enlightening, both with regard to the metabolic action of thyroid hormone in poikilotherms and its action in differentiating forms of such animals. MATERIAL AND METHODS Oxygen consumption was measured in a continuous-flow apparatus of the type used by Job (1955) in measuring the "standard" metabolism of trout. Four respirometer flasks (2.5-liter Fernbach culture jars) were used at any one time, one of these being used as a "blank." The four flasks were immersed in a large wooden tank in which the water level was maintained constant by means of an overflow. The flow of water through the flasks was so adjusted that Squalus embryos in groups of three removed about 0.5 to 1.0 ml. of oxygen. Rate of oxygen consumption was calculated from the flow rate, the difference in oxygen content between incur rent and excurrent water, and the wet weight of the animals 2 Q. O en --. UJ <-> > X o 25 0800 1200 1600 2000 0800 1200 JUNE 30 JULY I CLOCK TIME FIGURE 1. Serial determination of metabolic rate in untreated embryos of Squalus sitcklcyi over a 27-hour period. Two respirometers used with three pups in each. tested. Oxygen content of the water was determined by the unmodified Winkler technique. Water temperature never varied more than 1 C. during a single series of tests, and usually it did not change at all measurably. During most measure- ments of oxygen consumption the water temperature ranged from 13 to 14 C. However, extremes of 12 C. and 16 C. (on one unusually hot day) were recorded. The water in the large water bath was continuously aerated through a stone "air breaker," and preliminary tests showed that the oxygen content of the water was uniform at all points in the bath. Embryos were tested in groups of three, being placed in the respirometers one hour before the first measurement of oxygen consumption. The bath con- taining the respirometers was covered to shield the animals from most of the light and other extraneous factors. Frequent observation indicated that under these conditions spontaneous muscular movements were rare. Several prelimi- THYROID TREATMENT OF DOGFISH 111 TABLE I Metabolic rates of non-treated dogfish pups showing normal day-to-day variability in the laboratory. All runs made bet-ween 1:00 and 3:00 PM Date July3 July 4 July 6 July 8 July 10 No. of trials 4 4 3 3 3 Mean Oi consumption (cc./kg./hr.) 28.8 1.4 (std. dev.) 31.4 1.9 29.5 db .89 31.3 1.4 33.7 1.7 nary measurements were made on untreated pups, to ascertain the variability in oxygen consumption under laboratory conditions. Figure 1 illustrates the results of serial determinations on two groups of embryos, all taken from the same female, over a H-day period. The period of steadiest metabolic rate was in the afternoon; accordingly all measurements reported here were made in the afternoon only. In another preliminary test, metabolic rates were determined on pups from one female dogfish, over a period of one week immediately following transfer of the pups to the laboratory. The results (Table I) show that the average metabolic rate remained at about the same level over this period and that the variability in oxygen uptake (as indicated by the standard deviations) was quite small. TABLE II Experimental protocol for each injection group. Numbers in parentheses indicate the number of pups from that female Experiment Total no. of injections Females from which pups were taken Substances tested* Dose per injection 1 10 A(15) triac 10 Mg. 1-Tx 10 Mg. 2 9 B(11),C(9) triac 10 Mg- 1-Tx 10 /ig- PTU 50 M g. 3 9 D(10), E(10) triac 10 Mg- 100 Mg- 1-Tx 10 Mg- PTU 50 M g. 4 8 F(11),G(4), H(4), triac 10 Mg- I(4),J(5), K(7) 100 Mg. 1-Tx 10 Mg. T3 100 Mg. PTU 50 Mg- 5 5 L(19) 1-Tx 100 Mg- T3 100 Mg- PTU 50 Mg. 6 4 M(2S), N(10) triac 10 Mg. 1-Tx 10 Mg. * Abbreviations : triac, triiodothyroacetic acid; 1-Tx, 1-thyroxine; PTU, propylthiouracil ; T3, triiodothyronine. 112 AUSTIN W. PRITCHARD AND AUBRKY GORBMAN TABLE III Effects of repeated injections of thyroid hormones on oxygen consumption (cc./kg./hr.) of embryos of Squalus suckleyi.* Values in the table are means of 3 to 6 consecutive determinations, taken at 15-minute intervals. E = experimental. C = control Experi- ment No. of injections Triac 10 Mg. Triac 100 MS. 1-Tx 10 M g- 1-Tx 100 Mg. T3 100 Mg- PTU 50 Mg. 1 E C E/C X 100 32.40 30.79 105 33.52 32.52 103 1 4 E C E/C X 100 52.25 39.28 133 44.78 39.28 114 9 E C E/C X 100 46.82 44.60 105 2 E C E/C X 100 32.25 31.52 102 32.15 31.52 102 28.78 31.52 91 2 4 E C E/C X 100 41.90 35.80 117 40.90 35.80 114 36.00 35.80 101 9 E C E/C X 100 33.80 34.16 99 41.40 34.16 121 31.27 34.16 91 1 E C E/C X 100 33.10 33.20 99 33.40 33.20 101 33.30 33.20 100 32.70 33.20 98 3 4 E C E/C X 100 36.70 30.40 121 33.90 30.40 108 29.20 30.40 96 29.00 30.40 98 9 E C E/C X 100 35.87 32.96 109 35.50 32.96 108 33.62 32.96 102 38.09 32.96 115 2 E C E/C X 100 34.13 29.86 114 32.56 29.86 109 30.82 29.86 103 30.12 29.86 101 30.30 29.86 102 4 5 E C E/C X 100 37.02 30.41 122 33.45 30.41 110 33.97 30.41 112 34.63 30.41 114 32.51 30.41 107 8 E C E/C X 100 32.33 2749 118 33.66 27.49 122 31.86 27.49 116 32.28 27.49 118 29.44 27.49 107 * This table includes absolute values for oxygen consumption at the beginning (after 1 or 2 injections), the middle (after 4 or 5 injections), and end after 8 or 9 injections in each experiment. The complete course of each experiment is shown in Figures 4A, 4B and 4C, but these do not show absolute values. To tabulate all the absolute values would require an impractically long table. THYROID TREATMENT OF DOGFISH TABLE III Continued 113 Experi- No. of Triac Triac 1-Tx l-Tx T3 PTU ment injections 10 M g. 100 ng. 10 M g. 100 M g. 100 M g. 50 Mg. E 30.01 25.78 26.96 1 C 26.86 22.82 22.82 E/C X 100 112 113 118 5 E 39.92 42.96 37.07 5 C 35.78 35.78 35.78 E/C X 100 112 120 104 E 28.49 30.37 1 C 26.18 26.18 E/C X 100 109 110 6 E 35.34 32.82 4 C 29.78 29.78 E/C X 100 119 110 The animals used were "pups" removed from the uteri of Squalus sitckleyi females caught during July and August, 1958, at Friday Harbor, Washington, within 200 yards of the laboratory. The ovoviviparous young of this species remain in the uterus for two years. It could be estimated from the sizes of the yolk sacs that the embryos we used were approximately 19-23 months of age. Occasional "spontaneous" birth of pups of captive females was observed in late August. Embryos removed from the uterus were kept in apparently good con- dition for periods as long as several weeks in large, covered glass dishes (2-liter capacity) in groups of 4 or 5, in slowly flowing sea water. Whenever possible, all pups used in an experiment were taken from the same mother. In experiments requiring large numbers of pups it was necessary to combine litters from several mothers and these were distributed as evenly as possible into the different experi- mental groups (Table II). The limited number of embryos of equivalent de- velopment available at one time, and the limited capacity of the respirometers made it impossible to test all hormones at the same time. For this reason, six different experiments were performed during an eight-week period. In each experiment groups of embryos were injected intraperitoneally on alternate days with various doses (Table II) of hormones, propylthiouracil, or 0.7% NaCl solution, always in a volume of 0.05 cc. Oxygen consumption was determined on the clay after injection to avoid possible responses to handling. The compounds tested for their effect on oxygen consumption were 1-thyroxine (Tx). 1-triiodothyronine (3 : 5 : 3'-triiodo-l-thyronine, To), triiodothyroacetic acid (3:5:3' triiodothyroacetic acid, Triac) in doses of 10 micrograms or 100 micro- grams, and propylthiouracil in doses of 50 micrograms. EXPERIMENTS AND RESULTS Six experiments (Table III) were completed. The total number of injections, given at 2-day intervals, was as few as 4 or 5, but was usually (in four of the six experiments) 8 to 10. The shorter experiments were ended when accidental 114 AUSTIN W. PRITCHARD AND AUBREY GORBMAN blockage of the sea water occurred. Since such occurrences were obviously harmful, and their effects difficult to assay, respiratory measurements were accordingly not continued. In experiment 1 such a blockage killed all Triac- injected animals after the fifth injection (Fig. 2). The Tx-injected animals in this experiment showed an extreme but temporary respiratory depression (Fig. 2) at the same time, so may have had a brief experience of the same nature. Only one other extremely variant datum is seen in Figure 3, which shows the results of experiment 2. Here an exceptionally high respiratory rate was observed in saline solution-injected controls after three injections. Since these same animals in succeeding measurements showed relatively little variation in oxygen consump- 2 3456 NUMBER OF INJECTIONS 8 10 FIGURE 2. The effects of repeated injections of thyroid compounds on metabolic rate of dogfish embryos injection group 1. Solid line, control; dashed line, triiodothyroacetic acid (10/ug.); dotted line, 1-thyroxine (10/xg. ). tion, it is felt that the exceptional figure may have been due to some limited experience, possibly a brief interruption in water supply. The remaining data, summarized in the figures, and Table III, appear to show consistent trends of response, or lack of response, to the various forms of treatment. It may be seen in Figures 1 and 2, and Table III, that the oxygen consumption of control embryos varied throughout the periods of respiratory measurement, but not in any particular pattern. The nature of this variation, whether due to maturational or environmental factors, is not clear. However, whatever the basis for the variation in control respiratory metabolism, the changes were generally gradual. In the first experiment (Fig. 2) oxygen consumption increased gradu- THYROID TREATMENT OF DOGFISH 115 ally through most of the three-week period of observation ; in the second experiment (Fig. 3) this variation was, in general, less and showed no such constant trend. Fortunately, the general variation of the controls was paralleled by the hormone- injected embryos, and in addition, a relative difference from the controls was usually maintained, if it occurred at all. Accordingly, when the results are ex- pressed as per cent of the control oxygen consumption some conclusions appear to be offered (Table III, Fig. 4). The most potent stimulator of oxygen consumption in these tests was triiodo- thyroacetic acid (Fig. 4A). Despite all the variations to which such experiments seem to be prone, in all four experiments in which Triac was injected in 10- microgram quantities, it clearly induced an increase in oxygen consumption to maxima 17% to 33% above the control. These maxima were achieved 8 to 10 days after beginning the injections and thereafter oxygen consumption progressively 45 o u O 40 Q. CO O 35 o z UJ o O 30 o 34567 NUMBER OF INJECTIONS 8 FIGURE 3. The effects of repeated injections of thyroid compounds on metabolic rate of dogfish embryos injection group 2. Solid line, control; dashed line, triiodothyroacetic acid (10/ttg.) ; dotted line, 1-thyroxine (10/ug.). decreased. The larger dose of Triac (100 micrograms) was less effective than the smaller one (Table III). Thyroxine, in 10-microgram doses, was not as clearly a stimulator of oxygen consumption in the Sqitahts pups as was Triac. It consistently raised oxygen consumption in two of four experiments (Table III, Fig. 4B) above that of controls, but failed to do so in one, and in another did not produce a significant stimulation until the very end. However, in all thyroxine experiments oxygen consumption was rising at the end of the period of treatment, in comparison with the controls. In one test with 100-microgram quantities of thyroxine a 12% increase above the controls in respiratory metabolism was noted. Triiodothyronine, in 100-microgram quantities, in all instances stimulated oxygen consumption to levels as high as 18% to 20% above the controls (Table III). 116 AUSTIN W. PRITCHARD AND AUBREY GORBMAN UJ 130 0120 z o o 110 zlOO UJ o 90 a A TRIAC, lOpgm 34567 NUMBER OF INJECTIONS 8 UJ 130 ^120 ac | no O ^100 UJ B Thyroxine, -" > _._.X- -8-' 4567 NUMBER OF INJECTIONS 8 10 lil C PTU 120 110 ...o .. 0100 "2"' A .... '~ * - ~^.-:-i.:..o:^-^ ^ ^^^^ 890 *' x ^^ / N e * or 9 u 34567 NUMBER OF INJECTIONS 8 FIGURE 4; A, B, and C. The effects of repeated injections of thyroid compounds on metabolic rate of dogfish embryos. A, triiodothyroacetic acid, 10 /xg. each injection; B, 1- thyroxine, 10 /ug. ; C, propylthiouracil, 50 ^g- Points represent per cent of the control rate. Open circles, injection group 1; horizontal-barred circles, injection group 2; vertical-barred circles, injection group 3; filled circles, injection group 4. THYROID TREATMENT OF DOGFISH 117 Propylthiouracil had no particular effect on oxygen consumption relative to the saline-injected controls (Table III, Fig. 4C). DISCUSSION Results of this investigation indicate that, under the conditions of these experi- ments, 10 micrograms of triiodothyroacetic acid (Triac), given on alternate days, have acted as a stimulant of oxygen consumption in the near-term shark embryo (Fig. 4A). The unusual feature of this response is its diminution after 8 to 10 days despite continued injections of the hormone. In laboratory mammals (Foster, Palmer and Leland, 1936) and in clinical use (Means, Lerman and Salter, 1933) the continued administration of thyroxine or crude thyroid preparations is usually accompanied by a sustained increased respiratory rate. However, even in clinical experience it has been reported (Eppinger and Salter, 1935) that an initial rise in metabolism following a week of treatment with thyroid hormone may be followed by a sharp drop, even though treatment is continued. Thus, this pattern of metabolic response is not unprecedented, and may depend on particular physio- logical factors involved in the response to thyroid hormone. It is of especial interest that Triac has been found to be about 10 to 25 times as active as thyroxine in stimulating amphibian metamorphosis (Pitt-Rivers and Tata, 1959). A ten-fold larger dose of Triac was no more active than the 10-microgram quantity, and appeared, in fact, less active (Table III). Thyroxine, in either the 10- or 100-microgram dosage, was less clearly a metabolic stimulant. In two of four experiments animals receiving the 10- microgram dose remained consistently higher than controls in oxygen consumption by some 10% to 20%. In the other two experiments this superiority was either lacking or irregularly variable. However, in all four instances oxygen consump- tion was rising (relative to the saline-injected controls) at the ends of the experi- ments (Fig. 4B). This was the most variably effective metabolic stimulant and no explanation can be offered for this variability. Larger doses of thyroxine and triiodothyronine (Table III) produce a 12 to 20% increase in oxygen con- sumption by 10 to 16 days after beginning the injections of hormone. Propylthiouracil was neither a stimulant nor depressant for oxygen consumption in four different experiments which lasted about 18 days each. Almost all measures of oxygen consumption in dogfish pups treated with this antithyroid drug were within 10% of the control. It has been reported by Zaks and Zamkova (1952) that thiourea consistently reduces the oxygen consumption of young salmon and sturgeons below that of controls. Chavin and Rossmore (1956), working with thiouracil-treated young goldfish, found no effect on oxygen con- sumption. Interpretation of results of treatment with antithyroid drugs is always complicated by the fact that they are known to be toxic, even in small doses. Since the absence of a metabolic response to propylthiouracil might mean merely that no hormone is yet produced by the thyroid of these embryonic animals, five of them were injected with radioiodide (5 microcuries) and the rest were fixed for histological examination to investigate this possibility. The 24-hour thyroidal radioiodine uptake varied from 0.25% to 2%, a small but significant degree of accumulation. This compares favorably with thyroid uptakes of about 1.5% found by Gorbman and \Yaterman (unpublished) in pups of the Atlantic spiny 118 AUSTIN W. PRITCHARD AND AUBREY GORBMAN dogfish, Squalus acanthias. Vivien and Rechenmann (1954) who also treated shark pups (Scyliorhinns canicula) with I 131 , observed by radioautographic tech- niques that it is deposited in the thyroid, presumably in protein-bound form. The thyroid tissue examined histologically showed a slight increase in average cell height (about 25% ) and "vacuolization" of the colloid. This would appear to indicate that the pituitary-thyroid axis of mutual responsiveness is differentiated in these animals, and that it had responded in the PTU-treated animals to a change in thyroid hormone output by TSH secretion. However, despite this apparent decrease in endogenous thyroid hormone production there was no detectable change in oxygen consumption. It is possible that this decrease in endogenous thyroid hormone, if real, was much smaller in size than the 10 micrograms in the injected dose. In summary, it may be said that triiodothyroacetic acid has been shown in these experiments to be a temporary stimulant of oxygen consumption in near- term embryos of Squalus sitckleyi. Triiodothyronine proved slightly less active, and the metabolic stimulation by thyroxine was irregular. The thyroid glands of these animals appeared to be functioning at a low rate, and interruption of this function by propylthiouracil had no demonstrable effect on oxygen consumption. SUMMARY 1. The oxygen consumption rate of "near-term" pups of the dogfish, Squalus suckleyi, was determined at regular intervals during the course of repeated in- jections of physiological saline solution, thyroid hormones, or of anti-thyroid sub- stances. Up to 10 injections were given on alternate days. 2. Of the compounds tested, triiodothyroacetic acid at a dosage level of 10 micrograms per injection was the most consistent in raising the level of oxygen consumption. The effect, however, was transitory with oxygen consumption rising to a maximum (17% to 33%) level above the saline-injected controls after four injections, thereafter declining slowly to control levels. 3. L-thyroxine at a dosage level of 10 micrograms had a variable effect on oxygen consumption. In two of four experiments the oxygen consumption rate rose irregularly, reaching a level about 2G c / ( above the controls after 9-10 injections. In the remaining experiments, there was no clear tendency to remain above the controls. 4. Propylthiouracil, after 9 injections, had no consistent effect on metabolic rate in four experiments. 5. The results are discussed with reference to the possible level of thyroid function in these animals. LITERATURE CITED CHAVIX, W., AND H. W. ROSSMORE, 1956. Pituitary-thyroid regulation of respiration in the goldfish, Carassins auratus L. Anat. Rcc., 125: 599. EPPINGER, E. C., AND W. T. SALTER, 1935. The daily requirement in human hypothyroidism of purified human thyroid hormone at various metabolic levels. Aincr. J. Alcd. Sci., 190: 649-655. ETKIN, W., 1955. In: Analysis of Development, edited by B. H. Willier, P. \Yeiss, and V. Hamburger, Philadelphia : W. B. Saunders Co. ETKIN, W., R. W. ROOT AND B. P. MOFSHIN, 1940. The effect of thyroid feeding on oxygen consumption of the goldfish. Physiol. Zool., 13: 415-429. THYROID TREATMENT OF DOGFISH 119 FOSTER, G. L., W. W. PALMER AND J. P. LELAND, 1936. A comparison of the calorigenic potencies of /-thyroxine, rfMhyroxine, and thyroid gland. /. Biol. Client., 115: 467-478. FRY, F. E. J., 1957. In : The Physiology of Fishes, M. E. Brown, ed. New York, Academic Press. GORBMAN, A. (editor), 1959. Comparative Endocrinology. New York, John Wiley and Sons. HOAR, W. S., 1957. In : The Physiology of Fishes, M. E. Brown, ed. New York, Academic Press. HOAR, W. S., 1958. Effects of synthetic thyroxine and gonadal steroids on the metabolism of goldfish. Canad. J. Zooi, 36: 113-121. TOB, S. V., 1955. The oxygen consumption of Salvclinus fontinalis. Univ. of Toronto Biol. Ser. No. 61 : 33 pp. LEWIS, E. J. C, AND E. FRIEDEN, 1959. Biochemistry of amphibian metamorphosis : effect of triiodothyronine, thyroxine, and dinitrophenol on the respiration of the tadpole. Endocrinol., 65: 273-282. MATTY, A. J., 1957. Thyroidectomy and its effect upon oxygen consumption of the teleost fish, Pscudoscarus guacamaia. J. Endocrinol., 15: 1-8. MEANS, J. H., J. LERMAN AND W. T. SALTER, 1933. The role of thyroxin iodine and total organic iodine in the calorigenic action of whole thyroid gland. /. Clin. Investig., 12 : 683-688. MULLER, J., 1953. t'ber die Wirkung von Thyroxin und Thyreotropem Hormon auf den Stoffwechsel und die Farbung der Goldfisches. Zeitschr. vcri/l. Physiol., 35:1-12. PITT-RIVERS, R., AND J. R. TATA, 1959. The Thyroid Hormone. New York, Pergamon Press. SMITH, D. C., AND S. A. MATTHEWS, 1948. Parrot fish thyroid extract and its effect on oxygen consumption in the fish, Bathystoina. Amcr. J. Physiol., 153: 215-221. VIVIEN, JEAN, AND ROGER RECHENMANN, 1954. fitude sur la fonction thyroidienne de 1'embryon de selacien. C. R. Soc. Biol., 148: 170-172. ZAKS, M. G., AND M. A. ZAMKOVA, 1952. On the effect of thiourea on gaseous exchange in larval salmon and sturgeon. Dokladv Akad. Nank S.S.S.R., 48: 1101-1103. PERMEATION AND MEMBRANE TRANSPORT IN PARASITISM : STUDIES ON A TAPEWORM-ELASMOBRANCH SYMBIOSIS 1 C. P. READ,? J. E. SIMMONS, JR.,- J. W. CAMPBELL 2, 3 AND A. H. ROTHMAN - * Marine Biological Laboratory, Woods Hole, Massachusetts; Department of Pathobiology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland; and Department of Biology, The Rice Institute, Houston, Texas It was shown by Read, Simmons and Rothman (1960) that the amino acids L-valine and L-leucine enter the tapeworm, Calliobothrium verticillatum, by a process showing adsorption kinetics. The data obtained ruled out simple diffusion but did not permit a definite conclusion as to whether the permeation is a process of active transport. L-valine and L-leucine were each shown to competitively inhibit the entry of the other into the worm. Several other amino acids were shown to inhibit L-valine and L-leucine permeation, but it was not established that these inhibitions were competitive. The studies to be reported have shown that L-valine is actively transported, although further investigation has revealed that when Calliobothrium is treated as a member of a symbiotic relationship, the operation of the amino acid entry systems is indeed complex. Appreciation is expressed to Dr. G. Wertheim who lent technical assistance during part of this study, and to the gentlemen of the Supply Department of the Marine Biological Laboratory, Woods Hole, Massachausetts, who furnished many living dogfish. MATERIALS AND METHODS The methods of collection and handling of Calliobothrium from the dogfish, Mustelus canis, were similar to those used in a previous study (Read, Simmons and Rothman, 1960). The salt solution used in handling both worms and host tissues had the following composition: NaCl, 250 mM. ; KC1, 4.4 mM. ; CaGU, 5.1 mM.; MgCL, 2.9 mM.; urea 300 mM.; and tris (hydroxy methyl) amino methane-maleate buffer, 10 mM. (pH 7.2). All incubations were carried out in this medium, with appropriate experimental additions, at 10 C. In preparing tissues for experimental incubation, the worms were washed in several changes of the salt solution and incubated for 60 minutes at 10 C. before an experiment. Host tissues were removed in ice-cold salt solution and, before the experiment, were incubated at 10 C. for 30 minutes in a large volume of salt solution containing 10 mM. glucose. Throughout this period, the tissue was vigorously aerated with a sintered bubbler attached to a small air pump. For experimental incuba- 1 This work was supported by grants from the National Institutes of Health, U. S. Public Health Service (E-1508 and E-1384) and Smith, Kline and French Foundation. 2 Present address : Department of Biology, The Rice Institute, Houston, Texas. 3 Research Fellow, National Academy of Sciences-National Research Council. 4 U. S. Public Health Service Postdoctoral Fellow. 120 MEMBRANE TRANSPORT IN PARASITISM 121 tion, the worms or host tissues were lightly blotted on hard filter paper and transferred to the experimental medium. At the end of an experimental incubation, the worms or host tissues were rinsed by dipping twice in large volumes of the salt solution, blotted quickly on hard filter paper, and placed in a measured volume of 70% ethyl alcohol. It was previously shown that, with occasional shaking, free amino acids are extracted from worm tissues in 50 to 70 per cent alcohol in less than 24 hours (Read, Simmons and Rothman, 1960). In most cases extraction was carried out for over 48 hours but in no case for less than 24 hours. Actually, the concentration in the fluid of the tissues probably comes to equilibrium with the alcohol external to the tissues. The quantity of worm tissue with respect to the volume of the extracting fluid was kept sufficiently low so that an error of less than \% was introduced by the addition of the worm volume to that of the extracting fluid. Aliquots of alcoholic extracts were used for determination of radioactivity or analysis of amino acids. Many of the data are expressed in terms of the alcohol-extracted dry weight of tissue ; this was determined by heating the extracted tissue for 5 to 6 hours at 100 C. in tared foil pans. Drying for longer periods produced no significant change in dry weight values. Determinations of the alcohol-extracted dry weight/wet weight for 16 worms at the end of a 60-minute incubation in the salt solution at 10 C. gave a value of 0.302 0.016. Nineteen dogfish gut samples, identical with those used in experiments, gave an alcohol-extracted dry weight/wet weight value of 0.135 0.011 after a 30-minute, aerated incubation in salt solution with glucose at 10 C. Plating of extract samples and determinations of radio- activity were carried out as previously described (Read, Simmons and Rothman, 1960). Two-dimensional chromatography was carried out by a modification of the method of Levy and Chung (1953), as described by Campbell (1960). One- dimensional chromatograms were prepared on Whatman No. 52 paper using sec-butyl alcohol, formic acid, and water (75:15:10) as the solvent system. Amino acids were quantitatively estimated using the methods of Fowden (1951). Radioautographs of chromatograms w r ere prepared by exposing Eastman "no- screen" x-ray film to the chromatograms after removing the solvent. Histidine was determined by the method of Macpherson (1946). Nitrogen was determined by the micro-Kjeldahl method described by Lang (1958). Other details of methods will be described in context. EXPERIMENTAL Further characterization oj the ammo acid entry systems of Calliobothrium Several amino acids have been shown to inhibit the penetration of L-valine and L-leucine into Calliobothrium (Read, Simmons and Rothman, 1960). In the present study a number of experiments were carried out to determine whether these inhibitions are competitive in nature, and whether there is a reciprocal inhibitory effect of L-valine on penetration of certain of the inhibitory amino acids. An analysis of the inhibitory effects of L-serine, L-threonine, and L-alanine on valine entry showed that the inhibitions indeed are competitive in nature (Table I). Conversely, experimental analysis of the effect of L-valine on the 122 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN TABLE I Effect of L-serine, L-threonine, and L-alanine on the entry of L-valine into Calliobothrium. 