THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, University of Texas FRANK A. BROWN, JR., Northwestern University JOHN B. BUCK, National Institutes of Health T. H. BULLOCK, University of California, Los Angeles E. G. BUTLER, Princeton University J. H. LOCHHEAD, University of Vermont ARTHUR W. POLLISTER, Columbia University C. L. PROSSER, University of Illinois MARY E. RAWLES, Carnegie Institution of Washington WM. RANDOLPH TAYLOR, University of Michigan A. R. WHITING, University of Pennsylvania CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 115 AUGUST TO DECEMBER, 1958 Printed and Issued by LANCASTER PRESS, Inc. PRINCE 8C LEMON STS. LANCASTER, PA. 11 THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers $2.50. Subscription per volume (three issues), $6.00. Communications relative to manuscripts should be sent to the Managing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 1 and September 1, and to Dr. Donald P. Costello, P.O. Box 429, Chapel Hill, North Carolina, during the remainder of the year. Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., under the Act of August 24, 1912. LANCASTER PRESS, INC., LANCASTER, PA. CONTENTS No. 1. AUGUST, 1958 PAGE Annual Report of the Marine Biological Laboratory 1 BOOLOOTIAN, RICHARD A., AND ARTHUR C. GIESE Coelomic corpuscles of echinoderms 53 BOROUGHS, HOWARD, AND DELLA F. REID The role of the blood in the transportation of strontium 90 -yttrium 90 in teleost fish 64 BROOKBANK, JOHN W. Dispersal of the gelatinous coat material of Mellita quinquiesperforata eggs by homologous sperm and sperm extracts 74 BROWN, FRANK A., JR. An exogenous reference-clock for persistent, temperature-independent, labile, biological rhythms 81 EPPLEY, RICHARD W., AND CARLTON R. BOVELL Sulfuric acid in Desmarestia 101 GRIFFIN, D. R., A. NOVICK AND M. KORNFIELD The sensitivity of echolocation in the fruit bat, Rousettus 107 HODGSON, EDWARD S. Electrophysiological studies of arthropod chemoreception. 111. Chemo- receptors of terrestrial and fresh-water arthropods 114 MATHEWSON, ROBERT, ALEXANDER MAURO, ERNEST AMATNIEK AND HARRY GRUNDFEST Morphology of main and accessory electric organs of Narcine brasiliensis (Olfers) and some correlations with their electrophysiological properties . . 126 ROTHSCHILD, LORD, AND ALBERT TYLER The oxidative metabolism of eggs of Urechis caupo 136 \YATKINS, MARGARET J. Regeneration of buds in Botryllus 147 No. 2. OCTOBER, 1958 BLACK, ROBERT E., SAMUEL EPSTEIN AND ALBERT TYLER The oxidation of carbon monoxide by fertilized eggs of Urechis caupo shown by use of a C 13 label 153 FANGE, R., K. SCHMIDT-NIELSEN AND H. OSAKI The salt gland of the herring gull 162 FANGE, RAGNAR, AND JONATHAN B. WITTENBERG The swimbladder of the toadfish (Opsanus tau L.) 172 in iv CONTENTS FLEMISTER, LAUNCE J. Salt and water anatomy, constancy and regulation in related crabs from marine and terrestrial habitats. . . 180 FLICKINGER, REED A. Regional localization of neural and lens antigens in the frog embryo in relation to induction 201 HOYLE, GRAHAM Studies on neuromuscular transmission in Limulus . . 209 LANE, CHARLES E., AND ELEANOR DODGE The toxicity of Physalia nematocysts 219 MANWELL, CLYDE On the evolution of hemoglobin. Respiratory properties of the hemo- globin of the California hagfish, Polistotrema stouti 227 MUN, ALTON M. Toxic effects of normal sera and homologous antisera on the chick embryo 239 RYTHER, J. H., C. S. YENTSCH, E. M. HULBURT AND R. F. VACCARO The dynamics of a diatom bloom 257 SCHERBAUM, OTTO H., ALLAN L. LOUDERBACK AND THEODORE L. JAHN The formation of subnuclear aggregates in normal and synchronized protozoan cells 269 STUNKARD, HORACE W., AND JOSEPH R. UZMANN Studies on digenetic trematodes of the genera Gymnophallus and Parvatrema 276 \YKBB, H. MARGUERITE, AND FRANK A. BROWN, JR. The repetition of pattern in the respiration ol Uca pugnax 303 Abstracts of papers presented at the Marine Biological Laboratory : Tuesday Evening Seminars 319 Electrobiology Seminars 329 General Meetings 332 Lalor Fellowship Reports 371 No. 3. DECEMBER, 1958 ANDERSON, JOHN MAXWELL, AND JEANNE CAROL JOHANN Some aspects of reproductive biology in the tresh-water triclad turbel- larian, Cura foremanii 375 AUCLAIR, WALTER, AND DOUGLAS MARSLAND Form-stability of ciliates in relation to pressure and temperature 384 DAVENPORT, DEMOREST, AND KENNETH S. NORRIS Observations on the symbiosis of the sea anemone Stoichactis and the pomacentrid fish, Amphiprion percula 397 DENT, JAMES NORMAN, AND W. GARDNER LYNN A comparison of the effects of goitrogens on thyroid activity in Triturus viridescens and Desmognathus fuscus 411 Fox, WADE, AND HERBERT C. DESSAUER Responses of the male reproductive system of lizards (Anolis carolinen- sis) to unnatural day-lengths in different seasons 421 CONTENTS V HASTINGS, J. WOODLAND, AND BEATRICE M. SWEENEY A persistent diurnal rhythm of luminescence in Gonyaulax polyedra .... 440 HEILBRUNN, L. V., FRANCIS T. ASHTON, CARL FELDHERR AND WALTER L. WILSON The action of insulin on cells and protoplasm 459 HILL, ROBERT B. The effects of certain neurohumors and of other drugs on the ventricle and radula protractor of Busycon canaliculatum and on the ventricle of Strombus gigas 471 HUTCHISON, VICTOR H., AND CARL S. HAMMEN Oxygen utilization in the symbiosis of embryos of the salamander, Ambystoma maculatum and the alga, Oophila amblystomatis 483 ROYS, CHESTER C. A comparison between taste receptors and other nerve tissues of the cockroach in their responses to gustatory stimuli 490 SCHARRER, BERTA, AND MARIANNE VON HARNACK Histophysiological studies on the corpus allatum of Leucophaea maderae. I. Normal life cycle in male and female adults 508 VON HARNACK, MARIANNE Histophysiological studies on the corpus allatum of Leucophaea maderae. II. The effect of starvation 521 SCHNEIDERMAN, HOWARD A., AND LAWRENCE I. GILBERT Substances with juvenile hormone activity in Crustacea and other invertebrates 5.30 TERZIAN, LEVON A., AND NATHAN STABLER A study of some effects of gamma radiation on the adults and eggs of Aedes aegypti -. 536 WELSH, JOHN H., AND PEGGY B. PROCK Quaternary ammonium bases in the coelenterates 551 Vol. 115, No. 1 August, 1958 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE MARINE BIOLOGICAL LABORATORY SIXTIETH REPORT, FOR THE YEAR 1957 SEVENTIETH YEAR I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 10, 1957) .... 1 STANDING COMMITTEES II. ACT OF INCORPORATION 3 III. BY-LAWS OF THE CORPORATION 4 IV. REPORT OF THE DIRECTOR 6 Statement 7 Memorials 8 Addenda : 1. The Staff 12 2. Investigators, Lalor and Lillie Fellows, and Students 15 3. Fellowships and Scholarships 24 4. Tabular View of Attendance, 1953-1957 24 5. Institutions Represented 25 6. Evening Lectures 26 7. Shorter Scientific Papers (Seminars) 26 8. Members of the Corporation 28 V. Report of the LIBRARIAN 46 VI. REPORT OF THE TREASURER 47 I. TRUSTEES EX OFFICIO GERARD SVVOPE, JR., President of the Corporation, 570 Lexington Ave., New York City A. K. PARPART, Vice President of the Corporation, Princeton University PHILIP B. ARMSTRONG, Director, State University of New York, Medical Center at Syracuse C. LLOYD CLAFF, Clerk of the Corporation, Randolph, Mass. JAMES H. WICKERSHAM, Treasurer, 530 Fifth Ave., New York City EMERITI EUGENE DuBois, Cornell University Medical College G. H. A. CLOWES, Lilly Research Laboratory W. C. CURTIS, University of Missouri 1 2 MARINE BIOLOGICAL LABORATORY PAUL S. GALTSOFF, Woods Hole, Mass. Ross G. HARRISON, Yale University E. B. HARVEY, 48 Cleveland Lane, Princeton, N. J. M. H. JACOBS, University of Pennsylvania School of Medicine F. P. KNOWLTON, Syracuse University W. J. V. OSTERHOUT, Rockefeller Institute CHARLES PACKARD, Woods Hole, Mass. LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y. TO SERVE UNTIL 1961 D. W. BRONK, Rockefeller Institute G. FAILLA, Columbia University, College of Physicians & Surgeons E. NEWTON HARVEY, Princeton University 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 L. V. HEILBRUNN, University of Pennsylvania S. W. KUFFLER, Johns Hopkins Hospital C. B. METZ, Florida State University G. T. SCOTT, Oberlin College A. H. STURTEVANT, California Institute of Technology E. ZWILLING, University of Connecticut TO SERVE UNTIL 1959 E. G. BUTLER, Princeton University C. LALOR BURDICK, The Lalor Foundation, Wilmington, Delaware D. P. COSTELLO, University of North Carolina H. HIBBARD, Oberlin College M. KRAHL, University of Chicago D. MARSLAND, New York University, Washington Square College R. RUGH, Columbia University, College of Physicians and Surgeons H. B. STEINBACH, University of Minnesota TO SERVE UNTIL 1958 W. R. AMBERSON, University of Maryland, School of Medicine T. H. BULLOCK, University of California, Los Angeles AURIN CHASE, Princeton University ALBERT I. LANSING, Emory University DANIEL MAZIA. University of California S. MERYL ROSE, University of Illinois MARY SEARS, Woods Hole Oceanographic Institution ALBERT TYLER, California Institute of Technology TRUSTEES EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES GERARD SVVOPE, JR., Chairman E. G. BUTLER A. K. PARPART RUDOLPH KEMPTON J. H. WlCKERSHAM D. P. COSTELLO P. B. ARMSTRONG H. B. STEINBACH K. S. COLE EDGAR ZWILLING THE LIBRARY COMMITTEE MARY SEARS, Chairman E. G. BUTLER HAROLD F. BLUM J. P. TRINKAUS E. T. MOUL RALPH CHENEY THE APPARATUS COMMITTEE C. LLOYD CLAFF, Chairman ALBERT I. LANSING M. V. EDDS THE SUPPLY DEPARTMENT COMMITTEE RUDOLPH KEMPTON, Chairman ROBERT DAY ALLEN C. B. METZ L. V. HEILBRUXN THE EVENING LECTURE COMMITTEE P. B. ARMSTRONG, Chairman L. V. HEILBRUNN E. G. BALL W. D. MCELROY THE INSTRUCTION COMMITTEE S. MERYL ROSE, Chairman C. L. PROSSER L. H. KLEINHOLZ I. M. KLOTZ THE BUILDINGS AND GROUNDS COMMITTEE EDGAR ZWILLING, Chairman C. B. METZ RALPH WICHTERMAN SEARS CROWELL THE RADIATION COMMITTEE G. FAILLA, Chairman ROBERTS RUGH CLAUDE VILLEE MONES BERMAN WALTER L. WILSON ROGER L. GREIF 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 4 MARINE BIOLOGICAL LABORATORY 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; Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- setts, do liereb\ 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. 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 bv anv seven Trustees, to be held at BY-LAWS OF THE CORPORATION 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 selected for a term of five years and shall serve until his successor is selected and qualified; they shall appoint a Director of the Laboratory; and they may choose such other officers and agents as they may think best ; they may fix the compensation and define the duties of all the officers and agents ; and may remove them, or any of them, except those chosen by the members, at any time ; they may fill vacancies occurring in any manner in their own number or in any of the offices. The Board of Trustees shall have the power to choose an Executive Committee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient. They shall from time to time elect members to the Corporation upon such terms and conditions as they may think best. X. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. 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. MARINE BIOLOGICAL LABORATORY 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 seventieth 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. Crane Building The Officers of the Laboratory held several conferences during the winter (1957) with representatives of our architectural firm, Coolidge, Shepley, Richardson and Abbott, to develop plans for the rehabilitation of the Crane Building under the National Science Foundation Grant of $415,000. Planning was completed early in the summer ; the contracts and subcontracts were let in August. A de- tailed schedule of the operation was developed by the general contractor, the building was evacuated by the Laboratory immediately after Labor Day and the remodeling started. The schedule called for the completion of the job by May 1, 1958. Present indications are that the work will be completed on time, and that the equipment can be moved in for occupancy by the investigators not later than June 1. Out of this remodeling the Laboratory will have an essentially new building with facilities for any type of research in biology and the cognate sciences. The rearrangement of the standard facilities within the individual laboratories will result in a much more efficient use of available space. 2. New Research Laboratory In 1938 it was recommended by an ad hoc committee set up to formulate policy on the future development of the Marine Biological Laboratory that the wooden buildings should be replaced by a modern brick laboratory building. At the Annual Meeting of the Board of Trustees, August 16, 1957, the Officers of the Laboratory were authorized to seek funds to implement this recommendation. Applications were made to the Rockefeller Foundation, the National Science Foundation and the Public Health Service for the necessary funds. Early in December the Laboratory was notified of a grant from the Rockefeller Foundation of $738,500, providing one-half the necessary funds. Subsequently, grants were obtained from the Na- tional Science Foundation and the Public Health Service which shared equally in providing the other half of the cost of the new building and its equipment. Plan- ning for the new building is already under way, construction to start in the fall of 1959 with occupancy planned for the spring of 1961. It will be a three-story and basement building, almost entirely devoted to research and research service labora- tories. REPORT OF THE DIRECTOR 7 3. Housing The 1938 ad hoc Committee on the Development of the Laboratory also ex- pressed its concern with the problems of housing and adequate care of the large number of persons attracted to the community by reason of their Laboratory activi- ties. It was pointed out that the housing needs have, from the beginning, been recognized as one of the primary responsibilities of the Laboratory and that the arrangements then existing were not adequate. Since that report, three frame houses in the immediate vicinity of the Laboratory have been acquired and con- verted to dormitory use. Since World War II there has developed an acute short- age of housing for younger married investigators with children. Toward the end of the year (1957) the Laboratory made application to the National Science Foundation for a grant of $175,000 to erect 25 housekeeping cottages on the Laboratory's Devils Lane Property. Favorable action was taken by the National Science Foundation on this grant request. Plans have been developed for these cottages which will be erected for 1959 occupancy. Also, the Board of Trustees voted to discontinue any further sale of lots from the Devils Lane Property so that the Laboratory will retain title to the remaining 75 acres for future Laboratory use, either housing or scientific. 4. Grants, Contracts and Contributions The total income to the Laboratory from these sources of support amounted to $210,000 in 1957. This represents 32% of the total income and consists of the following accounts : American Cancer Soc. R-7G Fundamental Studies in Radiobiology $ 6,600.00 A.E.C. 1343 Program of Research on the Physiology of Marine Organisms Using Radioisotopes 9,545.00 N.I.H. 4359 Biological Research on the Morphology, Ecology, Physiology, Biochemistry and Biophysics of Marine Organisms . . 40,000.00 N.I.H' 5143 Training Program in Nerve-Muscle Physiology 40,342.00 National Science Found. G2142 Funds for Biological Research . . . 25,000.00 National Science Found. G3608 Optical Equipment 11,500.00 National Science Found. G3987 Centrifuge Equipment 10,000.00 O.N.R. 1497 Studies in Marine Biology 15,000.00 O.N.R. 09701 Studies on Isolated Nerve Fibers 7,670.00 O.N.R. 09702 Studies in Ecology 5,268.00 M.B.L. Associates 3,481.00 Abbott Laboratories 1,000.00 American Philosophical Society 2,500.00 Ciba Pharmaceutical Products, Inc 1,000.00 Eli Lilly Company 5,000.00 Merck and Company, Inc 1,000.00 Rockefeller Foundation 20,000.00 Sobering Corporation 1,000.00 Smith, Kline, and French Foundation 3,000.00 The Upjohn Company 1,000.00 $209,906.00 8 MARINE BIOLOGICAL LABORATORY 5. Boats Late in the year the Laboratory contracted with the Riverside Boat Company of Newcastle, Maine, for two 24-foot boats for trap work and inshore collecting. These boats will replace the old Sogitta and Tern, both of which, after years of service, outlived their usefulness. The new boats are to be delivered in May (1958). Respectfully submitted, PHILIP B. ARMSTRONG, Director MEMORIAL BENJAMIN M. DUGGAR by Win. Randolph Taylor Benjamin Minge Duggar, late Emeritus Trustee of this Laboratory, died 10 Sept. 1956 in New Haven, Conn. Dr. Duggar was born in Gallion, Alabama in 1872. His early education in private schools was followed by studies in civil engineering at the University of Alabama and the Mississippi Agricultural and Mechanical College, where his interest shifted to botany, and be received his B.S. in 1891. He then completed his work for the M.S. at Alabama Polytechnic, but, going to Harvard University, qualified there for the A.B. and A.M. degrees, transferring to Cornell University where he received the doctorate in 1898, completing his formal studies. He subsequently worked in several laboratories in Europe, further widening his experience. His government and academic appointments were numerous, but four institutions claimed his services as full Professor before retirement: first the University of Missouri, then Cornell, and then for much longer periods Washington University and the University of Wisconsin. His distinguished academic career was marked by a happy combination of physiology and pathology, in both of which fields he contributed notably in research and produced textbooks of exceptional merit, that in plant pathology remaining useful to this day. He contributed his share, also, to one of the most successful American elementary botanical text-books ever produced, that prepared by the Wisconsin group and still in use. During his period at Wisconsin the Department of Botany strengthened its position as one of the most notable in the country. His researches covered a considerable range of endeavor, but those on virus diseases, particularly the mosaic disease of tobacco, are most often remembered. Dr. Duggar does not seem first to have appeared at the Marine Biological Laboratory as a student or as an investigator, as is commonly the case. In 1909 he was appointed to what was termed the "Research Staff" "in botany, while Professor of Physiology at Cornell. In 1911 the course in botany was divided, the first three weeks dealing with the algae, the second with "The Physiology and Ecology of Marine, Strand and Bog Vege- tation" with Lewis Knudsen, also from Cornell, as his associate. Knudsen was replaced in 1912 by W. ]. Robbins, best known as the Director of the New York Botanical Gar- den, but the course was dropped in 1915. "Investigation Staff" replaced the old term for the advisory group, and Dr. Duggar served Botany on this board from 1926 to 1941. He was elected to the Corporation in 1911 and to the Trustees of the Laboratory in 1928, retiring Emeritus in 1944. During all these years he was frequently in residence through the summer, and always helpful to those at the Laboratory whose enquiries fell within his field of interest. REPORT OF THE DIRECTOR The discernment shown by Dr. Duggar respecting the affairs of the Marine Biological Laboratory was appreciated by other institutions, and he served as Trustee of the Bermuda Biological Station 1933-1937, and of the Woods Hole Oceanographic Institute from its inception in 1931 until 1938. Honorary degrees were bestowed on him by Washington University, the University of Missouri and the University of Wisconsin; he was elected to the most distinguished of our learned and professional societies. On retirement from Wisconsin Dr. Duggar promptly joined the research staff of the Lederle Laboratories of the American Cyanamid Company, and settled down to research on the discovery, production and evaluation of antibiotics from various Actinomycete bacteria. All reports from the company describe with admiration his quiet industry and the keen mind he placed most generously at the disposal of his fellow workers. His most spectacular success was in the discovery in 1945 of Aureomycin, a very effective anti- biotic, which has gone into extensive commercial production. He continued active in research until his final illness. Dr. Duggar lost his first wife in 1922; his second wife, several children and grand- children survive him. To them we wish to express our appreciation of his many con- tributions to science and our sympathy in the loss they have suffered. Mr. President, I move that a copy of this memorial be placed in the minutes of this meeting, and that a copy be sent to Mrs. Duggar. MEMORIAL E. S. G. BARRON by H. B. Steinbach E. S. G. Barren, "Achito" to many, died this summer and is buried in the cemetery at his beloved Woods Hole. His scientific studies achieved world-wide recognition as did the charming personality of the man responsible for them. While Barron was truly a scientist of the world, his ties to the Marine Biological Laboratory were strong and his affection for the area was great. He was elected a member of the Corporation in 1933, a trustee in 1949 and again in 1952. He served as instructor in the Physiology Course from 1945 until 1948 when he assumed the headship for a five-year period. Under his guidance the course continued its strong development and became especially well known on the international scene. He was largely instru- mental in obtaining much of the special equipment that is now in use. He conducted the special session of the course in honor of his revered teacher, Leonor Michaelis, and edited the volume "Modern Trends in Physiology and Biochemistry" which carried the fame of the MBL even farther than before. For the past several years Barron found it necessary to give up his attendance here to carry out a labor of love dear to his heart, spending his summers in teaching and con- sulting in South America as his contribution to the advancement of science in those areas, especially the country of his birth, Peru. While he was perhaps best known for his studies on oxidative mechanisms, Achito's interests and activities were very broad indeed, ranging from a classical work on bilirubinemia to the effects of ionizing radiations on crystalline proteins. However he was preeminently a