5 = concentration of L-valine; V = counts per minute per gram of alcohol-extracted dry tissue; N = number of samples Amino acid Inhibitor N l/S V 4 200 59,575 3673 5 X 10-W 4 500 36,200 2876 L-serine 4 1000 18,212 1004 4 2000 11,515 1529 4 200 96,900 4300 L-valine-C14 2 X 10-W L-serine 4 4 500 1000 41,850 1947 24,300 571 4 2000 16,812 857 4 200 116,600 10,148 None 4 500 61,222 5793 4 1000 46,700 2200 4 2000 31,775 1520 4 200 65,666 4843 5 X 10~W 4 500 36,368 6319 L-threonine 4 1000 18,769 852 4 2000 10,042 1102 4 200 73,450 5576 L-valine-C14 2 X 10-W L-threonine 4 4 500 1000 45,300 4529 27,937 1971 4 2000 16,875 1639 4 200 93,725 7925 None 4 500 64,550 5794 4 1000 40,920 3415 4 2000 28,300 3706 4 200 53,825 3594 5 X 10-W 4 500 31,070 2400 L-alanine 4 1000 22,400 842 4 2000 10,100 597 L-valine-Cl4 4 200 91,550 4132 None 4 500 75,902 2926 4 1000 41,850 4903 4 2000 28,475 2685 penetration of L-serine demonstrated that valine competitively inhibits the entry of this amino acid (Table II). Previous studies showed that at a concentration ratio of 2:1, glutamic acid did not inhibit the entry of L-valine. Therefore, it was surprising to find that aspartic acid is an effective inhibitor of L-valine entry. With L-valine at a concentration of 2 X 10~ n M, aspartic acid or proline at a concentration of 5 X 10~ 3 M, inhibited L-valine entry an average of 33 and 45%, respectively, in five experiments. A shortage of experimental material prevented a determination of whether or not aspartic acid is a competitive inhibitor. MEMBRANE TRANSPORT IN PARASITISM 123 L-lysine-C14 penetrates Calliobothriimi at a very low rate. A series of experi- ments were performed to determine whether L-lysine or L-valine affect the entry of the other. L-lysine entry was not significantly affected by L-valine and L-valine entry was not affected by L-lysine (Table II). The apparent stimulation of L-valine entry by L-lysine at a lysine/valine ratio of 2, previously reported (Read, Simmons and Rothman, 1960), is not considered to be significant in view of the results obtained when this broader range of concentration ratios was examined. When Calliobothrium was incubated for two minutes in 5 X 10~ 3 M L-valine- C14 and subsequently incubated for additional periods in salt medium without TABLE II Effects of certain amino acids on entry of L-serine, L-valine, and L-lysine. Data presented as in Table I Amino acid Inhibitor N l/S V 4 200 45,600 6111 5 X 10-W 4 500 29,500 8201 L-valine 4 1000 19,100 1124 4 2000 11,460 823 L-serine-C14 4 200 57,433 d= 2441 None 4 500 42,150 3300 4 1000 29,100 629 4 2000 20,200 4540 4 200 105,975 13,192 5 X 10-W 4 500 73,050 2563 L-lysine 4 1000 47,300 3379 4 2000 32,750 2958 4 200 97,025 12,604 L-valine-C14 2 X 10~ 3 M L-lysine 4 4 500 1000 72,025 3620 46,250 2418 4 2000 31,433 2240 4 200 103,775 14,202 None 4 500 61,300 963 4 1000 47,300 2588 4 2000 34,050 2952 4 200 14,970 1207 5 X 10- W 4 500 7552 1965 L-valine 4 1000 6247 1406 4 2000 5410 441 4 200 15,115 3924 L-lysine-C14 2 X 10~ 3 M L-valine 4 4 500 1000 7997 1534 6713 1408 4 2000 5290 805 4 200 15,358 1665 None 4 500 7930 1262 4 1000 6232 1836 4 2000 5142 909 124 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN CPB FIGURE 2, Chromatogram of free amino acids extracted from Calliobothrium after 40 minutes' incubation in presence of L-valine-C14. Solvent systems were sec-butyl alcohol, formic acid, water (75:15:10) = BFW and meta-cresol, phenol, borate buffer, ph 8.3 (60 : 30 : 15) = CPB. Black areas show presence of radioactivity, X being a non-ninhydrin- positive, unidentified metabolite. Table III. It is apparent that valine is concentrated against a gradient. Radio- autographs prepared from the chromatograms of tissue extract from the 40-minute incubations revealed that some L-valine is metabolized in this period. However, L-valine was the only radioactive ninhydrin-positive compound on the chromato- grams. A second radioactive spot was present but did not react with ninhydrin. A representative chromatogram is shown in Figure 2. It was not feasible to characterize the unknown metabolite further. On the other hand, radioautographs prepared from 2-dimensional chromatograms of worm extracts from 2-minute incubations in L-valine-C14 revealed a single radioactive spot which proved to be identical with valine by the "fingerprint" method. The amino acid entry systems of the host gut Agar et al. (1956) differentiated and studied (1) the absorption of amino acids by the rat intestine in vivo; (2) the transfer of amino acids from inner to 126 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN TABLE IV Uptake of L-valine-C14 by spiral valve tissue of Mustelus spiral valve. Incubation in 5 X 10~ 3 M L-valine for 2 minutes at 10 C. Values are counts per minute per gram alcohol-extracted dry -weight Spiral No. 2 80,300 85,900 93,500 74,700 79,000 87,000 88,800 82,000 Spiral No. 6 79,300 89,600 65,100 86,500 88,900 80,600 90,000 Mean 83,900 82,857 outer fluids using loops of rat intestine; and (3) the uptake of amino acids by intestinal tissue. In short experiments, the kinetics of the latter two showed rather good agreement, although, as might be expected, a lag was observed in transfer experiments. Since the removal of amino acids from the lumen of the gut by tapeworms and by the host mucosa, rather than transport of amino acids in the UJ OC UJ 0. (0 H o o MILLIGRAMS DRY WEIGHT FIGURE 3. The quantity of labeled valine taken up by various amounts of spiral valve tissue of Mustelus. Tissue weight is alcohol-extracted dry weight. MEMBRANE TRANSPORT IN PARASITISM 127 extra-intestinal host tissues, is the primary biological aspect to be considered here, experiments to study the uptake of amino acids by dogfish intestinal tissues were carried out. The preparations used were pieces from the lamina of the spiral valve cut approximately 1 cm. square. As many as 30 such pieces can be obtained from the lamina of a single spiral turn in the intestine of a sexually mature dogfish. Most of the experiments to be described were carried out with tissues from the fourth spiral posterior to the pyloric valve, although, as will be shown below, preparations from other spirals would probably yield quite comparable data. Calliobothrinm is found predominantly in the region of the fourth and fifth spirals. The methods of handling the dogfish tissue were de- TABLE V Reciprocal inhibition of L-serine and L-valine entry and L-lysine entry into spiral valve tissue of Mustelus canis. Data presented as in Table I. For experimental details, see text Amino acid Inhibitor No. l/S V 200 42,000 3725 None 4 500 28,700 4340 4 1000 14,900 1859 L-serine-C14 4 2000 7695 1236 4 500 10,666 1231 5 X 10-W 4 1000 6056 586 L-valine 4 2000 2966 561 4 200 68,980 db 8271 4 500 35,630 6643 None 4 1000 21,996 5036 L-valine-Cl4 4 2000 13,286 1306 4 200 32,666 2714 5 X lO" 3 ,!/ 4 1000 15,860 2286 L-serine 4 2000 9760 1699 4 200 27,293 646 L-lysine-C14 None 4 4 500 1000 17,551 3409 10,800 1184 4 2000 6575 1197 scribed earlier. It was reasoned that if preparations from different parts of single or separate spiral lamina of the intestine showed consistency in rate of amino acid uptake, with respect to weight of tissue, replicate samples from a single fish could be used in amino acid uptake studies. Initially, therefore, a determination of amino acid uptake was made with tissue samples removed from the second and sixth spirals. Data obtained with tissues from a single fish are presented in Table IV and show remarkably that there is no significant difference in the entry of L-valine into tissues from these two regions of the intestine nor into tissues from different parts of the same lamina. Further, the amount taken up is proportional to the dry weight of tissue used (Fig. 3). These findings showed that multiple sampling for kinetic studies is feasible with these preparations. 128 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN TABLE VI Effects of other amino acids on entry of L-valine into spiral valve tissue. V = counts /min.f gram alcohol-extracted dry tissue. N = Number of experiments Inhibitor Glycine L-leucine L-isoleucine L-methionine L-threonine L-lysine None N 4 4 4 4 4 4 4 98,771 57,692 44,326 40,253 71,655 79,363 91,799 6129 3406 1267 3294 4029 2326 1079 When the concentration of L-valine was varied, it was found that the entry of the amino acid into mucosal tissues follows an adsorption isotherm, and the apparent Michaelis constant for valine entry is about 5 X 10~ 3 . The addition of unlabeled L-serine at a concentration of 5 X 10~ 3 produced an inhibition of L-valine entry which was competitive in nature (Table V.) Conversely, L-valine com- petitively inhibits L-serine entry (Table V) which has an apparent Michaelis constant of about 4.5 X 10'". A number of other amino acids were tested as inhibitors of L-valine entry. Data obtained are summarized in Table VI. It was found that "preloading" the mucosa, by incubating for 40 minutes in non-radioactive L-valine, produced no effect on the subsequent entry rate of L-valine-C14. In balanced salt solution without added non-radioactive amino acid, L-valine does not leak out of the intestinal tissue to a significant extent. TABLE VII Relative concentrations of free amino acids in the fluid contents of the spiral valve of Mustelus canis. All values are related to a valine concentration of 1.00. mg. N = Mg. of alcohol-soluble nitrogen in the sample Amino acid Dogfish No. X S.E. 1 2 3 4 5 6 7 8 9 KI 11 12 Leucine 1.43 1.46 1.20 1.53 1.43 1.67 1.44 1.15 1.18 1.38 1.61 1.41 1.41 0.047 Phenylalanine Valine 0.77 1.00 0.77 1.00 0.53 1.00 0.71 1.00 0.47 1.00 0.58 1.00 0.62 1.00 0.54 1.00 0.84 1.00 0.57 1.00 0.75 1.00 0.59 1.00 0.64 0.034 1.00 Tyrosine Alanine 0.76 1.35 0.91 0.78 0.55 0.87 0.94 1.94 0.43 1.50 0.65 1.42 0.82 1.65 0.63 1.21 0.90 1.13 0.50 1.25 0.75 1.66 0.70 1.51 0.71 0.049 1.36 0.096 Threonine 0.58 0.42 0.35 0.65 0.78 0.49 0.88 0.75 0.83 0.72 1.07 0.84 0.70 0.060 Glutamic 1.98 1.34 1.61 4.24 2.66 3.63 7.65 2.70 2.42 1.99 2.87 2.92 3.00 0.49 Glycine & serine Aspartic Lysine Histidine 1.61 0.95 1.37 0.51 1.18 0.67 1.00 0.48 1.72 1.11 1.70 0.73 5.41 2.47 5.77 2.90 1.52 7.05 3.12 1.43 2.27 0.77 4.61 3.06 1.85 1.01 2.41 1.15 1.78 0.69 6.26 2.60 2.04 1.09 4.13 3.50 3.32 0.33 3.44 1.38 6.97 1.61 3.14 3.46 2.44 0.51 3.33 0.45 1.94 0.29 3.13 0.63 0.64 0.095 Arginine Cysteine Beta aminoiso- 0.46 0.57 0.53 0.84 0.18 0.39 0.70 0.19 0.82 0.60 0.52 0.34 0.86 0.35 0.26 0.59 0.33 0.21 0.46 0.078 0.19 0.090 0.081 0.037 butyric Beta alanine 0.24 1.94 2.21 0.99 0.55 1.44 0.62 0.23 mg. N 4.70 1.43 2.27 4.20 7.40 3.35 4.84 3.28 2.60 5.80 3.00 9.40 MEMBRANE TRANSPORT IN PARASITISM 129 Using dogfish gut preparations, attempts were made to show the inhibition of L-histidine uptake by other amino acids in experiments of 30-minute duration. L-histidine was taken up by the tissues and, during the experimental period, molar ratios of histidine in the tissue water/histidine in the external fluid of more than 2 developed. Initial concentration of histidine in external fluid was 1 mM. Addition of L-alanine, L-proline, L-valine, L-serine, or L-aspartic acid at a concentration of 2.5 mM. did not affect L-histidine uptake significantly. Histidine was removed from the medium at the rate of 20 to 40 micromoles per gram dry weight per 30 minutes. Free amino acids of the intestinal lumen Samples of the fluid contents of the spiral intestine were collected from a number of dogfish. There was great variation in the nutritional state of these animals. Some were freshly captured and some had been held in captivity for as long as 8 days without food. The samples were taken with a calibrated pipette from the middle portion of the spiral valve of living fish. The measured volume was immediately added to 10 volumes of 70% ethyl alcohol, mixed, and allowed to settle for several days. Free amino acids in the supernatant liquid were quanti- tatively determined. The analyses are summarized in Table VII. It is evident that there is great variation in the absolute quantity of alcohol-soluble nitrogen and of single amino acid components. However, study of the data shows that, for the most part, the molar ratios of one amino acid to another are strikingly constant. The dicarboxylic acids, aspartic and glutamic acid, show considerable variation, but it may be seen that the ratio aspartic/glutamic is relatively stable. The lysine/valine ratio also showed considerable variation. This may be associ- ated with the relatively low rate at which lysine penetrates the mucosa. The methods used did not allow a clear separation of glycine and serine and these two amino acids were estimated together. However, it was roughly estimated that serine made up about 75 per cent of this value. DISCUSSION The demonstration that certain amino acids, which inhibit the entry of L-valine into CaUiobothriiim, do so competitively, as previously shown with L-leucine (Read. Simmons and Rothman, 1960) suggests that this may also be the case with other inhibitory amino acids. Limitations of time and material have not allowed a complete analysis of the inhibitions of valine entry produced by cysteine, methionine. glycine, or proline. Furthermore, some of the amino acids which do not inhibit the entry of L-valine or L-leucine at a concentration ratio of 2 : 1 may very well inhibit at higher concentration ratios. D-valine was found to inhibit L-valine entry at a very high D/L ratio but had no significant effect when D-valine/L-valine was 2 (Read. Simmons and Rothman, 1960). Of great interest is the failure of mutual com- petition between L-lysine and L-valine over a wide range of concentration ratios. Since L-lysine entry appears to follow adsorption kinetics, the lack of interaction with L-valine entry suggests that the two compounds enter at different sites. The inhibition bv other amino acids of L-valine and L-serine entrv into the 130 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN intestinal tissue of the dogfish, shown to be mutually competitive in the case of L-serine and L-valine, is consistent with the observations of others who have reported inhibition of intestinal absorption of single amino acids by other amino acid species in warm-blooded vertebrates (Wiseman, 1955, 1956; Agar et al., 1956). A difference, however, is observed in the case of L-histidine uptake by dogfish and rat intestinal tissues. Agar ct al. (1956) found that L-histidine uptake was markedly inhibited by addition of equimolar concentrations of a number of single amino acids. Inhibition was not observed with dogfish tissues when several amino acids were singly added at twice the histidine concentration. Christensen and his co-workers (reviewed by Christensen, 1959) have described reciprocal inhibitions of amino acid uptake with Ehrlich ascites tumor cells and such relationships between amino acids are known to occur with Ncurospora (Mathieson and Catchside, 1955) CALLIOBOTHRIUM -600 ISO MUSTELUS SPIRAL VALVE -200 200 500 1000 -400 -200 -I (MOLAR) 200 500 1000 I/S FIGURE 4. A kinetic comparison of the uptake of three amino acids by Calliobothrhun and Mustclus spiral valve tissue. and certain bacteria (Cohen and Monod, 1957). It appears to be a very general phenomenon, perhaps as general as the distribution of special membrane mecha- nisms for amino acid entry. It seems unfortunate that some of the bacterial physiologists have chosen to refer to such entry systems as "permeases," a term implying that there is an entry enzyme. It is to be hoped that a term with such specific connotations is not widely adopted until considerably more understanding of mechanism is attained. There are definite differences in the amino acid entry systems of Calliobotliriinn and the dogfish intestinal tissue. In Figure 4 the worm and host tissue are compared with regard to their affinities for three amino acids. Several years ago the senior author reviewed the literature on the physiology of the small intestine with special reference to its peculiarities as a habitat (Read, MEMBRANE TRANSPORT IN PARASITISM 131 1950). It was concluded that there is a flow of organic compounds, with the notable exception of carbohydrates, from the tissues into the gut lumen and that much of this material is resorbed in areas of the gut distal to the point of secretion. It seemed obvious to infer that many of these materials are available to lumen- dwelling parasites. The present data on the free amino acids in the gut lumen of dogfish in highly variable states of nutrition, and the great stability of molar ratios of these amino acids lend weight to the above concept. As a part of a study of mammalian nutrition, Nasset and his colleagues (1955) demonstrated that molar ratios of free amino acids, one to another, in the small intestine of the dog are astonishingly constant and independent of the composition of protein ingested. The concentration ratios were essentially unchanged in dogs receiving no protein by mouth. This has broad implications in considering intestinal para- sitism. It reinforces the senior author's argument that in many chemical charac- teristics, the small intestine represents a relatively stable environment in a particular host. More specifically, it invites inquiry as to what effects constancy of the relative amounts of different amino acids might have on a particular intestinal parasite. It seems plain that if the environment offers a mixture of amino acids or other compounds of nutritional significance, and if these compounds compete with or otherwise affect the entry of one another into the tissues of the parasite, the ratios of amino acids in the mixture will be extremely important in determin- ing whether the nutritional requirements of the parasite can be maintained in a balanced state. The concept emerges that the ratios of nutrients may be critical in determining whether or not a given mixture of amino acids will represent a satisfactory food for a parasitic organism such as a tapeworm. Thus, ratios of nutrient concentration may be critical limiting parameters at the interface between host and parasite. Further, the ratios of rates of entry of amino acids may be manifestations of an important regulatory system governing the make-up of the amino acid pool in a given worm. As a homeostatic mechanism of importance in the physiology of the vertebrate, the competitions between amino acids would seem to represent part of a mecha- nism for regulating the composition of the amino acid mixture entering the portal system and hence the liver. Nasset (1957) has presented evidence that the relative concentrations of amino acids in the small gut are maintained by the secretion of endogenous nitrogenous material which is mixed with the ingesta. It would seem that the regulation of the composition of the amino acid pool for protein synthesis in the vertebrate begins at the mucosa. While it would appear that the host and the parasite are competing with each other as whole organisms and, from this standpoint, the competition of worm and host should be considered in terms of total absorption by worm and intestine, we may consider highly localized competition in terms of the entry systems for a particular amino acid. If other amino acids affect entry of this amino acid into the mucosa and the worm to differing extents, it is apparent that the concentration ratios of amino acids may undergo alteration in the immediate vicinity of the worm-mucosa system. If the rates involved do not undergo marked change, a new set of concentration ratios should be established. If this is indeed true, it may have wider implications in considering parasitisms in which the most obvious effects on the host are general unthriftiness or ill defined interferences with nutrition. It has not been feasible to study this experimentally in the dogfish-cestode system 132 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN hut it may be practical with other host-parasite combinations more amenable to lal (oratory control. It becomes increasingly apparent that the gut should not be considered a space outside the vertebrate body. The rapid changes in the properties of intestinal mucus indicating hydrolysis of components (Hartiala and Grossman. 1952) suggest that secretion, hydrolysis, and resorption must occur constantly. Lumen parasites are in a position to remove from this exocrine-enteric circulation compounds of nutritional value. If the data on rate of entry of individual amino acids into CaUiobuthriuin and host intestinal tissue are calculated on the basis of water content, making the assumption that the amino acids are in solution in this water, the tapeworm takes up the amino acids studied at a much higher rate than host intestinal tissue. However, the data on competitions indicate that rates of absorption for single amino acids are not directly applicable to complex mixtures. Stud}' of entry of single components from complex mixtures is obviously required. SUMMARY 1. The entry of CT4-L-valine into the tapeworm, CalliobotJiriitiu verticillatum., is competitively inhibited by L-serine, L-threonine, and L-alanine. Conversely, L-valine competitively inhibits the entry of C14-L-serine. 2. The entry of C14-L-valine is not significantly affected by L-lysine and, conversely. L-lysine entry is not affected by L-valine. 3. L-valine is concentrated against a gradient by Calliobothriuin in experi- ments of 40-minute duration. 4. The entry of C14-L-valine into mucosal tissues of the dogfish host, Must el us canis, is competitively inhibited by L-serine and, conversely, C14-L-serine entry is competitively inhibited by L-valine. L-leucine, L-isoleucine, L-methionine, L-threonine, and L-lysine also inhibit C14-L-valine entry but it has not been shown that inhibition is competitive. 5. In experiments of 30-minute duration, L-histidine uptake by dogfish mucosa was not affected by L-alanine, L-proline, L-valine. L-serine, or L-aspartic acid at the concentrations tested. 6. Quantitative analyses of free amino acids of the dogfish intestinal lumen showed variability in the absolute concentrations but great stability in the relative concentrations. 7. The data are discussed in terms of differences in amino acid entry systems of host and parasite, the significance of stability of amino acid ratios in the nutrition of host and parasite, and the necessity for evaluating host-parasite competitions in terms of entry of single components from complex mixtures. LITERATURE CITED AGAR, W. T., F. J. R. HIRD AND G. S. SIDHU, 1956. The absorption, transfer, and uptake of amino acids by intestinal tissue. Biochiin. Biopliys. Acta, 22: 21-30. CAMPBELL, J. W., 1960. The nitrogen and amino acid composition of three species of anoplo- cephalid cestodes : Monicsia cxpansa, Thysanosoina actinoides, and Cittotaenia perplexa. E.vp. Parasitol. (in press). CHRISTENSEN, H. N., 1959. Active transport, with special references to the amino acids. Perspectives in Biol. Mcd., 2: 228-242. MEMBRANE TRANSPORT IN PARASITISM 133 COHEN, G. N., AND J. MONOD, 1957. Bacterial permeases. Bact. Rev., 21 : 169-194. FOWDEX, L., 1951. The quantitative recovery and colorimetric estimation of amino-acids sepa- rated by paper chromatography. Biochcut. J., 48: 327-333. HARTIALA, K., AND M. I. GROSSMAN, 1952. Studies on chemical and physical changes in duodenal mucus. /. Biol. Chcin., 195 : 251-256. LAM;, C. A., 1958. Simple microdetermination of Kjeldahl nitrogen in biological materials. Anal. Chan.. 30: 1692-1697. LEVY, A. L., AND D. CHUXG, 1953. Two-dimensional chromatography of amino acids on buffered papers. Anal. Chew.. 25: 296-299. MACPHERSON, H. T., 1946. The basic amino acid contents of proteins. Biochcm. J., 40: 470-480. M ATHIESON, M. J., AND D. G. CATCHsiDE, 1955. Inhibition of histidine uptake in Ncnrospora crassa. J. Gen. Microhiol.. 13: 72-83. X ASSET, E. S., 1957. Role of the digestive tract in the utilization of protein and amino acids. J. Amcr. Mcd. Assoc., 164: 172-177. NASSET, E. S., P. SCHWARTZ AND H. V. WEISS, 1955. The digestion of proteins in rivo. J. Xiitr.. 56: 83-94. READ, C. P., 1950. The vertebrate small intestine as an environment for parasitic helminths. Rice I nst. P am phi.. 37(2) : 1-94. READ. C. P., J. E. SIMMONS, JR. AND A. H. ROTH MAX, 1960. Permeation and membrane transport in animal parasites : Amino acid permeation in cestodes from elasmobrachs. /. Parasit., 46: 33-41. WISEMAN, G., 1955. Preferential transference of amino-acids from amino-acid mixtures by sacs of everted small intestine of the golden hamster (Mesocricctns itnratns) . J. Parasit., 127 : 414-422. WISEMAN, G., 1956. Active transport of amino acids by sacs of everted small intestine of the golden hamster ( Mesocricctus tinnitus). J. Plivsiol.. 133: 626-630. PIGMENTED FAT CELLS IN A MUTANT OF DROSOPHILA MELANOGASTFR ' M. T. M. RIZKi Department oj Biology, Rccd College, Portland 2, Oregon The "red cell" mutant of Drosophila melanog aster is characterized by the presence of scattered pigmented cells in the thorax and head of the adult fly. This recessive mutant factor, re, is located on the second chromosome, and histological examination of the re phenotype by Jones and Lewis ( 1957) revealed that the red pigment was localized in granules in some of the pupal fat cells. The expression of the re factor was suppressed in the presence of mutant genes which interrupt the synthesis of the brown pigment of the eye: vermilion, scarlet, and cinnabar. On the other hand, the mutant gene brown which blocks the synthesis of the red component of the normal eye color did not interfere with the expression of the re gene as red cells in the thorax and head of the adult fly. Therefore Jones and Lewis (1957) concluded that the pigment in the red fat cells of re flies is related to the brown pigment component of the eye. This proposed relationship to insect eye pigments enhances the usefulness of the re mutant as a tool in probing the pigmentation process at a cellular level. The large pupal fat cells offer excellent material for experimental manipulations of individual cells. In addition, the implantation experiments of Beadle (1937a, 1937b) with eye discs and various tissues of Drosophila established the fatbody as a source of pigment precursors for the synthesis of brown eye pigment. Prelimi- nary observations of interaction between the re mutant and a mutant strain which develops melanotic masses in the fatbody suggested the present study. MATERIALS AND METHODS A stock homozygous for both the re and tit"' factors was made, and all experi- mental procedures utilized material from this stock. The wild type strain. Orc-R, was used to verify the morphological relationships of the various fat masses in the larval stages. The re mutant stock was kindly provided by Dr. E. B. Lewis in 1955, and the re gene has been maintained in our laboratory in combination with the tu w factor since that time. Melanotic tumorous masses occur in larvae homo- zygous for the recessive factor, tu w , located on the second chromosome. Detailed studies of this mutant stock have been reported previously (Wilson et al., 1955; Rizki, 1957). The tu w rc stock has been raised on Cream of Wheat medium with Fleischmann's yeast. Timed material for the experiments was collected in the following manner. Adult flies were placed in a half-pint bottle containing a paper teaspoon with Cream of Wheat medium heavily coated with a yeast-honey suspension. A fresh 1 This investigation was supported by a research grant RG 5285 from the National Institutes of Health, Public Health Service. 134 PIGMENTED FAT CELLS IN DROSOPHILA 135 food spoon was placed in the bottle twice daily, and after removal from the bottle the spoons were stored in an incubator at 23-25 C. Beginning at approximately 20 hours after a spoon had first been placed in the collection bottle, the newly emerged larvae were collected at intervals of one or two hours. These larvae were raised on Cream of Wheat-Fleischmann's yeast medium in crystallizing dishes in the incubator at 23-25 C. All ages of larvae and pupae are thus counted from the time of eclosion from the egg. In the starvation experiments, larvae were removed from the food dishes at 65 hours of age, rinsed in a saturated solution of NaCl, 2% solution of NaOCl, followed by repeated washing in six changes of distilled water. This procedure removed a considerable proportion of the adhering yeast and food particles. \Yashed larvae were placed on tissue paper strips (Kleenex brand) moistened with distilled water in petri dishes ; paper was also placed under the cover of the dish to prevent larvae from crawling out of the dishes. Care was taken to maintain the paper strip moist without excessive wetting. With each starvation experiment, a group of washed larvae from the same collection period was placed in petri dishes on paper strips to which a thin layer of Cream of Wheat medium and Fleischmann's yeast was added. These larvae served as the control fed material. The larvae removed from food at 65 hours of age generally pupated several hours before those which were left on food. No difference between the percentages of adults emerging in the two groups was noted. RESULTS No "red cells" appeared in the tit"' re homozygotes in the stock bottles or in the control fed series. However, the expression of the re factor was 100 per cent if the tu"' re larvae were removed from food at approximately 65 hours of age, that is, during the early third instar. In this case, the mutant pattern appearing in the adult flies was the same as that described by Jones and Lewis (1957) for the re stock. Red-pigmented cells were most abundant in the thorax and head, a few were found in the abdomen, and an occasional pigmented cell was seen in the appendages. In pupae shortly before hatching the red pattern in the thorax was striking. The red-pigmented cells occupied the haemocoel spaces between the flight muscles, and a dorsal striped pattern in the thorax was the result of alter- nating accumulations of red cells and regions of the muscle insertions (Figs. 1 and 2). The re factor exhibited no apparent influence on the development of melanotic masses in the posterior fatbody, the phenotypic character of the tit 11 ' factor. The penetrance of the tu"' factor varied between 90% and 95% during the course of the present investigation; however, the re phenotype was expressed in tu li 're flies which had been starved during development whether melanotic tumors were visible or not. Control fed larvae and starved larvae were examined at timed intervals after the beginning of the starvation period at age 65 hours. Starved larvae generally pupated two or three hours earlier than larvae which were feeding. No morpho- logical differences were noted between the two groups of larvae until after pupa- tion. During the early period of tanning of the puparium. a slightly yellow tinge was noticeable in the anteriormost fat masses of starved tit"' re pupae. This color 136 M. T. M. RIXKI PLATE I PIGMENTED FAT CELLS IN DROSOPHILA 137 was visible through the puparium which was still very light, but removal of this covering was necessary to reveal the extent of the coloring. Figure 9 is a camera lucicla drawing of a starved tit"' re pupa showing the location of the yellow masses underneath the puparium. and a photograph of a specimen removed from the puparium shortly after the pupal molt is labelled Figure 3. Dissection of pupae showed that the yellow color was localized in the most anterior pair of dorsal fat masses just posterior to the cerebral hemisphere, as well as those fat masses which are lateral to this first region and extend ventrally where they join in a commissure. From the ventral side of each of these extensions, a strand of fat cells passes anteriorly where they adhere to the paired salivary glands. The fat masses just described are the only fat cells which developed the yellow pigment. Larvae of the Ore-R strain have been dissected to establish morphological correla- tions of the fat masses in the mutant strain with the fat masses in the normal mate- rial. Dissection of the larvae of the wild type strain was considerably easier since manipulation of the fat masses in the starved tit "'re larvae tended to loosen the fat cells which did not seem to be held together so compactly as in the normal strain. Starvation also decreased the size of the fatbody cells as compared to fed material. In order to demonstrate the relationship of the intact fat masses, camera lucida drawings of the Orc-R strain are given in Figure 10. The common feature of these anterior fat masses is their proximity to the distal ends of the anterior pair of Malpighian tubules. As pupation progressed the cells of the fatbody became separated from one another, and were thus involved in the extensive reorganization of the body tissues which takes place during metamorphosis. The pigmented cells of the anterior fatbody were redistributed during this process and were found primarily in the head and thorax of the developing pupa. The color of the fat cells in the intact fatbody of starved tit"-' re pupae gradually changed from yellow to yellow-brown, and as the scattering of the separated cells occurred, the color deepened, and finally red pigment was apparent. The yellow color in the fatbody appeared before the imaginal discs had everted, and the red pigment in the fat cells preceded the appearance of the red eye color. FIGURE 1. Photomicrograph of a tit "'re pupa (starved) removed from the puparium. Red cells, RC, are distributed between the longitudinal muscles, M, of the thorax. A typical melanotic tumor, T, is apparent in the abdomen and two re fat cells are visible on the left margin of the tumor. (Darkfield illumination with a green filter. < 63.) FIGURE 2. A fully formed imago removed from the puparium to show the further dispersion of the red-pigmented fat cells (arrows). Note the retention of re cells in the mid- dorsal region and the further change in the distribution of re cells in the areas not occupied by the insertions of the dorsoventral muscles of flight. Very few re cells are seen among the fat cells of the abdominal region. The eyes are fully pigmented. ( Darkfield illumination, green filter, magnification X 60.) FIGURE 3. A tu w re (starved) prepupa removed from the pupal case, showing the position of the intact pigmented masses of fat, RCF. Note the intimate association of the anterior pair of Malpighian tubes, MP, with the RCF. The photograph represents the left dorsolateral view of the prepupa so that the left tracheal trunk is apparent and the right tracheal trunk can be visualized as a white band at the lower right margin of the photograph. The dark area under the left tracheal trunk is a melanotic tumor in the caudal fatbody, T. (Transmitted light from a Corning filter #CS 7-59. Magnification X 63.) FIGURE 4. The intact fat masses removed from a tn"-'rc prepupa (starved) showing the difference between pigmented fat masses, RCF, and unpigmented fat masses, F. This fat was treated with potassium metabisulfite to intensify the color. (Corning filter #CS 7-59; X 63.) 138 M. T. M. RIXKI PLATE II FIGURE 5. A pigmented fat cell isolated from an early prepupa (tit"' re starved) showing the intracellular distribution of pigment globules, PG. (Corning filter #CS 7-59.) FIGURE 6. A pigment globule removed from the cytoplasm of a red cell from a tit"' re prepupa (starved) showing the threadlike, DT, internal structures characteristic of these globules. A fat droplet is indicated at FG. (Corning filter #CS 7-59.) FIGURE 7. The re fat cells as seen through the body wall of a tit 11 ' re starved pupa corre- sponding in age to that given in Figure 1. Note the appearance of pigment in granular form. (Darkfield illumination, green filter.) FIGURE 8. Granular appearance of pigmented inclusions in a tit "'re starved imago corre- sponding in age to that in Figure 2. Bristles, B, and setae, S, are visible in this photograph of a red cell as seen through the body wall. ( Darkfield illumination, green filter. ) PIGMENTED FAT CELLS IN DROSOPHILA 139 Both the red and hrmvn pigment extracts from the eye of Drosophila are altered by oxidation and reduction (Ephrussi, 1942). It seemed desirable to determine whether the yellow pigment in the fat cells would undergo any changes /;; I'ltro. and a search was undertaken for conditions which might cause such an alteration. Anterior fat masses which had become yellow were dissected from starved pupae in \\addington Ringer- 109^ glucose. The various reagents to be tested were then added to this medium. The reducing rinse which had been pre- pared for use in the Feulgen reaction proved most satisfactory. It was then found t, ^ .-.:: -7t ;' /:' ' ij .. ::'..r *..'. .''$ :' t ' k '. ?" .v-h I s ' :- -a . ".. JV: ^ : FIGURE 9. Camera lucida drawing of a dorsal view of a tit ""re (starved) prepupa within the puparium illustrating the position of the brownish yellow anterior fat mass, RCF. convenient to add a few crystals of potassium metabisulfite to the drop of glucose- Ringer containing the isolated fat masses while they were under microscopic ob- servation on a white porcelain plate. The color of the fat cells showed a change from yellow to red within a minute. Figure 4 is a photograph of the isolated fat mass in which the re pigment had been intensified in this manner. The development of pigment in the starved tit"' re flies has been followed at the cellular level. Isolated cells from the anterior fatbodies of young pupae contained 140 M. T. M. RIZKI numerous yellow cytoplasniic globules. These globules were distinguishable from the fat droplets of the cells which are highly refractile and always spherical in fresh preparations. Isolated cells have been examined with darkfield illumination as well as brightfield illumination, and in addition, the use of a blue Corning * - , ''.-' ' '" " " * - r' - ' ' ' ' j^-** \"i*> ''V ;^^%^ff... ^/f^f0l^ '. ^^l^S^T^X -jX^Jhv-v.'^ ;-:, ":*? k '--i-iijfc" -'> .*" --^v-. '.-v-^-.f'.'-.' ' ' * : -*tev * : / -i\ &&: ^-IKMte ^t^lil ' -<':./'' -^ "^^^-3? ' - ' 1:''. "'-..' -^/<'iv! i :-- ' , 'A''.*:-' iKl'ji ^.,..-' BR FIGURE 10. Camera lucida drawings of a dissection of a late third instar larva of the Ore-R strain showing the relative positions of the fat masses ; the salivary glands, SG ; brain, BR; Malpighian tubes, MP. This specimen has been stained with Oil Red O. The diagram on the left is the ventrolateral view, and the same dissection has been turned over to show the lateral view of the fat masses in the drawing on the right. A, B, C, D, E are the regions of the fatbody which become pigmented in starved tn"'rc pupae. A, dorsal pair of fat masses; B, C, lateral fat masses ; D, fat mass attached to salivary gland ; E, fat cells forming the ventral commissure between the right and left masses of fat. Only the anterior region of C becomes pigmented. The club-shaped structures with concentric rings are the imaginal discs and the long tubular structure with branches is the tracheal trunk. Filter No. CS 7-59 proved most satisfactory for studying the cytoplasmic inclusions in the fat cells. With this filter, all yellow objects appeared bright red. In early tu w rc pupae (after starvation), this included the lightly tanned cuticle, isolated cells from melanotic masses, the granular structures in the Malpighian tubules, PIGMENTED FAT CELLS IX DROSOPHILA 141 and the yellow globules in the re fat cells. The yellow globules showed a definite threadlike internal structure when examined with this filter, while no structure was discernible in the fat droplets (Figs. 5 and 6). Fat cells from the more posterior regions of the pupa, i.e., cells other than the fatbody cells involved in the expression of the re phenotype. contained similar cytoplasmic inclusions in addition to the fat droplets. Whether these inclusions are structurally and func- tionally the same as those globules which become pigmented in the re cells remains to be examined. The cells containing the yellow globules were placed on a slide in \Yaddington- lO'/r glucose solution and several crystals of potassium meta- bisulfite were added under the coverslip in the vicinity of the cells. The yellow inclusions became red in color under these conditions. After the pigmented fat cells had become scattered throughout the thorax and head during the developmental processes occurring in pupal life, the color in the fat cells appeared more intense. In the late pupae and young adults, the pigmented structures in these cells were more granular in appearance (Figs. 7 and 8). DISCUSSIOX Two types of pigment are found in the eye of Drosophila, one brown and the other red. and many mutants are known which affect the production of these pigment components in the eye. Interference with brown pigment production results in a bright red eye color of the type found in the mutants vermilion, cinnabar, and scarlet while the phenotype of the brown eye mutant represents an interruption in the biochemical pathways leading to red pigment. The absence of both pigments occurs in the mutant, white eye. The literature on brown eye pigments in insects has been reviewed by Ephrussi (1942) Xolte (1952), and Kikkawa (1953). The synthesis of brown pigment proceeds through a pathway involving tryptophan, formylkynurenine, kynurenine, and hydroxykynurenine, and the known eye color mutants are blocks at successive stages in this synthetic chain. Transplantation of imaginal discs of mutant larvae into hosts of different geno- types has shown that in some cases the eye color is autonomous, whereas some mutant eyes do not themselves produce the prerequisites for brown pigment and are dependent upon other sources in the body for these precursors (Beadle and Ephrussi. 1936; Ephrussi, 1942; Ephrussi and Beadle, 1937). The presence of these pigment precursors has been demonstrated in the fatbody and the Mal- pighian tubules by transplantation experiments (Beadle, 1937a, 1937b). The time during which each of these tissues produced the pigment precursors was dependent upon the stage of development of the donor. Malpighian tubes showed activity through larval life from the earliest stages tested, appearance of active substances in the fatbody was not detected until after pupation, and in the eyes much later during pupal development (Beadle, 1937b; Clancy, 1940). The pigment granules in the re cells have been shown to be related to the brown eye pigment of Drosophila. Jones and Lewis (1957) found that the mutant factors, vermilion, cinnabar and scarlet, which interfere with brown pigment de- velopment in the eye, also prevent the formation of pigment in the re cells when each of these mutant factors is combined with the re gene. The mutant factor, brown, which blocks the synthesis of red pigment, does not interfere with the expression of the re gene in the fat cells. 142 M. T. M. RIZKI The explanation for the suppression of pigment in the fat cells when the re gene is combined with the tit"' factor may not he so direct. In the starvation experiments the penetrance of the tu' r factor was 90% 95%, and no difference in the expression of re was noted between the pupae with melanotic tumors and those that did not develop black masses. One point of comparison is the fact that both mutants have a common domain of expression, i.e., fat cells: re, the anterior fat mass and tit"', the posterior region of the fatbody. Tryptophan metabolism is not only related to the development of brown eye pigment and protein metabolism, but it also influences the expressivity and the penetrance of various melanotic tumor genes in nrosophila. Addition of tryptophan to the medium increases the frequency of melanotic tumors in strains carrying tumor genes (Hartung and Hartnett. 1951; Plaine and Glass, 1955), and Kanehisa ( 1956a, 1956b) reported an increase in tumor incidence by combining a tumorous factor with eye color genes. The appearance of pigmented fat cells in tn"'re pupae after starvation parallels the behavior of the vermilion mutants which develop brown eye pigment after the larvae have been starved (Beadle ct a/., 1938). Starvation of Drosophila adults results in a reduction in the size of the fatbody, and the reserves of fat and glycogen are rapidly depleted from the fat cells (Wiggles worth, 1949). In many insects the fatbodies may serve as storage sites for excretory products as well as food reserves. Wigglesworth (1942) has shown that starvation of Aedes larvae causes an increase in uric acid vacuoles in the fat cells and these deposits disappear from the cells after the feeding has resumed. The conditions imposed by starvation in the tit"' re larvae alter the metabolic pattern of the fat cell such that it differentiates as a pigmented cell. A similar effect, of course, is produced in the re mutant under normal feeding conditions. The presence of the tu w factor may restore the normal metabolic balance in the fat cell such that their phenotype resembles that of the wild type. The expression of the re phenotype is also dependent upon the action of another recessive gene, I\s, which causes an accumulation of the amino acid, lysine (Grell, 1958). It is thus obvious that the expression of the red cell phenotype is influenced by the interaction of a number of non-allelic genes. One suggestion may be made which will encompass the various aspects of the problem known at the present time. Any modification, genetic or environmental, which influences the normal pattern of protein synthesis will also alter the metabolic pool of various amino acids. Such changes which affect the availability of tryptophan may be reflected in the phenotypic expression of the re pigment. The larval fat cells of Drosophila form organized tissue masses, whereas soon after pupation the fatbody becomes separated into single cells or small clusters of cells. The cells of the caudal fat masses in tit"' larvae which are involved in the production of melanotic tumors in this strain resemble pupal fat cells in their tendency toward smaller cell aggregations and a loss of adherence to neighboring cells (Rizki, 1957). This precocious change in the structure of the caudal fat- body of tit"' larvae, as well as precocious changes in the blood cells, are processes which lead to tumor formation in the caudal fatbody prior to pupation. Therefore the hypothesis was presented that the melanotic masses in this tumorous strain of Drosophila represented an upset in the normal timed pattern of events occurring (luring metamorphosis. Under conditions favoring expression of the re gene, the PIGMENTED FAT CELLS IN DROSOPHILA 143 tit"' factor influences an earlier appearance of red pigment in the anterior fat cells. This pigmentation in the re mutant is not apparent until after the fat cells become isolated and scattered during the pupal stage (Jones and Lewis, 1957). However, the combination of re with the tn"' factor has shifted the time of development of the red pigment to a stage preceding this dispersion of the fat cells. Although no obvious explanation for the distribution of the red fat cells among other non- pigmented fat cells existed in the re strain, the morphological relationship of all the red cells in larval development becomes apparent in the tit"' re starved material. A measure of the dispersion of the cells of the anterior fat masses during early pupation is provided in this case by a mutant cytoplasmic marker. The localiza- tion of mutant characteristics in the tn"' caudal fat masses and the anterior re fat cells suggests that the cells of various regions of the fatbody may differ in their developmental physiology. It is interesting to note that the cellular ecology of the anterior and the posterior fatbody includes a common feature : the re fat cells are intimately associated with the distal ends of the anterior pair of Malpighian tubules, and the tn"' fat cells encircle the distal ends of the posterior Malpighian tubules. SUMMARY 1. Cytoplasmic pigment granules are found in some of the fat cells of the recessive mutant, re, of Drosophila melanogaster. These scattered red fat cells are located chiefly in the thorax and head, but a few occur in the abdomen and ap- pendages of the adult. Using genetic methods it had been shown previously that these pigment accumulations are related to the synthesis of the brown eye pigment of this insect (Jones and Lewis, 1957). 2. The pigmentation in the re fat cells is suppressed when the re gene is combined with the recessive factor, tit"'. This combination, however, in no way alters the expression of the characteristic pattern of tn"' as revealed by the pres- ence of melanotic tumors in the caudal fat masses of the homozygous tu' r re flies. After a period of larval starvation, the tn lc re flies develop both the red-pigmented fat cells and melanotic tumors. The time of appearance of the re pigment has been shifted under these nutritional and genetic conditions. The cytoplasmic pig- ment granules appear in the cells originating from the anteriormost section of the larval fatbody which is closely associated with the anterior Malpighian tubules. During the reorganization accompanying metamorphosis from the larval to the adult stage, these cells are redistributed mostly to the thoracic and cephalic regions while a few are found in the abdomen and appendages. An explanation is thus provided for the cytodifferentiation of pigmented and nonpigmented fat cells found side by side in the adult fly. 3. The nature of the pigment granules has been examined in in vitro prepara- tions at each of these periods of development, and of particular interest is the internal threadlike structure of these cytoplasmic inclusions during the early stages of pigment formation. LITERATURE CITED BEADLE, G. W., 1937a. Development of eye colors in Drosophila : fat bodies and Malpighian tubes as sources of diffusible substances. Proc. Nat. Acad. Sci., 23: 146-152. 144 M. T. M. RIZKI BEADLE, G. W., 19371). Development of eye colors in Drosophila: fat bodies and Malpighian tubes in relation to diffusible substances. Genetics, 22: 587-611. BEADLE, G. W., AND B. EPHRUSSI, 1936. The differentiation of eye pigments in Drosophila as studied by transplantation, Genetics, 21 : 225-247. BEADLE, G. W., E. L. TATUM AND C. W. CLANCY, 1938. Food level in relation to rate of development and eye pigmentation in Drosophilti melanogaster. Biol. Bull.. 75: 447-462. CLANCY, E. B., 1940. Production of eye color hormone by the eyes of Drosophila melanogaster. Biol. Bull., 78: 217-225. EPHRUSSI, B., 1942. Analysis of eye color differentiation in Drosophila. Cold Sprint/ Harbor Symp. Quant. Biol., 10: 40-48. EPHRUSSI, B., AND G. W. BEADLE, 1937. Development of eye colors in Drosophila : transplanta- tion experiments on the interaction of vermilion with other eye colors. Genetics, 22 : 65-75. GRELL, E. H., 1958. Genetics and biochemistry of "red cell." Proc. X Int. Cong. Genetics, 2: 104-105. HARTUNG, E., AND W. HARTNETT, 1951. A study of the relation of various dietary factors to tumor incidence in Drosophila melanogaster. Anat. Rcc., Ill : 3. JONES, J. C., AND E. B. LEWIS, 1957. The nature of certain red cells in Drosophila melano- gaster. Biol. Bull., 112: 220-224. KANEHISA, T., 1956a. Eye-colour genes and tumor incidence. Jap. J . Genetics. 31 : 144-146. KANEHISA, T., 1956b. Relation between the formation of melanotic tumors and tryptophane metabolism involving eye-colour in Drosophila. Annot. Zool. Jap.. 29: 97-100. KIKKAWA, H., 1953. Biochemical genetics of Boinb\.i- inori (Silkworm). Adi', w Genetics. 5: 107-140. NOLTE, D. J., 1952. The eye-pigmentary system of Drosophila III. The action of eye-colour genes. /. Genetics. 51 : 142-186. PLAINE, H. L., AND B. GLASS, 1955. Influence of tryptophan and related compounds upon the action of a specific gene and the induction of melanotic tumors in Drosophila melano- gaster. J. Genetics. 53: 244-261. RIZKI, M. T. M., 1957. Tumor formation in relation to metamorphosis in Drosophila melano- gaster. J. Morph., 100: 459-472. WIGGLESWORTH, V. B., 1942. The storage of protein, fat, glycogen and uric acid in the fat body and other tissues of mosquito larvae. /. Ex p. Biol., 19 : 56-77. WIGGLESWORTH, V. B., 1949. The utilization of reserve substances in Drosophila during flight. /. Exp. Biol., 26: 150-163. WILSON, L. P., R. C. KING AND J. L. LOWRY, 1955. Studies on the tu"' strain of Drosophila melanogaster: Phenotypic and genotypic characterization. Growth, 19: 215-244. EXPERIMENTAL STIMULATION OF GAMETOGEXESIS HYDROIDES DIANTHUS AXD PECTEX IRRADIAXS DURIXG THE WIXTER 1 HARRY J. TURNER, JR. AND JAMES E. HANKS ll'oods Hole ( >ccunognipliic Institution, Woods Hole. Massachusetts There is a long list of benthic marine invertebrates found in the Woods Hole, Mass, region that reproduce during the summer months. The majority of the species of organisms suitable for embryological experiments, listed by Costello et al. (1957), fall into this category. In addition, the extensive investigations of Redfield and Deevey (1952) have shown that most members of the fouling com- munity attach during the summer months at Woods Hole and in other localities where there is a considerable difference between the summer and winter tempera- ture extremes. The question arises as to which of the various environmental variables controls reproduction. Hutchins (1947) studied the world-wide distribution of a variety of benthic marine forms and came to the conclusion that the northward extension of the ranges of a number of organisms is regulated by the minimal summer temperature that permits propagation, thus suggesting that elevated temperatures either stimulate gametogenesis or induce spawning. Experimentally, Townsend (1940) obtained ripe gametes from the sea urchin Arbacia by holding specimens in aquaria at a temperature of 18 to 19 C. for one to two months in the late fall and winter. An ample supply of food was provided. Subsequently, Loosanoff and his co-workers have contributed greatly to the field by demonstrating the stimulating effects of elevated temperatures on the develop- ment of the gonads of certain commercial mollusks. Loosanoff and Davis ( 1950) succeeded in bringing the hard clam. I'enns incrccnaria, into reproducing condition during the winter months by gradually raising the temperature over a period of three weeks from that of the natural environment (5.0 to 7.0 C.) to 20 C. They mentioned that the same could be accomplished by placing the clams directly in water at 20 C. but some mortality occurred. Later (1952) these investigators studied the influence of temperature on the maturation of the gonad of the eastern oyster, Crassostrca I'irt/inica, and demonstrated a logarithmic increase in the rate of ripening of the gametes as temperatures were elevated from 15 C. to 30 C. They also determined that physiologically ripe gametes were formed earlier in the males than in the females under the same conditions. The importance of nutrition on gonad development was indicated by the failure of oysters in "poor" condition, i.e., containing little glycogen, to respond satisfactorily to the thermal stimulus. Experimental work on the effect of temperature on gonad development of 1 Contribution No. 1099 from the Woods Hole Oceanographic Institution. This investiga- tion was supported by the National Science Foundation NSF-G8905. 