biologist and, in his mind, all his studies were fundamentally directed at understanding cellular oxidations and their regulation. Shortly before his death, his plans for future work were keyed largely to a comprehensive comparative study of cellu- lar oxidations with the hope of finding critical keys to physiological regulations. Barron was born in Huari, Peru, in 1898. Following two years in France he came 10 MARINE BIOLOGICAL LABORATORY to this country in 1927, first as a Rockefeller Fellow and then as instructor in Johns Hopkins University. In 1930 he moved to the University of Chicago, his University until his death. During World War II, he did scientific work for both the AEC and the Medical Division of the Chemical Warfare Service. He was especially well fitted to carry out the important studies on effects of ionizing radiations and the biological actions of nitrogen mustards and related compounds. Achito was a remarkable teacher even though his position at Chicago did not involve conducting formal classes. He had a keen and incisive sense of humor and a fine critical attitude towards science. Many have benefitted from his wisdom and have been delighted with his conversation. He had a strong sense of the necessity for training minds in intellectual pursuits. This led him to his fruitful efforts in the Woods Hole course and in the training programs in South America. When he purchased his new home in Chi- cago some years ago, his greatest delight was that he had a large pleasant room with a big blackboard. Here he could invite his students and colleagues for seminars and dis- cussions and here many of the ideas for experiments by himself and collaborators were born. Achito, his wife Cora, and his son Richard constituted a family it was a pleasure and privilege to know. And while we are expressing our gratitude to Achito for his many contributions to us, we must include his wife and son for providing the setting for such a fruitful career. As an experimentalist, as a teacher, as one who travelled widely and spread the tradi- tions of science and inspired the young. Barren was at the height of his activity when he became ill and died. At such a time it is not trite to say that a man's death is untimely. MEMORIAL ROBERT CHAMBERS by B. W. Zweifach and G. H. A. Clowes With the death of Robert Chambers at the age of 75 on July 22nd, the Marine Bio- logical Laboratory lost one of the most illustrious members of its old guard marking as it were the passing of an era in which microscopy as a fine art was utilized to its fullest extent for the study of cellular behavior and protoplasmic structure. Chambers' associa- tions here in Woods Hole were long and deep-rooted. He first came to the M. B. L. in the summer of 1911 as a graduate investigator and in 1912 was on the teaching staff in Zoology and Embryology a course in those days associated with such names as Calkins, Lillie, Conklin, Morgan and Wilson. By 1914 it became apparent that Chambers' interests were not along the lines of conventional zoology and he was thereafter listed in the annual reports of the M. B. L. as an investigator in Physiology an indication that the science of cellular physiology had come of age. By training Robert Chambers was a histologist and embryologist. He was born and raised in Turkey, where his parents resided as missionaries. The rough, harsh life dur- ing his formative years left an indelible imprint on his makeup and was to a considerable extent responsible for his great compassion for the underdog and his willingness to champion humanitarian causes. It was at Roberts College that his interest in nature was crystallized and his future course indicated. Later, under the aegis of Hertwig and Gold- schmidt in Munich, where he received his Ph.D in 1908, Chambers was indoctrinated into the field of histophysiology and developed a keen interest in basic embryology. He returned to Canada, the early home of the Chambers family, and eventually joined Cornell Medical College in 1915. These were his most fruitful years his outstanding contribu- tions in large part derived from his ingenious researches at Woods Hole. His laboratorv REPORT OF THE DIRECTOR H here in Room 328, in association with the Eli Lilly group, was a beehive of activity where Chambers' dynamic personality infused all who worked with him. Few could keep pace with his amazing physical stamina and drive. At an early age Robert Chambers became virtually a legendary figure, not only because of his scientific stature but because of the anecdotes which grew up around his prodigious unconcern for practical matters. There are many here who knew him during these inspiring years as a most attractive and con- genial personality. Woods Hole was the center of the social and scientific life of Robert and Bertha Chambers. They practically raised their four sons at Bobtuckett Cottage and many of the delightful and entertaining experiences of the Chambers family have attained the stature of local folklore. Robert might be found at almost any time, day or night, in his M. B. L. laboratory and the Chambers family regarded the remainder of the year as an unavoidable intrusion into the Woods Hole continuum. In 1928 he transferred from the Anatomy Department at Cornell to the Department of Biology at New York University, where he maintained until his retirement in 1949 a research center which attracted students from every country of Europe, from Asia, and from South America, many of whom are today outstanding figures in scientific research. The magnitude of Robert Chambers' contribution becomes all the more impressive when it is considered that he published over 230 scientific articles, bearing in mind the fact that writing was extremely burdensome for Chambers. A great deal of what he did, he left for others to put into words. He unflaggingly, to the point of self-denial, gave his time and counsel to a never-ending stream of students, associates, cronies and visitors. Time was a meaningless entity to him. In 1912, at the M. B. L. seminar sessions, Chambers was greatly stimulated by a lecture in which G. L. Kite showed that it was possible to interfere with the develop- ment of marine ova with glass rnicrotools. In retrospect, we can see that this event proved to be the turning point in his scientific career. The potentialities of this approach appealed so much to Chambers that he developed and applied the microsurgical technique extensively, his name becoming synonymous with the micromanipulative method. In his early work, principally at Woods Hole, he clearly showed the importance of sol-gel transformations in relation to aster formation and cell division. There followed the beautiful demonstrations, accompanied by motion pictures, of the capacity of the cyto- plasm and cell surface to recover from various forms of microinjury in the presence of the proper ionic environment. He made the earliest measurements of the pH of the cytoplasm in intact cells, using indicator dyes. His enthusiasm was such that every aspect of cellular behavior intrigued him, the cohesion of blastomeres in developing embryos, the action of salts on protoplasm, the nature of vital staining, the interfacial tension at protoplasmic surfaces, the acid of injury, etc. Later, Chambers combined tissue culture with microtechniques. Especially note- worthy were his studies on malignant cells, the secretory activity of kidney tubules and chemotactic phenomena. During World War II, he devoted a goodly part of his energies to studies on capillary permeability and to the vascular sequelae of experimental shock. New and important concepts of circulatory homeostasis were originated. Numerous honors were bestowed upon Chambers. As early as 1926 he gave his first Harvey lecture on the living cell. During this period he received the Traill medal from the Linnean Society of London, the John Scott medal from the City of Philadelphia, the medal of L' Academic Nationale de Medecine of Paris, was made a Fellow of the Royal Microscopical Society of England, and was given an honorary LL.D. from Queens Uni- versity. He was active in the affairs of many societies, having been a Trustee of the Marine Biological Laboratory, a member of the Board of Directors of the Long Island Biological Association, President of the American Society of Zoologists, the Harvey Society, the Union of American Biological Sciences, and vice-president of the American Association of Anatomists. 12 MARINE BIOLOGICAL LABORATORY When one attempts to give an account of a man's life in a few hundred words, the impossibility of the task becomes increasingly apparent. In the case of Robert Chambers, his human qualities transcended even his outstanding scientific achievements. His later years were saddened by the loss of his oldest son, Robert, in World War II, and by the protracted illness and death of his wife Bertha. It would be a mere platitude to say that we shall miss him, but we hope that the imprint of his indomitable spirit will live on in those of us who were fortunate enough to know him and to contribute some small part to the fruits of his labor. 1. THE STAFF, 1957 PHILIP B. ARMSTRONG, Director, State University of New York, School of Medicine, Syracuse ZOOLOGY I. CONSULTANTS F. A. BROWN, JR., Professor of Zoology, Northwestern University LIBBIE H. HYMAN, American Museum of Natural History A. C. REDFIELD, Woods Hole Oceanographic Institution II. INSTRUCTORS THEODORE H. BULLOCK, Professor of Zoology. University of California, Los Angeles; in charge of course JOHN M. ANDERSON, Associate Professor of Zoology, Cornell University JOHN B. BUCK, Senior Biologist, National Institutes of Health CLARK P. READ, Associate Professor, School of Hygiene and Public Health, Johns Hop- kins University GROVER C. STEPHENS, Assistant Professor of Zoology, University of Minnesota MORRIS ROCKSTEIN, Associate Professor of Physiology, New York University College of Medicine CADET HAND, Assistant Professor of Zoology. University of California, Berkeley HOWARD A. SCHNEIDERMAN, Assistant Professor of Zoology, Cornell University III. LABORATORY ASSISTANTS ROBERT V. KIRCHEN, Columbia University PETER PICKENS, University of California EMBRYOLOGY I. INSTRUCTORS M. V. EDDS, JR., Professor of Biology, Brown University ; in charge of course N. T. SPRATT, JR., Professor of Zoology, University of Minnesota M. SUSSMAN, Associate Professor of Biological Sciences, Northwestern University J. P. TRINKAUS, Associate Professor of Zoology, Yale University P. B. WEISZ, Associate Professor of Zoology, Brown University E. ZWILLING, Program Director, National Science Foundation (on leave from University of Connecticut) II. LABORATORY ASSISTANTS R. G. BEARD, Carnegie Institution of Washington, Department of Embryology C. M. FULTON, Rockefeller Institute for Medical Research REPORT OF THE DIRECTOR 13 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, Woods Hole II. INSTRUCTORS W. D. MCELROY, Professor of Biology, Johns Hopkins University; in charge of course FRANCIS D. CARLSON, Assistant Professor of Biophysics, Johns Hopkins University BERNARD D. DAVIS, Professor of Pharmacology, New York University, College of Medi- cine DONALD GRIFFIN, Professor of Zoology, Harvard University HOWARD SCHACHMAN, Virus Laboratory, University of California, Berkeley ANDREW SZENT-GYORGYI, Institute for Muscle Research, Marine Biological Laboratory III. LABORATORY ASSISTANT ROGER THEIS, Rockefeller Institute BOTANY I. CONSULTANT WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan II. INSTRUCTORS HAROLD C. BOLD, Professor of Biology, Vanderbilt University ; in charge of course ROBERT \Y. KRAUSS, Associate Professor of Botany, University of Maryland RICHARD C. STARR, Associate Professor of Botany, Indiana University III. LECTURER RUTH PATRICK, Curator of Limnology, Academy of Natural Sciences of Philadelphia IV. COLLECTOR GINA ARCE, Vanderbilt University V. LABORATORY ASSISTANTS EUGENE Fox, Indiana University RAYMOND A. GALLOWAY, University of Maryland MARINE ECOLOGY I. CONSULTANTS PAUL GALTSOFF, U. S. Fish and Wildlife Service, Woods Hole ALFRED C. REDFIELD, Woods Hole Oceanographic Institution JOHN S. RANKIN, University of Connecticut 14 MARINE BIOLOGICAL LABORATORY II. INSTRUCTORS EUGENE P. ODUM, Professor of Zoology, University of Georgia ; in charge of course EDWIN T. MOUL, Associate Professor of Botany, Rutgers University JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution III. LABORATORY ASSISTANT JOANNE VAN DYK, University of New Hampshire THE LABORATORY STAFF, 1957 HOMER P. SMITH, General Manager MRS. DEBORAH LAWRENCE HARLOW, Librarian CARL O. SCHWEIDENBACH, Manager, Supply Department ROBERT KAHLER, Superintendent, Buildings and Grounds ROBERT B. MILLS. Manager, De- partment of Research Service GENERAL OFFICE IRVINE L. BROADBENT, Office Manager MRS. LILA S. MYERS NANCY WIGLEY GEORGIANA MARKS MARY A. ROHAN LIBRARY ALBERT K. NEAL NAOMI BOTELHO MAINTENANCE OF BUILDINGS AND GROUNDS ROBERT ADAMS EDMOND A. BOTELHO ARTHUR D. CALLAHAN ROBERT GUNNING JOHN H. HEAD DONALD B. LEHY RALPH H. LEWIS RUSSELL F. LEWIS ALTON J. PIERCE TAMES S. THAYER DEPARTMENT OF RESEARCH SERVICE GAIL M. CAVANAUGH JOHN P. HARLOW SEAVER R. HARLOW LUDIE A. JOHNSON SUPPLY DEPARTMENT DONALD P. BURN HAM MILTON B. GRAY GERALDINE E. KEELER ROBERT M. PERRY GEOFFREY J. LEHY ROBERT O. LEHY BRUNO TRAPASSO H. S. WAGSTAFF REPORT OF THE DIRECTOR 15 2. INVESTIGATORS, LALOR AND LILLIE FELLOWS, AND STUDENTS Independent Investigators, 1957 AIELLO, EDWARD, Assistant in Zoology, Columbia University ALLEN, M. JEAN, Associate Professor of Biology, Wilson College ANDERSON, JOHN MAXWELL, Associate Professor of Zoology, Cornell University ARMSTRONG, PHILIP B., Professor of Anatomy, State University of New York, College of Medicine, at Syracuse ARNOLD, WILLIAM A., Scientific Investigator, Oak Ridge National Laboratory BACON, DONALD F., Assistant in Department of Microbiology, Yale Medical School BANG, FREDERIK, Professor of Pathology, Johns Hopkins University School of Hygiene BARTON, JAY, II, Associate Professor of Biology, Collegeville, Indiana BENESCH, REINHOLD, Investigator, Marine Biological Laboratory BENNETT, MICHAEL, Research Worker, Columbia University, College of Physicians and Surgeons BENNETT, MIRIAM F., Instructor in Biology, Sweet Briar College BERG, WILLIAM E., Associate Professor of Zoology, University of California BERGER, CHARLES A., Chairman, Department of Biology, Fordham University BISHOP, NORMAN I., Research Associate, University of Chicago BRADY, ROSCOE, Section Chief, National Institutes of Health BRAAMS, RENIER, Research Associate, Yale University BRIDGMAN, JOSEPHINE, Professor of Biology, Agnes Scott College BROWN, FRANK A., JR., Professor of Biology, Northwestern University BRYANT, S. H., Professor of Pharmacology, University of Cincinnati, College of Medicine BUCKMANN, DETLEF, Zoologisches Institut, Saarstrabe 21, Mainz, Germany BULLOCK, THEODORE H., Professor of Zoology, University of California, Los Angeles BURGEN, ARNOLD, Professor of Physiology, McGill University CAMPBELL, MILDRED A., Instructor in Zoology, Smith College CARLSON, FRANCIS D., Associate Professor of Biophysics, Johns Hopkins University CASE, JAMES F., Assistant Professor of Zoology, Iowa State University CHAET, ALFRED B., Instructor in Physiology, Boston University School of Medicine CHANG, JOSEPH J., Member of Laboratory of Biophysics, National Institutes of Health CHASE, AURIN M., Associate Professor of Biology, Princeton University CHENEY, RALPH H., Professor of Biology; Director Physiology Division, Brooklyn College CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School CLARK, GORDON M., Research Associate, University of Michigan CLEMENT, A. C., Professor of Biology, Emory University CLOWES, G. H. A., Research Director Emeritus, Lilly Research Laboratories COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health COHEN, MELVIN J., Instructor in Biology, Harvard University COLLIER, JACK R., Instructor in Zoology, University of Vermont COLWIN, ARTHUR L., Associate Professor, Queens College COLWIN, LAURA HUNTER, Lecturer, Queens College CONNELLY, CLARENCE M., Associate, Rockefeller Institute 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 CROWELL, SEARS, Associate Professor of Zoology, Indiana University CSAPO, ARPAD L, Associate Professor, Rockefeller Institute for Medical Research GUSHING, JOHN ELDRIDGE, Associate Professor of Biological Sciences, University of California, Santa Barbara College DEVOE, ROBERT, Graduate Fellow, Rockefeller Institute for Medical Research DIETER, CLARENCE D., Head of Department of Biology, Washington-Jefferson College EDDS, MAC V., JR., Professor of Biology, Brown University EDWARDS, CHARLES, Professor of Physiological Optics, Johns Hopkins University ELLIOTT, ALFRED M., Professor of Zoology, University of Michigan 16 MARINE BIOLOGICAL LABORATORY ENGLE, RALPH L., JR., Assistant Professor of Medicine, Cornell University Medical College FAILLA, G., Professor, Columbia University FRYE, B. E., Postdoctoral Fellow, Princeton University GALL, JOSEPH G., Assistant Professor of Zoology, University of Minnesota GREEN, HOWARD, Assistant Professor of Chemical Pathology, New York University College of Medicine GREEN, MAURICE, Assistant Professor of Microbiology, St. Louis University School of Medicine GREENBERG, SYLVIA S., Damon Runyon Cancer Research Fellow, New York University GREGG, JAMES H., Associate Professor of Biology, University of Florida GREIF, ROGER L., Associate Professor of Physiology, Cornell University Medical College GROSCH, DANIEL S., Associate Professor of Genetics, N. C. State College GROSS, PAUL, Assistant Professor of Biology, New York University GRUNDFEST, HARRY, Associate Professor of Neurology, College of Physicians and Surgeons GUDERNATSCH, FREDERICK, 1300 York Avenue, New York 21 GUTTMAN, RITA, Assistant Professor of Biology, Brooklyn College HAND, CADET, Assistant Professor of Zoology, University of California, Berkeley HARVEY, ETHEL BROWNE, Research in Biology, Princeton University HARVEY, E. NEWTON, Professor Emeritus of Biology, Princeton University HAYASHI, TERU, Associate Professor of Zoology, Columbia University HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania HENLEY, CATHERINE, Research Associate, University of North Carolina HERVEY, JOHN P., Electronic Engineer, Rockefeller Institute for Medical Research HILL, ROBERT B., Instructor in Zoology, University of Maine HOBERMAN, HENRY D., Associate Professor of Biochemistry, Albert Einstein College of Medicine HOROWITZ, SAMUEL B., Research Fellow, Eastern Pennsylvania Psychiatric Institute HOYLE, GRAHAM, Glasgow University, Scotland HYDE, BEAL B., Assistant Professor of Plant Sciences, University of Oklahoma ISENBERG, IRVIN, Research Associate, Institute for Muscle Research JENNER, CHARLES E., Associate Professor of Zoology, University of North Carolina KALCKAR, BARBARA W., Biochemist, National Institutes of Health KEMP, NORMAN E., Assistant Professor of Zoology, University of Michigan KEMPTON, RUDOLF T., Professor of Zoology, Vassar College KLOTZ, IRVING M., Professor of Chemistry and Biology, Northwestern University KUFFLER, STEPHEN W., Professor of Ophthalmic Physiology and Biophysics, Johns Hopkins University LANSING, ALBERT I., Professor of Anatomy, University of Pittsburgh LAZAROW, ARNOLD, Professor and Head of Department of Anatomy, University of Minnesota LAWLER, H. CLAIR, Associate in Biochemistry, College of Physicians and Surgeons LAWRENCE, H. SHERWOOD, Associate Professor of Medicine, New York University College of Medicine LEIGHTON, JOSEPH, Assistant Professor of Pathology, University of Pittsburgh LEVY, MILTON, Professor and Chairman, Department of Biochemistry, New York University College of Dentistry LEWIN, RALPH A., National Institutes of Health LINDBERG, OLOV, Professor and Head of Wenner-Grens Institute, Sweden LITT, MORTIMER, Research Fellow in Bacteriology, Harvard Medical School LOCH HEAD, JOHN H., Professor of Zoology, University of Vermont LORAND, L., Assistant Professor of Chemistry, Northwestern University LOWENSTEIN, O. E., Professor of Zoology, University of Birmingham, England LUBIN, MARTIN, Associate in Pharmacology, Harvard Medical School MCELROY, W. D., Chairman, Biology Department, Johns Hopkins University MAAS, WERNER K., Assistant Professor of Pharmacology, New York University Medical College MARSHAK, ALFRED, Marine Biological Laboratory MARSLAND, DOUGLAS, Professor of Biology, New York University, Washington Square College MENKIN, VALY, Head of Experimental Pathology, Temple University School of Medicine METZ, CHARLES B., Associate Professor of Zoology, Florida State University METZ, CHARLES W., Professor of Zoology, University of Pennsylvania MIDDLEBROOK, W. ROBERT, Institute for Muscle Research REPORT OF THE DIRECTOR 17 MILLS, KENNETH S., Instructor of Biophysics, University of California Medical Center MOORE, JOHN W., Associate Chief, National Institutes of Health MULNARD, JACQUES G., Chef De Travau, University of Brussels, Belgium MULLINS, L. J., Associate Professor of Biophysics, Purdue University NACE, PAUL F., Associate Professor of Biology, Hamilton College, McMaster University, Ontario Niu, MAN-CHIANG, Associate, Rockefeller Institute for Medical Research ODUM, EUGENE P., Professor of Zoology, University of Georgia OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research PADAWER, JACQUES, Assistant Professor of Biochemistry, Albert Einstein College of Medicine PARPART, ARTHUR K., Professor and Chairman, Department of Biology, Princeton University PERSON, PHILIP, Chief, Dental Research, V. A. Hospital, Brooklyn PERT, JAMES H., Professor in Medicine, Cornell University Medical College PLOUGH, HAROLD H., Professor of Biology, Amherst College PROSSER, C. LADD, Professor of Physiology, University of Illinois READ, CLARK P., Associate Professor of Pathobiology, Johns Hopkins University REBHUN, LIONEL L, Instructor in Anatomy, University of Illinois College of Medicine RIESER, PETER, Research Associate, University of Pennsylvania ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of Medicine ROGERS, K. T., Assistant Professor of Zoology, Oberlin College ROSENBERG, EVELYN K., Assistant Professor of Pathology, New York University-Bellevue Medical Center ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College RUGH, ROBERTS, Associate Professor of Radiology, Columbia University SCHECHTER, VICTOR, Associate Professor of Biology, City College of New York SCHNEIDERMAN, HOWARD A., Associate Professor of Zoology, Cornell University SCHOFFENIELS, ERNEST, Research Associate, College of Physicians and Surgeons SCHUH, JOSEPH E., Professor and Chairman, Department of Biology, St. Peter's College SCHULMAN, MARTIN P., Assistant Professor of Biochemistry, State University of New York, College of Medicine at Syracuse SCOTT, DWIGHT B. McNAiR, Assistant Professor of Physiology, University of Pennsylvania Medical School SCOTT, SISTER FLORENCE MARIE, Professor and Chairman, Department of Biology, Seton Hill College SCOTT, GEORGE T., Professor of Zoology, Oberlin College SHANES, A. M., Physiologist, National Institutes of Health SHAW, EVELYN, Research Fellow, American Museum of Natural History SLIFER, ELEANOR H., Associate Professor of Zoology, State University of Iowa SMELSER, GEORGE K., Professor of Anatomy, College of Physicians and Surgeons SPEIDEL, CARL C., Professor and Chairman, Department of Anatomy, University of Virginia SPERELAKIS, NICK, Teaching Assistant, University of Illinois SPIEGEL, MELVIN, Assistant Professor of Biology, Colby College SPRATT, NELSON T., Professor of Zoology, University of Minnesota SPYROPOULOS, CONSTANTINE, National Institutes of Health STARR, RICHARD C., Assistant Professor of Botany, Indiana University STEELE, RICHARD H., Institute for Muscle Research STEINBACH, H. B., Professor of Zoology, University of Chicago STEPHENS, GROVER C., Assistant Professor of Zoology, University of Minnesota STEPHENSON, WILLIAM K., Assistant Professor of Biology, Earlham College STETTEN, DEWnr, Associate Director in Charge of Research, National Institutes of Health STOREY, ALMA G., Professor Emeritus, Mount Holyoke College ; STONE, WILLIAM, JR., Massachusetts Eye and Ear Infirmary STUNKARD, HORACE W., Research Biologist, U. S. Fish and Wildlife Service SUSSMAN, MAURICE, Associate Professor of Biological Sciences, Northwestern University SZENT-GYORGYI, ALBERT, Chief Investigator, Institute for Muscle Research SZENT-GYORGYI, ANDREW G., Investigator, Institute for Muscle Research TASAKI, TCHIJI, Chief, Special Senses Section, National Institutes of Health 18 MARINE BIOLOGICAL LABORATORY TAYLOR, WILLIAM RANDOLPH, Professor of Botany, University of Michigan TE\VINKEL, Lois E., Professor of Zoology, Smith College TRINKAUS, JOHN PHILIP, Associate Professor of Zoology, Yale University TROLL, WALTER, Assistant Professor, New York University College of Medicine TWAROG, BETTY MACK, Research Fellow, Harvard University TWEEDELL, KENYON S., Arsistant Professor of Zoology, University of Maine ULLBERG, SVEN G. F., Royal Veterinary College, Stockholm, Sweden DEViLLLAFRANCA, GEORGE W., Assistant Professor of Zoology, Smith College VILLEE, CLAUDE A., Associate Professor of Biological Chemistry, Harvard Medical School VINCENT, WALTER S., Instructor in Anatomy, State University of New York, Medical Center at Syracuse WAINIO, WALTER W., Associate Professor of Biochemistry, Rutgers University WEBB, H. MARGUERITE, Assistant Professor of Physiology, Goucher College WEIGLE, WILLIAM O., Research Associate, University of Pittsburgh School of Medicine WESTHEIMER, GERALD, Assistant Professor of Physiological Optics, Ohio State University WHITING, ANNA R., Lecturer in Zoology, University of Pennsylvania WHITING, P. W., Professor of Zoology Emeritus, University of Pennsylvania WICHTERMAN, RALPH, Professor of Biology, Temple University WILBER, CHARLES G., Chief, Comparative Physiology Branch, Army Chemical Center WILLEY, C. H., Chairman, Department of Biology, New York University WILSON, DONALD M., Teaching Assistant, University of California, Los Angeles WILSON, T. HASTINGS, Assistant Professor of Biological Chemistry, Washington University School of Medicine 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 WOODS, KENNETH R., Research Associate, Cornell University Medical College WRIGHT, PAUL A., Associate Professor of Zoology, University of Michigan ZWEIFACH, BENJAMIN W., Associate Professor of Pathology, New York University-Bellevue Medical Center ZWILLING, EDGAR, Associate Professor of Genetics, University of Connecticut Beginning Investigators, 1957 ALSUP, PEGGY, University of Pennsylvania BENSUSAN, HOWARD B., Western Reserve University BURKE, JOSEPH, S.J., Fordham University CAGLE, JULIEN, Princeton University CASCARANO, JOSEPH, University of Minnesota Medical School CERT, JEAN A., University of California CHANCE, ELEANOR K., University of Pennsylvania DINGLE, A. D., McMaster University GANGI, DOMINICK P., Upstate Medical Center, State University of New York HARDIMAN, CLARENCE W., Florida State University KANE, ROBERT E., Johns Hopkins University MASHIMA, HIDENOBER, Rockefeller Institute for Medical Research MASON, DAVID T., Reed College Moos, CARL, Northwestern University NAGLER, ARNOLD L., Bellevue Medical Center Ross, SAMUEL M., State University of New York, College of Medicine at Brooklyn RUGGIERI, GEORGE, St. Louis University SCHWARTZ, JAMES H., New York University College of Medicine SMITH, ROBERT G., Washington University Medical School STROHMAN, RICHARD C., Columbia University THEIS, ROGER E., Rockefeller Institute for Medical Research TURNER, BARBARA, Johns Hopkins University School of Medicine WESSELLS, NORMAN KEITH, Yale University REPORT OF THE DIRECTOR 19 Research Assistants, 1957 ALBERT, MORRIS, Boston University AMATNIEK, ERNEST, Columbia University AUCLAIR, WALTER, New York University, Washington Square College BARN HART, B. J., Indiana University BARNWELL, FRANKLIN H., Northwestern University BARROW, PATIENCE C, University of Toronto BENOIT, RICHARD, Massachusetts Eye and Ear Infirmary BLANCHARD, ROBERTA, Woods Hole, Mass. BRUCK, STEPHEN D., duPont de Nemours & Company GATHER, JAMES N., Emory University CLARK, WILLIAM R., JR., Boston University CORNER, M., Rockefeller Institute CROWLEY, ELIZABETH M., University of Pittsburgh DIBBELL, DAVID G., University of Pennsylvania DOUGLAS, STEVEN, Cornell University ERDMAN, HOWARD E., North Carolina State College FEINBERG, HARRIET ADELE, University of Pennsylvania FELDMAN, RICHARD, Rockefeller Institute for Medical Research Fox, J. EUGENE, Indiana University FRIEDMAN, LEONARD, Rutgers University GEBHART, JOHN H., National Institutes of Health GIFFORD, CAMERON E., Harvard University GIFFORD, CHARLES A., University of Minnesota GORDON, ROBERT, Massachusetts Institute of Technology GORKENANT, INGEBURG, Woods Hole, MaSS. GOUDSMIT, ESTHER M., University of Michigan GRINNELL, ALAN, Harvard University HIATT, HOWARD, Harvard Medical School INGLIS, LAURA H., Hahnemann Medical College JONES, HELEN, Massachusetts Eye and Ear Infirmary JOSEPHSON, ROBERT K., University of California KARAKASHIAN, STEPHEN J., Drew University KAUFMAN, SHARON L., Smith College KEREVYI, THOMAS, Harvard Medical School KERNAN, RODERICK P., Rockefeller Institute for Medical Research KIRCHEN, ROBERT V., Columbia University KOPMAN, AARON, Queens College KRASSNER, STUART, Johns Hopkins University LEVI, COLETTE P., Northwestern University LIEBERMAN, HARRY, New York Univcrsity-Bellevue Medical Center LORING, JANET, Harvard Medical School LUHRS, CARO, Harvard Medical School MATHESON, GAIL E., Yale University McCANN, FRANCIS, University of Connecticut METCALF, CARROLL, Colby College MORRISON, ELAINE, Massachusetts Eye and Ear Infirmary NASS, SYLVAN, New York University OLSON, JOANNE M., University of Minnesota PAULSEN, ELIZABETH, Rutgers University PLUMB, MARY ELLEN, Vassar College POLLOCK, BRIAN, Brooklyn V. A. Hospital REICH, MELVIN, Rutgers University RICHARDS, ELMER G., University of California ROBERTSON, MRS. C. W., United States Fish and Wildlife Service ROOT, RICHARD, University of Michigan ROOT, ELIZABETH, University of Michigan 20 MARINE BIOLOGICAL LABORATORY ROSENBLUTH, RAJA, Columbia University Ross, SHIRLEY EILEEN, Washington University ROSSILLO, LUDWIG A., St. Peter's College RUBINOFF, IRA, American Museum of Natural History SCHINSKE, ROBERT, University of Minnesota SCHELTEMA, AMELIE H., University of North Carolina SHAY, JONATHAN, Temple Medical School SHEPARD, DAVID, University of Chicago SIMMONS, JOHN E., Johns Hopkins University SMILEY, SHELDON, New York State University at Syracuse STADLER, JOAN, Swarthmore College STAUB, HERBERT W., Rutgers University TITUS, CHARLES C., Western Reserve University TREMOR, JOHN, University of Michigan WAITE, RICHARD E., University of Pennsylvania WARWICK, ANNE C., Johns Hopkins University WEISBLUM, BERNARD, State University of New York WELLINGTON, FREDERICA, Harvard Medical School WHITBECK, ELAINE, Smith College WYTTENBACH, CHARLES R., Carnegie Institute Library Readers, 1957 ALLFREY, VINCENT G., Associate, Rockefeller Institute for Medical Research AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland School of Medicine BALL, ERIC G., Chairman, Division of Medical Sciences, Harvard Medical School BERNHEIMER, ALAN W., Associate Professor of Microbiology, New York University College of Medicine BLOCK, ROBERT, Associate Editor, Biological Abstracts, University of Pennsylvania BODANSKY, OSCAR, Sloan-Kettering Institute BROOKS, MATILDA M., Research Associate in Physiology, University of California CHANUTIN, ALFRED, Professor of Biochemistry, Medical School, University of Virginia CLARK, ELIOT R., Professor Emeritus of Anatomy, University of Pennsylvania School of Medi- cine COHEN, SEYMOUR S., Professor of Biochemistry, Children's Hospital DEANE, HELEN WENDLER, Harvard Biological Laboratories DIXON, FRANK J., JR., Chairman, Department of Pathology, University of Pittsburgh School of Medicine DuBois, ARTHUR D., Associate Professor of Physiology, University of Pennsylvania School of Medicine EICHEL, HERBERT J., Hahnemann Medical College EISEN, HERMAN N., Professor of Medicine, Washington University GABRIEL, MORDECAI L., Associate Professor of Biology, Brooklyn College GAFFRON, HANS, Professor of Biochemistry, University of Chicago GOLDTHWAIT, DAVID A., Western Reserve University GREEN, JAMES W., Associate Professor of Physiology, Rutgers University JACOBS, M. H., Emeritus Professor of General Physiology, University of Pennsylvania School of Medicine KAAN, HELEN W., Indexer, National Research Council KARUSH, FRED, Associate Professor of Immunology, University of Pennsylvania LIONETTI, FABIAN J., Associate Professor of Biochemistry, Boston University School of Medicine LONDON, IRVING M., Professor and Chairman, Department of Medicine, Albert Einstein College of Medicine LOVE, Lois H., Research Associate, National Research Council MCDONALD, SISTER ELIZABETH, Chairman, Department of Biology, College of Mt. St. Joseph MOORE, GEORGE M., Professor and Chairman of Zoology, University of New Hampshire NOVIKOFF, ALEX B., Research Associate Professor of Pathology, Albert Einstein College of Medicine REPORT OF THE DIRECTOR 21 PICK, JOSEPH, Professor of Anatomy, New York University-Bellevue Medical Center ROOT, WALTER S., Professor of Physiology, College of Physicians and Surgeons ROSE, S. MERYL, Professor of Zoology, University of Illinois SCHLESINGER, R. WALTER, Director, Department of Microbiology, St. Louis University School of Medicine SCOTT, ALLAN, Professor of Biology and Chairman of Department, Colby College SHERMAN, FRANK E., Assistant Professor of Pathology, University of Pittsburgh STEINHARDT, JACINTO, Director, Operations Evaluation Group, Massachusetts Institute of Tech- nology SULKIN, S. EDWARD, Professor and Chairman, Department of Microbiology, University of Texas, Southwestern Medical School WAGNER, ROBERT R., Assistant Professor of Medicine, Johns Hopkins University School of Medicine WARNER, ROBERT C, Associate Professor of Biochemistry, New York University College of Medicine WHEELER, GEORGE E., Instructor of Biology, Brooklyn College WHITEHOUSE, MICHAEL W., Instructor of Biochemistry, University of Pennsylvania School of Medicine YNTEMA, CHESTER L., Professor of Anatomy, State University of New York College of Medi- cine ZORZOLI, ANITA, Associate Professor of Physiology, Vassar College LALOR FELLOWS, 1957 BACON, DONALD, Yale Medical School BISHOP, NORMAN, University of Chicago BRYANT, S. H., University of Cincinnati BUCKMANN, DETLEF, Zoologisches Institut, Mainz, Germany BURGEN, A. S. V., McGill University EDWARDS, CHARLES, Johns Hopkins University ENGLE, RALPH, Cornell University Medical College LORAND, L., Northwestern University LINDBERG, OLOV, Wcnner-Grens Institute, Sweden LUBIN, MARTIN, Harvard Medical School SCHULMAN, MARTIN, State University of New York, College of Medicine at Syracuse STEPHENSON, W. K., Earlham College WHITEHOUSE, MICHAEL, University of Pennsylvania School of Medicine WILSON, T. HASTINGS, Washington University School of Medicine WOODS, KENNETH, Cornell University Medical School Lillie Fellow, 1957 Niu, MAN-CHIANG, Rockefeller Institute for Medical Research Students, 1957 BOTANY ABELES, FRED B., Cornell University ARNOLD, ELIZABETH L, University of Rochester ARONSON, FLORA P., Brooklyn College BOUCK, GEORGE B., Columbia University COOK, PHILIP W., University of Vermont CZELUSNIAK, MARILYN M., Smith College FRANKEL, JOSEPH, Yale University HERSKOWITZ, JULIA, Antioch College KEELER, CARL R., JR., Northwestern University KLEPPER, ELIZABETH, Vanderbilt University MARINE BIOLOGICAL LABORATORY KUENZLER, EDWARD J., University of Georgia MORELAND, RALPH E., JR., Indiana University MUSCHIO, HENRY M., Fordham University PAIR, HYANGJU, Wellesley College PAOLI, GISELA, Chatham College PARKER, BRUCE C., Yale University PROTA, CARL D., Fordham University RICE, ELEANOR, Wheaton College TEWS, LEONARD C., Indiana University WALSER, STEPHANIE L., Radcliffe College EMBRYOLOGY ARKLESS, RICHARD, University of Pennsylvania Medical School CASTON, J. DOUGLAS, University of North Carolina GOERINGER, GERALD C., Johns Hopkins University GRIFFIN, JOE LEE, Princeton University HANKS, JAMES E., University of New Hampshire HERSH, GEORGE L., University of California KARAKASHIAN, STEPHEN J., Drew University KERR, NORMAN S., Northwestern University KESSEL, RICHARD G., State University of Iowa i KIRCHEN, ROBERT V., Columbia University KRAM, FLEURETTE L., Northwestern University LOVE, DAVID S., University of Colorado LOWE, JANET M., University of Minnesota MATHIESEN, GEORGE C., Harvard University t MELLON, DEFOREST, JR., Yale University NELSON, SHIRLEY, Northwestern University ROSEWATER, JOSEPH, Harvard University SPARANO, BENJAMIN M., Fordham University TALBOT, WILLIAM H., Rockefeller Institute 9 TYSON, GRETA E., University of New Hampshire VAN DYK, N. JOANNE, University of New Hampshire WALCOTT, CHARLES, Cornell University WATKINS, MARGARET J., University of Minnesota WHITE, JEAN ANN, Mount Holyoke College WYLIE, RICHARD M., Harvard University PHYSIOLOGY CLARK, ALVIN JOHN, Harvard University Cox, RODY P., University of Pennsylvania DAVIDSON, MORTON, Bellevue Medical College DUBNAU, DAVID A., Columbia University ERWIN, JOSEPH A., Syracuse University FAHN, STANLEY, University of California School of Medicine FELIX, MARIE D., Cornell University Medical School HAFT, DAVID E., University of Rochester School of Medicine HALPEREN, SIDNEY, University of Texas KAHLBROCK, MARGIT, Columbia University *KIRSCH, JACK F., Rockefeller Institute MCCLUSKEY, ROBERT T., New York University-Bellevue Medical Center MAZUR, PETER, Princeton University MEDINA, HEITOR S., Inst. de Biolojia Curitiba, Paroni, Brazil MINDICH, LEONARD E., Rockefeller Institute NAGLER, ARNOLD L., Bellevue Medical School OTERO, Luis R., University of Puerto Rico RABINOWITZ, LAWRENCE, University of California RAWITSCHER, ERIKA, American Museum of Natural History REPORT OF THE DIRECTOR 23 ROBERTS, PATRICIA R., Duke University SCHNEIDER, JOHN H., University of Wisconsin SIGER, ALVIN, Johns Hopkins University STERN, DANIEL N., Albert Einstein College of Medicine STONE, NANCY J., Columbia University TAKEUCHI, IKUO, Princeton University WEEKS, BOYD M., University of California WILLIAMS, FRANK ROBERT, Oberlin College WILLIAMS, FREDERICK M., Yale University WILLIS, JOHN S., Harvard University INVERTEBRATE ZOOLOGY ASHER, DAVID M., Harvard University AUGENFELD, JOHN M., University of Wisconsin BECKER, JOYCE E., Evansville College BRANNING, ARLEEN, City College of New York BRAVERMAN, MAXWELL H., University of Illinois CAMP, DONALD B. M., Acadia University CLARKE, ARTHUR H., JR., Cornell University COLEMAN, CHASE, Vassar College CONCANNON, BRO. JOSEPH, St. John's University COOPER, MADELINE, American Museum of Natural History COOPER, KENNETH K., American Museum of Natural History CROWELL, JANE, Oberlin College DIAMOND, JARED M., Harvard University DOBBEN, PHYLLIS A., Rocky River 16, Ohio DOBBS, HARRY D., Wofford College EGLOFF, DAVID A., Amherst College GFELLER, SISTER MARION D., Marquette University GUZE, CAROL D., Washington University HAFENER, PAUL A., JR., Franklin and Marshall College HECHTEL, GEORGE J., Yale University HILD, DAVID H., Wesleyan University HORVATH, NANCY, 10121 S. Parnell Avenue, Chicago 38, Illinois HORWITZ, JUDITH, Radcliffe College ISAAC, DONALD E., University of California JENSEN, DONALD DALE, Yale University JOHNSON, B. THOMAS, University of California JORDAN, ELKE, Goucher College KAUFMAN, JOHN H., University of California KRASSNER, STUART, Johns Hopkins University LANE, ROSEMARY M., Dalhousie University LEISY, ELSA, University of California LONGACRE, HARRIETTS, Mount Holyoke College LORENZO, MICHAEL A., St. Louis University LOWE, MILDRED E., Tulane University MCMANUS, LAWRENCE ROBERT, Cornell University MENAKER, MICHAEL, Princeton University NEWBERRY, ANDREW TODD, Stanford University POULSON, THOMAS L., University of Michigan PRAGER, JAN C, University of Cincinnati REESE, ERNST S., University of California ROOT, RICHARD B., University of Michigan Ross, SHIRLEY E., Washington University SHERMAN, IRWIN W., City College of New York SMITH, S. CLARKE, Wabash College SMITH, SUSAN, Earlham College THOMPSON, JANE F., University of Massachusetts THOMPSON, MARTHA JANE, Oberlin College 24 MARINE BIOLOGICAL LABORATORY TROTTER, NANCY L., Brown University VITOLS, ANDRIS T., University of Minnesota WILHELM, ROBERT C, Cornell University WILLIS, JOHN S., Harvard University WITTRY, SISTER ESPERANCE, College of St. Catherine WOOD, LANGLEY H., Cornell University Yow, FRANCIS W., Emory University ECOLOGY ABELES, FRED, Cornell University BARBER, RICHARD I., Brown University BARTH, ROBERT H., JR., Harvard University BLUNT, SISTER MARION XAVIER, Marquette University BOTHNER, RICHARD C., Fordham University ELLSWORTH, JOANNE, Elmira College GIFFORD, CAMERON E., Harvard University RANDALL, DONALD, Oberlin College STORY, LAWRENCE P., Drew University 3. FELLOWSHIPS AND SCHOLARSHIPS, 1957 Lucretia Crocker Scholarship : GEORGE B. BOUCK, Botany Course Conklin Scholarship : ROBERT KIRCHEN, Embryology Course Merkel Jacobs Scholarship : MARGIT KAHLBROCK, Physiology Course Calkins Scholarship : THOMAS L. POULSON, Invertebrate Zoology Course Bio Club Scholarships : ARLEEN BRANNING, Invertebrate Zoology Course IRWIN W. SHERMAN, Invertebrate Zoology Course Linton Memorial Fund : C. D. DIETER, Washington-Jefferson College 4. TABULAR VIEW OF ATTENDANCE, 1953-1957 1953 1954 1955 INVESTIGATORS TOTAL 310 298 250 Independent 176 180 162 Under Instruction 37 20 9 Library Readers 46 52 54 Research Assistants 51 46 25 STUDENTS TOTAL 136 134 148 Zoology 55 56 56 Embryology 30 29 30 Physiology 31 28 30 Botany 11 12 19 Ecology 9 9 13 TOTAL ATTENDANCE 446 432 398 Less persons represented as both investigators and students 5 446 427 398 7956 304 184 20 50 50 140 55 28 30 18 9 444 2 442 1957 326 186 23 42 75 139 55 27 30 18 9 465 3 462 REPORT OF THE DIRECTOR 25 INSTITUTIONS REPRESENTED TOTAL 155 136 129 130 129 By investigators 90 104 95 97 94 By students 65 32 34 33 35 SCHOOLS AND ACADEMIES REPRESENTED By investigators 2 3 3 5 By students 1 1 2 1 1 FOREIGN INSTITUTIONS REPRESENTED By investigators 15 11 8 9 11 By students 6 13 6 6 5 5. INSTITUTIONS REPRESENTED, 1957 Amherst College American Museum of Natural History Boston University School of Medicine Brooklyn College Brown University Bryn Mawr College Chatham College Children's Hospital of Philadelphia City College of New York Colby College College of Mt. St. Joseph on the Ohio Columbia University, College of Physicians and Surgeons Columbia University, Zoology Dept. Cornell University Cornell University Medical School Corporation of Roman Catholic Clergymen Duke University Albert Einstein College of Medicine Elmira College Emory University Florida State University Fordham University Hahnemann Medical College Harvard University Harvard University Medical School Indiana University Institute for Muscle Research Johns Hopkins University Johns Hopkins University Medical School Eli Lilly and Company Marquette University National Institutes of Health New York University Heights New York University College of Medicine New York University, Washington Square College North Carolina State College Northwestern University Oberlin College Princeton University Purdue University Radcliffe College Rockefeller Institute for Medical Research Rutgers University Saint Joseph's College St. Louis University St. Louis University, School of Medicine Single Cell Foundation Sloan-Kettering Institute Southwestern Medical College State University of Iowa State University of New York, College of Medicine at Syracuse Syracuse University Temple University University of Chicago University of Florida University of Illinois University of Illinois, College of Medicine University of Maine University of Michigan University of Minnesota University of New Hampshire University of North Carolina University of Oklahoma University of Pennsylvania University of Pennsylvania Medical School University of Pittsburgh University of Rochester University of Vermont University of Virginia, School of Medicine University of Wisconsin U. S. Fish and Wildlife Service Vassar College Veterans Administration Hospital of Brooklyn Wabash College Washington and Jefferson College Washington University School of Medicine Wellesley College Wesleyan University Wheaton College Wilson College Yale University 26 MARINE BIOLOGICAL LABORATORY SUPPORTING INSTITUTIONS AND AGENCIES, 1957 Abbott Laboratories Eli Lilly and Company American Cancer Society Merck and Company, Inc. American Philosophical Society National Institutes of Health Associates of the Marine Biological Labora- National Science Foundation tory Office of Naval Research Atomic Energy Commission The Rockefeller Foundation Ciba Pharmaceutical Products, Inc. Schering Corporation The Grass Foundation Smith, Kline and French Foundation Kellogg Foundation The Upjohn Company The Lalor Foundation FOREIGN INSTITUTIONS REPRESENTED, 1957 Zoologisches Institut, Mainz, Germany University of Oslo, Sweden McGill University, Montreal, Canada