145 146 HARRY J. TURNER, JR. AND JAMKS K. HANKS other henthic marine invertebrates appears to lie lacking. The investigations to be* described were undertaken to determine if certain organisms occupying ap- proximately the same geographical range as that of the oyster and the hard clam would respond in a similar fashion. The serpulid polychaete, Hydroidcs dianthus (-- H. Itc.nn/onns) (Verrill), ranges from Cape Cod to Florida (Pratt, 1948) and is found from the low tide mark to depths of several fathoms. It reproduces from the middle of June to the end of October (Grave, 1933) and the ripeness of the gametes may be easily determined by removing the worms from their tubes and observing the products issuing from the nephridiopores (Grave, 1933). The bay scallop, Pectcn irradians Lamarck, ranges from Cape Cod to Texas (Turner, 1953). It is a hermaphroditic species that spawns from mid-June to mid-August in the Woods Hole region (Belding. 1931). The ripening of the reproductive products may be determined on macroscopic examination by the development of a bright orange color in the ovary at the distal extremity of the visceral mass. MATERIAL AND METHODS Specimens of H. dianthus were obtained from the supply department of the Marine Biological Laboratory in the middle of December. They had been freshly collected from the adjacent waters where the temperature was approximately 8 C C. The specimens were attached to old J'cniis and Pectcn shells in a community which included Astrangia, sulphur sponges and dead barnacle shells. The tubes ranged between 5 and 7 cm. in length, well beyond the size of earliest sexual maturity (Grave, 1933). Bay scallops were collected from the Eel Pond, Woods Hole, in December and January, in shallow water at low^ tide. They averaged a little over 3 cm. in the longest dimension and were the young of the year (Belding. 1931). There were no year-and-one-half old specimens in the Eel Pond and those usually found in nearby localities had been almost completely depleted by commercial fishing. Consequently, larger specimens were not available in adequate numbers for experi- mental work. At the beginning of the experiments, a number of specimens of H. dianthus were removed from their tubes and placed in warm (23 C.) sea water to deter- mine if they would extrude ripe gametes. No gametes were extruded and the worms were preserved in saturated aqueous mercuric chloride with 5 r /c acetic acid for histological study. Similarly a number of scallops were opened and ex- amined for the characteristic orange color of the ripe ovary. None showed this character and a number of appropriate anatomical portions were preserved in the same manner as were the worms. The worms were subjected to an elevated temperature in aerated still water. Several tube-encrusted shells, containing about two dozen worms, were placed in a non-toxic five-gallon polyethylene container of warmed sea water held at 23 C. by a constant temperature bath. The water was changed every three days and continuously aerated. Suspensions of the planktonic alga, Phacodahctylluin tricornutum ( -- Nitzschia clostcrinin, fonua ininiitissinia), w r ere added daily for food. The amount added was adjusted to the quantity that the worms would just consume in 24 hours. A control was set up and maintained in an identical manner except that the container was placed in a tank supplied by the laboratory sea water STIMULATION OF GAMETOGENESIS 147 system in which the temperature declined slowly from an initial level of 8 C. The illumination of the experimental group and the control group was practically identical. Growth of the tubes was determined in most cases by measuring the new white addition at the mouth beyond the discolored and fouled original tubes. In cases where the tubes were not sufficiently discolored to make new growth clearly distinguishable, a gram of powdered alizarin was added to the sea water and kept in suspension for 24 hours. The worms readily took up the alizarin and combined it with the calcareous material of the new growth, forming a prominent purple ring of alizarin lake. This treatment provided a very satisfactory method of marking the tubes and apparently had no deleterious effect on the worms. Specimens were removed on the third, fifth, seventh, and tenth days, tested for extrusion of gametes and preserved for histological examination. The experiment was repeated four times during December, January and early February. Preliminary experiments indicated that bay scallops could not be maintained successfully in still water so they were treated in a different manner. In late January a number of specimens were placed in a 3' X 3' X 8" Fiberglas tank supplied with running warmed sea water at a rate of -J gallon per minute. The apparatus used to warm the water was similar to that described by Loosanoff (1949) but modified to use electric power instead of gas. The temperature was maintained at 23 C. No food was added because the laboratory facilities for rearing phytoplankton were inadequate to supply the vast quantities required in running sea water systems. Consequently the scallops had to subsist on such quantities of food materials as remained in the water after it had passed through the intake line, reserve tank, and lengthy distributing system. A control was set up in a similar manner except that it was supplied with sea water at the ambient temperature which was holding steady at approximately 3 C. The light- ing conditions over both groups were identical. Specimens were removed weekly, examined grossly for gonad development and preserved for sectioning. RESULTS Hydroides diaiitJuis Xone of the worms shed gametes when first obtained, nor did any taken from the control group which was maintained at a temperature approximating that of the natural environment at any subsequent time. In the experimental group which was maintained at 23 C., gametes were first obtained after seven days of warming. Males shed copious quantities of reproductive material consisting largely of spermatocytes and spermatids with a few tailed spermatozoa. By this time, females shed numerous eggs which were very much smaller than normal size and could not be fertilized. On the tenth day all males tested produced normal spermatozoa and the females shed eggs, the majority of which were of normal size. These eggs were successfully fertilized, underwent normal cleavage ac- cording to the usual time schedule (Costello ct ol., 1957), and produced vigorous trochophores. During the ten days of warming all the worms increased the length of their tubes from three to five millimeters, indicating that they were vigorous and healthy. Worms in the control group showed no measurable growth. The sequence of events during gametogenesis of the tube worm was followed 148 HARRY J. TURNER, JR. AND JAMES E. HANKS in histological sections. The winter gonad consists of a series of pairs of syncytial masses of germ cells, one pair to each of the abdominal segments. Each segment contains a mass of reproductive material on either side at the ventro-lateral aspect of the wall of the coelom. Figure 1, A is a typical cross-section of a worm pre- c . E 1mm FIGURE 1. Transverse sections of H. diaiitlnts. Delafield's haematoxylin and eosin stain. A, Specimen collected in December. Germinal masses indicated by arrows. B, Female held at 23 C. for five days. A few ovocytes free in the coelom. C, Male held at 23 C. for three days. Numerous spermatocytes free in the coelom. D, Female held at 23 C. for seven days. Numerous ovocytes partially grown free in the coelom. E, Male held at 23 C. for ten days. Mature spermatozoa in the coelom. F, Female held at 23 C. for ten days. Mature ova in the coelom. STIMULATION OF GAMETOGENESIS 149 served in mid-winter. There was little evidence of sexual differentiation at this time although some specimens contained masses with slightly enlarged nuclei, suggestive of primitive ovocytes. All specimens examined contained a few mitotic figures in the germinal masses, indicating that the germinal material was pro- liferating slowly. Sexual differentiation became apparent in males after three days of warming. Cells broke away from the germinal masses into the coelom where the maturation divisions took place (Fig. 1, C). This process continued for the next seven days and by the tenth day the coelom w r as packed with mature spermatozoa, as shown in Figure 1, E. No significant changes in the female gonads were observed until the fifth day of warming. At this time the nuclei began to enlarge as typical germinal vesicles and a few ovocytes broke free into the coelom (Fig. 1, B). By the seventh day, there were more free ovocytes in the coelom and some had shown appreciable growth (Fig. 1, D). By the tenth day, the coelom was completely filled with ovocytes, the majority of which were of mature size (Fig. 1. F). There was remarkable uniformity in the rate of development of the gonads in all specimens examined and this was repeated in each of the four experiments run in sequence. At no time did any of the animals taken from the low temperature controls show any evidence of gametogenesis. Pcctcn irradians The reaction of the bay scallops to the elevated temperature was slower and more erratic than that of the tube worm. Approximately 5^ died during the course of the experiment. A few specimens failed to show any development of the gametes, and in many, gametogenesis proceeded for a short time and then ceased. The majority of the specimens examined eventually produced tailed spermatozoa and ova which appeared to be structurally mature on histological examination. However, normal spawning did not occur so that physiological maturity could not be determined. The bay scallop is hermaphroditic. The gonad consists of cylindrical, branch- ing, tubular follicles ramifying through the visceral mass in the blood space sur- rounding the digestive tract. The follicle wall consists of a single layer of squamous epithelium with germ cells scattered along the inside. Male and female germ cells develop in separate follicles. Sections of specimens taken from the natural environment in January showed no significant gametogenesis. The follicles of the male gonad as they appeared in sections are shown in Figure 2, A. The follicle walls of the female gonad (Fig. 2, B) appeared to be thicker because of the larger sizes of the ovogonia but there was no evidence of significant development. Marked changes occurred after the scallops had been subjected to a tempera- ture of 23 C. for one week. Rapid proliferation of the germinal material in the male resulted in a multi-layered lining of the follicles, consisting of spermatogonia and spermatocytes (Fig. 2, C), while the germ cells of the female gonad (Fig. 2, D) enlarged considerably and develop characteristic germinal vesicles with prominent nucleoli (Fig. 2, D.) 150 HARRY J. TURNER, JR. AND JAMES E. HANKS At the end of the third week of warming', the ovarian portion of the gonad acquired the orange pigmentation characteristic of the ripe ovary and the testicular portion took on a light, cream color with a plump appearance. Histological sections showed that the male follicles were lined with many layers' of spermatozoa with the tails projecting out into the much reduced lumens (Fig. 2, K). The ovarian follicles were much enlarged and were completely filled with ova of mature size -'4 5asS UEfc*"^ 1mm FIGURE 2. Sections of gonads of P. irradians. Delafield's haematoxylin and eosin stain. A, Male gonad of specimen collected in January. B, Female gonad of same specimen. C, Male gonad of specimen held at 23 C. for one week. D, Female gonad of the same specimen. E, Male gonad of specimen held at 23 C. for three weeks. F, Female gonad of same specimen. STIMULATION OF GAMETOGENESIS 151 (Fig. 2. F). None of the specimens taken from the control group showed any evidence of gametogenesis during January and February. DISCUSSION It is clear from the foregoing that the tube worm, Hydroidcs dianthus, and the hay scallop, Pcctcn irradians, respond to artificially elevated temperatures during the winter in a manner similar to that shown by many commercial molluscs, as described by Loosanoff, by developing their gametes out of season. All of the tube worms subjected to a temperature approximating that of the natural environ- ment during the normal reproductive period developed ripe gametes within ten days. These underwent normal fertilization, cleavage, and developed into viable larvae. The response of the bay scallop was somewhat erratic and the development of the gametes was much slower. It is quite probable that the experimental con- ditions were not entirely satisfactory, particularly in regard to nutrition. Oysters and clams maintained in the laboratory sea water system grow thin and watery after a time, indicating that suspended nutrient material is sparse. Consequently, the scallops held in running sea water at the elevated temperature undoubtedly had to synthesize the reproductive materials from stored substances which may have varied considerably among individuals at the time they were collected. Individuals in which gametogenesis failed to go on to completion were probably those with inadequate supplies of reserve nutrients, similar to the oysters in "poor" condition described by Loosanoff and Davis (1952). In any event it is clear that temperatures approximating those existing during the normal spawning period will stimulate gametogenesis in both P. irradians and H. dianthus if imposed experimentally during the winter months when the temperature of the natural environment is approaching the seasonal minimum. SUMMARY 1 . Temperatures approximating those existing during the normal summer reproductive period will stimulate gametogenesis in Hydroidcs dianthus and Pcctcn irradians if artificially imposed during the coldest winter months. 2. H. dianthus will produce mature gametes in ten days if held in aerated sea water at 23 C. and fed adequate quantities of Phaeodactyllum ti-icoruiitiini. 3. P. irradians may produce gametes that appear to ,be mature on histological examination in three weeks if held in running sea water warmed to 23 C. but in many cases development fails to go to completion. 4. Failure of gametogenesis to reach completion in some" specimens of P. irradians may be due to inadequate food supply under the conditions of the experiment. LITERATURE CITED (BELDING, D. L., 1931. The Scallop Fishery of Massachusetts. Marine Fisheries Series No. 3. Commonwealth of Mass., Dept. of Cons., Div. of Fish, and Game, Marine Fish. Sec. COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Alethods for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory, \Yoods Hole, Mass. 152 HARRY J. TURNER, JR. AND JAMES E. HANKS GRAVE, B. H., 1933. Rate of growth, age at sexual maturity, and duration of life of certain sessile organisms, at Woods Hole, Massachusetts. Biol. Bull., 65: 375-386. HUTCHINS, L. W., 1947. The bases for temperature zonation in geographical distribution. licol. Monofir., 17: 325-335. LOOSANOFF, V. L., 1949. Method for supplying a laboratory with warm sea water in winter. Science, 110: 192-193. LOOSANOFF, V. L., AND H. C. DAVIS, 1950. Conditioning /'. nierccnaria for spawning in winter and breeding its larvae in the laboratory. Biol. Bull., 98: 60-65. LOOSANOFF, V. L., AND H. C. DAVIS, 1952. Temperature requirements for maturation of gonads of northern oysters. Biol. Bull.. 103: 80-96. PRATT, H. S., 1948. A Manual of the Common Invertebrate Animals, Revised Edition. The Blakiston Co., Philadelphia. REDFIELD, A. C., AND E. S. DEEVEY, JR., 1952. Pt. II. The Biology of Fouling. In: Marine Fouling and its Prevention. United States Naval Institute, Annapolis. Chap. 5. The Seasonal Sequence, 48-76. TOWNSEND, G., 1940. Laboratory ripening of Arbacia in winter. Biol. Bull., 79: 363. TURNER, H. J., JR., 1953. A review of the biology of some commercial molluscs of the East Coast of North America. In : Sixth Report on Investigations of the Shellfisheries of Mass., Dept. of Nat. Res., Div. of Marine Fisheries, 39-74. A NEW SPECIES OF CHIRIDOTEA (CRUSTACEA: ISOPODA) FROM NEW ENGLAND WATERS ROLAND L. WIGLEY r. S. Department of the Interior, I : islj and Wildlife Service, Bureau of Commercial fisheries, Biological Laboratory, Woods Hole, Alassachusctts The genus Chiridotca is unique to the eastern coastal region of the United States and southeastern Canada. Members of this genus are small (usually < 1 cm.), broad, depressed valviferous forms that inhabit sandy areas and charac- teristically burrow just beneath the sediment surface. The species described herein constitutes the fourth known species of this genus. Those previously described are: C. cocca (Say, 1818); C. tuj'tsi ( Stimpson, 1853); and C. alnivra Bowman, 1955. Included in Bowman's paper is a revision of the generic characteristics of this group. The description of this new form is based on specimens collected on Georges Bank, a relatively shallow portion of the continental shelf east of Massa- chusetts, U. S. A. These specimens were encountered while processing collections of benthic invertebrates taken by the R/V Albatross III and the R/V Delaware for the Woods Hole Laboratory, Bureau of Commercial Fisheries, U. S. Fish and Wildlife Service. Material E.raniined. Holotype, adult female with oostegites developed, 7.5 mm. in length, deposited in the U. S. National Museum (Catalogue No. 104282) ; allotype, adult male 6.0 mm. in length (U.S.N.M. Catalogue No. 104280) ; para- types, 1 male 5.0 mm. in length and 1 ovigerous female 6.5 mm. in length (U.S.N.M. Catalogue No. 104281 ) ; all type specimens were collected August 6, 1959, by means of a grab-type bottom sampler on Georges Bank at lat. 41 48' N., long. 67 53' \Y. ; water depth 15 fathoms, sand substrate, bottom water tempera- ture 57.0 F. (R/V Delaware, cruise number 59-9, station 21). One female, 7.5 mm. in length, collected December 7, 1955, at lat. 40 51' N., long. 68 55' W r . ; substrate coarse sand, water depth 36 fathoms, bottom water temperature 47.2 F. (R/V Albatross III, cruise number 70, collection 3). One male, 6.5 mm. in length, collected December 14, 1955. at lat. 41 40' N., long. 67 36' W. ; substrate gravelly sand, water depth 28 fathoms, bottom water temperature 45.7 F. (R/V Albatross III, cruise number 70, collection 38). Five females, body lengths 4.0, 5.0, 6.5, 7.5, 7.5 mm., and two males, body lengths 7.0 and 7.0. collected August 24, 1957, at lat. 41 22' N.. long. 68 20' W.; substrate coarse sand, water depth 24 fathoms (R/V Albatross III, cruise number 101, station 64). Three specimens, 4.0, 4.5, 4.5 mm. in length, collected August 24, 1957, at lat. 41 34' N., long.. 67 28' W. ; substrate coarse sand, water depth 23 fathoms (R/V Albatross III, cruise number 101, station 90). Diagnosis. Medium-sized Chiridotca with short antennae; flagellum of antenna 2 is much shorter than the peduncle ; antenna 1 usually not reaching beyond the peduncle of antenna 2 ; outer margin of pereion epimeral plates 5-7 153 ROLAND L. WKiLEY Fic.rkKS 1-9. Chiridotea urcnifolu n. sp. 1 FIGURE 1. Dorsal view of female holotype. NEW CHIRIDOTEA FROM NEW ENGLAND 155 E in 6 E E m d FIGURE 2. FIGURE 3. Pereiopod 1, <$. Pereiopod 2, <$. 156 ROLAND L. WIGLEY FIGURE 4. Antenna 1, d. with relatively few or no setae. This species most closely resembles C. cocca (Say) but differs in that it is smaller, the pereiopods are more slender, the body is thinner and less convex dorso-ventrally, the pleotelson is narrower and more evenly tapered, and the anterior lobes of the antero-lateral sections of the head are shorter and have margins devoid of setae. Other differences are mentioned later in this paper under the heading Discussion. Description. Anterior margin of head broadly and shallowly excavated each side of the rostrum. Lateral anterior projections on the head are rounded or obtusely pointed. Pronounced, but comparatively shallow, V-shaped notch in each antero-lateral margin of the head ; margin anterior to the notch is without FIGURE 5. Antenna 2, NEW CHIRIDOTEA FROM NEW ENGLAND 157 E E OJ O E E CVJ 6 E CVJ 6 E E C\J 6 FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. Left mandible, Maxilla 1, rf- Maxilla 2, times its width at the base; sides of the pleo- telson taper somewhat irregularly from the base to the apex; lateral margins near the apex are very finely denticulate and beset with setae. General body proportions are : body width 0.43 times body length ; abdomen length 0.45 times body length ; head length about 0.17 times body length. Antenna 1 is short, extending only to about the end of the peduncle of antenna 2 ; the flagellum consists of 1 segment and it usually bears 4 pairs of inflated setae on the anterior margin. Antenna 2 is only slightly longer than antenna 1, its flagellum is made up of 3-5 segments. First segment of the peduncle is expanded ; second segment with the distal two-thirds expanded ; third segment elongate and slender, approximately equal in length to the flagellum. Maxilliped palp is composed of 3 segments. Maxilla 1 with the inner branch possessing a large, plumose seta and a minute seta. Mandible is without a molar process. Propodus of pereiopod 1 is 1.2 times as long as broad; posterior margin of dactyl 1 bears 46 fine setae ; a few small setae are present on the external lateral surface of the pro- podus, near the posterior margin. The posterior (ventral) margin of the carpus of pereiopod 1 is armed with only one stout spine. Pereiopods 1. 2, and 3 are generally similar in conformation and embellishment ; likewise, pereiopods 4, 5, 6, and 7 resemble one another. Color. Basic color of the body and appendages ranges from light tan or a pinkish hue to nearly white with the integument partially covered with dark chromatophores. The chromatophores are black or deep violet and are distributed somewhat unevenly over the body and the larger exposed appendages. Chromato- phores are not evident on the pleopods or the inner mouth parts. In the material studied, much variation was observed in the pigmentation pattern from one specimen to another ; however, in all specimens examined the chromatophores are consistently more densely concentrated on the uropods and pleotelson than on other parts of the body. Differences in pigmentation appear to be unrelated to size, sex. or season of capture. Range. Georges Bank (east of Massachusetts. U. S. A.) in water depths 15 to 36 fathoms and water temperature 45.7 to 57.0 F. Discussion.- -Shape of the head in C. arcnicola is quite like that found in C. aluiyra. The posterior portion is comparatively long and somewhat narrowed, and the antero-lateral lobes are rather short. The notch separating the antero- lateral margin into two lobes is shallow, as compared to related species. C. arcnicola is distinguished from all other species in this genus by the absence of setae on the anterior lobe of the antero-lateral margin of the head. The propodus of pereiopod 1 in C. arcnicola is relatively short and wide, similar to that of C. cocca, but it lacks the setation on the outer central, lateral surface found in that species. The dactyl of pereiopod 1 has only a few thin setae on the occluded margin ; in this feature it resembles C. alinyra and C. cocca. In C. arenicola the size and number of setae on the outer margin of epimeral plates 5-7 varies considerably from one specimen to another ; however, these setae are generally shorter and much less numerous in this species than in other members of the genus. A slight sexual dimorphism in this feature is apparent XF.VY CHIRIDOTEA FROM NEW ENGLAND 159 in the few specimens available for study. The setae appear to be longer and more numerous in large males than in adult females and small males. The shape of the pleotelson of C. arcnicola is intermediate between that of C. cocca, which is broad and tapers irregularly, and that of C. titftsi, which is rather narrow and evenly tapered. The eye is dark and distinct in some specimens and faint in others. Variation in this characteristic occurs at random among the sexes, specimens of various sizes, and in different collections (some of which have been in preservative for two months and others nearly two years). In the specimens at hand, the eye appears to be proportionately much smaller in C. arenicola than that depicted for C. cocca by Bousfield (1956). To my knowledge neither C. alinyra nor C. cocca have been reported from offshore waters, and they have not been observed in our collections of benthic invertebrates from the Georges Bank area. C. arcnicola and C. tnftsi are the only representatives of this genus found in our samples taken during the past few years. Of these two species the latter is far more numerous than arcnicola ; a ratio of approximately 20:1, based on the total number captured. Our records indicate that C. tnftsi is rather widely distributed over Georges Bank and nearly always within the 50-fathom isobath. C. arcnicola seems to be more restricted, having been taken only near the shoals on the north-central part of Georges Bank and on the western end near Great South Channel. A factor which appears to be important in affecting the distribution of these two species is the particle size composition of the substrate. C. arcnicola has been taken most frequently from areas where the predominant sediment fraction is a coarse sand (0.5-1.0 mm.). Conversely, C. tnftsi is most commonly found where the predominant sediment fraction is a medium sand (0.25-0.5 mm.). Also, there is some evidence that C. arcnicola either burrows more deeply into the sand or for some other reason is more difficult to evict from the substrate than C. tnftsi. This inference is based on two factors : ( 1 ) in dredges that normally scrape only the surface of the sea bed, hundreds of tnftsi have been caught, compared to only two specimens of arcnicola; (2) in grab-samplers that usually dig 3 to 6 inches into the sea floor, 10 specimens of arcnicola have been taken, versus 7 specimens of tuftsi. Some of the more common Crustacea with which C. arcnicola has been found associated are the amphipods : Acginina longicornis (Kroyer), Ampclisca spinipes Boeck, Auiphiporcia rirginiana Shoemaker, Ericthonins rnbricornis Stimpson. Hanstorins arcnarins (Slabber), Leptocheirus pinguis (Stimpson), Plwtis dentata Shoemaker, Pontogoicia incnnis (Kroyer). Podoccropsis nitida (Stimpson). Sviuplcnstcs glabcr (Boeck), and Tiuctony.r nobilis (Stimpson); the mysid : \coin\sis aincricana (Smith); and the decapods: Cancer borcalis, Stimpson. Cancer irroratns Say, Crangon septemspinosa Say, Dichelopandahts Icpto- ccrns (Smith), and Pagnrns acadianns Benedict. These species are the most abundant crustaceans that were taken in the same bottom-grab samples and dredge hauls with C. arcnicola. It will be recognized that many of these associates are infauna forms ; however, all of the larger species except one are epibenthic. Some species listed above are exceedingly tolerant and are adaptable to diverse environ- mental features, but others listed here are restricted to very specific types of habitats. 