University of Brussels, Belgium Glasgow University, Scotland Royal Veterinary College, Sweden University College, London, England Utrecht University, The Netherlands Wenner-Grens Institute, Sweden McMaster University, Hamilton College, Can- University of Birmingham, England ada 6. EVENING LECTURES, 1957 July 5 BENTLEY GLASS "In pursuit of a gene" July 12 K. LINDERSTROM-LANG "Deuterium exchange of proteins in aqueous solution" July 19 OLOV LIXDBERG "Functional-structural correlations in mito- chondria" July 26 ALBERT 1. LANSING "Chemical morphology of the elastic fiber" August 2 JAMES D. EBERT "The acquisition of biological specificity" August 9 J. C. ECCLES "The behavior of nerve cells" August 16 FRANCIS J. RYAN "Mutation as an error in gene duplication" August 23 SEYMOUR S. COHEN "The chemical pathology of the virus in- fected cell" 7. TUESDAY EVENING SEMINARS, 1957 July 2 CHARLES B. METZ "The enhancement of starfish sperm motility and respiration by metals and metal bind- ing agents" NORMAN E. KEMP "Differentiation of cortical cytoplasm and extra-cellular membranes of oocytes. in- cluding changes at fertilization" REPORT OF THE DIRECTOR 27 LAURA HUNTER COLWIN and ARTHUR L. COLWIN "Lytic and other activities of the individual spermatozoon during the early events of sperm entry (Hydroides, Saccoglossus, and several other invertebrates)" July 9 A. M. SHANES "Ion movement in vertebrate nerve" WILLIAM STEPHENSON "Relationships between ion movements and membrane potential changes in muscle" G. HOYLE "Nervous control of muscular contraction in arthropods" W. H. FREYGANG, JR "Evidence for electrical inexcitability of neuron soma" July 16 T. R. TOSTESON, S. A. FERGUSON and L. V. HEILBRUNN "Further studies of the antimitotic and car- cinostatic action of ovarian extracts" L. V. HEILBRUNN, FRANCIS ASHTON, CARL FELDHERR and W. L. WILSON . . "The action of insulin on living cells" FRANCIS ASHTON "Magnetic studies on cells and protoplasm" CARL FELDHERR "The metachromatic reaction in various types of protoplasm" PETER RIESER "Effect of x-rays on fibrinogen" PAUL R. GROSS, SYLVAN NASS and WILLIAM PEARL "Mechanisms of sol-gel transformations in the cytoplasm" July 23 R. E. BENESCII and R. BENESCH "Sulfur linkages in hemoglobins" A. CHASE "Uricase inactivation by urea" L. LORAND "Clotting of blood : a study of the polymeri- zation of proteins" H. K. SCHACHMAN "Structural considerations on bushy stunt virus" July 30 LUIGI PROVASOLI .' "Effect of plant hormones on sea weed" DU-KIHT McNAiR SCOTT '\ hanges in RNA during synchronous di- vision of E. coli" TAY S. ROTH "Observations on the RNase system of rat liver" BERNARD DAVIS "Bacterial permease systems" August 6 JOSEPH GALL "Thymidine incorporation into the macro- nucleus of Euplotes (Protozoa)" BEAL B. HYDE "The effect of Versene on the sulfhydryls of chromatin" C. W. METZ "Interactions between chromosomes and cy- toplasm during early embryonic develop- ment in Sciara (Diptera)" MARINE BIOLOGICAL LABORATORY August 13 BOSTWICK H. KETCHUM "Marine ecology and its place in biological research" EUGENE P. ODUM "Studies on simple natural ecosystems" JOHN H. RYTHER "On the efficiency of photosynthesis in the sea" THOMAS S. AUSTIN "The ecology of the biota of the equatorial Pacific" August 20 L. LORAND, J. MOLNAR and C. Moos .... "Biochemical studies of relaxation in gly- cerinated muscle" F. D. CARLSON and A. SIGER "Creatine phosphate and adenosintriphos- phate breakdown in iodoacetate poisoned muscle" A. G. SZENT-GYORGYI and CAROLYN COHEN "Structural aspects of muscle proteins" T. HAYASHI, R. STROHMAN and R. ROSENBLUTH "Myosin and actin interaction, and construc- tion" 8. MEMBERS OF THE CORPORATION, 1957 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 DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota 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 MACNAUGHT, MR. FRANK M., Woods Hole, Massachusetts 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 REPORT OF THE DIRECTOR 29 2. REGULAR MEMBERS ABELL, DR. RICHARD G., 7 Cooper Road, New York City, New York ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York ADOLPH, DR. EDWARD F., University of Rochester, School of Medicine and Dentis- try, Rochester, New York ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota ALLEN, DR. M. JEAN, Department of Biology, Wilson College, Chambersburg, Pennsylvania 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., c/o Dr. A. Lurff, Institut Pasteur, 28 Rue du Dr. Roux, Paris 15e, France ARMSTRONG, DR. PHILIP B., State University of New York College of Medicine, Syracuse 10, New York ARNOLD, DR. WILLIAM A., Oak Ridge National Laboratory, Oak Ridge, Tennessee ATWOOD, DR. KIMBALL C, 68.] Outer Drive, Oak Ridge, Tennessee 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., Zoological Laboratory, University of Pennsylvania, Philadel- phia, Pennsylvania BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medi- cal School, Boston 15, Massachusetts BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School of Hygiene, Baltimore 5, Maryland BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire BARD, DR. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland BARTH, DR. L. G., Department of Zoology, Columbia University, New York City, New York BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana, Illinois BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa City, Iowa BECK, DR. L. V., Department of Physiology and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh 13, Pennsylvania BEERS, DR. C. D., University of North Carolina, Chapel Hill, North Carolina 30 MARINE BIOLOGICAL LABORATORY BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana BENESCH, DR. REINHOLD, Marine Biological Laboratory, Woods Hole, Massachu- setts BENESCH, DR. RUTH, Marine Biological Laboratory, Woods Hole, Massachusetts BENNETT, DR. MIRIAM, Department of Biology, Sweet Briar College, Sweet Briar, Virginia BERG, DR. WILLIAM E., Department of Zoology, University of California, Berkeley, California BERMAN, MR. MONES, Sloan-Kettering Institute, 410 E. 68th Street, New York 21, New York BERNSTEIN, DR. MAURICE, Virus Laboratory, University of California, Berkeley 4, California BERNHEIMER, DR. ALAN W., New York University College of Medicine, New York 16, New York BERTHOLF, DR. FLOYD M., College of the Pacific, Stockton, California BEVELANDER, DR. GERRIT, New York University School of Medicine, New York 16, New York BIGELOVV, DR. HENRY B., Museum of Comparative Zoology, Harvard University, Cambridge, 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, c/o Biological Abstracts, 3815 Walnut Street, Philadelphia 4, Pennsylvania BLUM, DR. HAROLD F., 24 Rue de Babylone, Paris VII, France BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center, 444 East 68th Street, New York 21, New York BODIAN, DR. DAVID, Department of Epidemiology, Johns Hopkins University, Baltimore 5, Maryland BOELL, DR. EDGAR J., 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, 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 & 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 BROWN, DR. FRANK A., JR., Department of Biological Sciences, Northwestern University. Evanston, Illinois REPORT OF THE DIRECTOR 31 BROWNELL, DR. KATHERINE A., Ohio State University, Columbus, Ohio BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of Health, Bethesda, Maryland (10405 Muir Place, Kensington, 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, 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, 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, Md. CARPENTER, DR. RUSSELL L., Tufts College, Medford 55, Massachusetts CARSON, Miss RACHEL, 204 Williamsburg Drive, Silver Spring, Maryland 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., Boston University School of Medicine, 80 E. Concord Street, Boston 18, Massachusetts CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical School, Coral Gables, Florida CHANG, DR. JOSEPH J., National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland 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, MR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts CLARK, DR. A. M., Department of Biological Sciences, University of Delaware, Newark, Delaware CLARK, DR. E. R., 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 Laboratory, Cambridge 38, Massachusetts CLELAND, DR. RALPH E., Indiana University, Bloomington, Indiana CLEMENT, DR. A. C., Department of Biology, Emory University, Emory, Georgia CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana COE, DR. W. R., 183 Third Avenue, Chula Vista, California COHEN, DR. SEYMOUR S., Department of Physiological Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 32 MARINE BIOLOGICAL LABORATORY 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 COOPERSTEIN, DR. SHERWIN J., Department of Anatomy, Western Reserve Uni- versity Medical School, Cleveland, Ohio COPELAND, DR. D. E., 1027 N. Manchester Street, Arlington 5, Virginia COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine COPLEY, DR. ALFRED L., Centre National cle Transfusion Sanguine, 6, Rue Alex- andra-Cobonel, Paris XVe, France CORNMAN, DR. IVOR, Hazleton Laboratories, Box 333, Falls Church, Virginia COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro- lina, Chapel Hill, North Carolina CRANE, MR. JOHN O., Woods Hole, Massachusetts CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire GROUSE, DR. HELEN V., Goucher College, Baltimore, Maryland CROWELL, DR. P. S., IR., Department of Zoology, Indiana University, Bloomington, Indiana CSAPO, DR. ARPAD I., Rockefeller Institute for Medical Research, 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., Department of Pharmacology, New York University Col- lege of Medicine, New York 16, New York DAWSON, DR. A. B., Harvard University, Cambridge 38, Massachusetts DAWSON, DR. T. A., 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, 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, Pennsylvania DOODS, DR. G. S., West Virginia University School of Medicine, Morgantown, West Virginia DOLLEY, DR. WILLIAM L., Department of Biology, Randolph-Macon College, Ash- land, Virginia REPORT OF THE DIRECTOR 33 DONALDSON, DR. JOHN C, University of Pittsburgh School of Medicine, Pitts- burgh, Pennsylvania DOTY, DR. MAXWELL S., Department of Biology, University of Hawaii, Honolulu, T. H. DuBois, DR. EUGENE F., 200 East End Avenue, New York 28, New York 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, Wilmer 25, Johns Hopkins Hospital, Baltimore 5, Mary- land EICHEL, DR. BERTRAM, Bureau of Biological Research, Box 515, Rutgers Univer- sity, New Brunswick, New Jersey EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania ELLIOTT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann Arbor, Michigan EVANS, DR. TITUS C., State University of Iowa, Iowa City, Iowa FAILLA, DR. G., College of Physicians and Surgeons, Columbia University, New York City, 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 Uni- versity, 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 FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois, Urbana, Illinois FREYGANG, DR. WALTER H., JR., 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 GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn, New York GAFFRON, DR. HANS, Research Institutes, University of Chicago, 5650 Ellis Ave- nue, Chicago 37, Illinois GALL, DR. JOSEPH G., Department of Zoology, University of Minnesota, Minne- apolis 14, Minnesota 34 MARINE BIOLOGICAL LABORATORY GALTSOFF, DR. PAUL S., Woods Hole, Massachusetts GASSER, DR. HERBERT S., Rockefeller Institute, New York 21, New York GEISER, DR. S. W., Southern Methodist University, Dallas, Texas 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 GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, Emory University, Georgia GOODRICH, DR. H. B., Wesleyan University, Middletown, Connecticut GOTTSCHALL, DR. GERTRUDE Y., 315 E. 68th Street, New York 21, New York GOULD, DR. H. N., Biological Sciences Information Exchange, 1113 Dupont Circle Building, Washington, D. C. GRAHAM, DR. HERBERT, U. S. Fish and Wildlife Service, Woods Hole, Massa- chusetts 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 GRAY, DR. IRVING E., Duke University, Durham, North Carolina GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New Brunswick, New Jersey GREEN, DR. MAURICE, Department of Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania GREGG, DR. JAMES H., University of Florida, Gainesville, Florida GREGG, DR. J. R., Department of Zoology, Columbia University, New York 27, New York GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical Col- lege, New York 21, New York GROSCH, DR. DANIEL S., Department of Zoology, 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 Sur- geons, 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 1, Michigan GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn, New York GUYER, DR. MICHAEL F., University of Wisconsin, Madison, Wisconsin HAJDU, DR. STEPHEN, U. S. Public Health Institute, Bethesda 14, Maryland HALL, DR. FRANK G., Duke University, Durham, North Carolina HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St. Louis, Missouri HAMILTON, DR. HOWARD L., Iowa State College, Ames, Iowa HANCE, DR. ROBERT T., Box 108, R. R. #3, Loveland, Ohio HARDING, DR. CLIFFORD V., JR., 705 N. Wayne Street, Apt. 305, Arlington 1, Virginia REPORT OF THE DIRECTOR 35 HARMAN, DR. MARY T., Box 68, Camden, North Carolina HARNLY, DR. MORRIS H., Washington Square College, New York University, New York City, New York HARRISON, DR. Ross G., Yale University, New Haven, Connecticut HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research, 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 HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New Jersey HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 663 North Oak Street, Buffalo 3, New York HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institute of Oceanog- raphy, University of California, La Jolla, California HAYASHI, DR. TERU, Department of Zoology, Columbia University, New York City, New York HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesley 81, Massachusetts HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, Phila- delphia, 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 HENSHAW, DR. PAUL S., 17th Floor, 501 Madison Avenue, New York 22, New York HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts HESS, DR. WALTER N., Hamilton College, Clinton, New York HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio HILL, DR. SAMUEL E., 135 Brunswick Road, Troy, New York HINRICHS, DR. MARIE, Board of Education, Bureau of Health Service, 228 North LaSalle Street, Chicago, Illinois HISAW, DR. F. L., Harvard University, Cambridge 38, Massachusetts HOADLEY, DR. LEIGH, Harvard University, Biological Laboratories, Cambridge, Massachusetts HODGE, DR. CHARLES, IV, Department of Zoology, Temple University, Philadelphia, Pennsylvania HOFFMAN, DR. JOSEPH, National Heart Institute, National Institutes of Health, Bethesda, Maryland HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia, Pennsylvania HOLLAENDER, DR. ALEXANDER, P. O. Box W, Clinton Laboratories, Oak Ridge, Tennessee 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, Bogata, Colombia, South America HUTCHENS, DR. JOHN O., Department of Physiology, University of Chicago, Chi- cago 37, Illinois 36 MARINE BIOLOGICAL LABORATORY HYDE, DR. BEAL B., Department of Plant Sciences, University of Oklahoma, Nor- man, Oklahoma HYMAN, DR. LIBBIE H., American Museum of Natural History, New York City, 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, Pennsylvania JACOBS, DR. WILLIAM P., Department of Biology, Princeton University, Princeton, New Jersey JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina JOHNSON, DR. FRANK H., Biology Department, Princeton University, Princeton, New Jersey JONES, DR. E. RUFFIN, JR., Department of Biology, University of Florida, Gaines- ville, Florida KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachusetts 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, Pennsylvania KAUFMANN, DR. B. P., Carnegie Institution, Cold Spring Harbor, Long Island, New York KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann Arbor, Michigan KEMPTON, DR. RUDOLF T., 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., Carleton College, Northneld, Minnesota 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 KLEINHOLZ, 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 KOPAC, DR. M. J., New York University, Washington Square College, New York City, New York 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 REPORT OF THE DIRECTOR 37 KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Baltimore, Maryland KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois KUFFLER, DR. STEPHEN, Department of Ophthalmology, Johns Hopkins Hospital, Baltimore 5, Maryland KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York LACKEY, DR. JAMES B., University of Florida, College of Engineering, Gainesville, 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 LANGE, DR. MATHILDA M., Box 307, Central Valley, New York LANSING, DR. ALBERT I., Department of Anatomy, University of Pittsburgh Medi- cal School, Pittsburgh 13, Pennsylvania LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts- burgh, Pennsylvania LAVIN, DR. GEORGE I., 3714 Springdale Avenue, Baltimore, Maryland LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota, Medi- cal School, Minneapolis 14, Minnesota LEDERBERG, DR. JOSHUA, Department of Genetics, University of Wisconsin, Madi- son 6, Wisconsin 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 LEHMANN, DR. FRITZ, Zool. Inst, University of Berne, Berne, Switzerland LESSLER, DR. MILTON A., Department of Physiology, Ohio State University, Co- lumbus, Ohio LEVINE, DR. RACHMIEL, Michael Rees Hospital, Chicago 16, Illinois LEVY, DR. MILTON, Biochemistry Department, New York University School of Dentistry, New York 10, New York LEWIN, DR. RALPH A., Marine Biological Laboratory, Woods Hole, Massachusetts LEWIS, DR. I. F., 1110 Rugby Road, Charlottesville, Virginia LING, DR. GILBERT, Eastern Pennsylvania Psychiatric Inst., Henry Avenue and Abbottsford Road, Philadelphia 29, Pennsylvania LITTLE, DR. E. P., 150 Causeway Street, Anderson Nichols & Company, Boston 24, Massachusetts LLOYD, DR. DAVID P. C., Rockefeller Institute, 66th Street & 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., Presbyterian Hospital, 620 West 168th Street, New York 32, New York LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York LORAND, DR. LASZLO, Department of Chemistry, College of Liberal Arts, North- western University, Evanston, Illinois 38 MARINE BIOLOGICAL LABORATORY LOVE, DR. Lois H., 4233 Regent Street, Philadelphia 4, Pennsylvania LOVE, DR. WARNER E., 1043 Marian Drive, Baltimore, Maryland LUBIN, DR. MARTIN, Department of Pharmacology, Harvard Medical School, Bos- ton 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. WILLIAM G., Department of Biology, Catholic University of America, Washington, D. C. MACDOUGALL, DR. MARY S., Mt. Vernon Apartments, 423 Clairmont Avenue, Decatur, Georgia 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 MACKLIN, DR. CHARLES C., 37 Gerard Street, London, Ontario, Canada 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, Marine Biological Laboratory, Woods Hole, Massachu- setts MARSLAND, DR. DOUGLAS A., New York University, Washington Square College, New York City, New York MARTIN, DR. EARL A., Department of Biology, Brooklyn College, Brooklyn, New York MATHEWS, DR. A. P., Glenwood Boulevard, Schenectady, New York MATHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College, Williamstown, Massachusetts MAYOR, DR. JAMES W., 8 Gracewood Park, Cambridge 58, Massachusetts MAZIA, DR. DANIEL, Department of Zoology, University of California, Berkeley 4, California MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania MEIGS, MRS. E. B., 1736 M Street, N. W., Washington, D. C. MEINKOTH, DR. NORMAN A., Department of Biology, Swarthmore College, Swarth- more, Pennsylvania MEMHARD, MR. A. R., Riverside, Connecticut MENKIN, DR. VALY, Agnes Barr Chase Foundation for Cancer Research, Temple University Medical School, Philadelphia, Pennsylvania REPORT OF THE DIRECTOR 39 METZ, DR. C. B., Oceanographic Institute, Florida State University, Tallahassee, Florida METZ, DR. CHARLES W., University of Pennsylvania, Philadelphia, Pennsylvania MIDDLEBROOK, DR. ROBERT, Institute for Muscle Research, Marine Biological Lab- oratory, Woods Hole, Massachusetts MILLER, DR. J. A., Basic Science Building, Emory University, Georgia MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire, Durham, New Hampshire MOE, MR. HENRY A., Secretary General, 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 W., Laboratory of Biophysics, NINDB, National Institutes of Health, Besthesda 14, Maryland MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick, New Jersey MOUNTAIN, MRS. J. D., 9 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 MUSSACCHIA, 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 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 NELSON, DR. LEONARD, Department of Anatomy, University of Chicago, Chicago, Illinois NEURATH, DR. H., Department of Biochemistry, University of Washington, Seattle 5, Washington NEWMAN, DR. H. H., 173 Devon Drive, Clearwater, Florida NICOLL, DR. PAUL A., Indiana Contract, Box K, A. P. O. 474, San Francisco, California Niu, DR. MAN-CHIANG, Rockefeller Institute for Medical Research, 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, Pennsvlvania 40 MARINE BIOLOGICAL LABORATORY OSTER, DR. ROBERT H., University of Maryland, School of Medicine, Baltimore 1, Maryland OSTERHOUT, DR. W. J. V., Rockefeller Institute, 66th Street and York Avenue, New York 21, New York OSTERHOUT, MRS. MARION IRWIN, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York PACKARD, DR. CHARLES, Woods Hole, Massachusetts PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio PARMENTER, DR. CHARLES L., Department of Zoology, University of Pennsylvania, Philadelphia, Pennsylvania PARPART, DR. ARTHUR K., Department of Biology, Princeton University, Prince- ton, 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 PETTIBONE, DR. MARIAN H., Department of Zoology, University of New Hamp- shire, Durham, New Hampshire PHILPOTT, MR. DELBERT E., 496 Palmer Avenue, Falmouth, Massachusetts PICK, DR. JOSEPH, Department of Anatomy, New York University-Bellevue Medi- cal Center, New York City, New York PIERCE, DR. MADELENE E., Vassar College, Poughkeepsie, New York PLOUGH, DR. HAROLD H., Amherst College, Amherst, Massachusetts POLLISTER, DR. A. W., Columbia University, New York City, New York POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut PRATT, DR. FREDERICK H., 105 Hundreds Road, Wellesley Hills 82, Massachusetts PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti- more 12, Maryland PROSSER, DR. C. LADD, 401 Natural History Building, University of Illinois, Ur- bana, Illinois PROVASOLI, DR. LUIGI, Department of Biology, Haskins Laboratories, 305 E. 43rd Street, New York 17, New York QUASTEL, DR. JUDA H., Department of Biochemistry, McGill University, Montreal, Canada 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 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, Emory, Georgia READ, DR. CLARK P., Johns Hopkins University, Baltimore, Maryland REBHUN, DR. LIONEL I., Department of Anatomy, University of Illinois, College of Medicine, Chicago, Illinois RECHNAGEL, DR. R. O., Department of Physiology, Western Reserve University, Cleveland, Ohio REPORT OF THE DIRECTOR 41 REDFIELD, DR. ALFRED C, Woods Hole, Massachusetts REINER, DR. J. M., Columbia-Presbyterian Medical Center, 622 W. 168th Street, New York 32, 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, Entomology Department, University Farm, University of Minnesota, St. Paul, Minnesota RICHARDS, DR. OSCAR W., American Optical Company, Research Center, South- bridge, Massachusetts RIESER, DR. PETER, Marine Biological Laboratory, Woods Hole, 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 ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pittsburgh Medical School, Pittsburgh 13, Pennsylvania Rossi, DR. HAROLD H., Department of Radiology, Columbia University, New York 32, New York ROTH, DR. JAY S., Department of Biochemistry, Hahnemann Medical College, Philadelphia 2, Pennsylvania ROTHENBERG, DR. M. A., Chief, Chemical Laboratories, Dugway Proving Ground, Dugway, Utah RUGH, DR. ROBERTS, Radiological Research Laboratory, College of Physicians and Surgeons, New York City, New York RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden RUTMAN, DR. ROBERT J., Department of Zoology, University of Pennsylvania, Philadelphia, Pennsylvania RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole, Massachusetts SANDEEN, DR. MURIEL I., Department of Zoology, Duke University, Durham, North Carolina SAUNDERS, MR. LAWRENCE, R. D. 7, Bryn Mawr, Pennsylvania SCHAEFFER, DR. ASA A., Department of Biology, Temple University, Philadelphia, Pennsvlvania 42 MARINE BIOLOGICAL LABORATORY SCHARRER, DR. ERNST A., Albert Einstein College of Medicine, 1710 Newport Avenue, New York 61, New York SCHECHTER, DR. VICTOR, College of the City of New York, New York City, 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, Minne- apolis 14, Minnesota SCHNEIDERMAN, DR. HOWARD A., Department of Zoology, Cornell University, Ithaca, New York SCHOLANDER, DR. P. F., Institute of Zoophysiology, University of Oslo, Oslo, Norway SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massachusetts SCHRADER, DR. FRANZ, Department of Zoology, Columbia University, New York City, New York SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia University, New York City, New York SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington, Indiana SCOTT, DR. ALLAN C., Colby College, Waterville, Maine SCOTT, DR. D. B, McNAiR, Department of Biochemistry, University of Pennsyl- vania Hospital, Philadelphia, Pennsylvania SCOTT, SISTER FLORENCE MARIE, Seton Hill College, Greensburg, Pennsylvania SCOTT, DR. GEORGE T., Oberlin College, Oberlin, Ohio SEARS, DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massa- chusetts SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and Surgeons, New York City, New York 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, DR. EDWARD H., Woods Hole Oceanographic Institution, Woods Hole, Massachusetts REPORT OF THE DIRECTOR 43 SMITH, MR. HOMER P., General Manager, Marine Biological Laboratory, Woods Hole, Massachusetts SMITH, MR. PAUL FERRIS, Marine Biological Laboratory, Woods Hole, Massa- chusetts SMITH, DR. RALPH I., Department of Zoology, University of California, Berkeley 4, California SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington, Indiana SONNENBLICK, DR. B. P., 40 Rector Street, Newark 3, New Jersey SPEIDEL, DR. CARL C., University of Virginia, University, Virginia SPIEGEL, DR. MELVIN, Department of Biology, Colby College, Waterville, Maine SPRATT, DR. NELSON T., Department of Zoology, University of Minnesota, Minne- apolis 14, Minnesota STARR, DR. RICHARD C., Department of Botany, Indiana University, Bloomington, Indiana STEINBACH, DR. HENRY BURR, Department of Zoology, University of Chicago, Chicago 15, Illinois STEINBERG, DR. MALCOLM S., Department of Embryology, Carnegie Institution of Washington, Baltimore 5, Maryland STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota, Min- neapolis 14, Minnesota STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois STOREY, DR. ALMA G., Department of Botany, Mount Holyoke College, South Hadley, Massachusetts STRAUS, DR. W. L., JR., Johns Hopkins University, Baltimore 18, Maryland 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 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 Neuro- logical 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 TRACY, DR. HENRY C., P. O. Box 54, Oxford, Mississippi TRACER, DR. WILLIAM, Rockefeller Institute, 66th Street and York Avenue, New York 21, New York 44 MARINE BIOLOGICAL LABORATORY TRINKAUS, DR. J. PHILIP, Osborn Zoological Laboratories, Yale University, New Haven, Connecticut TROLL, DR. WALTER, Department of Internal Medicine, New York University College of Medicine, New York City, New York TWEEDELL, DR. KENYON S., Department of Zoology, University of Maine, Orono, Maine TYLER, DR. ALBERT, 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 W., Department of Zoology, Smith College, North- ampton, Massachusetts VILLEE, DR. CLAUDE A., Harvard Medical School, Boston 15, Massachusetts VINCENT, DR. WALTER S., Department of Anatomy, State University of New York School of Medicine, Syracuse 10, New York WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New Brunswick, New Jersey WALD, DR. GEORGE, Biological Laboratory, 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 Laboratory, Yale University, New Haven, Connecticut WEBB, DR. MARGUERITE, Department of Physiology and Bacteriology, Goucher College, Towson, Maryland WEISS, DR. PAUL A., Laboratory of Developmental Biology, Rockefeller Institute, New York 21, New York WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pennsylvania WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut WHITAKER, DR. DOUGLAS M., Rockefeller Institute for Medical Research, New York 21, New York WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania WHITING, DR. ANNA R., University of Pennsylvania, Philadelphia, Pennsylvania WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, Pennsylvania WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts WIERCINSKI, DR. FLOYD J., Department of Physiology, Hahnemann Medical Col- lege, Philadelphia, Pennsylvania WILBER, DR. C. G., Medical Laboratories, Applied Physiology Branch, Army Chem- ical Center, Maryland WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore, Maryland WILSON, DR. J. W., Brown University, Providence 12, Rhode Island REPORT OF THE DIRECTOR 45 WILSON, DR. WALTER L., Department of Physiology, University of Vermont Col- lege of Medicine, Burlington, Vermont WITSCHI, DR. EMIL, Department of Zoology, State University of Iowa, Iowa City, Iowa WOLF, DR. ERNST, Pendleton Hall, Wellesley College, Wellesley, Massachusetts WOODWARD, DR. ARTHUR A., Army Chemical Center, Maryland (Applied Physiol- ogy Branch, Army Chemical Corps, Medical Laboratory) WRIGHT, DR. PAUL A., Department of Zoology, University of Michigan, Ann Arbor, Michigan WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton, Massachusetts YNTEMA, DR. C. L., Department of Anatomy, State University of New York Col- lege of Medicine, Syracuse 10, New York YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings- ton, Rhode Island ZIRKLE, DR. RAYMOND E., Department of Radiobiology, University of Chicago, Chicago 37, Illinois ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie, New York ZWEIFACH, DR. BENJAMIN, New York University-Bellevue Medical Center, New York City, New York ZWILLING, DR. E., Department of Genetics, University of Connecticut, Storrs, Connecticut 3. ASSOCIATE MEMBERS ALDRICH, Miss AMY OWEN ALTON, DR. AND MRS. BENJAMIN H. ARMSTRONG, DR. AND MRS. P. B. BACON, MRS. ROBERT BARBOUR, MR. Lucius BARB, MR. ROBERT P. BARTOW, MR. AND MRS. CLARENCE BARTOW, MRS. FRANCIS D. BARTOW, MR. AND MRS. PHILIP BELL, MRS. ARTHUR BRADLEY, MR. ALBERT L. BRADLEY, MRS. CHARLES CRANE BROWN, MRS. THORNTON BURLINGAME, MRS. F. A. CAHOON, MRS. SAMUEL CALKINS, MR. G. NATHAN, JR. CALKINS, MRS. GARY N. CARLETON, MRS. WINSLOW CLAFF, MR. AND MRS. C. LLOYD CLARK, DR. AND MRS. ALFRED HULL CLARK, MRS. LEROY CLARK, MR. W. VAN ALAN CLOWES, MR. ALLEN W. CLOWES, MRS. G. H. A. CLOWES, DR. AND MRS. GEORGE, JR. COLTON, MR. H. SEYMOUR CRANE, Miss LOUISE CRANE, MRS. W. CAREY CRANE, MRS. W. MURRAY CROWELL, MR. PRINCE S. CURTIS, DR. W. D. DANIELS, MR. AND MRS. F. HAROLD DAY, MR. AND MRS. POMEROY DRAPER, MRS. MARY C. DREYER, MRS. FRANK ELSMITH, MRS. DOROTHY ENDERS, MR. FREDERICK EWING, MR. FREDERICK FASEY, MRS. PAULINE M. FAY, MRS. BRUCE CRANE FRANCIS, MR. LEWIS, JR. FROST, MRS. EUGENIA 46 MARINE BIOLOGICAL LABORATORY GALTSOFF, MRS. EUGENIA GlFFORD, MR. AND MRS. JOHN A. GlLDEA, DR. AND MRS. E. F. GREEN, Miss GLADYS W. HAMLEN, MR. J. MONROE HARRELL, MR. AND MRS. JOEL E. HARRINGTON, MR. AND MRS. A. W. HARRINGTON, MR. ROBERT D. HlRSCHFELD, MRS. NATHAN HOUSTON, MR. AND MRS. HOWARD E. JEWETT, MRS. GEORGE F. KEITH, MR. AND MRS. HAROLD C. KING, MR. FRANKLIN KOLLER, MRS. LEWIS LEMANN, MRS. SOLEN B. LOBB, MRS. JOHN LURDON, MR. W. R. McKELOY, MR. JOHN MARVIN, MRS. WALTER T. MAST, MRS. S. O. MEIGS, MRS. EDWARD B. MEIGS, DR. AND MRS. J. WISTER MITCHELL, MRS. JAMES McC. MIXTER, MRS. JASON MOSSER, MRS. FLORENCE M. MOTLEY, MRS. THOMAS NEWTON, Miss HELEN K. NICHOLS, MRS. GEORGE NIMS, MRS. E. D. PACKARD, DR. AND MRS. CHARLES PACKARD, MRS. LAURENCE B. PARK, MR. MALCOLM S. PENNINGTON, Miss ANNE H. REDFIELD, MRS. ALFRED REZNIKOFF, DR. PAUL RIGGS, MRS. LAWRASON RIVINUS, MR. AND MRS. F. MARKOE ROOT, MRS. WALTER ROZENDOAL, MR. H. M. RUDD, MRS. H. W. DWIGHT SANDS, Miss ADELAIDE G. SAUNDERS, MRS. LAWRENCE SHIVERICK, MRS. MARY STONE, MR. AND MRS. S. M. SWIFT, MR. AND MRS. E. KENT SWOPE, MR. AND MRS. GERARD, JR. SWOPE, Miss HENRIETTA H. TILNEY, MRS. ALBERT A. TOMPKINS, MR. AND MRS. B. A. WEBSTER, MRS. EDWIN S. WHITELY, Miss MABEL W. WlCKERSHAM, MR. AND MRS. JAMES H. WILLISTON, Miss EMILY WOLFINSOHN, MRS. WOLFE V. REPORT OF THE LIBRARIAN In 1957, seventy-six new journals were acquired, bringing the total number of currently received titles to 1635. Of these titles, there were 490 (15 new) Marine Biological Laboratory subscriptions; 617 (14 new) exchanges and 192 (21 new) gifts; 90 (9 new) were Woods Hole Oceanographic Institution subscriptions; 191 (7 new) were exchanges and 55 (10 new) were gifts. During the past ten years, we averaged 60 new journals per year. The ever growing number of new journals being issued far exceeds the number which cease publication. The Laboratory purchased 151 books, received 61 complimentary copies (4 from authors and 57 from publishers), and accepted 13 miscellaneous gifts. The Institution purchased 39 titles and received 10 gifts. The total number of books accessioned amounted to 274. By purchase and by gift the Laboratory completed 13 journal sets and partially completed 19. The Institution completed 4 sets and partially completed 3. There were 3920 reprints added to the collection, of which 2055 were of current issue. At the close of the year, the Library contained 67,961 bound volumes and 206,125 reprints. REPORT OF THE TREASURER 47 The Library sent out on inter-library loan 243 volumes and borrowed 115 for the convenience of the scientists. It is hoped that a copying machine may be pur- chased in the near future so that short papers may be reproduced for out-of-town loans, thus eliminating some of the depreciation on our volumes. A process such as this could also be utilized for summer service. Reprint collections were received from the estate of Dr. Arthur Weysse and from the University of Pittsburgh ; many books, journal numbers and papers were received from Drs. Ethel B. Harvey, C. Ladd Prosser, Rufus R. Humphrey, Phineas W. Whiting, Paul S. Galtsoff, Ralph Wichterman, and the Tompkins-McCaw Library, Medical College of Virginia. Dr. Alfred W. Senft kindly donated back volumes and a current subscription to the "New England Journal of Medicine." Grateful acknowledgment is herewith extended to the donors of these very accept- able presentations. With a larger sum available for the purchase of books, and with the many sug- gestions so willingly submitted by the Library Advisory Committee, we were in a position in 1957 to add many new titles to the shelves. An increase in the binding budget also enabled us to have bound 275 back volumes, bringing the total to 1110 for the year. This same degree of progress is anticipated in 1958. Respectfully submitted, DEBORAH L. HARLOW, Librarian VI. REPORT OF THE TREASURER The market value of both the General Fund and the Library at December 31, 1957, amounted to $1,461,278 as compared with the total of $1,472,265 as of December 31, 1956. The average yield on the securities was 3.84% of market value and 5.60% of book value. The total uninvested principal cash in the above accounts as of December 31, 1957, was $2,248. Classification of the securities held in the Endowment Funds appears in the auditor's report. The market value of the pooled securities as of December 31, 1957, was $247,629 with uninvested principal cash of $102. The book value of the securities in this account was $236,735. The average yield on market value was 3.88% and 4.06% of book value. The proportionate interest in the Pooled Fund account of the various Funds as of December 31, 1957, is as follows: Pension Fund 17.608% General Laboratory Investment 57.866 Other : Bio Club Scholarship Fund 1.687 Rev. Arsenious Boyer Scholarship Fund 2.064 Gary N. Calkins Fund 1.933 Allen R. Memhard Fund 374 F. R. Lillie Memorial Fund 6.515 48 MARINE BIOLOGICAL LABORATORY Lucretia Crocker Fund 7.054 E. G. Conklin Fund 1.194 M. H. Jacobs Scholarship Fund 850 Jewett Memorial Fund 626 Anonymous Gift 2.229 The Pooled Fund includes the Jewett Memorial Fund and an anonymous Gift Fund which were additions during 1957. The Jewett Memorial Fund was created by gifts in memory of the late George Frederick Jewett. Mr. Jewett as well as his father and mother and the other members of his family have been keenly interested in the Laboratory since its inception. It has not yet been determined how the Jewett Fund and the fund created by the anonymous gift will be used, but the views of the Jewett family and the donor of the latter fund will be given first consideration. Considerable activity was recorded in the special custodian account owing to the purchase of short-term Government bonds to activate available cash which would otherwise remain idle in our regular cash accounts pending payment of construction expenses. Income earned was $646.40. Inasmuch as the MBL Club loan was reduced to $2,052, the securities pledge to cover this loan was reduced to $3,000. Donations from MBL Associates for 1957 were $3,481 as compared with $5,255 in 1956. Unrestricted gifts from foundations, societies and companies amounted to $33,000. For the rehabilitation of the Crane Building, the National Science Foundation advanced $415,000 in 1957. Construction began in September and is scheduled for completion in May of 1958. In April of 1957 we paid off the David House Mortgage in the amount of $5,000. Lynbrand, Ross Bros. & Montgomery have examined our books and submitted financial statements for examination. Following is a statement of the auditors. To the Trustees of the Marine Biological Laboratory, Woods Hole, Massachusetts: We have examined the balance sheets of Marine Biological Laboratory as at December 31, 1957, the related statements of operating expenditures and income for the year then ended, and statement of current fund for the year ended December 31, 1957. Our examination was made in accordance with generally accepted audit- ing standards, and accordingly included such tests of the accounting records and such other auditing procedures as we considered necessary in the circumstances. In our opinion, the accompanying financial statements present fairly the assets, liabilities and funds of Marine Biological Laboratory at December 31, 1957, and the expenditures and income for the year then ended. LYBRAND, Ross BROS. & MONTGOMERY Boston, Massachusetts May 22, 1958 JAMES H. WICKERSHAM, Treasurer REPORT OF THE TREASURER 49 MARINE BIOLOGICAL LABORATORY BALANCE SHEET December 31, 1957 Investments Investments held by Trustee : Securities, at cost (approximate market quotation $1,461,278) $1,002,682 Cash 2,248 1,004,930 Investments of other endowment and unrestricted funds : Pooled Investments, at cost (approximate market quotation $247,629) 236,735 Less temporary investment of current fund cash 5,728 231,007 Other investments (Note A) 67,323 Cash 11,263 Accounts receivable 5,038 314,631 Plant Assets Land, buildings, library and equipment (Note B) 2,517,845 Less allowance for depreciation (Note B) 1,026,681 1,491,164 Construction in progress 103,856 Cash 34,560 U. S. Treasury bills, due 1/30/58, at cost (face value $350,000) 346,815 1,976,395 Current Assets Cash 142,160 U. S. Treasury bills, at cost : $40,000 face value due 2/13/58 39,649 Temporary investment in pooled securities 5,728 Accounts receivable (U. S. Government $19,605) 36,274 Inventories of specimens and Bulletins 57,282 Prepaid insurance and other 13,531 $3,590,580 Notes : A The Laboratory has guaranteed a note of approximately $2,400 of the M.B.L. Club and has pledged as security therefor bonds with an original cost of $3,000 included in other investments. B The Laboratory has since January 1, 1916, provided for reduction of book amounts of plant assets and funds invested in plant at annual rates ranging from \% to 5% of the original cost of the assets. 50 MARINE BIOLOGICAL LABORATORY MARINE BIOLOGICAL LABORATORY BALANCE SHEET December 31, 1957 Endowment Funds Endowment funds given in trust for benefit of the Marine Biological Laboratory .. $1,004,930 Endowment funds for awards and scholarships : Principal $ 64,415 Unexpended income 2,428 66,843 Unrestricted funds functioning as endowment 206,378 Retirement fund 46,233 Pooled investments accumulated gain or (loss) ' (4,823) 314,631 Plant Liability and Funds Funds expended for plant, less retirements $2,551,469 Less allowance for depreciation charged thereto 1,026,681 1,524,788 Unexpended plant funds 381,375 1,906,163 Accounts payable 70,232 1,976,395 Current Liabilities and Funds Accounts payable 43,409 Unexpended balances of gifts for designated purposes 8,744 Advance payments on research contracts 94,217 Current fund 148,254 $3,590,580 REPORT OF THE TREASURER 51 MARINE BIOLOGICAL LABORATORY STATEMENT OF OPERATING EXPENDITURES AND INCOME Year Ended December 31, 1957 Operating Expenditures Direct expenditures of departments : Research and accessory services $146,859 Instruction 35,237 Library, including book purchases 32,712 Biological Bulletin 16,995 231,803 Direct costs on research contracts 129,983 Administration and general 54,526 Plant operation and maintenance 81,156 Dormitories and dining services 143,322 Plant additions from current funds 59,581 700,371 Less depreciation included in plant operation and dormitories and dining services above but charged to plant funds 36,351 664,020 Income Direct income of departments : Research fees 43,418 Accessory services (including sales of biological specimens $67,562) 103,718 Instruction fees 16,980 Library fees and income 8,239 Biological Bulletin, subscriptions and sales 19,846 192,201 Reimbursement and allowance for direct and indirect costs on research contracts 151,444 Dormitories and dining services income 108,349 451,994 Investment income used for current expenses : Endowment funds 83,984 Current fund investments 1,645 Gifts used for current expenses 127,301 Sundry income 175 Total current income 6o5,099 Excess of income 1 ,079 MARINE BIOLOGICAL LABORATORY STATEMENT OF CURRENT FUND Year Ended December 31, 1957 Balance January 1, 1957 $147,175 Excess of income over operating expenditures 1957 1,079 Balance December 31, 1957 $148,254 52 MARINE BIOLOGICAL LABORATORY MARINE BIOLOGICAL LABORATORY SUMMARY OF INVESTMENTS December 31, 1957 Cost Approximate % of Market Total Quotations Investment % of Income Total 1957 Securities held by Trustee: General endowment fund : U S Government bonds . $ 81,000 9.7 $ 81,000 6.8 $ 2,359 Other bonds 420,980 50.2 403,589 33.7 12,246 Preferred stocks . 