160 ROLAND L. WIGLEY Judging from the information available at this time, it seems likely that C. arcnicola will be found most closely associated with some of the burrowing amphipods listed above, such as Amphiporeia, Hanstorius, and Tinctony.v. KEY TO THE SPECIES OF CmRiooTEA 1 1 . Flagellum of antenna 2 much shorter than peduncle, segments 5 or less ; antenna 1 nearly as long as antenna 2 2 Flagellum of antenna 2 longer than peduncle, 812 segmented ; antenna 1 much shorter than antenna 2 3 2. Antenna 1 extends beyond peduncle of antenna 2 ; margin of anterior lobe of the antero-lateral margin of head is setose C. coeca Antenna 1 does not extend beyond peduncle of antenna 2 ; margin of anterior lobe of the antero-lateral margin of head is not setose C. arenicola, n. sp. 3. Posterior margin of dactyl of pereiopod 1 armed with strong spines ; pleotelson narrow, tapering evenly from base to apex C. tiiftsi Posterior margin of dactyl of pereiopod 1 armed with a few fine setae ; pleotelson broad, sides nearly parallel at the basal half C. alinyra LITERATURE CITED BOUSFIELD, E. L., 1956. Malacostracan crustaceans from the shores of western Nova Scotia. Proc. Nova Scotiaii hist. Sci., 24: 25-38. BOWMAN, T. E., 1955. The isopod genus Chiridotea Harger, with a description of a new species from brackish waters. /. Wash. Acad. Sci., 45 : 224-229. SAY, T., 1818. An account of the Crustacea of the United States. /. .lead. Nut. Sci. Phila.. 1 : 424-425. STIMPSON, W., 1853. Synopsis of the marine Invertebrata of Grand Manan. Smithsonian Contr. Knsunns tan ) was used for this metabolic study of islet tissue because of the accessibility of the islets and the general availability and hardiness of this species. In a previous study ( Lazarow, Cooperstein, Bloomfield and Friz, 1957), the oxygen uptake of islet tissue was measured under varying conditions of pH, tonicity, and electrolyte composition, and the baseline conditions under which maximal respiration of islet tissue occurs were defined. The present paper reports further characterization of the over-all metabolic pathways of islet tissue achieved by studying the effects of specific exogenous substrates and inhibitors on the respiration of islet slices. M. \TKRIAL AND AlKTIIODS After the toadfish were killed by a blow on the head, the principal islets were dissected from the mesentery and the capsules of the islets were removed. The latter is an important step in the procedure, since any acinar tissue that may be present underneath the capsule, as well as any acinar tissue that may be present 1 This investigation \vas supported by research grants A-l'o 1 ' and A-1887 from the National Institute of Arthritis and Metabolic Diseases, Public Health Service. -Present Address: Carlsberg Laboratories, Copenhagen, Denmark. 161 162 CARL T. FRIZ, ARNOLD I.AZAROW AND S. J. COOPERSTEIX within the islet but attached to the capsule by trabeculae, is removed when the capsule is stripped. In each experiment a single islet was cut into eight slices and these slices were distributed between the experimental and control groups. The Cartesian diver microrespirometer was used to measure the oxygen uptake of each toadfish slice, in a manner previously described (Lazarow ct a/., 1957). A modification ( Laza- row ct al., 1957) of the method of Lowry, Rosebrough, Farr and Randall (1951) was used to measure the protein content of each slice. The metabolic activity was expressed as millimicroliters (m/J.) of oxygen per microgram (ju.g.) of pro- tein per hour. The metabolic activity of toadfish liver tissue was also studied in order to compare it with that of toadfish islet tissue. The Warburg manometer was used to make these measurements, and the oxygen uptakes were also expressed in millimicroliters per microgram of protein per hour. A constant amount of phosphate buffer at pH 7.4 (0.054 Jl/) was present in all of the experiments in this study. The respiration of islet tissue was studied in a medium containing phosphate plus substrate or inhibitor, and the oxygen uptakes thus obtained were compared to those observed in either a medium con- taining phosphate plus saline or a medium containing phosphate alone. 3 The various solutions of substrates and inhibitors were prepared at twice the desired final concentration and mixed with an equal volume of 0.108 M phosphate buffer so that the final phosphate concentration was 0.054 M . RESULTS Effect of substrates The substrates studied included glucose, pyruvate (lithium salt), a-ketogluta- rate, glutamate, succinate, and isocitrate. When glucose was added to the medium /;; I'itro (Table I ) neither islet (/> -- 0.21 ) nor liver ( f> -- 0.9) respiration was altered. It should be noted that the endogenous metabolic activity of liver tissue slices was considerably higher than that of islet tissue. In a similar series of experiments using pyruvate as the substrate, respiration of islet slices was compared in the following media: (a) a hypotonic medium containing phosphate ; ( b) an isotonic medium containing phosphate plus NaCl ; and (c) a medium containing phosphate plus pyruvate (Table II ). If one assumes that the added pyruvate readily enters the islet cells, then the respiration of islet slices in the pyruvate-containing medium should be compared with that in the hypotonic medium because (a) and (c) would be of equal tonicity. When these media are in fact compared, it is noted that pyruvate has no effect on the respira- tion of islet tissue. If, on the other hand, one assumes that the added pyruvate does not enter the islet cells, then media (b) and (c) should be compared since 3 Whereas Lazarow ct ol. (1957) reported that optimal respiration of islet slices was observed in a hypotonic medium (0.054 M phosphate), in the present study optimal respiration of islet slices was sometimes observed in an isotonic medium whereas at other times no differ- ence was noted between respiration in hypotonic and isotonic media. Evidence indicates that part of this discrepancy may be due to the varying temperature of the water in which the fish were kept. Further work is now in progress in order to clarify these differences. METABOLISM OF TOADFISH ISLET TISSl I. 163 TAFJLE I Effect of glucose on the respiration of toad fish islet and liver s Tonicitv of Islet Li\ ci medium Medium (equiv. XaCl cone.* M /liter) No. of deter. Ave. mjil. ( )_'. Hg. prot. hr. ) was calculated using the following equation : difference /> = \ N l N 2 - where a\ and <7 2 are the standard deviations of the control (without substrate) and experimental (uith substrate) groups, respectively; Ni and A\> are the corresponding number of determinations in each group. they would be of approximately equal tonicity. Comparison of the respiration in these media indicates that pyruvate addition inhibits by 23% (p -- 0.019). The addition of a-ketoglutarate (Table III ) increased the oxygen uptake of islet slices by approximately 33 % when compared with either a hypotonic (p -- 0.07) or isotonic (/> -- .008) medium. Table IV shows that a 4470 increase in oxygen uptake was observed when islet slices were placed in a phosphate-glutamate medium (/> == 0.016). This is to be compared to a 30-36^/c increase observed when toadfish liver slices were placed in the same medium (/> = <0.001 ). Oxygen uptake was also increased when succinate ( Table Y ) was added either to toadfish islet or to toadfish liver tissue. The increase in islet tissue respiration in an isotonic phosphate-succinate medium was 47 r r ( f> -- 0.01 ) when compared to respiration in an isotonic phosphate-saline medium and 100'v ( [> -- 0.001) when TABLI-; II Effect of pyruvate on the respiration of toadfish islet slices Tonicitv of N-i if- Medium medium (equiv. XaCl cone. Xo. of deter. Ave. mjil. Oi/ Mg. prot. hr. a .\f liter) a 0.054 ,17 PO 4 0.0755 43 1.7 0.70 1) 0.054 M PO 4 + 0.0645 .17 0.140 14 2.2 0.69 XaCl c 0.054 .17PO 4 + 0.040- 0.1155-0.140 28 1.7 0.70 0.0645 .17 pyruvate 164 CARL T. FRIZ, ARNOLD LA/AKOW AND S. J. COOPHRSTKIN TABU-: III /'Iff fit of a-ketoglutarate on the respiration of load fish islet slices Medium Tonicitv of medium (equiv. NaCl cone. No. of deter. Ave. niAil. < > MK- prot. lir. a M liter) 0.054 .17 PO 4 0.0755 13 2.1 0,83 0.054 .17 PO 4 + 0.0645 M Nad 0.140 13 2.0 0.60 0.054 M PO 4 + 0.0645 .17 fv-kftnijiit. irate 0.140 14 2.7 0.84 compared to respiration in the hypotonic medium. The addition of succinate increased the respiration of toadfish liver slices by about 75 r /f (/> = : < 0.001). In contrast to a-ketoglutarate, glutamate, and succinate, another Krebs cycle intermediate, isocitrate, did not stimulate islet respiration (Table Yl). I'.fjcct oj inhibitors The use of inhibitors on whole cells, extracts, homogenates, and tissue slices has provided much detailed information concerning- the occurrence and components of complex enzyme systems, such as the glycolytic and tricarboxylic acid systems. \ arious inhibitors were used in this study in order to assess the importance of these enzyme systems for the metabolism of islet tissue. TABU- IV Effect of glutamate on the respiration of toadfish islet mid liver slices Tonicitv of Islet Liver medium Medium (equiv. NaCl cone. No. of Ave. m/il. O" No. of Ave. m/al. O*/ .U liter) deter. Aig. prot., hr. deter. Mg. prot./hr. a 0.054 M PO 4 0.0755 14 1.6 0.66 9 i ~> 0.40 0.054 M PO 4 + 0.140 11 1.6 0.64 9 2.3 0.23 0.0645 .17 NaCl 0.054 .17 PO 4 + 0.140 13 2.3 0.78 9 3.0 0.30 0.043 .17 glutamate Table YII shows the per cent inhibition produced by the various inhibitors. Fluoride added to a phosphate medium was found to inhibit respiration of islet slices by 35 f /r ( /> == 0.0063), iocloacetate inhibited by 53% (p -- <0.001 ). malonate by 43% (/> = <0.001 ), and azide by 50 r v ( /> == 0.001 ). DISCUSSION Although the addition of glucose did not stimulate islet tissue respiration, this does not mean that glucose can not be utilized by islet tissue. There are a number of reasons why an added substrate might not stimulate respiration, such as its failure to readily enter the cell or the presence of sufficient endogenous substrate to saturate the enzyme system concerned. This is emphasized by our finding that MKTABOLISM OF TOADFISH ISLET TISSUE 165 TABLK \' Effect of siiccinatc on the respiration of toad fish islet and lii'er slices [slel Liver Tonicitv of Tonicity of Medium medium (equiv. XaCl Xo. of deter. Ave. m/jl. 02/Mg. prot. hr. (7 medium (equiv. XaCl No. of deter. A\ r. rn/nl. ( ).. Mg . pint. hr. a cone. cone. M. liter) M liter) 0.054 M PO 4 0.0755 14 1.1 0.61 0.0755 9 2.9 0.37 0.054 M PO 4 + 0.140 14 1.5 0.64 0.140 9 2.7 0.37 0.0645 M XaCl 0.054 M PO 4 + 0.172 13 2.2 0.77 0.140* 9 4.9 0.89 0.0645 .1/surrinate * 0.043 M surrinate used in this case. the addition of glucose did not stimulate liver respiration, despite the known occurrence of the glycolytic system in the liver of most species. Our studies with fluoride and iodoacetate provide suggestive evidence that at least two enzymes of the glycolytic scheme (enolase and phosphoglyceraldehyde dehydrogenase) are also present in islet tissue. Neither of the inhibitors can be considered specific, but phosphoglyceraldehyde dehydrogenase is extremely sensitive to iodoacetate ( MeyerhofT and Kiessling, 1933; Adler, Euler and dumber, 1938) and at least one of the actions of fluoride is to inhibit enolase ( Warburg and Christian, 1941 ). Of the enzymes involved in the tricarboxylic acid cycle, the presence of a-ketoglutarate oxidase and succinoxidase are indicated bv the stimulation of oxy- gen uptake observed following the addition of their respective substrates. The presence of succinoxidase is further indicated by the observed inhibition by malonate (43 r r ). This inhibition can be compared with the 50-6CK4 inhibition of rat liver homogenates reported by Holtkamp and Hill ( 1951 ) and Pardee and Potter (1949) as well as the 70 c r inhibition of rat brain homogenates (using 0.02 M malonate ) reported by Pardee and Potter (1949). Finally, the participation of succinoxidase in islet tissue respiration is also consistent with the previous demonstration of the presence of succinate-cytochrome c reductase in this tissue ( Lazarow and Cooper- stein, 1951 ). Neither pvruvate nor isocitrate stimulated islet respiration but this could be- due to failure of these substrates to penetrate to their site of utilization. The TABLE VI Effect of isocitrate on the respiration of toad fish islet slices Medium Tonicitv of medium (equiv. XaCl cone. Xo. of deter. A vi'. nifil. (>.' Mg. prot./hr. a M liter) 0.054 M PO 4 0.0755 12 2.2 0.58 0.054 M PO 4 + 0.645 .17 XaCl 0.140 12 2.2 0.57 0.054 M PO 4 + 0.032 M isocitrate 0.140 12 2.3 0.45 166 CARL T. FRIZ, ARNOLD LAZAROW AND S. L COOPERSTEIN Glucose Glycogen II Glucose -I- phosphate It Glucose - 6 - phosphate If Fructose - 6 - phosphate it Fructose- 1,6 - diphosphate I t 3 Phosphoglyceraldehyde ^=^ Dihydroxyacetone phosphate 53% inhibition by 0.01 M lodoacetate 1,3 dip/iosphoglycenc acid It 3 phosphoglyceric acid it 2 phosphoglyceric acid 1 35% inhibition by 001 M fluoride Phosphoenol pyruvate It Pyruvate Oxalacetate +Acetyl-Co A Malate Fumarate 47% stimulation by succmate, 43% inhibition by 001 M malonate glutamate ~ " proteins 44% stimulation by glutamate Succinate 35% stimulation by a-ketoglutarate A = Electron transport chain [50% inhibition by 0001 M azidel FIGURE 1. A simplified scheme of carbohydrate metabolism, which indicates the evidence obtained for its operation in islet tissue metabolism. MKTABOLISM OF TOADFISH ISLET TISSUE 167 TARI.K VII Effect of inhibitors on the respiration of toadfish islet tissue. (Each inhibitor was studied in a separate series of experiments and compared with controls run at the same time) Medium No. of deter. Ave. ni/jl. < > ^g. prot., hr. a % inhibition 0.054 .17 PO 4 13 2.0 0.85 0.054 .17 PO 4 + 0.01 ,17 fluoride 14 I..? 0.37 35 0.054 .17 PO 4 16 1.9 0.74 0.054 .17 PO 4 + 0.01 .17 iodoacetatL- 13 0.9 0.45 53 0.054 .17 PO 4 12 2.1 0.43 0.054 .17 PO 4 + 0.01 .17 malonatt- 12 1.2 0.37 43 0.054 .17 PO 4 8 2.0 0.79 0.054 .17 PO 4 + 0.001 J7a/ide 8 1.0 0.24 50 enzymes of the tricarboxylic acid cycle are localized within the mitochondria, and it has heen reported (Schneider, Striebich and Hogeboom, 1956) that the mito- chondrial membrane may not be permeable to citrate. The ability of islet to utilize glutamate is of considerable interest since this compound is a key link between the tricarboxylic acid cycle and protein synthesis. The existence of cytochrome oxidase in islet tissue has been reported previously ( Lazarow and Cooperstein, 1951) and its participation in the electron transport system is indicated by the inhibition of respiration produced by azide, 4 a known inhibitor of cytochrome oxidase (Keilin, 1936). Figure 1 depicts a simplified scheme of carbohydrate metabolism, and indicates the evidence obtained for its operation in islet tissue metabolism. SUMMARY AND CONCLUSIONS The effects of substrates and inhibitors on the metabolic activitv of toadfish *> islet slices were measured in the Cartesian diver microrespirometer under varying experimental conditions. Of the substrates added, a-ketoglutarate, glutamate, and succinate increased islet respiration while glucose, pyruvate, and isocitrate were ineffective. lodoacetate, fluoride, malonate, and azide inhibited islet respiration. LITERATURE CITED ADLKK, E., H. Y. EULER AND G. GUNTHER, 1938. Dehydrasen und Jodessigsaure. Skand. Arch. f. Physiol, 80: 1-15. DIAMARE, V., 1899. Studii comparative sulle isole di Langerhans del pancreas. Intern. Monatschr. f. Anat. u. Physiology, 16: 155-209. HOI.TKAMP, D. E., AND R. M. HILL, 1951. Comparison of malonate and malondialdehyde in in vitro oxygen uptake studies. Arch. Biochcm. Biophys.. 34: 216-218. KF.II.IX, D., 1936. The action of sodium azide on cellular respiration and on some catalytic- oxidation reactions. Proc. Roy. Soc. London, Scr. B. 121: 165-173. 4 Studies with cyanide have not been carried out to date because these are very difficult to perform in the Cartesian diver. 168 CARL T. FRIZ. ARNOLD LAZAROW AND S. J. COOPERSTEIN I.A/Ako\v, ARNOLD. AMI S. J. Cooi'KKSTi-.i \, 1951. Studies on the isolated islet tissue of fish. 1. The cytochrome oxidase and sticcinic dehydrogenase c< intents of normal toadfish (Opsanus tan}. />';/. />//.. 100: 101-198. '.. \XAKO\V, ARNOLD, S. J. COOPKKSTEIN, I). K. IILOOMFIKLD AND C. T. EKIX, 1957. Studies on the isolated islet tissue of fish. II. The effect of electrolytes and other factors on the oxygen uptake of pancreatic islet slices of toadfish, using the Cartesian diver micro- respirometer. />/<)/. Hull., 113: 414-425. l.mvKY, O. H., N. J. ROSEBROUGH, A. L. FAKK AMI R . J. RANDALL, 1951. Protein measurement with the Folin phenol reagent. /. Hiol. L'licin., 193: 205-275. MEVEKHOFF, O., AND \\". KIKSSLINI;, 1933. t v her die phosphorylierten Zwischenprodukte und die letzten Phasen der alkoholischen Garung. Biochein. Zcitschr.. 267: 313-348. I'AKDKK, A. B., AND Y. R. POTTER, 1949. Malonate inhibition of oxidations in the Krehs tri- carboxylic acid cycle. /. Hiol. Chan.. 178: 241-250. SCHNEIDER, W. C., M. J. STRIKBUH AND (i. H. HOGEBOOM, 1956. Cytochemical studies Yll. Localization of endogenous citrate in rat liver tractions. ./. Hiol. Chan.. 222: 969-977. \\'.\RBn<(;, O., AND \\ . CHRISTIAN, 1-41. Isolierung und Kristallisation des Garungsferments Enolasc. Hioclicm. /.citschr., 310: 384-421. A STUDY OF REPRODUCTION IX THE LXTERTIDAL BARXACLE, MITELLA POLYMERUS, IX MONTEREY BAY, CALIFORNIA 1 GALEX HOWARD H1LGARI) :! Hopkins Marine Station of Stanford ['inrersily. California The goose barnacle, Mitclla polymerus (Sowerby, 1S33), sometimes referred to as Pollicipes polymerus or the leaf barnacle, is distributed along the exposed rocky coast of Western North America from British Columbia to the middle of Baja California (Cornwall, 1925). It is generally abundant here in the upper two-thirds of the intertidal belt ( Ricketts and Calvin, 1952), though it is occa- sionally found below this level where there is considerable surging wave action. Along the central California coast, clusters of Mitclla patch the exposed rocky regions, the barnacles usually attaching themselves to rock, to Mytilus calijornianus, or to other individuals of Mitclla. Individuals are seldom isolated and one sec> among the mussel beds or on the rocks rosette-shaped clusters in which large barnacles are at the center and smaller barnacles grade toward the periphery. In other aggregations, individuals of nearly the same size are packed closely together, frequently with such uniformity in size and orientation that their valves form a geometric pattern, and their closely packed bodies make a strong but resilient mat against the pounding surf. Often where the animals are attached to rock beneath the mussel beds, their stalks extend up eight inches or more to the surface. Occa- sionally, solitary animals occur on tables of rock exposed to strong wave action ; in these the stalks remain short and stubby while the shells and bodies grow. While Mitclla polymerus is abundant, conspicuous, and well-known taxonomi- cally, almost nothing is known of its reproductive biology. Xussbaum ( 1890) has described the anatomy of M. polymerus in some detail. The only published studv of reproduction and development in this genus is that of Batham (1944-45), on the New Zealand species, Mitclla sphwsus. Since Batham's study of the repro- ductive cycle was carried out at latitude 44 52' South, a comparison of her results with the situation occurring in M . polvmcnis at Monterey Bay ( 3640' North i seemed particularly interesting. The following study of reproduction in M. poly- merus includes: the anatomy of the reproductive system, the relationship between size and sexual reproduction, the seasonal reproductive cycle, the rate of egg and embryo development, an estimate of fecundity, and evidence concerning self- fertilization. 1 This investigation was carried out as partial fulfillment for the degree. Master of Arts. - The author wishes to express her gratitude to Dr. Donald P. Abbott, under whose guidance this work was carried out ; and to acknowledge with appreciation his generous help with arrangement and discussion of the data. 'Author's current address: Department of Biology. Stanford University, Stanford. Cali- fornia. 169 170 GALEN HOWARD HILGARD TIIK REPRODUCTIVE SYSTEM The gross reproductive anatomy of Mitclla polymerus is shown in Figure 1. Ovarian tissue is found in the upper portion of the peduncle. From the ovaries, a pair of oviducts lead up into the body proper, emptying into the glandular oviducal atria in the bases of the first thoracic cirri. Fggs pass clown the oviducts to the FIGURE 1. Gross anatomy of the reproductive system of M. polymcrns, exposed by dis- section from the left side. ad. muse. = adductor muscle; dig. gl. = digestive gland; fil. ap. -filamentary appendages; gut = gut ; m. c. = mantle cavity; o. ap. oviducal aperture; o. at. oviducal atrium ; o. duct = oviduct ; ov. ovaries ; ped. peduncle ; pen. penis ; test. testes ; s. ves. = seminal vesicle ; v. def. = vas deferens. atria, where they receive a protective coating which glues them together into masses. The egg masses are then extruded through the oviducal apertures and come to lie in the mantle cavity on either side, where they are pressed flat to form the two ovigerous lamellae. REPRODUCTION IN MITELLA POI.YMKRl'S 171 The numerous small testes are found on either side of the gut, extending ventrally into the coxopodites of the first four pairs of thoracic appendages and dorsally into the numerous paired filamentary appendages. Fine efferent ducts connect the testes to the paired vasa deferentia, which lead to the paired storage organs or seminal vesicles. These in turn join posteriorly at the base of the penis, and form a single duct extending to its tip. Copulation was not observed, but sperm are deposited in the mantle cavity. The embryos are brooded in the ovigerous lamellae in the mantle cavity until they are hatched out as nauplius larvae. The gross reproductive anatomy of .17. polymer us is similar to that of M. spinosus; but M. spinosus has no filamentary appendages and the testis is de- scribed as (p. 370) "a median structure lying closely in the U-bend of the gut," with a pair of ducts leading from either side of it which expand to form the seminal vesicles (Batham, 1944 1-5). [MATERIALS AND METHODS FOR THE STUDY OF REPRODUCTION Nearly all living material used in the present study was collected in or very near Monterey Bay, California, and studies were carried out at the Hopkins Marine Station of Stanford University, Pacific Grove. For the study of the reproductive cycle, a population of Mitella polynients was sampled at approximately monthly intervals for a period of fifteen months. All individuals were collected within an area approximately fifteen feet square on granite rocks and adjacent beds of Alytilus californianus on the northern shores of Mussel Point, Pacific Grove, California, at an intertidal level corresponding to the upper middle horizon of Zone Three of Ricketts and Calvin ( 1952). Care was taken to insure that all barnacles large enough to be reproductively mature were collected in close proximity to other individuals of a reproductive size, and to avoid taking isolated individuals which had lacked the opportunity for cross-fertilization. Animals were anaesthetized for four hours in a solution of magnesium chloride isotonic with sea water. This was sufficient to relax the cirri and peduncle. The animals were then preserved in 10% formalin buffered with borax. Individuals of all sizes were collected and examined. For most of the repro- ductive data, the largest common animals available were used. All of these were of a reproductive size, and for purposes of consistency, large animals which ranged in breadth (distance from rostrum to carina ) from 27.5 mm. to 32.5 mm. were used (occasionally larger animals are found). When maturing, the ovarian tissue in the peduncle (Fig. 1 ) undergoes marked and visible changes. Tiny eggs appear and grow, accumulating yolk until they are passed up through the oviducts and are extruded into the mantle cavity. Preliminary observations showed that throughout the ovarian tissue, eggs are generally of about the same size and stage of development at any one time ; an exception to this is provided where a new batch of tiny eggs appears in the ovary before the previous batch of very much larger eggs is extruded. With a compound microscope and calibrated ocular micrometer, the greatest diameters of five to ten eggs were measured (in each animal), and an average ovarian egg size was re- corded for the individual. Where two batches of eggs were present at once in 17J GALEN HO\VARI> HIMiAUI) an ovary (i.e.. large and small), this situation was quite evident, and the two were treated separately. The average egg sizes were then grouped into three useful classes for purposes of comparison : small eggs (diameter 0.016 mm. to 0.065 mm. ), medium eggs (diameter 0.066 mm. to diameter 0.09 C ' mm.), and large eggs (diameter 0.100 mm. to diameter 0.127 mm.). The few individuals in which no eggs could he seen were treated separately. As the male gonads of Mitclla mature, they also show easily visible changes: testes in the filamentary appendages and throughout the body lose their trans- lucency and become opaque white with sperm. Sperm then travel through efferent tubules and vasa deferentia to the paired seminal vesicles, where they are stored. As more and more sperm accumulate in the seminal vesicles, these, too, change from translucent to opaque white and increase in diameter. Diameter of the seminal vesicles affords a fairly good index for the maturation of the male repro- ductive organs. \Yidth was measured with a pair of dividers at a point just back of the anterior sharp bend in the seminal vesicle ( Fig. 1 ) . Conditions of the male organs were finally grouped into three categories : ( 1 ) seminal vesicles translucent and apparently empty of sperm; (2) seminal vesicles ranging in width from one to two mm.; and (3) seminal vesicles more than two mm. in width (ranging up to a maximum observed width of 3.S mm. ). Fertilized eggs and developmental stages present in ovigerous lamellae were also studied. Embryos were examined in the ovigerous lamellae from animals taken in the monthly samples. In a pair of ovigerous lamellae taken from any one parent, embryos are all at about the same stage of development. As the fertilized egg develops, it increases slightly in size, but not enough for size to yield a good criterion for stage of development. Major morphological changes can be studied fairly readily, and it proved practicable for the purpose of the present problem to divide embryonic development into three stages : ( 1) early stages, with neither limb buds nor nauplius eye; (2) middle stages, with limb buds developing but no nauplius eye ; and ( 3 ) late stages, with well-formed limbs and median eye present. Studies were also made of lamellae removed from the parent and raised in vitro. Ovigerous lamellae were isolated from their parent barnacles, placed in clear glass dishes of filtered sea water, and kept at a constant 13 C. in a water bath or a refrigerated room. Sea water was changed approximately daily, at which times the embryos were examined in small sections of the ovigerous lamellae plucked off with glass needles. Many embryos raised in vitro were observed through development to hatching, and many larvae were raised beyond this through several naupliar molts. SIZE AND SEXUAL REPRODUCTION Figure 2 shows the occurrence of ovigerous lamellae in animals of different sizes at selected times during the breeding season (May through December). Animals with a breadth of more than 27.5 mm. were always found to be sexually mature during the reproductive season ; all individuals examined bore ovigerous lamellae, or enlarging ova in the ovaries, or both. In the individuals below 27.5 mm. breadth, the percentage of animals with ovigerous lamellae can be seen to decrease more or less directly with decreasing size. For individual months, the tendency is not always clear, and this may be due to sampling deficiencies (c.l O O . rt ot rt .- V s- V V FK.CKE 2. Variation in reproductive activity with size of animals. Vertical bars indicate percentage of individuals of each size class which contained ovigerous lamellae. Size classes are designated as follows: I, breadth less than 17.2 mm.; II, breadth 17.2 to 22.5 mm.: Ill, breadth 22.(> to 27.5 mm. ; and IV. breadth 27.5 to 32.5 mm. THE REPRODUCTIVE CYCLE ix THE MITELLA POLYMERUS POPULATION Each month, 10 to 25 large animals were collected, measured, and examined for reproductive condition. The number of individuals carrying ovigerous lamellae was noted ; conditions of the female and male gonads, and stage of development of the embryos (where present) were studied. The distribution of egg sizes and larval stages is shown in Table I and Fig- ure 3. Table I shows for each sample the number and per cent of individuals which contained given egg size classes and which brooded particular embryonic stages. However, in Figure 3, each separate mass of eggs or embryos is treated as a sepa- rate unit. For example, in cases where a given individual contained simultaneously small eggs, large eggs, and late embryos, these appear in Figure 3 in three different horizons in the same date column. It is apparent in Figure 3 that between November, 1956. and January, 1957, breeding waned and was discontinued until the spring of 1957. Then, during March, eggs began to enlarge, but did not exceed the upper limits of the class of small eggs. ly April, the first medium and large ovarian eggs were present, and 174 CAI.KX HOWARD HII.(,.\K1) the first fertilized egg mass appeared. Egg production continued through the year to January, 195S, when the number of ovigerous lamellae present in the population dropped significantly. The season of reproductive activity for the population cov- ers three-quarters of the year, and it is of interest that all stages of egg and embryo development were found throughout the season. On the average, during the height of the breeding season, between 50 r /r and 6Q c / f of the population of large animals are carrying ovigerous lamellae at any one time. Data for the male gonads are plotted in Table I and in Figure 4. Sperm is present in at least some members of the population throughout the year. While there is seasonal variation in the condition of the male gonads, this is less well defined than is that of the female gonads. In the fall of 1956 and through January, Dare oj collection 3IOCT IO56 3 1 NOV. 1957 8 JAN. 1956 M --O <* S^ U-l 3 u O 00 oo g v g o Late Intermeaiate IvnbryoS Earty Embryos large eggs (0 1 am.. 100 -.127 nun ) Mecuum c^s (Diam,. 066- 099mm) Smaff eggs (Diam., 0)6 -065mm) absent sampfe) 14 10 10 o/o oj- total no of eua masses. A> of individuals examined.. Sin of sample 12 14 IO 15 IO 25 13 15 15 12 IO FIGURE 3. Distribution of egg and embryo masses of various stages of development in large M. pulyiiicrns. 1957, some seminal vesicles were very thick with sperm, while others appeared spent. In February and March, 1957, most of the vesicles had a meager amount of sperm, presumably building up, and some were empty. In April, most vesicles were quite full with sperm, others had a meager amount, and none were empty, and from this point on through the summer and fall of 1957, the mean vesicle width was high (well over two mm.). Empty vesicles were not observed again until January, 1958. Thus during the winter months, the quantity of sperm present in the seminal vesicles of most of the population was considerably less than the amount present during the rest of the year. There is a suggestion of waves of reproductive activity during the breeding sea- son, in the data presented in Figure 3 and Table I. This appears most clearly in Table I in the column showing the percentage of animals which are carrying REPRODUCTION IX MITELLA POLYMERUS 175 TABI.K I variation in egg size, seminal vesicle width, and stage of development of brooded embryos in M. polymer us Dates 1956 Oct. Nov. 30 1957 Jan. Feb. 11 Mar. 11 Apr. 18 May 18 Jun. 28 Jul. 24 Sept. 5 Nov. 7 Dec. 5 I95X Jan. 31 19 8 Xo. of animals examined : 12 14 10 15 10 22 25 13 15 15 12 10 18 Xo. and per cent of animals \vith : Eggs absent No. 3 2 1 4 1 , % 25 14 10 27 10 Small eggs Xo. 3 9 9 11 9 11 5 1 1 2 4 9 % 25 65 90 73 90 50 20 8 7 13 40 SO Medium eggs No. 3 9 10 6 3 2 8 4 9 % 21 41 40 46 20 13 67 40 50 Large eggs Xo. 6 6 5 3 1 1 % 50 24 38 20 7 10 Both small and Large eggs Xo. 2 4 1 8 10 4 1 % 9 16 8 53 67 33 10 Xo. and per cent of ani- mals with ovigerous lamellae : Xo. 4 6 1 13 9 8 10 5 7 2 % 33 43 5 52 69 53 67 42 70 11 X'o. and per cent of ani- mals with ovigerous lamellae bearing : Early embryos No. 1 1 5 3 1 2 % 8 5 20 23 7 13.5 Middle embrvos No. 3 5 4 2 2 5 1 % 21.5 20 31 13.5 17 50 5.5 Late embrvos No. 3 3 3 2 7 6 3 2 1 Of /o 25 21.5 12 15 46 40 25 20 5.5 Conditions of seminal vesicles present in the population X T o. of animals examined : 12 13 18 15 10 21 23 13 14 14 12 10 18 X'o. of animals with seminal vesicles in following conditions : Apparently empty 1 1 1 4 3 2 Width 1-2 mm. 5 6 5 9 7 4 1 2 2 5 4 9 7 \\ 'idth 2-3.8 mm. 6 6 12 2 17 22 11 12 i ') 8 1 9 176 GALEN HOWARD HILGARD MONTEREY BAY, CALIF, o 1956 '957 1958 Oct. Nov. Deo, Jan. Felj. Mar. Apr. May Tun. JuC. Au.g, Sept. Oct, Nov. Dec. Jem 17 Sdore temperatures, average everj 72,cay5 at: 14 Hopkins Marine Station, Mean(o)nnc( extreme (.) wi'cttks oj seminal vestcUs in 5a.-m.ptes of farae taken at intervals tkrouodoat tke jecu% (mm., or empty) No, of ani-ma.ls examined - 10 io '3 H- 14- 11 10 Percentages of ianipies erf Carge M,po(ymerixs 700 ovujerows Caweffae at" various times ctxnVtq t At year No. of anima 10 15 'J '5 li (0 M.SPINOSU5) NEWZEAiAND Numfcer of Reproductive siie barnacles ^ (M, spinosus) carrying ovigemvis (ameffae JQ out of 5 ampte oj 1 25 (Data from Batfumij 1945) 5 r ' Surjace sea. temperatu.re5^ naont^i(v averages from ii> j.OJ weekly readmos on the eastern coast of Otaqo Penninsuta-j (C?) Oct Nov. Dec, Jan. Feb. Mar Apr. May Jun, Ju[, Aiy. Sept, Oct- Nov. Dec, Dcxte FIGURE 4. Temperature and reproductive activity in M. polymerus in Monterey Bay and in M. spinosus in New Zealand. REPRODUCTION IX MITELLA POLYMERUS 177 ovigerous lamellae at different times during the year. The highest values, i.e., those for June, September, and December (69%, 67%, and 70%, respectively), alternate with the lower values shown for May, July, and November (53%, 53%, and 42%, respectively), and therefore suggest that the population as a whole may be reproducing in waves, more or less simultaneously. As will be indicated later, this phenomenon is very likely related to the successive waves of reproductive activity shown by individual barnacles during the breeding season. However, not all individuals start to reproduce at precisely the same time, and waves of activity do not proceed in all individuals at exactly the same rate. The differences between alternate highs and lows and their deviations from the mean for the population are not statistically significant ; and a larger sample might be expected to yield a smoother plateau for reproductive activity of the population. THE REPRODUCTIVE CYCLE IN INDIVIDUALS The reproductive cycle in the population of M. [>oly merits represents a summa- tion of the reproductive processes in individual barnacles. The animals are hermaphroditic and ovoviviparous, brooding their young for a period of time in the mantle cavity. It is accordingly of interest to examine the relative degrees of development of the two gonads in individual animals, and to relate the ovarian egg size with the stage of development of brooded embryos present simultaneously in individual barnacles. Ovcm'cm egg size classes' small -medium large 55. Percent of rndivtdixaLs less titan 1 mm, in cC{a.nt. sperm. 1-itnw./ in cCt'am, Sjjerm Z-3.Xrn.ru m cCiarvt . julC of sperm. 10 FIGURE 5. Egg sizes and seminal vesicle widths found simultaneously in large individuals. Left-hand side : numbers within the blocks represent numbers of individuals examined ; right- hand side : numbers above blocks represent percentages of individuals with seminal vesicles of given sizes. 178 GALKX HOWARD HIUIAKD For studies of tlu j two gonads, the large animals taken at intervals throughout the study period were examined. Animals were grouped into four categories according to egg size : animals with small eggs, those with medium eggs, those with large eggs, and those with hoth small and large eggs. The seminal vesicle width of each animal of a given egg category was noted. The data for all animals examined are shown in Figure 5. In Figure 5, the bars on the right hand side represent the frequency of occur- rence of the various conditions of the seminal vesicle which may be found in indi- TAHI.K II Cm-relation of size of ovarian egg with developmental stage of brooded embryos present in the same individual. Numbers of individuals showing each combination of conditions are indicated bv numbers within the squares. Heavy vertical lines enclose the "peak" months of the breeding season. Kgg size Embryos 31 Oct. 30 Nov. 14 Jan. 1 1 Feb. 11 Mar. 18 Apr. IX May 28 June 24 July 5 Sept. 7 Nov. 5 Dec. 8 Jan. Absent 2 4 1 4 6 3 1 Small & Early Large Middle Late 5 4 1 Absent 2 5 9 11 9 11 1 o Small Early Middle 2 4 1 1 1 1 3 Late ] 2 1 1 Absent 1 9 3 4 1 8 Medium Early Middle 1 1 4 1 4 1 1 2 2 1 Late 1 2 1 1 1 2 1 Absent 4 5 4 2 1 Large Early Middle Late 2 1 1 1 1 Absent 3 2 1 3 1 Eggs Early Absent Middle Late 12 14 10 14 10 22 25 13 15 15 12 10 18 viduals with eggs of any given size category. We see from the graph that sperm is absent only in animals where eggs are small or absent. Under all other ovarian conditions, sperm is always present, and in the majority of barnacles with medium or large eggs, the seminal vesicles are full. Table II contains data illustrating the conditions which may occur simul- taneously in the ovary and in the brooded ovigerous lamellae, in the same individual at different times of the year. From these data, the information on large animals collected during the breeding season (October to November, 1956; and May, 1957 REPRODUCTION IX MITHU.A POLYMERUS 179 to January, 1958) and containing both ovarian eggs and ovigerous lamellae, has been selected and is shown graphically in Figure 6. It is evident from Figure 6 that there is a general relationship between ovarian egg size and brooded embryo stage in the same individual, particularly during the height of the breeding season. In general, where eggs are small in the ovaries, embryos are in an early stage of development in the mantle cavity ; where eggs are medium-sized in the ovaries, embryos are in the middle stage of development in the mantle cavity: and where eggs in the ovaries are large (nearly ready to be extruded), embryos in the mantle cavity are advanced (nearlv ready to be lib- erated). The parallel diagonal lines drawn through successive peak conditions show clearly the synchronized pattern of development of ovaries and embryos during the peak months of the reproductive season. Certainly such a condition, in which eggs and embryos show more or less parallel development, appears to No.of i'ndi- victuals ex- .7fnrud . Developmental stage , of, e-m&rjos in ovtcjerous (ameuac ; FIGURE 6. Developmental stages, within individuals, of ovarian eggs and brooded embryos, where both are present simultaneously. Numbers pointing to black bars show percentages of given stages present during the "peak" months of the reproductive season ; numbers within the larger bars show percentages of given stages present during the total reproductive season. represent the most efficient timing pattern for an ovoviviparous organism which produces successive broods of young in a single breeding season. However, it is clear from the graph and the table that precise coordination between rates of egg enlargement and larval development does not exist for all animals examined during the reproductive season. For example, some animals with small eggs in the ovaries were found brooding middle or late stage embryos. In such cases, it appears that the interval between successive broods of larvae is greater than in cases where eggs and embryos develop in phase, and that ovarian egg development is retarded with respect to embryonic development. Table II shows clearly that these animals with relatively retarded ovarian development tend to be localized in time, predominating during the later months and not the peak months of the reproductive season. Thus, the five cases in which late embryos accompany small 180 GALEN HOWARD HILGARD ovarian eggs occurred in October and November of 1956, December of 1957, and January of 1958, suggesting that at this time development of the ovaries had slowed down or stopped for the season, and that the final batch of nauplii would soon have been shed. Also, the combination of small eggs with middle embryonic stages occurred, with one exception, either at the beginning (May, 1957) or near the end (November, 1956 and December, 1957) of the breeding season. Animals with medium eggs and no ovigerous lamellae occurred in April and May, 1957 (when they were presumably giving rise to their first batch of eggs of the season) and in November, 1956 and 1957 (when they may represent individuals in which the ovarian growth has slowed down toward the close of the season). In con- trast, a few animals contained medium-sized eggs along with early embryos in the ovigerous lamellae, suggesting a relative acceleration of egg development. How- ever, in all three cases, the eggs measured fell close to the lower size limits of the medium egg size class, and the apparent acceleration is exaggerated by the position- ing of size-class limits in the grouping of data. TABLE III Apparent conditions of ovaries (ovarian eggs) with relation to embryos (found simultaneously in an individual) Dates Number of times ovaries appeared in following conditions: Accelerated Synchronized Retarded 31 Oct. 1956 2 1 30 Nov. 1956 1 5 14 Jan. 1957 11 Feb. 1957 11 Mar. 1957 no reproductive activity 18 Apr. 1957 18 Mav 1957 1 9 3 28 fun. 1957 1 6 1 24Jul. 1957 5 Sept. 1957 7 Nov. 1957 1 6 7 3 1 2 2 5 Dec. 1957 2 5 8 Jan. 1958 1 1 Table III summarizes the condition of the ovaries with relation to embryonic stages. We see that during the early and middle months of the reproductive sea- son, most of the animals examined appeared to show synchrony in rates of ovarian and embryonic development. Toward the end of the reproductive season, a relative lag in ovarian egg development became noticeable. By combining data for the months of May through September (the main reproductive period ) and for the period of October through December (the end of the breeding season), we can show that the prevalence of animals with synchronized ovaries during the former months, and of animals with retarded ovaries during the latter months, is indeed statistically significant. REPRODUCTION IX MITELLA POLYMERUS 181 While animals with apparently well synchronized brood development are prob- ably producing batches of eggs at a relatively efficient rate, there is evidence that even in such animals some time does elapse between broods. We might expect, in a perfectly synchronized animal, that immediately following the liberation of a batch of larvae, a second batch of eggs would be extruded, and that such an animal would be carrying ovigerous lamellae virtually all of the time. However, at no time during the reproductive period were all reproductively mature members of the population carrying ovigerous lamellae ; even during the height of the breeding season, only 50% to 60% of the population were brooding embryos. The occurrence of a very few slow developers during this time could hardly account for such a discrepancy ; and it must be assumed that even during the height of reproduction, some time does elapse between the hatching of a batch of larvae and the extrusion of the next batch of eggs from the same individual. NUMBER OF BROODS AND LARVAE LIBERATED BY MITELLA POLYMERUS, AND ITS RELATIVE FECUNDITY No direct observations are available on the number of broods produced by an adult barnacle per year. However, by combining all the lines of evidence at hand, we can arrive at a hypothetical figure for the number of batches of larvae liberated by a large individual during one reproductive season. Three lines of evidence are considered here : the duration of the reproductive season, the rate of development of embryos in the ovigerous lamellae, and the developmental stages of eggs and embryos present at any one time in an individual during the reproductive season. We have already seen that the reproductive period for most barnacles extends through about eight months or about 240 days (Fig. 3). The approximate dura- tion of the lamellar brood period was determined in the laboratory. Several batches of larvae were raised /;/ vitro at 13 C., starting with what appeared to be the two-cell stage, and continuing to hatching. Periodic examination of these ovigerous lamellae showed that embryos remain in the "early" stage for the first nine days, are in the "middle" stage from the tenth through the fifteenth day, and remain in the "late" stage from the sixteenth day to hatching, which occurs on the twenty-ninth to the thirty-first day. Development from fertilization to hatching of the nauplius thus averages about thirty days. This rate is substantiated for field conditions in Figure 3, where we find the first large eggs being extruded in April and the first late embryo just one month later. It is interesting to note that the brood period for "normal" M. spinosus, raised under conditions where tempera- ture was not controlled but averaged 14 C. to 15 C., was thirty to thirty-two days (Batham, 1946). The developmental stages present simultaneously in individuals have been shown in Figure 6 and Table II. Here, the simultaneous presence in single animals of three stages (small eggs, large eggs, and late embryos, especially in animals taken in July and September) strongly suggests that an animal may give rise to at least three batches of embryos during one reproductive season. Tables II and III show that during the first six months of the reproductive season, egg and embryo stages tended to be more or less synchronized in individuals ; and during the last two months of the breeding season, the majority of animals carried broods which tended to be out of phase by two stages. In a perfectly synchronized animal, in which GALEN HOWARD HILGARD ovarian eggs and lamellar embryos develop in phase, we might assume that the interval between the average small egg and the average early embryo is equal to the lamellar brood period, or thirty days. In animals one step out of phase, that is, in animals in which medium embryos accompany small eggs, or late embryos accompany medium eggs, we might assume that the interval between broods is thirty-eight days (or thirty days plus the difference between the average age of an early embryo and the average age of a middle stage embryo eight days). In animals two steps out of phase, in which late embryos accompany small eggs, or (specifically in November, 1957) no embryos accompany medium eggs, we might assume that the interval between successive broods is at least 48.5 days (or thirty days plus the difference between the average age of an early embryo and the average age of a late embryo 18.5 days). Through the breeding season (eight months), these various intervals were found to be more or less localized in time. That is, for approximately the first six months, most of the population seemed to be producing broods either in phase or one stage out of phase. Hence, at the assumed rate of one brood for every thirty to thirty-eight days, a minimum of 4.7 broods and a maximum of six broods could have been extruded by an individual during this time. For the last two reproductive months, a majority of the population carried broods two steps out of phase. At the assumed rate, then, of one brood for every 48.5 days, one to 1.2 broods may have been extruded by an individual during these two months. During the TOTAL reproductive period, then, it appears that a single large barnacle could have liberated from five to 7.2 broods of larvae. If an average animal gives rise to six broods in a season, each brood developing in the mantle cavity for thirty days, we would expect such an animal to be carrying- embryos for approximately 180 days out of the total 240 days, or approximately 7S% of the total time. Since no single individual was followed during the breed- ing season, no data are available to provide a direct means of checking this figure. However, 59% of the large individuals collected during the eight-month repro- ductive season bore ovigerous lamellae. This suggests that perhaps large indi- viduals contain ovigerous lamellae for only about 59 c /c of the total reproductive period, or 142 out of 240 days. On this basis, then, the average animal probably produced only four or five broods during the season. There seem to be two good reasons for the discrepancy between the two esti- mates for average number of broods per season. The first, already mentioned, is that even in well-synchronized animals, some time did elapse between successive lamellar broods. There was no direct measurement of the duration of this mini- mum interval, and thirty days was used as the minimum time for the enlargement and extrusion of eggs ; but actually, the period during which eggs remain in the <>vary is probably a few days longer. A suggestion providing some independent support for this is seen in the data in Figure 3, where on March 11, 1957, all ovarian eggs were in the small size class (though some approached the upper limits of this class), and on April 18, 1957, 36 days later, one individual was just extruding eggs from the oviduct. The other possible reason for the discrepancy be- tween the two estimates of number of broods produced per year is shown by the fact that many stages of development were found in the population at any one time, and that the whole population did not produce broods synchronously. Ft is probable REPRODUCTION IX MITELLA POLYMERUS 1ol\inerus may liberate from 104.000 to 240,000 larvae from a single brood contained in one pair of ovigerous lamellae ; the slightly smaller M. spinosus liberates approximately 3000 larvae per brood (Batham, 1944-45). It appears that a single adult M. polyments may produce roughly 52 to 280 times as many larvae per year as a single adult M. s 100 > TptaC no, in eac k sarnpie excLJan. '. Con,tro(5 (adjacent) Distance from nearest neighbor of a. reproductive sizx : 2."-4" 4.1- fe" fc.7-8" 8.J-/0" 70.f"-74" 55 29 30 32. 25 27 FIGURE 7. Summary graph showing percentages of large ,17. pnlyincnis containing ovigerous lamellae when separated from other large specimens by given distances. SELF-FERTILIZATION For a study of self-fertilization, a series of large and relatively isolated barnacles, situated at various measured distances from their nearest neighbors of reproduc- tive size, were collected and inspected for the presence of ovigerous lamellae. These samples were collected at different times throughout the reproductive sea- son. The data, grouped for the months of collection, and for the distances by which the sexually mature barnacles were separated, are presented in Table IV and Figure 7. It can be seen that over 509r of the control animals for each month (except January, 1960) carried ovigerous lamellae; animals separated by two to four inches appeared to carry ovigerous lamellae less frequently, but the differences are not statistically significant. There is a statistically significant drop in the presence of embryos in animals separated from their nearest neighbor of a repro- ductive size by over four inches, and an absence of embryos in animals separated by more than eight inches. Thus it appears that eight inches was the maximum distance over which copulation of large animals collected could take place, and that animals separated by greater distances failed to receive sperm. Despite the 184 GALEN HOWARD HILGARD TABLE IV Numbers of barnacles of reproductive size isolated from other such barnacles by given distan<-< \, showing presence ( +) and absence ( ) of ovigerous lamellae Date Distance from nearest neighbor of reproductive size: Controls (adjacent) 2"-4" 4"-6" 6"-8" S"-10" 10"-14" + + ! + + + 1958 12 8 3 2 1 3 5 2 8 1959 20 July 7 Aug. 2 Nov. 10 5 6 4 4 (i 3 3 2 8 2 6 1 6 10 1 8 1 6 12 8 8 7 6 3 6 10 1960 2 Jan. 30 8 6 5 8 Summary, excluding Jan. I960 32 23 10 19 3 27 1 31 23 27 simultaneous maturity of male and female gonads, and despite the evidence for self-fertilization in other barnacle species (Barnes and Crisp, 1956), self-fertiliza- tion apparently does not take place in M. polymerus. It also appears that formation of ovigerous lamellae does not occur in the absence of fertilization. DISCUSSION On the subject of reproduction in cirripeds, a good deal has been written. Some accounts, such as those of Darwin (1851), Broch (1922), and others are concerned with the questions of hermaphroditism, the existence of complemental males, etc., rather than reproductive anatomy, reproductive cycles, and fecundity. Comparison of the seasonal reproductive cycle of M . polywierus in Monterey Bay with that of M. spinosits on the New Zealand coast (Batham, 194445) shows some interesting features. Figure 4 summarizes reproductive and temperature data for the two species. In Monterey Bay, from November, 1956, through the greater part of April, 1957, shore temperatures remained between 11.2 C. and 12.4 C., but showed a definite rise toward 13 C. during February and March. From May, 1957, through about one-half of November, shore temperatures re- mained above 13 C., rising to over 14 C. in May, July, and August, and to above 16 C. during September and October. Thus, the year may be roughly divided into the colder winter months and the warmer spring, summer, and fall months. In M. polymerus, the increase in mean seminal vesicle width during the early spring roughly parallels the rise in shore temperature ; the decrease in mean seminal vesicle width in the winter follows roughly the winter decrease in temperature. Likewise, the earliest occurrence of embryos follows closely behind the seasonal rise in temperature, and embryos continue to be present in the population until just after temperatures begin to drop during the winter. There is thus a clear corre- spondence between reproduction and shore temperature. The three peaks seen REPRODUCTION IN MITELLA POLYMERUS 1S5 in the occurrence of embryos in the latter half of 1957, while not statistically sig- nificant, show a relationship with the temperature peaks of May, July, and Sep- tember which is perhaps suggestive. The data given by Batham (1944-45) for M. spinosns, from the open coast of Otago Peninsula, New Zealand, and the corresponding temperature curve (from two later years and a neighboring vicinity ; Batham, 1958) show a similar relation- ship between temperature and reproductive activity. As might be expected for a related species occurring at a slightly higher latitude but in the southern hemi- sphere, the reproductive cycle is a rough mirror image of that of M. polymer us in Monterey Bay, California, though the breeding season is somewhat shorter. Other barnacle species have been observed to be reproductively active pri- marily during the months of warmer water temperatures : Balanus crcnatus, studied on the Atlantic coast of Canada (Bousfield, 1952-53) and in San Francisco Bay. California (Herz, 1933) ; Balanus improrisus, studied on the Atlantic coast of Canada (Bousfield. 