501,980 85,788 59.9 10.2 484,589 71,713 40.5 6.0 14,605 3,370 Common stocks 251,097 29.9 641,355 53.5 28,312 838,865 100.0 1,197,657 100.0 46,287 General Educational Board ment fund : U S Government bonds endow - 25,000 15.3 25,000 9.5 749 Other bonds 70,530 43.0 68,813 26.1 2,327 Preferred stocks 95,530 27,281 58.3 16.7 93,813 24,337 35.6 9.2 3,076 1,130 Common stocks 41,006 25.0 145,471 55.2 5,608 163,817 100.0 263,621 100.0 9,814 Total securities held by Trustee $1,002,682 Investments of other endowment and un- restricted funds : Pooled investments : $1,461,278 67,323 Total investments of other en- dowment and unrestricted funds $ 304,058 Total investment income , Custodian's fee charged thereto Income of current funds temporarily invested in pooled securities $56,101 U S Government bonds = , B 833 Other bonds 138,302 58.4 141,416 57.1 3,105 Common stocks 138,302 98,433 58.4 41.6 141,416 106,213 57.1 42.9 3,938 5,676 236,735 100.0 $ 247,629 100.0 9,614 Other investments : U. S Government bonds 2,970 131 Common stocks 43,600 23,444 Real estate and mortgage 20,753 23,575 $33,189 89,290 (574) (204) Investment income distributed to funds $88,512 COELOMIC CORPUSCLES OF ECHINODERMS 1 RICHARD A. BOOLOOTIAN - AND ARTHUR C. GIESE Hopkins Marine Station of Stanford University, Pacific Grove, California Although a variety of corpuscles have been described during the last century by investigators of echinoderm perivisceral fluid, disagreement exists among the descriptions of different authors and a re-investigation of the problem with newer methods is desirable before the corpuscles of echinoderm perivisceral fluid can be properly characterized. These newer methods are primarily observation through the phase contrast microscope, so effective in Gregoire's studies (1953) on insect blood, and observation of cells unaltered by contact with air, glass or chemicals which Hensill (1949) found so useful in his study of crab blood. In addition, the study of all the transformations of a cell of a given type under gradually altered con- ditions discloses changes from one cell type to another in some instances. Further- more, a comparative study made possible a useful tentative classification of the cells found in fifteen species of echinoderms representing all the living classes of Echinodermata. MATERIALS AND METHODS The animals were collected in the vicinity of the Monterey Peninsula at low tide in some cases and by dredging in others. The animals were used as soon after col- lection as possible since starvation is known to alter clotting (Glavind, 1948). Cell types of each species were determined by the examination of fluid drawn from the perivisceral cavity with the aid of a siliconized syringe. A drop of the fluid was placed on a siliconized cover slip which was inverted over a depression slide, and examined immediately at magnifications of 43 X and 97 X and photographed periodically. The optical equipment consisted of a Spencer 18 ML phase microscope equipped with a Spencer phase turret condenser, bright contrast objectives and wide field oculars. The source of illumination was an Ortho-Illuminator-B (American Op- tical Co.), using 100-300 watt bulbs. The photomicrographic equipment used was a Kine-Exacta model VX camera coupled to a Leitz Micro-Ibso attachment. Exposures were made on Microfile film which was developed in D-ll developer and printed on single weight glossy surface DuPont Varigram paper. Since contact with air is known to alter the morphology of cells, the perivisceral fluid was taken up into evacuated capillaries. The capillaries were prepared by 1 Supported in part by National Science Foundation Grant GS-482 and Public Health Grant RG-4578 (C). We are also indebted to Dr. A. R. Moore for his sustained interest and suggestions, to Dr. L. Blinks for accommodations and suggestions, to Dr. R. L. Bolin for extending use of facilities and for helpful criticism, and to Mr. A. Farmanfarmaian for counsel and advice. - Now at the Department of Zoology, University of California at Los Angeles. 53 54 RICHARD A. BOOLOOTIAN AND ARTHUR C. GIESE pulling 5-mm. Pyrex tubing in such a manner that the capillary diameters never ex- ceeded 1 mm. The inner walls of the capillaries were coated with silicon (G.E. Dri-Film) by aspirating the reagent and subsequent drying. They were then flame-sealed at one end, evacuated, and flame-sealed at the other end in 7.5-cm. segments. Silicon was used because it coats the glass and prevents cytolysis of cells coming in contact with clean glass (Jacques ct at., 1946). Each capillary was scratched with a carborun- dum point half a centimeter from one end. The scratched end inserted through the peristome (echinoids) or a dermal branchia (asteroid) can be broken at the scratch by a slight pressure, and the body fluid is aspirated into the capillary. In the case of holothuroids a longer capillary, scratched in the center, was inserted into the interambulacral margin of the animal and broken in the middle in the same manner. The open tip of the capillary was covered with silicon grease upon removal. Then the capillary was placed on a slide in a channel filled with glycerine and covered with a cover slip. With this method it is possible to study types for at least five minutes before clotting appears, and to observe any changes which occur during this time. Furthermore, the capillary tubes can be rotated and the nature of the corpuscles as- certained in three dimensions. Clots also can be studied effectively in such prepara- tions. This method readily lends itself to photography. In order to determine which coelomic cells, if any, were phagocytic, one ml. of finely ground carmine suspension in sea water was injected by a syringe through the peristomial membrane in echinoids, through a dermal branchia in asteroids and through the body wall in holothuroids. At various time intervals, ranging from ten minutes to five days, hanging drop and capillary-tube preparations of the peri- visceral fluid were examined and photographed. CLASSIFICATION OF CORPUSCLES OF ECHINODERM BODY FLUIDS The results of the present study, documented in succeeding sections, revealed thirteen types of fairly distinct cells (see Tables I and II). Some of these cor- puscles appear to be phyletic in distribution, e.g., the bladder amebocytes (Fig. 1) and the filiform amebocytes (Fig. 2), the first of which occur in thirteen of the spe- cies examined and the latter in twelve of the species examined. As will be dis- cussed later, these two cell types are different phases of the same cell, e.g., in Pis- aster ochraceus. The small spherical amebocytes (Fig. 3) are found in three of the asteroids investigated and in the ophiuroid and the crinoid. The fusiform cor- puscle (Fig. 4), the vibratile corpuscle (Fig. 6), the eleocyte (Fig. 7), and hyaline hemocyte (Fig. 8) are found in the sea urchins only. The colorless spherical ame- bocyte (Fig. 5) is common both to the sea urchins and sand dollars. The other types of cells have a rather limited distribution. The large spherical corpuscle (Fig. 9) and the red corpuscle (Fig. 10) are found in the sand dollar and the cri- noid. The lobular corpuscle (Fig. 11), on the other hand, is limited to the crinoid only. The hyaline plasma amebocyte (Fig. 12) is found in the starfish Poraniopsis. Cells "staining" with osmic acid (Fig. 13) are observed only in the sand dollar. CORPUSCLES OF ASTEROIDS The fluid within the spacious coelomic cavity of the asteroids contains coelomo- cytes of fewer types than occur in other classes. Two main types of cells have been COELOMIC CORPUSCLKS OF ECHINODERMS 55 ~ r- V = .ti ^ .a X Z2 u'~ sii* X CN * ctf u 1 1 O i . 1 O X i i i i ^- X X C' C. ffl X X 0- .Son 13 ! X X X OO -- " u , ^ rt *"* X X X *"" 5 X X X X .0 C >8 i~3_ u b oJaj f X X X X X IJ-; !&! u rt ^S X X X X X -f _ o> = 31 ||| X X X X X re; tn o , s s o. O -" 4J X X X X X X X X X X X X C-J ^^ ^ U, ra || S X X X X X X X X X X X X X ^ JS E u S3 a to to Ji s R Species Astro pecten californicus Mediastcr aequalis a | ."> O R Patiria miniata P;yc no podia he! ia nthoides Pisaster ochraceus Pisaster giganteus Pisaster brevispinus Strongyloccntrotus purpurai Strongyloccntrotus francisca A rongyloccntrotlts frag U is Dendraster cxccntricus Gorgonocephaliis eucnemis Heliometra glacialis Stichopns californicus 0) -0 ~ OJ M (^ ^ R x, I a I 03 ^ a -Ci ' 56 RICHARD A. BOOLOOTIAN AND ARTHUR C. GIESE TABLE II properties of coelomic corpuscles Cell type Range or size in M* Color Granules Vacuoles Function Citation Bladder amebocyte 9-51 colorless, gray numerous black several phagocytic Kindred, 1921 Filiform amrlx 11 8-55 gray several black two-many clot, phagocytic Kindred, 1921 Small spherical amebocyte 4-8, 7-35 green, yellow, red black and red occasional clot Cuenot, 1888 Fusiform corpuscle 2-12X6-30 gray ? Cuenot, 1891 Colorless splierical amebocyte 8-1 2 X 13.6-28 pale yellow lobular ii li|iius calif ornicus, the species studied here. Phagocytes are found in all species so far studied. Various names such as cells with elongated pseudopodia (Herouard), hyaline ameboid corpuscles (Ohuye), and bladder amebocytes (Kindred, 1924) have been applied to them. The term, bladder amebocyte (Fig. 1), is preferable since the large bladder-like projections are readily observable when viewed three-dimensionally. The colorless spherule amebocytes (Fig. 5) were abundant in Stichopus cali- f ornicus. Hamann (1883) designated these as plasma wandering cells. Cuenot (1891), who identified them as muriform cells, considered the proteinaceous spher- ules to be food reserves. The homogeneous amebocytes, which lack inclusions, have been reported by Hamann (1883) and Becher (1907). This type of cell is rare and Hyman (1955) considers it a developmental stage of other cell types. It was not found in Stichopus. Theel, Kawamoto, and Ohuye observed crystal-containing cells in several spe- cies of holothuroids. The crystals are in the cytoplasm and are mostly rhomboidal in shape. No crystal-containing cells were observed in Stichopus. A cell type which has not been previously reported by investigators in holo- thuroids is the filiform amebocyte (Fig. 2). In Stichopus californicus these cells are actively involved in clot formation and also exhibit phagocytosis. DISCUSSION Many types of coelomic corpuscles have been described by various investigators of echinoderm body fluids, most of whom fixed and stained the cells or used live cells without preventing degenerative changes following contact with glass or air. As a consequence their results were not entirely convincing. In the present study in which pains were taken to avoid the above pitfalls, many of the same cell types COELOMIC CORPUSCLES OF ECHINODERMS 61 were seen. However, more confidence may now be attached to the cell types de- scribed by the earlier workers, since their appearance has been checked with live cells under conditions which at least delay changes in cells occurring with clotting or agglutination. Such coelomic cells as were not seen in the preparation made here, but which have been described by previous workers, may constitute additional cell types since the species used in the present study were not the same as theirs. Only future work using the same species of organism, can resolve this uncertainty. In the special case of the hemocytes hemoglobin-containing cells of certain holothuroids no question exists of their reality, even though they were not observed in the species of holothuroid used here (Stichopus calif ornicus) , since hemocytes have been ob- served in live specimens and recorded many times by various authors. Some types of coelomocytes were observed in the species examined here which had not been previously described, e.g. the red corpuscles of the sand dollar and the crinoid, and the lobular corpuscles of the crinoid. The existence of bladder amebocytes need no longer be questioned, even though the bladders appear to be petaloid rather than vesicular in fixed preparations (Good- rich, 1919). Examined in three dimensions, the bladder-like nature of the ecto- plasmic extrusions is readily observable. It was possible to resolve one controversy which occurs in the literature con- cerning the possible identity of the bladder amebocytes and the filiform amebocytes in asteroids. Theel (1919) and Kindred (1924) state these are merely phases of one another but cite no convincing evidence, and others question this conclusion. In observations on body fluids of several asteroids, the fresh sample showed a predominance of bladder amebocytes, but upon standing, the same preparation shows a predominance of filiform amebocytes. If the filiform amebocytes represent a pre-coagulation change, it should be possible to prevent this with an anti- coagulant such as cysteine. Cysteine-treated coelomic fluid was found to contain only bladder amebocytes when examined at various time intervals in Pisaster ochraceous body fluid. This experiment was repeated eight times with the same results. In the control, samples of coelomic fluid were treated with sea water equal in volume to the sample of anticoagulant and upon standing, both phases were seen. Whether such transformation occurs in all echinoderm coelomic fluids in which such cells are found remains to be seen. Some problems are presented by the present study of echinoderm coelomic fluid which may be of special interest to comparative and cellular physiologists. The function of the echinochrome-containing eleocytes and the various types of amebo- cytes still remains a challenge. The function of the vibratile corpuscles of the sea urchins, with the possibility that they represent parasites, is another example of an intriguing problem. The bladder amebocytes and the explosive amebocytes should serve as interesting material for a further study of ameboid movement. The mechanism of the transformation of bladder amebocytes to filiform amebocytes offers still another perplexing problem. The data so far gathered do not permit evolutionary speculations concerning the origin and diversification of the different types of coelomocytes. However, it cannot escape mention that a greater diversity of cell types appears in the body fluid of the more highly specialized forms, such as the echinoids, than in the 62 RICHARD A. BOOLOOTIAN AND ARTHUR C. GIESE asteroids. A more complete survey of the coelomic corpuscles of other species of each class, especially of the classes studied sparingly at present, may yield informa- tion making possible more generalizations than can be made now. SUMMARY 1. The cellular elements from the body fluid of 15 different species of echino- derms were studied by phase contrast microscopy. Thirteen types of corpuscular elements were identified and the distribution, properties, characteristics and, where possible, functions, were determined. 2. Some types of coelomocytes were observed in the species examined here which had not been previously described, e.g. the red corpuscles of the sand dollar and crinoid, and the lobular corpuscles of the crinoid. Some of the coelomo- cytes formerly described were also found in the species described. Among these are the controversial bladder amebocytes in which the presence of bladder has been questioned. Present studies verify the bladders as real structures easily seen in three dimensions. The bladder amebocyte undergoes a transformation into the filiform amebocyte which represents a pre-coagulation change. 3. A greater diversity of cell types was observed in the body fluid of the more highly specialized forms such as the echinoids than in the less specialized asteroids. LITERATURE CITED AWERINZEW, S., 1911. Uber die Pigment von S. droebachicnsis. Arch. Zool. Exp. Gen., ser. 5, 8 : i-viii. BECKER, S., 1907. Rhabdonwlogus ruber Keferstein und die Stammform der Holothurien. Zeitschr. iviss. Zool., 88: 545-689. BEHRE, E., 1932. A preliminary notice on the histology of the body fluid of Mcllita quinquies- perjorata. Anat. Rec., 54 (suppl.) : 92. BOLIEK, M., 1935. Syncytial structures in sponge larvae and lymph plasmodia of sea urchins. /. Elisha Mitchell Sci. Soc., 51 : 252-288. BOOKHOUT, C. G., AND N. D. GREENBURG, 1940. Cell types and clotting reactions in the echinoid, Mellita quinquiesperjorata. Biol. Bull., 79 : 309-320. CUENOT, L., 1888. fitudes anatomiques et morphologiques sur les ophiures. Arch. Zool. Exp. Gen., ser. 2, 6 : 3-82. CUENOT, L., 1891. fitudes sur le sang et les glandes lymphatiques dans la serie animale (2* partie: Invertebres). Arch. Zool. Exp. Gen., ser. 2, 9: 13-90, 364-475, 593-670. DURHAM, H. E., 1888. The emigration of ameboid corpuscles in the starfish. Proc. Roy. Soc. London, ser. B, 43: 327-330. ENDEAN, R., 1958. The coelomocytes of Holothuria leucospilota. Quart. J. Micr. Sci., 99: 47-60. GEDDES, P., 1879-1880. Observations sur le fluid perivisceral des oursins. Arch. Zool. Exp. Gen., 8: 483-496. GEDDES, P., 1880. On the coalescence of ameboid cells into plasmodia and on the so-called coagulation of invertebrate fluids. Proc. Roy. Soc. London, ser. B, 30: 252-255. GLAVIND, J., 1948. Studies on coagulation of crustacean blood. Nyt. Nordisk Forlag, Copen- hagen, 12-137. GOODRICH, E. S., 1919. Pseudopodia of the leukocytes of invertebrates. Quart. J. Micr. Sci., 64: 19-27. GREGOIRE, C. H., 1953. Blood coagulation in arthropods. III. Reactions of insect hemolymph to coagulation inhibitors of vertebrate blood. Biol. Bull., 104: 372-393. GRIFFITHS, A. B., 1892. Sur 1'echinochrome : un pigment respiratoire. C. R. Acad. Sci. Paris. 115: 419-420. HAMANN, O., 1883. Beitrage zur Histologie der Echinodermen. I. Die Holothurien, Pcdata, und das Nervensystem der Asteriden. Zeitschr. zviss. Zool., 39: 145. COELOMIC CORPUSCLES OF ECHINODERMS 63 HAMANN, O., 1889. Anatomic der Ophiuren und Crinoiden. Jen. Zeitschr. Naturwiss., 23: 233-388. HENSILL, J., 1949. Studies on blood coagulation in decapod Crustacea. Thesis, Stanford University. HEROUARD, E., 1889. Recherches sur les holothuries des cotes de France. Arch. Zool. Exp. Gen., ser. 2, 7: 535-704. HOGBEN, L., AND J. VAN DER LiNGEN, 1928. On the occurrence of hemoglobin and erythrocytes in the perivisceral fluid of a Holothurian. /. E.rf>. Bioi. 5: 292-294. HOWELL, W. H., 1885. The presence of hemoglobin in invertebrates. Johns Hopkins Univ. Circ. 5 (43). HOWELL, W. H., 1886. Notes on the presence of hemoglobin in echinoderms. Studies Biol. Lab. Johns Hopkins Univ., 3: 289-291. HYMAN, L., 1955. The Invertebrates. IV. The Echinodermata. AlcGraw-Hill Book Co., New York. JACQUES, B., E. FIDLAR, E. T. FELDSTED AND A. E. MACDONALD, 1946. Silicones and blood coagulation. Can. Med. Assoc. J., 55: 26-31. KAWAMOTO, N., 1921. The anatomy of Caudina chilcnsis with special reference to the peri- visceral cavity, the blood and water vascular system in their relation to the blood circulation. Sci. Rep. Tohoku Imp. Univ. Biol., 2: 239-264. KINDRED, J., 1921. Phagocytosis and clotting in the perivisceral fluid of Arbacia. Biol. Bull., 41: 144-152. KINDRED, J., 1924. The cellular elements in the perivisceral fluid of echinoderms. Biol. Bull., 46: 228-251. KOBAYASHI, S., 1932. The spectral properties of haemoglobin in the Holothurians, Caudina chilcnsis and Molpadia roretzii. Sci. Rep. Tohokn Imp. Unir. Biol.. 7: 211-227. KUHL, W., 1937. Die Zellelemente in der Liebeshohlenfliissigheit des Seeigels Psammechinus miliaris und ihr Bewegung physiologisches Verhalten. Zeitschr. Zclliorsch. mikro. Anat., 27: 1-13. KUHN, R., AND K. WALLENFELS, 1939. Uber die chemische Xatur des Stoffes den die Eier des Seeigels (Arbacia pustulosa) absondern, um die Spermatozoen anzulocken. Ber. Dtsch. Chew. Gcs., 72: 1407-13. LIEBMAN, E., 1950. The leucocytes of Arbacia pnnctnlata. Biol. Bull.. 98: 46-59. LISON, L., 1930. Recherches histophysiologiques sur les amebocytes des echinodermes. Arch. de Biol., 40: 175-203. MACMUNN, C. A., 1885. On the chromatology of the blood of some invertebrates. Quart. J. Micr. Sci., 25: 469-490. OHUYE, T., 1934. On the coelomic corpuscles in the body fluid of some invertebrates. I. Reaction of the leucocytes of a holothuroid, Caudina chilcnsis, to a vital dye. Sci. Rep. Tohoku Imp. Univ. Biol, 9: 47-52. OHUYE, T., 1936a. On the coelomic corpuscles in the body fluid of some invertebrates. IV. Reaction of the coelomic corpuscles of a holothuroid Molpadia roretzii with reference to those of Caudina chilcnsis. Sci. Rep. Tohoku Imp. Unir. Biol., 11 : 207-222. OHUYE, T., 1936b. On the coelomic corpuscles in the body fluid of some invertebrates. V. Reaction of the coelomic corpuscles of an echinid Temnopleurus hardwickii to vital dyes and some chemical reagents. Sci. Rep. Tohoku Imp. Unir. Biol., 11: 223-238. REICHENSPERGER, A., 1912. Beitrage zur Histologie und zum Verlauf der Regeneration bei Crinoiden. Zeitschr. u'iss. Zool., 101 : 1-69. SCHINKE, H., 1950. Bildung und Ersatz der Zellelemente der Leibeshnhlenfliissigkeit von P. miliaris. Zeitschr. Zellforsch. mikro. Anat., 35: 311-331. THEEL, H.. 1896. Remarks on the activity of ameboid cells in the echinoderms. Festschr. Lilljeborg, Uppsala, 47-58. THEEL, H., 1919. Om amoebycyteroch andra kroppar i. perivisceralhalan hos echinodermer. I. Asterias rubens. Arkiv. Zool., Stockholm, 12: 1-38. THEEL, H., 1921. On amebocytes and other coelomic corpuscles in the perivisceral study cavity of echnioderms. III. Holothuroids. Arkiv. Zool., Stockholm, 13: 1-40. VAN DER HYDE, H. C., 1922. Hemoglobin in Thyonc briareus Lesueur. Biol. Bull., 42: 95-98. THE ROLE OF THE BLOOD IN THE TRANSPORTATION OF STRONTIUM 90 -YTTRIUM 90 IN TELEOST FISH 1 ' 2 HOWARD BOROUGHS 3 AND DELLA F. REID Hawaii Marine Laboratory, University of Ha^vaii, Honolulu, Hawaii As the result of global fallout and the introduction of radioactive wastes from nuclear reactor plants into the oceans, marine organisms are being subjected to an environment which is potentially hazardous to themselves and to other members of the ecosystems involved. During the last few years, a study has been made in this laboratory of various aspects of the metabolism of radiostrontium by marine fish. These fish may pick up strontium directly from sea \vater, by way of the skin, gills, or by swallowing the water (Boroughs, Townsley and Hiatt, 1956). They may also take up this element from their food. In any event, the transporta- tion of strontium within the fish, including its excretion, depends upon its trans- portation by the blood, except for the strontium which is unabsorbed from the digestive tract. It is the purpose of this paper to report on certain aspects of the transportation of strontium 90 -yttrium t)0 in teleost blood. MATERIALS AND METHODS The species used in this experiment was Tilapia mossambica, a teleost fish. In- dividuals weighed between 50 and 110 grams each. They were kept in tanks supplied with running sea water. Two concentrations of Oak Ridge Sr 90 -Y 90 were prepared by dilution with saline solution approximately isotonic with Tilapia blood. Those fish which were to be bled a day or more after injection were given 100/>ic of Sr 90 , while the fish killed at shorter time intervals were given only 10 ju,c. In both instances the dose injected was 0.2 ml. The injections were made, and blood was withdrawn with the fishes' opercula in water. Separate fish were used for each time interval studied instead of using a single fish for repetitive bleedings. All the fish were handled as gently and uniformly as possible, and their eyes were covered with the hand. We believe this procedure results in a minimum of trauma. The Sr 90 -Y 90 dose was injected directly into the ventricle of the heart. At predetermined time intervals of 5, 15, 30, and 45 minutes and 1, 4, and 8 days, as much as possible of each fish's blood was withdrawn through the kidney sinus. A red blood cell count was made each time a fish was injected and again \vhen blood was removed. 1 Contribution No. 108 Hawaii Marine Laboratory, University of Hawaii. 2 This work was supported in part by contract No. AT(04-3)-56 between the U. S. Atomic Energy Commission and the University of Hawaii. 