1952-53) ; and Chthainalus stellatus, studied in Great Britain (Crisp, 1950). While such a correlation between temperature and reproductive activity might seem obvious and only reasonable, a number of barnacles reproduce primarily when water temperatures are low or at a minimum. This group includes the following : Balanus balanoides, studied on the Atlantic coast of Canada (Bousfield, 1952-53), at Woods Hole, Massachusetts (Fish, 1925), and in Great Britain (Moore, 1935; Crisp and Patel, 1960) ; Balanus haincri, studied on the Atlantic coast of Canada (Bousfield, 1952-53), and in Great Britain (Crisp, 1954) ; Balanus porcatus, studied in Great Britain (Crisp, 1954) ; and Balanus glandula, studied at Van- couver, B. C., and La Jolla, California (Barnes and Barnes, 1956) and in San Francisco Bay, California (Herz. 1933). There is evidence that still another group of barnacles reproduce (perhaps with some variation in rate) throughout the entire year. These include Elminius modest us, studied in Great Britain (Crisp and Davies, 1955), Verruca stroemia, studied in Great Britain ( Pyefinch. 1948), and Balanus tintinabulum, observed at La Jolla, California (Coe, 1932 J. Bousfield (1952-53) has studied the distribution and spawning seasons of the barnacles of the Atlantic coast of Canada, and reviewed the evidence supporting temperature as a principal factor governing reproduction in cirripeds. He clearly indicated that there is variability in reproductive period within a given species at different latitudes within its geographic range, and showed that reproductive activity tended to occur at times when temperatures were similar, regardless of latitude. The relationship of temperature and food supply to rate of reproduction has been studied by Crisp and Davies (1955) in the barnacle Elminius modestus. By growing these barnacles on glass slides and observing development through the translucent bases, these workers were able to follow, in i'n'0, the development of both ovarian eggs and lamellar embryos. They found that (p. 379) "the time interval occupied by successive broods varies among individuals, and with the season of the year. Rate of development of embryos seems to be a function of temperature alone, but regeneration of the ovary depends on nutrition and food supply." Crisp (1959), working with Balanus balanoides, showed that the rates (iAI.KX HOWARD HIi.CARb of development of the early embryonic stages (through the limb bud stage) are temperature-dependent up to 12-14 C., but that the later stages vary little in rate of development between 3 C. and 12 C. Further points of comparison may be brought out between M. polymer us, M. sphwsus, and other barnacles. The present study indicated that sexual maturity is not necessarily a function of size of the animals alone. Results of studies on .17. spinosus and litininnts modestus showed similarly that in populations of Miialler barnacles, sexually mature individuals are found, but less frequently than in populations of larger barnacles. Self-fertilization apparently does not occur in large isolated individuals of M. polynicrns, and cross-fertilization appears to be the rule. Crisp (1954) and Crisp and Patel (I960) pointed out that cross-fertilization also appears obligatory in B. crenatiis, Elmiiihts inodcstns, and in B. balanoidcs. However, self-fertilization very probably can occur in at least three species of acorn barnacles. Barnes and Crisp (1956) experimentally isolated individuals of Chthamalns stellatus, Verruca strucnria, and Balaniis pcrjoratns and found that they frequently produced ovigerous lamellae. They also observed that fertilized eggs found in such isolated individuals are frequently less viable than cross-fertilized eggs. The genetic advantage of cross-fertilization is well known, and it appears that the Mitclla polymerus popu- lation, composed usually of closely-packed individuals, is well adapted for cross- fertilization. SUMMARY 1. The gross structure of the reproductive system of M. poly merits is de- scribed and compared with that of the southern hemisphere species, M, spinosus (studied by Batham, 1944-45). 2. Size and reproductive activity in M. polymerus are related. All animals over 27.5 mm. in breadth (distance from rostrum to carinaj are found to repro- duce ; smaller animals arc- found to contain developing embryos less frequently. Xo sexually mature animals less than 17.2 mm. in breadth were found. 3. A fifteen-month study of the reproductive cycle in the population is de- scribed. Reproductive activity is evident during the greater part of the year. For the year of 1957, developing embryos were present in the population for a period of eight months during which time the shore temperature ranged from 12.3 C. to 17 C. The reproductive season for the southern hemisphere species likewise occurs during the warmest months ; thus the yearly cycle of M. polymerus shows a perhaps expected mirror image of the situation occurring in the southern hemisphere. 4. Within individuals, male and female gonads mature at approximately the same time during the year, and during the greater part of the year, an individual contains both developing eggs and seminal vesicles full of sperm. 5. Stages of development of ovarian eggs and brooded embryos found simul- taneously in individuals are compared. During the early and middle months of the reproductive season, ovarian eggs and brooded embryos tend to be in similar stages of development (that is, small eggs are found with early stage embryos, large eggs with late stage embryos, etc.). During the later reproductive months, a relative lag in ovarian egg development is evident. REPRODUCTION* IX MITEI.LA POLVMERUS 1< S " 6. Embryos were raised in z>itro under controlled temperatures. The em- bryos took an average time of thirty days for development from fertilized egg to free-swimming larva. 7. Estimates are given of the number of broods of young and the number of larvae liberated by a large individual during a year, and these are compared with the results of Batham (1944-45) for <\L spinosus. Studies of the larval brood period, the stages of eggs and embryos found simultaneously in individuals, and the length of the reproductive season allow an hypothesis that five to seven broods may be liberated by a large individual during a year. Three to four broods appears more probable for an average large animal. A pair of ovigerous lamellae may contain from 104,000 to 240,000 larvae. Batham's data (1944-45) showed that probably two broods, each containing approximately 3000 larvae, are liberated by M. spinosus during a year. Thus M . polymer us may liberate from 52 to 280 times as many larvae per year as a single large M. spinosus. 8. The possibility of self-fertilization in M. polymerus is studied. Relatively isolated large animals are found to carry ovigerous lamellae less frequently than those adjacent to each other ; and large animals isolated from each other by over eight inches were never found carrying embryos. From this evidence, it appears that self-fertilization does not occur in this species, and that cross-fertilization is necessary for the formation of ovigerous lamellae. LITERATURE CITED BARNES, H., AND M. BARNES, 1956. The general biology of Balanus f/Iandula Darwin. Pac. Set., 10(4) : 415-430. BARNES, H., AND D. J. CRISP, 1956. Evidence of self-fertilization in certain species of bar- nacles. /. Mar. Biol. Assoc., 35: 631-639. BATHAM, E. J., 1944-45. Pollicipcs spinosus Quoy and Gaimard. I. Notes on biology and anatomy of the adult barnacle. Trans. Roy. Soc. Nczi 1 Zealand, 74: 359-374. BATHAM, E. J., 1946. Pollicipcs spinosus Quoy and Gaimard, II. Embryonic and larval development. Trans. Roy. Soc. JVYrc 1 Zealand, 75: 405-418. BATHAM, E. J., 1958. Ecology of Southern New Zealand exposed rocky shore at Little Papanui, Otago Peninsula. Trans. Roy Soc. Nezv Zealand, 85 (Part 4) : 647-658. BOUSFIELD, E. L., 1952-53. The distribution and spawning seasons of barnacles on the Atlantic coast of Canada. Ann. Rep. Nat. Mus. Canada, Bull. No. 132: 112-154. BROCH, H., 1922. Studies on Pacific cirripeds. I' id. Mccld. Dansk Naturli. Foren. Kobenhavn., 73: 215-358. COE, W. R., 1932. Season of attachment and rate of growth of sedentary marine organisms at the pier of the Scripps Institution of Oceanography, La Jolla, California. Univ. of California Press, Berkeley. CORNWALL, I. E., 1925. A review of the Cirripedia of the coast of British Columbia with a glossary, and key to genera and species. Contr. Lanad. Biol., 2: 469-502. CRISP, D. J., 1950. Breeding and distribution of Chthainalns stcllatus. Nature, 166: 311-312. CRIST, D. J., 1954. The breeding of Balannx porcatus (da Costa) in the Irish Sea. /. Mar. Biol. Assoc.. 33: 473-494. CRISP, D. J., 1959. The rate of development of Balanus balanoidcs (L.) embryos in ritro. J. Anini. Ecol, 28: 119-132. CRISP, D. J., AND P. A. DAVIES, 1955. Observations in rit'n on the breeding of Elininins modcstns grown on glass slides. /. Mar. Biol. Assoc., 34: 357-380. CRISP, D. J., AND B. S. PATEL, 1960. The moulting cycle in Balanus balanoides (L.). Biol. Bull., 118: 31-47. INS GALEN HOWARD HILGARD DARWIX, C., 1851. A Monograph on the Subclass Cirripedia. I. Lcpaclidae. Ray Society, London. FISH, C. J., 1925. -Seasonal distribution of the plankton of the Woods Hole region. Bull. U. S. Bur. Fish.. 41: 91-179. i IKKZ, L. E., 1933. The culture and morphology of the later stages of Balanus crenatus. Ph.D. thesis, Stanford University. (Published in part, in Biol. Bui!., 64: 432-442, 1933.) MOORE, H. B., 1935. The biology of Balanus balanoides. III. The soft parts. /. Mar. Biol. Assoc., 20: 263-274. NUSSBAUM, M., 1890. Anatomische Studien an Californischen Cirripedien. Bonn, Germany. PYEFINCH, K. A., 1948. Notes on the biology of cirripedes. /. Afar. Biol. Assoc., 27: 464-503. RICKETTS, E. F., AND J. CALVIN, 1952. Between Pacific Tides. Stanford Univ. Press, Stan- ford, Calif. OBSERVATIONS ON THE NUTRITION OF THE RHYNCHOCOELAN LINEUS RUBER (O. F. MULLER) J. B. JENNINGS Department of Zoology, University of Leeds, England Feeding and digestion in the Rhynchocoela have received relatively little attention apart from brief accounts by \Yilson (1900), Reisinger (1926), Coe (1943) and Hyman (1951). These indicate that the group is carnivorous, preying upon a variety of invertebrates which are captured by means of the extensible proboscis and swallowed whole, and that digestion may be either extracellular or partially intracellular. No further details of rhynchocoel nutrition are available and to remedy this deficiency, that of the common British species Linens rnber (O. F. Miiller) has been investigated. MATERIALS AND METHODS Specimens of Linens ntbcr were collected from beneath rocks embedded in sand at mid-tide level at Cremyll, Plymouth. After starvation for two days to induce a readiness to feed and to clear the gut of remnants of previous meals, individuals were presented with representatives of the fauna of their habitat, and the methods of capture and ingestion of the selected prey observed. The course of digestion was followed by histological examination of individuals fixed at progressive intervals after feeding upon either the natural food or easily identifiable test foods such as frog erythrocytes and raw, optically active starch grains. Fixation was in Susa at 30 C. and sections cut at 8 /* were stained \vith the haematoxylin and eosin. Feulgen, periodic acid-Schiff (P.A.S.), Alcian blue (for mucin), benzidine-peroxide (for haemoglobin), and Lugol's iodine techniques. Changes in the pH of the gut contents during digestion were followed by feeding particles of fish muscle stained with 0.5% sea water solutions of various indicators and observing subsequent color changes by periodically flattening the fed indi- viduals and examining by both reflected and transmitted light. Food reserves were studied after fixation in Flamming (for fats) and 90% alcohol containing \% picric acid (for carbohydrates and proteins). Sections of individuals fixed in the latter reagent were stained by the Best's carmine, P.A.S. and modified Millon's methods. OBSERVATIONS The food and feeding mechanism Linens ruber feeds mainly on small annelids and crustaceans but particles of any dead organic material will be taken, providing it is not too decomposed. The oligochaete Clitellio arcnarins was particularly common in the habitat and at the time of collection (July-August) appeared to form the bulk of the food. 189 J. 15. JKXXIXCS Living prey is detected by the eye-spots and a starved Linens will respond to animals moving within 2 cm. of the head. Dead or injured animals emitting de- composition products or body fluids can be located at greater- distances and here it is presumably chemoreceptors in the cephalic ciliated grooves which are stimu- lated. Detection of living prey is followed by immediate everMon of the proboscis 1 through the proboscis pore at the anterior tip of the bod}-, and this occurs with such explosive force that as the proboscis strikes the prey it becomes coiled around it in a tight spiral grip. It does not penetrate the prey, since it lacks stylets or similar piercing organs, but the tightness of its grip may rupture the integument and cause loss of body fluids or gut contents. The grip is aided by sticky secretions from the proboscis epithelium and immediately it is secured the proboscis begins to retract and draws the prey, usually struggling violently, back towards the mouth. This lies ventrally 2-3 mm. behind the proboscis pore, and as the prey is drawn towards it the body anterior to the mouth is raised and extended until it can grasp the prey by curling downwards over it. This movement of the anterior tip of the body continues downwards and backwards and forces the prey into the mouth which gapes open to receive it. The proboscis then gradually relinquishes its grip and withdraws into the rhynchocoel as movements of the mouth, aided by contractions of the anterior body musculature, force the prey into the gut. Inges- tion of small animals is completed in 15 to 20 seconds but with larger prey, such as annelids one-third to one-half the length of the feeding individual, it may take as long as 30 minutes, and in such cases the first part to be swallowed is partially disintegrated before ingestion is complete. Small animals usually die within sec- onds of entering the gut, but active errant polychaetes with armored jaws may survive long enough either to force their way to the exterior through the gut and body walls or propel themselves down the length of the gut to emerge unharmed at the anus. This is particularly liable to happen when the prey is ingested head first, but in the majority of cases the proboscis seizes an animal about its middle and consequently draws it back to the mouth bent upon itself in the shape of a U. It is then ingested in this form and is unable to escape from the gut before being killed. During capture and ingestion Linens extends to its fullest length and produces copious sticky mucoid secretions from the ventral surface. This enables a firm hold to be retained upon the substratum whilst dealing with the prey,, and even if the latter is partially buried it can be drawn from its retreat and swallowed without causing the Linens to shift position. Inert masses of food, such as animal remains or the test foods used in this investigation, do not stimulate eversion of the proboscis but are seized directly by the mouth and swallowed piecemeal. The structure of the c/nt The gut consists of three histologically distinct regions, namely the mouth and buccal cavity, the foregut, and the intestine. It runs the length of the body from mouth to anus without coiling, is ciliated throughout and lacks both multicellular lands and musculature. 1 Details of the proboscis and the mechanism of its eversion are given by Hyinan (1951) and are not included here. NUTRITION OF LINEUS RUBER 191 The mouth consists of a ventral subterminal invagination of the epidermis some 200 //, deep and opening directly into the buccal cavity. It is fringed with large cilia and externally has a lobed appearance due to folds in its walls which allow expansion during ingestion. The invaginated epidermis contains acidophil, P.A.S.- and Alcian blue-positive gland cells whose secretions probably facilitate passage of food, and the entire mouth region is surrounded by concentrations of similar sub-epidermal gland cells. The buccal cavity is lined by ciliated cuboidal cells lO-12/i tall and these are backed by masses of acidophil and basophil gland cells, the majority of which stain with Alcian blue and appear to have the same function as those around the mouth. The walls of the cavity are much folded and ascend diagonally backwards to become continuous with those of the foregut beneath the proboscis sheath. The foregut (Fig. 1) runs posteriorly for one-tenth the length of the animal and its walls are considerably thickened, especially ventrally where the wall may be up to 300 fj. in depth. They are throw-n into small simple folds and consist of a single layer of ciliated cuboidal cells, 10-12 /i tall, lining the lumen and lying upon acidophil syncytial tissue containing numerous gland cells, free nuclei and occasional large lacunae. In the anterior portion the gland cells consist of P.A.S.- and Alcian blue-positive acidophils and basophils in approximately equal amounts, together with a number of others which are intensely basophilic but give no reaction to Alcian blue. The proportion of the latter increases along the length of the fore- gut to the median portion where all the gland cells are of this type. The gut wall then gradually decreases in thickness and gland cell content as it nears the intestine and terminates in a constriction (Fig. 1) separating the latter from the foregut. The intestine is the longest part of the gut and runs from its junction with the foregut in the anterior part of the body direct to the anus at the extreme posterior end. It bears paired and serially repeated lateral pouches or caeca throughout its length, apart from a short unpouched region immediately before the anus. The intestinal wall or gastrodermis (Fig. 3) is made up of two types of cells arranged in a single layer upon a thin basement membrane. The larger and more numerous of these are attenuated columnar cells, 50-55 ^ tall and 8 /A wide, with granular basophilic cytoplasm containing various acidophil inclusions and basal vesicular nuclei. The free distal borders of the cells bear cilia which in unfed individuals are of uniform appearance and size, but in the presence of digesting food the cilia lose their uniformity and coalesce into pseudopodia-like processes which extend out into the lumen (Fig. 5). This peculiar modification of the cilia is correlated with entry of food material into the cells and is dealt with later. The second type of cell found in the gastrodermis is glandular and occurs between the bases of the columnar cells. These gland cells (Figs. 2 and 3) are 40-50 p. tall and 5-6 /A wide, unciliated and contain up to thirty acidophil pro- teinaceous spheres, each 0.5 /A in diameter, which are discharged into the intestinal lumen when food enters from the foregut. They are most numerous in the anterior part of the intestine, where there may be as many as one gland cell to every three columnar, but this ratio is graded down the length of the intestine to approximately one in twenty in the middle region and one in fifty or more beyond until the gland cells disappear, finally, in the short unpouched region before the anus. 192 T. B. 1ENN1N',- * ^>^R f& "*^ FIGURE 1. Longitudinal section of Linens showing the posterior portion of the foregut (left) and the constriction which separates this from the intestine (right). Haematoxylin and eosin. Scale : 1 cm. = 50 AI. FIGURE 2. ' Longitudinal section of the intestine in Linens showing part of a newly in- gested Clitcllio lying intact and undamaged in the lumen. Acidophil gland cells are prominent in the gastrodermis in the lower portion of the figure. Haematoxylin and eosin. Scale : 1 cm. = 100 M- FIGURE 3. A portion of the gastrodermis in Linens showing ciliated columnar cells inter- spersed with darker acidophil gland cells. Intestine empty. Haematoxylin and eosin. Scale : 1 cm. = 50 fj.. FIGURE 4. The gastrodermis in Linens 30 minutes after a meal of frog erythrocytes. The intestinal lumen (top) contains a homogeneous mass of digested haemolyzed erythrocytes which stains heavily with Feulgen and almost obscures the ciliary processes. The columnar cells are loaded with engulfed spherical masses identical in nature with the contents of the lumen. Feulgen and light green. Scale : 1 cm. = 50 /*. FIGURE 5. The gastrodermis in Linens 30 minutes after a meal of raw starch grains. The cilia have coalesced into pseudopodia-like processes and a few starch grains already engulfed are ranged along the distal margins of the columnar cells. Lugol. Scale : 1 cm. = 50 ,u. FIGURE 6. The gastrodermis in Linens 60 minutes after a meal of raw starch grains. The columnar cells are loaded with grains, many of which are as yet unchanged and still exhibit the characteristic black cross in polarized light. The lumen contains occasional free grains and on the left portions of two gregarine trophozoites with prominent nuclei. Section stained with haematoxylin and eosin and photographed by polarized light. Scale: 1 cm. = 40^. NUTRITION OF LINEUS RUBER I'M The lateral pouches have the same structure as the rest of the intestine and arc merely simple extensions to increase its area, not specialized digestive caeca. Tlic course of digestion Ingested food passes rapidly through the buccal cavity into the foregut where it is held for a few seconds before its passage intact into the intestine (Fig. 2). Living food usually dies during the brief pause in the foregut and this is due, no doubt, to acid secretions from the numerous basophilic gland cells present here, for when particles of fish muscle stained with bromo-cresol purple or chlor-phenol red were fed, their pH value fell from 7.0 to 5.5 as they passed through the fore- gut, and sections of newly fed individuals showed the majority of the glands to be discharged. There is no trituration or break-up of the food in the foregut but digestion begins immediately it enters the intestine. A series of individuals fixed at inter- vals after ingestion of the oligochaete Clitellio showed that as early as fifteen minutes after feeding the gland cells had discharged their spheres and digestion was well advanced. The Clitellio lay in the main median portion of the intestine with the epidermis deeply eroded and the entire body starting to fragment. The cilia of the columnar cells were still uniform in appearance and apparently creating currents in the gut contents to distribute the fragmenting food, for pieces of tissue were already passing into the lateral pouches. Digestion progressed rapidly with time and 30 minutes after feeding the intestine contained a heterogeneous mass of heavily eroded pieces of tissue, intact and fragmented setae, nephridia (which resisted digestion for longer than other tissues and stood out from these with sur- prising clarity) and diatoms, algal chains, etc. released from the oligochaete gut. The gastrodermal cilia were now beginning to lose their uniformity and coales.ce into pseudopodia-like processes stretching into the lumen of the intestine, whilst semidigested material from the latter was appearing as acidophil spheres in the distal portions of the columnar cells bearing these structures. These spheres passed back deeper into the cells and their number increased rapidly with time. Sixty minutes after feeding, the material in the lumen was almost homogeneous, with setal fragments and diatom cases being the only recognizable elements in it, whilst the columnar cells of the gastrodermis were packed with spheres of food under- going intracellular digestion. The spheres decreased in size and affinity for stains ( especially the Millon reagent for protein) as they passed down the cells to dis- appear finally in the basal region, the cells presumably then passing the products of digestion to other tissues whilst taking up more semidigested material distally until the lumen was emptied. This occurred some six hours after feeding and the amount of intracellular material then rapidly decreased. After a further three hours the columnar cells contained only a few acidophil inclusions, their cilia had resumed their normal shape and size, and the gland cells were again full of en- zymatic spheres. Indigestible residues were collected in the short unpouched region of the intestine near the anus, being swept there, probably, by the reconstituted cilia, and observations on living specimens showed that they were expelled even- tually by a sudden contraction of the posterior body musculature. A parallel series of feeding experiments, using fish muscle stained with indi- cators, showed that the initial drop in pH as the food passes through the foregut 194 J. B. JENNINGS is maintained in the intestine during digestion. In some cases it fell even further, to pH 5.0, and when sufficient indicator-stained material entered the columnar cells to be visible in neutral saline squashes, the intracellular digestion was seen to be progressing in a similarly acid medium of pH 5.0-5.5. It was not clear from the Clitellio-ied series how semidigested material enters the columnar cells, but the pseudopodia-like appearance of the coalesced cilia and spherical compact form of the material when within the cells suggested a form of phagocytosis. This possibility was investigated by feeding Linens on frog eryth- rocytes and raw, optically active starch grains made palatable by mixing with frog plasma, to ascertain whether such discrete particles were in fact taken into the columnar cells. In the series fed on erythrocytes, however, haemolysis occurred as they entered the intestine, the break-up including the majority of the nuclei, and 30 minutes after feeding the lumen contained a semidigested mass which gave a strong reaction with Feulgen, due to released nuclear materials, and with the benzidine-peroxide reaction for haemoglobin. The cilia had coalesced into the usual processes and many of the cells contained spherical masses with the same staining properties as the material in the lumen (Fig. 4). These apparently phagocytosed masses passed back into the cells as more appeared distally, and gradually decreased in size and their reaction to Feulgen and benzidine-peroxide as intracellular digestion progressed. Digestion of the blood meal was completed in six hours and the intracellular spheres disappeared without leaving residues of haematin or other insoluble pigments from the degradation of the haemoglobin. Final confirmation of the occurrence of phagocytosis came from the series fed on starch grains. Thirty minutes after feeding the cilia had formed filamentous processes extending into the lumen, and a few intact grains, staining blue with Lugol and still exhibiting the characteristic black cross in polarized light, had al- ready been taken into the cells and were ranged along their distal margins (Fig. 5). The number of such grains increased rapidly and 60 minutes after feeding packed the columnar cells (Fig. 6). Only grains 5 ^ or less in diameter were engulfed and larger ones remained in the lumen where they gradually lost their optical activity, fragmented and stained brown with Lugol. The fragments then passed into the cells and joined the previously engulfed grains which were undergoing intracellular digestion, losing their identity and disappearing towards the bases of the cells. Parasites of tJie gut Approximately 75 % of the Linens examined contained in the intestine an acephaline eugregarine identified as Urospora nenicrtes (Koelliker). Trophozoites (Fig. 6) 150-180 /j. long and 15-20 ^ wide, with basophil, P. A. S. -positive cyto- plasm and prominent nuclei, occurred in all parts of the lumen, and the intracellular stages, strikingly prominent with P.A.S., were common in the columnar cells. The gregarine did not appear to harm Linens in any way, apart from a few occasions when infected columnar cells reacted against developing intracellular stages and caused them to degenerate into masses of yellowish brown crystals. Such cells then burst, either hi situ or after being shed into the lumen, and the crystals were eliminated with the faeces. NUTRITION OF LINEUS RUBER 195 The food reserves Fat forms the principal food reserve in Linens and occurs as intracellular glob- ules up to 5 /x in diameter in the parenchyma and, to a lesser extent, in the columnar cells of the intestine. There are no specific protein reserves and the only demon- strable carbohydrate reserve is in the form of tiny granules of glycogen scattered throughout the parenchyma, musculature, and columnar cells. DISCUSSION The main points of interest in the nutrition of Linens ruber lie in the feeding mechanism and the digestive processes. In the case of the former a simple but effective method of capturing the food, supplemented by slight modification of the anterior portion of the alimentary canal into a thick-walled glandular foregut for its reception and killing, enables this rhynchocoelan to prey successfully upon animals far more active and elaborate than itself. In this respect Linens resembles the tur- bellarian flatworms where similarly simple feeding mechanisms make available prey ranging from protozoa to molluscs and tunicates (Jennings, 1957; 1959a). In the Turbellaria it is the pharynx which forms the principal element of the feeding mechanism and this organ is thus analogous in function to the rhynchocoelan pro- boscis as seen in Linens. The only disadvantage apparent in the type of feeding found in Linens is the possibility of escape by the prey before the secretions of the foregut can take effect but this is overcome, no doubt, in those fhynchocoelans which possess a proboscis armed with stylets and poison glands by killing or paralyzing the prey at the moment of capture. Digestion in Linens follows a pattern observed in other Acoelomata (Jennings, 1957; 1959b) in that both extracellular and intracellular processes are concerned, but here the intestinal wall is ciliated and consequently the semidigested food would be expected to enter by absorption. In fact, however, it enters by a form of phagocytosis, as is proved beyond doubt by the appearance in the columnar cells of starch grains which retain their form and optical activity after entry and so must have passed into the cells as solid discrete particles. This method of taking- material into the columnar cells involves temporary modifications in the form and behavior of the cilia during the digestion of each meal, and the protoplasmic pseudopodia-like processes formed from the coalescence of neighboring cilia are probably concerned in the engulfing of semidigested food, although this has not been observed histologically. The need for the intestine to be ciliated probably arises from its length and the absence of musculature, which together create a need for some method of distributing fragmenting food in the early stages of digestion and collecting residues near the anus at the end. Contractions of the body musculature appear to be insufficient for anything but the final expulsion of the collected resi- dues and hence ciliary currents are used. The reason for the retention of phago- cytic uptake of food under these conditions is unknown, for there is no apparent reason why extracellular digestion should not be carried to a point where the semi- digested food is soluble enough for absorption, and this presents an interesting subject for further investigations. 196 J. B. JENNINGS SUMMARY 1. The rhynchocoelan Linens ruhcr feeds on small annelids and crustaceans which are captured by the unarmed proboscis and swallowed whole. 2. The alimentary canal is differentiated into three regions : a buccal cavity, a foregut where the prey is killed by 'acid secretions, and an intestine where it is digested. 3. Digestion is the result of both extracellular and intracellular processes and occurs in an acid medium of pH 5.0-5.5. The enzymes responsible for the initial extracellular breakdown come from gland cells in the intestinal wall and digestion is completed within the columnar cells of the latter. Semidigested food enters these columnar cells by a phagocytic process and this involves temporary modifi- cations in the form and function of their cilia. 4. The food reserves consist of fat deposits in the parenchyma and, to a lesser extent, in the columnar cells of the intestine. LITERATURE CITED COE, W. R., 1943. Biology of the nemerteans of the Atlantic coast of North America. Trans. Conn. Acad. Arts Sci., 35: 129-328. HVMAN, L. H., 1951. The Invertebrates. Vol. II: Platyhelminthes and Rhynchocoela. Mc- Graw-Hill Book Co., Inc., New York. JENNINGS, J. B., 1957. Studies on feeding, digestion and food storage in free-living flatworms (Platyhelminthes: Turbellaria). Biol. Bull., 112: 63-80. JENNINGS, J. B., 1959a. Observations on the nutrition of the land planarian Orthodcnuis tcrrcstris (O. F. Miiller). Biol. Bull., 117: 119-124. JENNINGS, J. B., 1959b. Studies on digestion in the monogenetic trematode Polystoma intcgerriinuin. J. Helminth., 33: 197-204. REISINGER, E., 1926. Nemertini. /;;: P. Schulze (ed.), Biologic der Ticrc Deutschlands, Lief. 17. WILSON, C. B., 1900. The habits and early development of Cercbratulus lactcus (Verrill). Quart. J. Micr. Sci,, 43: 97-198. HERMAPHRODITISM IN THE SEA SCALLOP, PLACOPECTEX MAGELLANICUS (GMELIX) ARTHUR S. MERRILL AND JOHN B. BURCH 1 U. S. Department of the Interim-, Fish and U'ildlifc Scnicc, Bureau of Commercial Fisheries, Biological Laboratory, Woods Hole, Mass.; and Museum of Zoology, University of Michigan, Ann Arbor, Midi. While hermaphroditism is of common occurrence among normally dioecious mollusks, a careful search of the literature failed to reveal any published record of this condition for the commercially important sea scallop, Placopecten inageUani- cns. Consequently, observations on the occurrence of the phenomenon in this species are of some interest and importance. Our first hermaphroditic sea scallop was collected on September 17, 1959, while doing routine investigatory work relative to the scallop fishery aboard the chartered scalloper Whaling City in the western part of Georges Bank (68 45' AY. Long., 43 03' N. Lat.). September is within the spawning season for the sea scallop on Georges Bank (Posgay and Norman, 1958). However, spawning had not yet started at this locality and all specimens, including the hermaphrodite, were ripe and full. After close macroscopic examination the hermaphroditic gland was fixed for histological study in Newcomer's (1953) fluid and stained either by means of the Feulgen reaction or with Harris' hematoxylin and eosin. Some Feulgen-stained sections were counterstained with light green (yellowish). Pieces of tissue were cleared in chloroform, embedded in paraffin, and sectioned at 10 micra. For com- parison normal male and female gonads were likewise treated except that in these Benin's fluid was the fixative. In the normal gonad the size and shape are similar in both sexes. The single gonad is tongue-shaped and occupies most of that portion of the body ventral to the foot. It extends dorsally to form a thin layer over the surface of a portion of the digestive gland. The genital organ is large and plump when ripe but after spawning it becomes much smaller, shriveled, and quite flaccid. The sexual prod- ucts are easily seen through the tissue of the gonads, the ova giving the female gonad a bright coral red appearance at maturity, the sperm producing a whitish- cream coloration in the male gonad. The hermaphrodite mentioned above has the male and female parts located in different regions of the same gonad. The proximal part forms the ovary while the testis lies distal to it. The boundary between the two regions is indefinite with quite irregular islets of one tissue occurring within the" tissue of the other (Fig. la). This is even more pronounced histologically (Fig. 2a). The gonad is unspent and, relative to the degree of development, compares favorably with normal unspent gonads. 1 Grant 2E-41, National Institute of Allergy and Infectious Diseases, U. S. Public Health Service (in part). 197 I OS ARTHUR S. MERRILL AND JOHN B. BURCH The ovarian follicles are closely crowded and are filled with mature ova which take the shape of polyhedrons because of their tightly packed condition. The lumina of the follicles are completely filled. Kach follicle is lined on the outside by a single cell layer of squamous epithelium. Inside the follicles the individual ova are separated by a non-granular intracellular substance sandwiched between and separating the cell membranes. This matrix is rather uniformly distributed and of little variation in thickness. Actually, it may not be intracellular in the strict sense but a component of the cell membranes. Mature ova average about 45-50 micra in diameter in fixed material. Their germinal vesicles are large and clear, and contain a fine network of chromatin and several conspicuous nucleoli (Fig. 2b). Connective tissue or follicular cells are rarely found between either male or female follicles in this individual. The testicular follicles are tightly packed and are covered by the same kind of squamous cell layer as found covering the ovarian follicles. Inside the follicle and adjacent to the covering epithelium is a layer of spermatogonial cells. This layer of primordial germ cells is one to several cells thick (usually two or three, but sometimes up to ten or more). Progressing inward toward the lumen of the follicle are the primary and secondary spermatocytes, followed by spermatids, then mature sperm at or near the center. The tails of the spermatids are usually located in the most central part of the follicle. In portions of the testis where spermato- IMGURK la. Hermaphroditic I'lacopcclcn magellanicus. Mantle and gill tissue folded back to show the large bisexual gonad (X2/3). Ib. Hermaphroditic gonad of P. magellanicits. Male portion has spawned, female (bulbous) part still unspawned (X2). (Note: gonad fixed in Bouin's and preserved in alcohol before photo taken, this resulting in loss of color.) HERMAPHRODITISM IX THE SEA SCALLOP 199 L 1 \1 L? I* b b b b b 7 a b FIGURE 2a. Histological section through central portion of the hermaphroditic gland of specimen shown in Figure la (X 50). a 1 , Portions of female follicles filled with ova. a 2 , Por- tions of male follicles containing all stages of spermatogenesis. 2b. Enlarged portion of Fig- ure 2a showing part of a male and a female follicle and their boundary (X 250). b 1 , Mature sperm, b-, Spermatids. b 3 . Secondary spermatocytes. b 4 . Primary spermatocytes. b 5 , Sper- matogonia. b 6 . Epithelial lining, b 7 . Portion of a mature ovum. genesis is completed, the follicles are completely filled, from epithelial lining to epithelial lining, with mature sperm. However, even in such follicles, there are occasional cells at the epithelium which we take to be primordial germ cells but which may perhaps be follicle cells. The number of spermatogonial divisions before synapsis can not be determined, but there is no evidence in this specimen, or in normal unisexual males, to suggest that there are not more than two (e.g., see Coe and Turner, 1938, Fig. 17, p. 103). Since whole bunches of spermatogonia, ten or more cells deep, are sometimes seen to project out toward the lumen of the follicle, they probably go through a series of divisions before synapsis. In pulmonate gastropods, where the number of spermatogonial divisions can be accurately determined, there are normally six such divisions prior to spermatocyte formation (Burch, 1959). Spermatogonia average about 5 micra in diameter in sectioned material, primary spermatocytes about 2.5 micra in diameter, and the width of each sperm head is about 1.7 micra. The individual follicles in this hermaphroditic gland are either all male or all female ; there are no ambisexual follicles. In the proximal end of the gland most follicles are female, and in the distal end most are male. But, in a wide central area both male and female follicles are widely and indiscriminately dispersed (Fig. 2a). The size and appearance of the cells, as far as can be ascertained, are identical to similar stages in normal individuals of this species. Atypical spermatogenesis as found by Coe and Turner ( 1938) was not observed. A second hermaphrodite was found on November 21, 1959, in a sample from the eastern part of Georges Bank (66 45' W. Long., 41 23' N. Lat.). The ARTHUR S. MKRRII.L AXU JOHX I',. MURCH arrangement of gonadal tissue in this specimen was similar to that of the first. However, the spermary \vas mostly spent while the ovary still remained large and plump (Fig. 11)) with no sign of having started to spawn. This differs from the observed spawning habit of the normally monoecious great scallop, Pec ten ina.v units. In this species the products are released within a few hours of each other, with either the eggs or the sperm being shed first (Mason, 1958). Also, the arrange- ment of the genital tissues within the gonad of /'. nw.viinus is the reverse of that of the two hermaphroditic P. magellanicus (e.g., the testis proximal and the ovary distal). Of colleagues who have investigated various aspects of the scallop fishery, only Dickie- (personal communication) has advised of having observed this phe- nomenon. He remembered seeing hermaphroditic sea scallops on two separate occasions. Once during the summer of 1956 he was shown several preserved mature hermaphroditic gonads from Georges Bank by one of the scallop-vessel skippers. Another time (July 22, 1949) he found a 95-mm. predominantly female hermaphrodite from "Hour Ground" off Digby, Nova Scotia. Hermaphroditism is the usual condition in most of the Pectinidae (Coe, 1945) ; P. magellanicus is an exception. Hermaphrodites in other unisexual genera of pelecypods are not uncommon and it seems likely that the occasional deviations in the developmental processes which produce these hermaphrodites are due to the failure of the sex-differentiating mechanism to function normally, as has been suggested by Coe and others. According to the classification of Coe (1942) these abnormalities would be termed accidental functional ambisexuals, in which the primary sex factors go astray and fail to activate or suppress either the male or female hereditary mechanism at some early stage of development. This results in various amounts of both kinds of tissue being produced. Young's (1941) suggestion that hermaphroditism in normally bisexual species may be due to aberrant chromosomal behavior during gametogenesis has not yet been corroborated by cytological or experimental evidence. Similar suppositions were advanced to account for production of male and female cells in the ovotestis of the pulmonate gastropod L\mnaca stognalis apf>rcssa ( =- L. s. JHfjnlaris) by Crabb (1927) and in Physa gyrina by Mahoney (1940). Perrot (1930), how- ever, showed that this was not the case for L. stagnalis and Burch and Bush (1960) have shown the observations on P. gyrina to be in error. In the commercially important bivalves in which sex has been extensively studied, and which are dioecious, occasional occurrence of hermaphrodites is nor- mal. Thus, to list a few examples common to the North Atlantic coastline, Thorson (1936) remarked that Mytilus c dulls has a considerable percentage of hermaphrodites. Loosanoff (1936) in his sexual studies of the quahog (Jl/rr- cenaria mercenaria) found two cases of functional hermaphroditism among several hundred mature clams. Also, Turner 11 (personal communication) mentioned having seen a functional hermaphrodite in this species. He observed it to release the sperm first, and then the eggs. Coe and Turner (1938) found three cases of hermaphroditism on examining about a thousand soft-shelled clams (Mya arenaria). In the case of P. magellanicus about 3000 gonads were inspected after the first and - Lloyd M. Dickie, Biologist, Biological Station, St. Andrews, New Brunswick, Canada. 3 Harry J. Turner, Jr., Marine Biologist, Woods Hole Oceanographic Institution. HERMAPHRODITISM IN THE SKA SCALLOP 201 before the second hermaphrodite was found. The low frequency of occurrence accounts for this condition seldom being observed in the sea scallop. LITERATURE CITKU BURCH, JOHN B., 1959. Chromosomes of aquatic pulmonate snails (Bassommatophora). Ph.D. Dissert., Univ. Michigan, Ann Arbor, Pp. 97. BURCH, JOHN B., AND LINDA L. BUSH, I960. Chromosomes of I'hysa i/yrina Say (Mollusca: Pulmonata). /. dc Couch.. 100: 49-54. COE, \V. R., 1942. Sexual differentiation in mollusks. 1. Pelecypods. Quart. }\cr. Biol., 18: 154-164. COE, W. R., 1945. Development of the reproductive system and variations in sexuality in Pccten and other pelecypod mollusks. Trans. Connecticut Acad. Arts Sci., 36: 673-700. COE, WESLEY ROSWELL, AND HARRY J. TURNER, JR., 1938. Development of the gonads and gametes in the soft-shell clam (Mya arcnaria). J. Morph., 62: 91-111. CRABB, E. D., 1927. The fertilization process in the snail Lvuniacn stin/nalis apprcssa Say. Biol. Bull., 53: 67-97. LOOSANOFF, VICTOR, 1936. Sexual phases in the quohog. Science, 83: 287-288. MAHONEY, F. J., 1940. Spermatogenesis with special reference to certain extranuclear struc- tures in the pulmonate Physa (/yrina Say. Unir. Colorado Stud.. 26: 81-83. MAMIX, JAMES, 1958. The breeding of the scallop, Pccten ina.rinnis (L.), in Manx waters. /. Mar. Biol. Assoc.. 37: 653-671. NEWCOMER, E. H., 1953. A new cytological and histological fixing fluid. Science. 118: 161. PERROT, J.-L., 1930. A propos du nombre des chromosomes dans les deux lignees germinales du Gasteropode hermaphrodite Limnca stpgnalis ( variete rhodani). Rev. Smssc Zool., 41: 693-697. POSGAY, J. A., AND K. DUANE NORMAN, 1958. An observation on the spawning of the sea scallop, Placopecten magellanicus (Gmelin), on Georges Bank. Limnol. Oceanogr., 3: 478. THORSON, GUNNAR, 1936. The larval development, growth, and metabolism of Arctic marine bottom invertebrates compared with those of other seas. Mcdd. Gronland. 100(6) : 1-155. YOUNG, R. T.. 1941. An hermaphroditic Mytilus. Xau/ilus, 54: 90-91. ANTIGENS OF ARBACIA SI'KKM EXTRACTS L CHARLES B. METZ AND KURT KOHLER - Oceano graphic Institute, Plorida State University, Tallahassee, Florida, and Marine Biological Laboratory, }\'oodx Hole, Mass. The initial steps in fertilization appear to involve interactions of the sperm and egg surfaces at the molecular level (see Tyler, 1948, 1949; Metz, 1957a, 1957b). Most of the present information concerning such interaction has been obtained from studies of egg and sperm extracts. Among sperm extracts those with action on eggs have commanded most interest. In the sea urchin and certain other forms, extracts prepared by a variety of methods precipitate the egg jelly layer, agglutinate eggs and neutralize the sperm agglutinating action of the fertili- zin obtained from eggs. These effects of the extracts may result from the action of the sperm-surface receptor substance, antifertilizin, with which fertilizin com- bines in the sperm agglutination reaction (e.g. Tyler, 1948; Metz, 1957b). Whether or not these effects are to be identified with antifertilizin, absorption experiments have shown that such extracts contain some antigens in common with those of the sperm surface (Kohler and Metz, 1959a, 1959b, 1960). Further examination to reveal a more complete spectrum of antigens in such extracts seemed desirable. Accordingly, in the present investigation, Arbacia sperm extracts were examined for antigens by means of agar diffusion and immunoelectrophoretic tech- niques. The study revealed a maximum of four antigens in extracts prepared by f feeze-thawing sperm. MATERIAL AND METHODS Arbacia f>iuictulatii from the vicinity of the Florida State University Marine Laboratory, Alligator Point, Florida, and from Woods Hole, Massachusetts, were used in the study. Semen was usually obtained from the animals by electrical stimulation (Harvey, 1956). The spermatozoa were separated from the seminal plasma by centrifugation (approximately 3000 X g ; 20 minutes) at 4 C. The packed sperm were resuspended once in sea water and settled again by low speed centrifugation. The final supernatants following such washing regularly failed to give precipitation bands when diffused against anti-sperm serum. Standard sperm suspensions were prepared by diluting the packed sperm with three volumes of sea water. Sperm extracts were prepared from such suspensions of washed sperm by a variety of methods. These included the established methods for preparing agents 1 Contribution from the ( )ceanographic Institute, Florida State University. Aided by grants from the National Science Foundation, the National Institutes of Health and the Research Council of Florida State University. - Fulbright Fellow. Present address: Max-Planck-Institut fur Virusforschung, Tubingen, Germany. 202 ANTIGENS OF ARBACIA SPERM EXTRACTS 203 which act upon the egg jelly layer, namely, heating sperm to 100 C. (Frank, 1939) and freeze-thawing sperm (Tyler, 1939). The latter extracts are called "frozen-thawed extracts" below. Other methods are described with the individual experiments. Antisera were prepared by injecting rabbits with sperm (25% washed sperm in sea water). The immunizing antigens were administered through intravenous, intraperitoneal and subscapular injections. In the last instance the antigen was injected as an emulsion in Freund's adjuvant (Difco). Several axiti-Arbacia sera were examined. With the exceptions noted in the text the experiments reported here were performed with serum from the hyper-immune rabbit "#33." This rabbit received three injections of antigen in Freund's adjuvant (Difco) over a period of five months and was bled two weeks subsequent to the final injection. The immune serum obtained regularly agglutinated sperm to dilutions of 2~ 8 to 2~ 10 . No sera were pooled. Agar diffusion and electrophoresis. Agar diffusion experiments using the technique of Ouchterlony (1948) were performed in 2% agar containing Mer- thiolate (0.01%) as a preservative. The reaction plates were incubated at room temperature for several weeks. Immunoelectrophoretic analysis (Wunderly, 1957 ) was performed using 2% agar blocks prepared in 0.05 ionic strength veronal buffer, pH 8.5 and containing 0.01% Merthlolate. Wells in the agar block were filled with antigen prepared as follows : after dialysis against 0.05 ionic strength veronal, the antigen was heated to 45 C. and mixed with an equal volume of melted (45 C.) 4% agar, also in veronal buffer. The mixture was then pipetted into the antigen wells of the agar slab and allowed to solidify. Agar slabs meas- uring approximately 20 X 6.05 cm. with antigen wells of 0.3-0.4 ml. capacity were used in these experiments. To achieve electrophoretic migration the preparations were subjected to a current of 25 ma for about six hours. To improve the resolution of precipitin bands the agar blocks were fixed in 2% acetic acid, stained with Amidoschwarz (0.1% in acetate buffer, pH 4.0 solu- tion) and destained in methanol-water-acetic acid (45:45: 10). RESULTS Agar diffusion precipitin tests were performed on extracts prepared by freeze- thawing washed Arbacid sperm and subsequently centrifuging the extracts in a clinical centrifuge (approximately 3000 X g) . When diffused against anti-whole sperm serum such extracts yielded a maximum of four precipitin bands. Proceed- ing from the antigen to the antibody well in the agar plate, these four bands are designated a, b, c, and d. As seen in Figure 1, some variation in band number was found in repeated tests (e.g. two bands in Figures Ia5, Ib2, Ib3; three bands in Ib4, Ic6; four bands in Icl, Id3). Antisera other than #33 gave one to two bands. These differences in tests with different "frozen-thawed extracts" using serum #33 are attributed to differences in antigen concentration. However, the possibility of qualitative differences has not been eliminated. Differences in the band number using other antisera may reflect differences in concentration of anti- body as well as antigen in the tests. In view of the fact that the sperm were washed sufficiently before extraction to remove seminal plasma antigens, the pre- 204 CHARLES B. METZ AND KURT KOHLEK cipitin bands in the extracts must have arisen from antigens extracted from the sperm cells. In this connection it is of interest that undiluted seminal plasma forms three hands when diffused against anti-whole sperm serum (Fig. Ia4, Ibl). Two of these seminal plasma hands join but do not cross two of the "frozen-thawed extract" bands. It appears, then, that the seminal plasma shares at least two antigens with the extract. In fact these seminal plasma antigens may have diffused from the FIGURE 1. Ouchterlony agar diffusion tests. The center wells of all plates contain anti- Arbacia sperm serum No. 33. All surrounding wells were filled with extract and fluid of Ar- bacia punctulata. Each well received 0.5 ml. of the sample : ( a ) ( 1 ) supernatant over frozen- thawed sperm after centrifugation at 26,000 X g for 20 minutes; (2) residue of No. (1), resuspended in sea water and centrifuged at low speed; (3) supernatant over Mickle-clisinte- grated sperm; (4) seminal plasma; (5) "frozen-thawed extract" of sperm, low speed centri- fuged; (6) body fluid; (b) (1) seminal plasma; (2) "frozen-thawed extract" of whole sperm, low speed centrifugation; (3) "frozen-thawed extract" of whole sperm, low speed centrifuga- tion; (4) "frozen-thawed extract," low speed centrifugation; (5) supernatant over Mickle- disintegrated sperm, low speed centrifugation; (6) supernatant over Mickle-disintegrated sperm, low speed centrifugation; (c) (1) "frozen-thawed extract," low speed centrifugation; (2) basic protein, pH 0.9 extract; (3) supernatant over washed sperm after standing (aging), low speed centrifugation ; (4) supernatant over citric acid-extracted sperm, low speed centrifu- gation ; (5) supernatant over urea-treated sperm; (6) frozen-thawed extract, low speed cen- trifugation; (d) (1) isolated heads, "frozen-thawed extract," low speed centrifugation; (2) isolated tails, "frozen-thawed extract," low speed centrifugation; (3) whole sperm, "frozen- thawed extract," low speed centrifugation ; (4) isolated heads, "frozen-thawed extract," low speed centrifugation; (5) acid extract (pH 3) of sperm, low speed centrifugation; (6) acid extract (pH 1.9) of sperm, low speed centrifugation. sperm. However, the one band that has been clearly demonstrated in the super- natant of aging sperm is band a of "frozen-thawed extract" (Fig. Ic3). It should be noted that the three bands just described do not constitute the complete antigenic spectrum of Arbacia seminal plasma. As seen in Figure 2cl, immunoelectrophoresis resolved seven bands in this material. Three of these correspond in position to three precipitin bands in the sperm extract (Fig. 2c ) . It should be clear from these results that extracts prepared by freeze-thawing ANTIGENS OF ARBACIA SPERM EXTRACTS 205 sperm are not solutions of a single macromolecule. On the assumption that each precipitin hand represents a single antigen, the extracts can contai