3 Present address : Institute Interamericano de Ciencias Agricolas, Turrialba, Costa Rica. 64 Sr" -Y so IN TELEOST FISH BLOOD 65 Immediately after removing the blood from the fish, triplicate 0.1-ml. samples were pipetted onto circles of one thickness of absorbent tissue on aluminum planchettes. Three-tenths-ml. aliquots of the remaining blood were centrifuged for 10 minutes at 2100 rpm in calibrated small bore hematocrit tubes in an Inter- national clinical centrifuge. The separated blood in one tube was used for measur- ing the radioactivity in the plasma and also that associated with the cells. From a second tube the plasma was removed without disturbing the packed cells. Five- hundredths ml. of these cells were washed by re-suspending them twice in fresh saline solutions. All the saline washings were pooled. In a third tube, the same volume of saline-washed cells was lysed with distilled water. The ghosts were washed with distilled water until no further radioactivity could be removed from them. The lysing solution containing the cell contents was added to the distilled water wash for measurement of the radioactivity of the cells exclusive of that bound to the stroma. Separated organs and tissues were ashed and prepared for counting as pre- viously described (Boroughs, Townsley and Hiatt, 1956). Radioactivity was measured with a thin window G-M tube using a commercial sealer. Counts were corrected for coincidence whenever necessary. In order to get an approximation of mixing time, Sr R5 was injected in the heart. Ten, 20, and 30 minutes later, blood was removed from the ventral aorta and from the kidney sinus, and 0.1-ml. samples were counted in a well scintillation counter with the aid of a single channel pulse height analyzer. RESULTS AND DISCUSSION Preliminary experiments Since very little is known about fish blood, we were at the outset faced with problems which were not pertinent to the main idea of this research. The first problem to be overcome was the bleeding, because apparently very few biologists have successfully removed blood directly from teleost fish (Prosser, personal com- munication). In general, fish have been bled by cutting the tail and allowing the blood to drip. Even more refined methods have involved the use of heparin, citrate, or other anticoagulants. We have found it difficult to withdraw unclotted blood from Tilapia if the fish had been kept out of water for even a short time. There is probably a dehydration of the blood in some species of fish as a result of asphyxiation (Hall, Gray and Lepkovsky, 1926). If Tilapia were stressed by prolonged chasing with a net, by rough handling or by repeated bleeding, removal of blood was difficult even though they were not taken from the water. The cell/ plasma ratio increased as it did with asphyxiation. We had previously observed red blood cell counts which varied between 1 and 4 X 10 6 /mm 3 in this species of fish, and other workers (Young, 1949) have ob- served similar large variations with other teleost fishes. Table I is a summary of the rbc counts of the fish used in this experiment and shows that these variations are not intrinsic and that it is possible to remove fish blood that has a reasonably small fluctuation in the rbc count. This blood does not clot even on prolonged standing at room temperature. The tremendous shift in the number of red blood cells observed in fish blood 66 HOWARD BOROUGHS AND DELLA F. REID Time interval between injection and killing 5 min. 5 min. 5 min. 15 min. 15 min. 30 min. 30 min. 45 min. 1 hr. 1 hr. 1 hr. 2 hr. 2 hr. 2 hr. 4hr. 4 hr. 8 hr. 8 hr. 1 day 1 daj' 2 days 2 days 4 days 4 days 8 days 8 days TABLE I Red blood cell count in Tilapia mossambica RBC/mm. 3 of blood Counted before Counted before dose injected blood withdrawn 1.444 1.150 1.375 1.350 1.209 1.548 1.175 1.125 1.200 1.162 1.050 1.223 1.148 1.150 1.151 1.209 1.199 1.011 1.312 1.649 1.100 1.298 1.103 1.271 1.150 1.018 X 10 6 X 10 6 X 10 6 X 10 6 X 10 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10" X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 X 10 6 1.627 1.423 1.400 2.050 1.374 1.525 1.400 1.460 1.600 1.384 1.025 1.347 1.326 1.220 1.169 1.137 X 10 6 X 10 6 X 10 6 X 10 6 X 10" X 10 6 X 10 6 X 10 10 6 10 6 X 10 X 10 X 10 6 X 10 6 X 10 6 X 10" X X 1.102 X 10 6 1.396 X 10 6 1.598 X 10 6 .199 X 10 6 .298 X 10 6 .362 X 10 6 .273 X 10 6 .175 X 10 6 could mean that the plasma, or some portion of it, either leaves the circulatory system or is in effect removed by some pocketing device. The increase in red blood cells may also result from the introduction into the blood stream of cells previously sequestered in an organ or tissue. Studies on fish blood volume and mixing time using either classical techniques or radioisotopes would be of little value if the fish were stressed. The circulation of fish blood is distinguished from that of higher animals in that oxygenated blood does not necessarily return to the heart. All the blood from the heart goes to the gills, but from the gills the blood may go to the head. TABLE II The mixing time of Tilapia blood Blood source Ventral aorta Ventral aorta Ventral aorta Kidney Kidney Kidnev Minutes elapsed 10 20 30 10 20 30 Counts/min. 249 79 51 40 45 50 Dose: 8477 cpm in 0.2 nil. injected into ventricle of heart. r^-Y 110 IN TELEOST FISH BLOOD 67 Q. O 140 120 100 80 60 40 20 O 120 o 100 80 60 40 20 Whole Blood Plasma a. o o o o 120 100 80 60 20 15 45 min. 2 HOURS III 1 o i i i i o i 4 12 | MRS. 2 3 4 5 6 7 8 DAYS AFTER DOSE FIGURE 1. The disappearance of Sr^-Y 90 from the whole blood and plasma of Tilapia inossainbica. 68 HOWARD BOROUGHS AND DELLA F. REID back to the heart, or to the remainder of the body. This means that mixing is a more complicated process in fish than it is in the higher animals. The results of studying mixing time in a single fish are shown in Table II. It can be seen that the bulk of the Sr s3 injected into the heart remained in the anterior portion of the fish, and that it required about 30 minutes for the blood from the ventral aorta and that from the kidney to reach the same level. Since we lack precise information about blood volume, we have assumed that it is roughly proportional to body weight. We have done this not only on the basis of our own work, but because Martin (1950) has suggested a similar relation- ship for other teleost fishes. Rate of disappearance of Sr 90 -Y 90 from the blood Figure 1 shows the rate of disappearance of Sr 90 -Y 9u from whole blood and plasma. The numbers have been corrected for body weight. The activity is given in counts/min./ml. whole blood and cpm in the plasma present in 1 ml. of whole blood. Each point on the curve represents the average activity from at least two fish. It can be seen that practically all the radioactivity in the whole blood is carried in the plasma, and that the formed elements can be responsible for only a very small amount. The two curves are practically superimposable. The small inserts on this graph show the appearance of radioactivity during the first few hours, and the larger graph extends the curves to 8 days. Since all the radioactivity was injected into the heart at zero time, at first glance it may seem odd that the amount of radioactivity recoverable from the blood increases up to 30 minutes. However, Table II indicates that this apparent increase is a reflection of the mixing time. At least two processes are occurring during this time which make it extremely difficult to find out exactly how much radioactivity is in the blood system. First, the isotopes are being excreted as soon as they appear in the blood, at first principally by way of the gills. Second, radioactivity is rapidly accreted by the various organs and tissues, and thus the concentration is decreasing continuously. We would like to emphasize that it is the resultant of these proc- esses that is being measured. The radioactivity was very rapidly lost from the blood during the next 30 minutes, and after 24 hours, only between 0.8 and 1.6 per cent of the injected dose remained in the blood, assuming a blood volume of 2-4 per cent of the body weight. The shape of the curves shows that more than one rate process is involved in the disappearance of the radioactivity from the blood. It must be emphasized at this point that the above samples were counted at least three weeks after the fish was killed, so that we were observing the radioactivity in an equilibrium mixture of Sr 90 -Y 90 . Strontium 110 has a half-life of about 28 years and a maximum beta energy of 0.61 Mev. It decays to form radioactive Y 90 which has a half-life of 2.54 days and a maximum beta energy of 2.18 Mev. Secular equilibrium exists when the Y 90 decays as fast as it is formed, and the radioactivity of such a mixture is the sum of the radioactivity of the separate isotopes. In an equilibrium mixture, therefore, no decay of radioactivity would be ob- servable during this experiment unless the two isotopes were separated by either biological or physico-chemical processes. Such a fractionation can be detected by following the counting rate of a sample daily. No changes in this rate will be Sr^-Y 90 IN TELEOST FISH BLOOD 69 observed if no fractionation has occurred. If the rate increases, Y 90 has been removed and is building up to its equilibrium value at which point it will level off. If the rate decreases, the bulk of the radioactivity must be due to the Y 90 which is decaying, and the counts will decrease until a level is reached which is a function of the amount of Sr 90 present. The role of the blood fractions in the transport of Sr 90 -Y 90 The increase in the counts/minute of the whole blood and plasma in Figure 2 is due to the build up of Y 90 . There are two simple explanations for the loss of yttrium from the blood. One is that the yttrium was lost prior to its appearance in the blood initially, that is, adsorbed to the glassware used in making the dilutions o plasma v whole blood 8 9 10 II 12 13 14 15 16 FIGURE 2. The increase with time of radioactivity in samples of whole blood, plasma, and the dose, indicating the build-up of Y 90 . and injections. The second explanation is that the yttrium was lost to various organs and tissues through which the blood passed. These explanations are not mutually exclusive and we believe that both processes occur. In Figure 3, the curve labelled "dose" was obtained by counting planchettes prepared from the Sr 90 -Y 90 present in the syringe used for injections. It can be seen that over a period of time, the cpm increased, indicating that some Y 90 was lost from the equilibrium mixture. This Y 90 was lost to the glassware. The curve for whole blood and plasma, however, increased to a much higher value, indicating that additional Y 90 had been removed after the dose was injected. Figure 3 shows the rate of radioactive decay of the washed and unwashed cells, the saline washings, the washed ghosts, and the distilled water washings which include the cell contents. The decay of the unwashed cells suggests that both Sr 90 and Y 90 were associated with the cells. The decay of the washed cells, saline 70 HOWARD BOROUGHS AND DELLA F. REID wash, and ghosts, however, suggests that the Sr 90 is readily removable either from or through the cell wall. The activity remaining in the washed cells and ghosts indicates it to be Y 90 , because the decay rates are very similar to the rate for pure Y 90 . All these conclusions are in harmony with the findings of Thomas et al. (Thomas, Litovitz, Rubin and Geschickter, 1950). who showed that radiocalcium. metabolically similar to strontium, was carried in the plasma of rabbit blood. 800 600 400 ~~i 1 1 1 1 1 r 1 O n O -LI O ^ O l I 1 O O i L_u *%. n w a s h e d cells 130 - LL) I- ID Z a: UJ a. tn i- O O 100- di st. H was h , XX * FIGURE 3. The radioactive decay of washed and unwashed cells, the saline and distilled water wash, and the cell ghosts. The decay of washed cells and ghosts indicates that they pick up Y 90 rather than Sr 80 . 90 Retention and distribution of Sr go -Y Figure 4 shows the retention by the fish of the injected Sr 90 -Y 90 as a function of time. The upper curve represents the entire fish, and the other curves represent, respectively, the bone, integument, gills, muscle, and visceral organs. Each point is the average of at least two fish, and the samples were counted at secular equilibrium. These results may be compared with those obtained previously by Sr^-Y 90 IN TELEOST FISH BLOOD 71 LJ k"^^ .gills pmuscle ^rJ .2 -T -r- -T- HRS. 1 3 4 DAYS 1 1 I 567 t 8 Time interval % injected dose remaining (samples at secular equilibrium) Total of fish* Bones Integument Gills Muscle Visceral organs 5 min. 98.2 13.2 13.4 13.4 3.1 3.4 15 min. 95.9 30 min. 93.2 39.3 8.2 17.3 11.9 4.0 1 day 92.1 61.7 19.2 4.9 4.5 1.4 4 days 81.5 53.7 17.1 4.7 4.2 1.4 8 days 76.6 50.2 16.2 7.4 3.3 1.4 Including blood. 72 HOWARD BOROUGHS AND DELLA F. REID Biological fractionation of Sr 90 -Y Three fish were injected with Sr 90 -Y 90 and killed five minutes, 30 minutes, and one day later. Since the amount of separation of the two isotopes by the glassware was unknown, it is not possible to draw a curve showing the rate of decay of the radioactivity in the various organs that would be a true measure of the decay due to the fractionation by the organs themselves. The planchettes were counted one day after the fish were killed, and this value was taken as a base line. They were then counted until secular equilibrium had been re-established. Table IV shows the percentage increase or decrease in radioactivity in the various organs with respect to the radioactivity present at one day. TABLE IV" Fractionation of intracard tally injected 5V 90 -]' 90 by organs and tissues of Tilapia mossambica Organ or tissue sample % Decrease of activity 1 day to secular equilibrium Organ or tissue sample f "c Increase of activity 1 day to secular equilibrium Liver 53.1 Gills 73.8 Gall bladder 42.5 Stomach 22.3 Heart 38.6 Brain 18.3 Kidney Spleen Gonads 18.8 10.6 8.5 Muscle Intestine Eyes Urinary Bladder Skin 16.3 10.4 6.9 5.6 5.0 Urine 28.5 Blood clots Scales Fat 26.8 14.1 11.4 Feces 2.1 It can be seen that the first two columns represent the organs which concen- trated Y 90 more than they did Sr 90 , while the last two columns represent organs that favored the Sr. In general, the more vascular organs and tissues preferred yttrium. SUMMARY 1. Blood can be easily removed without clotting from the heart or kidney sinus of fishes if the fish are handled gently and their opercula are kept immersed. 2. Blood so removed has a uniform number of red blood cells/mm 3 . 3. The mixing time of Sr 90 -Y 90 injected in the ventricle of Tilapia mossambica, a teleost fish, is approximately 30 minutes. 4. Sr 90 -Y 90 rapidly disappears from the blood. At 24 hours, only between 0.8 and 1.6 per cent of the injected dose remains in the blood. 5. The disappearance of radioactivity from the blood depends on more than a single process. 6. Almost all of the Sr 90 in whole blood is carried by the plasma. 7 '. Very little Sr 1 ' is found either in the cells or on the cell walls. 8. Yttrium 90 , on the other hand, is present in the stroma. Sr^-Y 90 IN TELEOST FISH BLOOD 9. The pattern of internal distribution of intravascularly injected Sr 90 -Y 90 is the same as that which was found for either intramuscular or oral administration in the same species. 10. Vascularized tissues have a greater avidity for Y m) than they have for Sr 90 LITERATURE CITED BOROUGHS, H., S. J. TOWNSLEY AND R. W. HIATT, 1956. The metabolism of radionuclides by marine organisms. I. The uptake, accumulation, and loss of strontium s9 by marine fishes. Biol. Bull, 111: 336-351. HALL, F. G., I. E. GRAY AND S. LEPKOVSKY, 1926. The influence of asphyxiation on the blood constituents of marine fishes. /. Biol. Chem., 67 : 550. MARTIN, A. W., 1950. Some remarks on the blood volume of fish. Studies Honoring Trevor Kincaid. Univ. of Washington Press, pp. 125-140. THOMAS, R. O., T. A. LITOVITZ, M. I. RUBIN AND C. F. GESCHICKTER, 1950. Dynamics of calcium distribution. Time distribution of intravenously administered radiocalcium. Amer. J. Physiol., 169: 568-575. YOUNG, R. T., 1949. Variations in the blood cell volume of individual fish. Copeia. Sept. 15, 1949, No. 3. DISPERSAL OF THE GELATINOUS COAT MATERIAL OF MELLITA QUINQUIESPERFORATA EGGS BY HOMOLOGOUS SPERM AND SPERM EXTRACTS 1 JOHN W. BROOKBANK Department of Biology, University of Florida, Gainesville, Florida Live sperm or sperm extracts of a number of animal species have been found to possess the property of solubilizing or dispersing the secondary and tertiary envelopes normally surrounding the unfertilized eggs of these species, thus fa- cilitating the approach of the sperm to the egg surface. Groups in which this phenomenon has been demonstrated include amphibians (Hibbard, 1928; Wintre- bert. 1929 and 1933), mammals (see reviews by Duran-Reynals, 1942; Meyer, 1947; Meyer and Rapport, 1952), gastropods (Tyler, 1939 and 1948; von Medem, 1942), and bivalves (Berg, 1949). In addition, a number of \vorkers have de- scribed the solubilization of the gelatinous coat material (fertilizin) of echinoid eggs by live sperm or sperm extracts. Hartmann ct al. (1940) extracted the residue of methanol-precipitated seminal fluid of Arbacia pushdosa with sea water and reported that the resulting solution was capable of dispersing the gelatinous coat material of unfertilized Arbacia eggs. This extract was also capable of neutralizing the sperm agglutinating property of Arbacia fertilizin, and thus pos- sessed antifertilizin activity. Monroy and Ruffo (1947) described an acid extract of sea urchin sperm which was reported as acting to dissolve the fertilizin of unfertilized eggs. Others have described a decreased viscosity of fertilizin solu- tions in the presence of live sperm or sperm extracts (Lundblad and Monroy. 1950; Vasseur, 1951 ; Monroy and Tosi, 1952; Monroy et al., 1954). It has been emphasized (Tyler and O'Melveny, 1941 ; Krauss. 1950; Monroy and Tosi, 1952; Monroy et al., 1954) that apparent dispersal of the gelatinous coat of unfertilized eggs by sperm or sperm extracts, as well as the decrease in viscosity observed when live sperm or extracts are added to fertilizin solutions, can be accounted for by precipitation of fertilizin by antifertilizin present in the extracts or on the surface of the live sperm. Therefore, any investigation of supposed lytic or dispersing agents from sperm must include experiments which demonstrate that the activity of the agent is separable from the activity of antifertilizin. Ishida (1954) has presented evidence that a fertilizin-dissolving factor is released at fertilization from the sperm of Heiniccntrotus pulchcrrimus. Treatment of the sperm with fertilizin, which rendered the sperm non-fertilizing, did not prevent the solution of the fertilizin coat of the eggs by these sperm. This latter observation tends to eliminate antifertilizin as the agent responsible for removing the fertilizin from the eggs. However, though the sperm concentration employed in the experiments was not stated, sperm carbon dioxide might have been responsible for the solubilizing action 1 This investigation was supported in part by a research gfant (RG 4659 s ) from the Na- tional Institutes of Health of the Puhlic Health Service. 74 GELATINOUS COAT DISPERSAL BY SPERM 75 of the sperm. Heated (100 C. for one minute) sperm failed to cause dispersal of the fertilizin coat. This failure of heated sperm to cause dispersal of the gelatinous coat has been ascribed, by Ishida, to the clenaturation of a dispersing agent on the sperm. Alternatively, the denaturation of respiratory enzymes, re- sulting in loss of motility and decreased carbon dioxide production, could account for the failure of heated sperm to solubilize the fertilizin of the eggs. The present report bears evidence that a factor, which is distinct from anti- fertilizin and which is capable of causing the dispersal of the gelatinous coat of unfertilized eggs, is present in sperm extracts and on the surface of live sperm and of the sand dollar, Mellita quinquiesperforata. MATERIALS AND METHODS Animals were collected by dredging on the shallow banks surrounding the University of Florida Marine Laboratory at Sea Horse Key. The animals were transported to Gainesville and kept in the laboratory at 12 C. in sea water supplied with a continuous Mow of washed, compressed air. Under these con- ditions the sand dollars remained alive for approximately two weeks. Eggs and sperm were obtained by injecting the animals with an isotonic KC1 solution (Tyler, 1949). For use in experiments in which live sperm were employed, the "dry" sperm were diluted to a concentration of 5 ( / f with filtered sea water. Where separation of sperm from the surrounding Muid was desired, the suspensions were centrifuged at 2900 X gravity in a Sen-all SS-1 centrifuge for 10 minutes. The supernatant Muids were collected and tested for dispersing activity on the gelatinous coat of fresh unfertilized eggs. The sedimented sperm were suspended in the original volume of fresh sea water and tested for their ability to disperse the gelatinous coat of the eggs. Sperm extracts were prepared from washed sperm in the following manner. Two volumes of sea water were added to the sperm following centrifugation and re- moval of seminal Muid, and the resulting 30% suspension was frozen at -- 20 C. for 2 to 12 hours. The frozen suspension was then homogenized in an ice bath, using a Potter homogenizer with a motor-driven pestle. After homogenization, the sus- pension was centrifuged at 11,000 X gravity for 15 minutes in a Servall SS-1 centri- fuge. This procedure yielded a gray precipitate, which was discarded, and an opal- escent supernatant Muid, which was used as the final sperm extract preparation. In assaying for the dispersing action of sperm and sperm extracts on the gelati- nous coat of the eggs, advantage was taken of the presence of echinochrome granules in the gelatinous coat. As can be seen in Figure la, where the outer boundary of the coat has been outlined with antifertilizin, the granules normally have a rather regular position in the gelatinous coat. The locus of this position could be described as a spherical shell lying midway between the outer surface of the gelatinous coat and the surface of the egg. In practice, a small number of freshly shed eggs were transferred with a pipette from the vessel in which they had been allowed to settle, to fresh sea water, and used in the various tests. Dispersal of the egg coat could be followed by noting the length of time required for the echinochrome granules to fall to the bottom of the culture dish, due to the dispersal of the gelatinous matrix in which they were embedded. The time at which the granules were released repre- sented the time at which approximately half the gelatinous coat had been dispersed. 76 JOHN W. BROOKBANK and was taken as the end-point of the reaction. Naturally, every effort was made to insure that the eggs used in the experiments possessed comparable amounts of gelati- nous coat material outside the layer of granules. In practice, this was not particu- larly troublesome since the egg coat of Mellita eggs is quite rigid and not readily soluble in sea water, and since handling of the eggs was restricted to a single transfer to fresh sea water following shedding. la Ib * ft FIGURE la. Unfertilized egg of Mellita treated with heated (70 C. for 10 minutes) sperm extract. Final magnification: 175 X. FIGURE Ib. Unfertilized egg of Alellita treated with heated sperm extract for 30 minutes, followed by treatment with unheated extract for 15 minutes. Final magnification: 175 X. RESULTS When one drop of 5% sperm suspension was added to one drop of egg suspen- sion (containing about 200 eggs), the gelatinous coat was dispersed in approxi- mately 10 minutes. Elevation of the fertilization membrane did not result in the dispersal of the gelatinous coat of control eggs which were washed and removed to sea w r ater following exposure to the sperm suspension. When the above sperm sus- pension was centrifuged at 2900 X gravity for 10 minutes, and the supernatant fluid decanted and tested, it was found to be inactive (no dispersal of the gelatinous coat occurred even after 12 hours of exposure to the sperm supernatant). The sedi- mented sperm, on the other hand, remained able to disperse the gelatinous coat ma- terial after being diluted to the original concentration with sea water. GELATINOUS COAT DISPERSAL BY SPERM 77 Acidification of a 5% sperm suspension to pH 4, followed by return to pH 8 after 2-4 minutes, with subsequent centrifugation at 2900 X gravity to recover sperm and supernatant fluid, resulted in the loss of the capacity of the sperm to disperse the fertilizin coat. The sperm, in most instances, remained motile following the treat- ment. The supernatant fluid under these conditions occasionally showed slight anti- fertilizin activity, as evidenced by the formation of a slight precipitation membrane on the gelatinous coat, but remained inactive with respect to the dispersal of the gelatinous coat. The fertilizing capacity of such acid-treated sperm was reduced, perhaps due to the loss of antifertilizin from the sperm surface (Tyler and O'Mel- veny, 1941), or to the loss of the ability to disperse the gelatinous coat, or both. Sperm extracts, prepared as described in the previous section, were also ca- pable of dispersing the gelatinous coat of unfertilized eggs. The final extracts, pre- TABLE I The effect of temperature and pH on dispersing activity of Mellita sperm extract. Activity of extracts assayed at 25 C. Date of preparation of extract Exposed to pH Tested at pH Dispersal time Antifertilizin activity Dec. 13 7 7 30' Dec. I') 7 7 30' Dec. 5 4 8 fan. 17 4 8 Dec. 5 9 8 30' fan. 3 9 8 30' Jan. 17 8 8 15' Dec. 5 8 8 15' Jan. 3 8 8 10' Dec. 5* 8 8 + + + Dec. 19* 8 8 + + + Dec. 19* 8 8 + + + * Indicates heated extract (70 C. for two minutes). - Indicates no dispersing activity evident. pared from frozen-thawed sperm, had a pH varying between 6.9 and 7.1, depending on the particular preparation. Extracts at this pH range were capable of dispersing the gelatinous coat in 30 minutes at 25 C. Control eggs in sea water at pH 6.9 showed no release of echinochrome granules for 6 hours or more. Exposure of active sperm extracts to pH 4 for 3-5 minutes, followed by return to the original }>H, inactivated the dispersing factor. The optimum pH for dispersing activity ap- peared to be 8, since dispersal occurred in approximately 10 minutes at this pH. Alkalinization to pH 9, followed by return to pH 8 after 3-5 minutes, partially in- activated the dispersing factor, dispersal occurring after 30 minutes in these prepa- rations. Heating to 70 C. for two minutes in a water bath completely inactivated the dispersing factor. The heated extracts possessed definite antifertilizin activity (Table I). The heat stability of antifertilizin from sperm of sea urchins (Frank, 1939), and the key-hole limpet (Tyler, 1939) has been previously described. Weak antifertilizin activity was also evident in untreated extracts at pH 8. A faint precipitation membrane appeared on the surface of the fertilizin coat about two 78 JOHN W. BROOKBANK minutes after the addition of extract, but disappeared after 5 minutes under the in- fluence of the dispersing factor. This point was further illustrated by experiments with heated extracts possessing stronger antifertilizin activity. The heated ex- tracts (70 C. for 5-10 minutes) formed definite precipitation membranes on the fertilizin coat after two minutes (Fig. la). The precipitation membrane so formed did not contract to the egg surface, but remained in the position in which it was originally formed for a*- long as 24 hours (with no dispersal of the egg coat). Ad- dition of unheated sperm extract at pH 8 caused the disappearance of the precipita- tion membrane, and, after 10-15 minutes, the dispersal of the gelatinous coat (Fig. Ib). Experiments involving the addition of extract to eggs up to five minutes prior to the addition of sperm showed that fertilization is not enhanced by this treatment. The fertilization membrane was elevated within three minutes regardless of the presence or absence of the extract. The sperm are apparently supplied with enough dispersing factor to make their way through the gelatinous coat material. More- over, the gelatinous coat is not dispersed by the dilute suspensions usually employed for insemination. In this respect, the situation parallels that of hyaluronidase of mammalian sperm, in that hyaluronidase added to inseminates does not enhance fertilization (Chang, 1947; Leonard et al., 1947). The Mellita sperm extracts were not tested on eggs of other species, and the de- gree of specificity of the dispersing agent is therefore not known at this time. An extract of frozen Arbacia piinctulata sperm (5% suspension) failed to cause the re- lease of the echinochrome granules of Mellita eggs, but showed strong antifertilizin activity (the precipitation membrane with enclosed granules contracted to the egg surface in 5 minutes). Addition of extract of Mellita sperm, at pH 8.0, caused the gradual disappearance of the precipitation membrane formed by the Arbacia anti- fertilizin, and release of the echinochrome granules following dispersal of the fer- tilizin coat. In this connection, it is of interest that a fresh suspension of Mellita sperm was capable of fertilizing eggs treated with Arbacia antifertilizin. in the presence or absence of extract of Mellita sperm, indicating the ability of live sperm to penetrate precipitation membranes. Further experiments were performed in the hope of discovering the means by which the Mellita sperm extract accomplished the dispersal of the gelatinous coat. A fertilizin solution, prepared by acid (pH 4) treatment of Mellita eggs, with a sperm-agglutination titer of 1 : 1000, was gently shaken with an equal volume of sperm extract at pH 8 for 60 minutes at 25 C. No decrease in titer of the fertilizin solution was evident at the end of the experiment. The sperm extract used in this experiment had been previously shown to disperse the gelatinous coat in 10 minutes, and showed no sperm-agglutinating property. The experiment indicated that no degradation of the fertilizin molecule resulting in loss of agglutinating activity oc- curred in the presence of the extract. Most probably, the dispersal of the gelatinous coat by sperm extract is accomplished by depolymerization of the gelatinous coat material, and not by splitting of individual fertilizin molecules. DISCUSSION Since the dispersing factor is heat-labile under conditions where antifertilizin is stable, it seems reasonable to consider them to be separate substances. This con- GELATINOUS COAT DISPERSAL BY SPERM 79 elusion is supported by the failure of strong antifertilizin solutions to cause dis- persal of the gelatinous coat, even though a precipitation membrane forms and, in some instances, contracts to the surface. One can distinguish, therefore, between the dispersal of the gelatinous coat and its precipitation. Further, acid treatment of sperm or sperm extracts, a procedure not infrequently used for antifertilizin extrac- tion from whole sperm (Tyler and O'Melveny, 1941), results in the inactivation of the dispersing factor, again indicating that the dispersing factor and antifertilizin are separate substances. Since the respiration of such acid-treated sperm is most probably normal (Tyler and O'Melveny, 1941), carbon dioxide is probably not involved in the dispersal of the gelatinous coat. The observed temperature and pH sensitivity of the dispersing factor suggest that it is protein in nature, possibly an enzyme. The dispersing factor of Mellita sperm apparently does not act on fertilizin in solution, but only serves to liquify or disperse the gelatinous coat. If the fertilizin, in the gel state, is bound by cross linkages involving the area of the molecule ca- pable of combining with antifertilizin, as Tyler (1948) suggests, the dispersing factor may operate by breaking such cross linkages, thereby releasing fertilizin from the gel. Further, the dissolution of fertilizin-antifertilizin precipitation membranes by extracts containing the dispersing factor may be due to the breaking of linkages at the fertilizin-antifertilizin combining site. Further experiments are necessary be- fore the relationship of the dispersing factor to the fertilizin-antifertilizin reaction can be stated with certainty. SUMMARY 1. A factor causing the dispersal of the gelatinous coat of Mellita eggs was shown to be present on the surface of Mellita sperm and in frozen-thawed extracts of sperm suspensions. 2. The factor was separable from antifertilizin on the basis of temperature and pH sensitivity. 3. The factor did not degrade fertilizin in solution, but released this substance from the gel surrounding the egg. 4. Active extracts were capable of dissolving fertilizin-antifertilizin precipitation membranes, formed on the surface of the fertilizin coat of unfertilized eggs in the presence of Arbacia or Mellita antifertilizin. LITERATURE CITED BERG, W. E., 1949. Some effects of sperm extracts on the eggs of Mytilus. Amer. Nat., 83: 221-226. CHANG, M. C, 1947. Effects of testis hyaluronidase and seminal fluids on the fertilizing ca- pacity of rabbit spermatozoa. Proc. Soc. Exp. Biol. Med., 66: 51-54. DURAN-REYNALS, F., 1942. Tissue permeability and the spreading factors in infection. Bact. Rev., 6: 197-252. FRANK, J. A., 1939. Some properties of sperm extracts and their relationship to the fertilization reaction in Arbacia punctnlata. Biol. Bull., 76: 190-216. HARTMANN, M., O. SCHARTAU AND K. WALLENFELS, 1940. Uber die Wechselwirkung von Gyno- und Andro-Gamonen bei der Befruchtung der Eier des Seeigels. Naturwiss., 28: 144. HIBBARD, H., 1928. Contribution a 1'etude de 1'ovogenese, de la fecondation et de 1'histogenese chez Discoglossus pictits. Otth. Arch. Biol., 38: 251-326. 80 JOHN W. BROOKBANK ISHIDA, J., 1954. Jelly-dissolving principle released from sea-urchin sperm at the time of fertilization. /. Fac. Sci. Tokyo, Sect. IV , Zoology, 7 : 53-59. KRAUSS, M., 1950. On the question of hyaluronidase in sea urchin spermatozoa. Science, 112: 759. LEONARD, S. L., P. L. PERLMAN AND R. KURZROK, 1947. Relation between time of fertilization and follicle cell dispersal in rat ova. Proc. Soc. Exp. Biol. Med., 66: 517-518. LUNDBLAD, G., AND A. MoNROY, 1950. Mucopolysaccharase activity of sea-urchin sperms. Ark. f. Kemi, 2: 343-347. VON MEDEM, F. G., 1942. Beitrage zur Frage der Befruchtungs-stoffe bei marinen Mollusken. Biol. Zentr., 62 : 431-446. MEYER, K., 1947. The biological significance of hyaluronic acid and hyaluronidase. Physiol. Rev., 27 : 335-359. MEYER, K., AND M. M. RAPPORT, 1952. Hyaluronidases. Adv. in Ensymol., 13 : 199-236. MONROY, A., AND A. RUFFO, 1947. Hyaluronidase in sea-urchin sperm. Nature, 159 : 603. MONROY, A., AND L. Tosi, 1952. A note on the jelly-coat-sperm interaction in sea urchins. Experientia, 8: 393-394. MONROY, A., L. Tosi, G. GIARDINA AND R. MAGGIO, 1954. Further investigations on the inter- action between sperm and jelly-coat in the fertilization of the sea urchin egg. Biol. Bull., 106: 169-177. TYLER, A., 1939. Extraction of an egg membrane lysin from sperm of the giant key-hole lim- pet (Mcgathura crenulata). Proc. Nat. Acad. Sci., 25: 317-323. TYLER, A., 1948. Fertilization and immunity. Physiol. Rev., 28: 180-219. TYLER, A., 1949. A simple, non-injurious method for inducing repeated spawning of sea urchins and sand dollars. Coll. Net, 19: 19-20. TYLER, A., and K. O'MELVENY, 1941. The role of antifertilizin in the fertilization of sea urchin eggs. Biol. Bull, 81 : 364-374. VASSEUR, E., 1951. Demonstration of a jelly-splitting enzyme at the surface of the sea-urchin spermatozoon. Exp. Cell Res., 2 : 144-146. WINTREBERT, P., 1929. La digestion de 1'enveloppe tubaire interne de 1'oeuf par des ferments issus des spermatozoides, et de 1'ovule, chez Discoglossus pictus. Otth. C. R. Acad. Sci. Paris, 188 : 97-100. WINTREBERT, P., 1933. La fonction enzymatique de 1'acrosome spermien du Discoglosse. C. R. r. Biol., 122 : 1636-1640. AN EXOGENOUS REFERENCE-CLOCK FOR PERSISTENT, TEMPERATURE-INDEPENDENT, LABILE, BIOLOGICAL RHYTHMS * 2 FRANK A. BROWN, JR. Department of Biological Sciences, Northwestern University, Evanston, Illinois The phenomenon of persistent rhythmicity of one or more of their vital processes is widespread among animals and plants. By persistent rhythmicity is meant that the rhythm still continues when conditions are held constant with respect to all fac- tors generally conceded to influence the organisms. Reviews of this subject have included those by Biinning (1936, 1956a, 1956b), Jores (1937), Kalmus (1938), Welsh (1938), Park (1940), Kleitman (1949), Calhoun (1944, 1945-46), Korringa (1947), Webb (1950), Caspers (1951), Cloudsley-Thompson (1953), Bruce and Pittendrigh (1957), and Brown (1957d, 1958). The broad distribution of such rhythmicity is suggestive of an hypothesis that all living things have potentially the means of persistent rhythmicity provided it has a period close to that of one of the natural geophysical rhythms. The organis- mic rhythms usually are essentially temperature-independent in their frequencies, whether the periods are solar-daily, lunar or annual. Most of the observed rhythms are clearly endogenous, and are labilely adaptable in form and phase relationships to the needs of the organism. Much has been learned, particularly in recent years, as to the properties, including modifiability, of this endogenous rhythmicity. The fundamental problem, however, that of the tim- ing mechanism of the rhythmic periods, has largely eluded any eminently reasonable hypotheses in terms of cell physiology or biochemistry. The problem was already a difficult one when only solar-daily cyclicity was under consideration, but especially in recent years it has been found that one and the same organism may simultaneously possess overt daily and lunar tidal cycles of two bodily processes. Further, the possession of persistent lunar monthly (Brown, Bennett and Webb, 1958) and even annual cycles (Biinning and Miissle, 1951 ; Biinning and Bauer, 1952; Brown, 1957c) in constant conditions has emphasized the magnitude and complexity of this basic problem. Added to the property, temperature-independence, in indicating the unconven- tional character of the rhythm-timing mechanisms, are the repeated demonstrations of the immunity of the frequency-determining mechanism to most metabolic poisons. Recently, evidence has been rapidly accumulating pointing to the possession by living organisms of basic metabolic cycles of the natural geophysical frequencies, 1 This study was aided by a contract between the Office of Naval Research, Department of the Navy, and Northwestern University, NONR-122803. 2 The author wishes to express his appreciation to the several students who worked many hours in assisting to obtain and process the data used here. Thanks are especially due to Messrs. W. D. Korte, who handled the carrot experiments, E. F. Lutsch, F. H. Barnwell, E. J. Macey, H. Gibson, Jr., and Misses J. Strunk and B. Getting. 81 82 FRANK A. BROWN, JR. produced in the organism by an external cyclic stimulus still operative in so-called laboratory constant conditions (Brown, 1957; Brown, Shriner and Webb, 1957; and Brown, Webb and Macey, 1957). These cycles are not phase- or form-labile. The problem of a common explanation for persistent rhythmicities of all the well- known natural frequencies including the year becomes at once more susceptible to reasonable working hypotheses as to their mechanism when it is firmly estab- lished that protoplasm in "constant conditions" is, fundamentally, exogenously rhythmic. For the study to be reported here, the potato and carrot were selected as organ- isms neither of which appears to possess any obscuring, labile, endogenous rhythms. It was considered that such organisms would reveal most readily any extant basic protoplasmic cyclicities and also permit easier analysis of any mechanisms they involved. On the basis of this hypothesis, of an exogenous reference clock providing the timing of cyclic periods, the often-described endogenous rhythms would be con- sidered a consequence of the evolution by the organism of adaptive labile cyclic changes, utilizing the basic exogenous cycle-timing mechanism. The endogenous mechanisms could be inherited. The only inherited aspect of the exogenous cyclicity would be the fundamental protoplasmic responsive systems which are involved. MATERIALS AND METHODS The potatoes, Solanum tuberosum, were of the Idaho variety and were pur- chased from local grocery stores. The carrots, Daucus carota, were similarly pur- chased from local stores. Using a cork-borer, small cylinders, 2.2 cm. in diameter and about 1.5 cm. tall, were prepared from the potatoes in such a manner that each carried an eye on the center of its upper surface. These were permitted to heal their cut surfaces before being set, in shallow water, in respirometer vessels where the same individual organisms were retained up to three or more months. These always gave rise to sprouts and usually also to a root system, and in some instances even developed new tubers up to a centimeter or more in diameter during their sojourn in the respirometers. For the carrots, short cylindrical sections, about the size of the potato-cylinders, were cut and allowed to heal over before being placed in respirometers. The respirometers have been described earlier. These were originally designed by Brown (1954) and later modified (Brown, 1957a) to permit maintenance of constant pressure. Five independent barostat-respirometer ensembles, each with 4 respirometers recording as a unit, were in nearly continuous operation during the period of study, Feb. 1, 1956 through Feb. 28, 1958. The potatoes in the respirometers were in constant illumination (estimated at 0.05 ft. c. at the site of the plants) from incan- descent lamps supplied by a voltage-regulated line. The temperature, 20 C., was maintained constant by the respirometers being immersed in a large non-stirred, copper water-bath (the barostat) deeply immersed in an outer, stirred, steel (55-gal. drum) water bath, with the latter cycling with a few-minute period within a 0.05 C. range. The pressure was kept constant, 28.5 in. Hg, through hermetically seal- ing the respirometer-recorder-containing barostat and then aspirating the system to this level. Oxygen and CO 2 tensions were maintained essentially constant through EXOGENOUS BIOLOGICAL RHYTHMICITY 83 use of the principle of continuous CX-replacement together with CO 2 absorbents, and there were clearly no regularly cyclic fluctuations in these substances. Also, the sealed, water-included systems allowed for no changes in humidity. With a single exception (12 days) the copper tanks, or barostats, remained sealed for periods ranging from 2 to 8 days, with an average of 4.46 days. At these intervals the organisms were exposed for 15-20 minutes to laboratory conditions which were relatively constant over the year. No work was done within 15 feet of outside windows ; the laboratory fluorescent illumination at table top was about 45 ft. c. (The carrot study was carried out wholly in a dark-room without any natural illumination.) The room temperature was relatively constant, about 75 F., except for slightly higher values during the summer months. The barostats were opened at various hours of the day from 8 AM to 10 PM. Excluding those days the respirometers were opened to renew the O 2 and the CO 2 absorbent, a total of 2485 uninterrupted calendar days of data were obtained. The recording systems of the respirometers possessed two points of slight me- chanical frictional resistance, a) a two-point pivotal, spring-scale bearing, and b) the point of contact of the ink-writing pen with the slowly moving paper. These resulted in random, spurious apparent intra-hour fluctuations in rate of CX-con- sumption. Since the principle of operation of the recorder was one with which the hourly values of O 2 -consumption were obtained by calculating the differences be- tween consecutive hourly markers on a continuing trend-line denoting cumulative 0.,-consumption, these spurious fluctuations in apparent rate could, and undoubtedly did, produce larger hour-to-hour differences than bore any significance. Hence, time units of less than three hours (three-hour "moving means") were never used in determining the mean rates centered on any given hour. By this means the random mechanically induced error was reduced to about one-third its single-hour influence. For most of the study reported here, a weighted (1:2:3:3:3:2: 1) seven-hour "moving mean" was used. This reduced by essentially 90% the ran- dom fluctuations while retaining all the precision of measurement of average, actual, O 2 -consumption for this longer interval, as modified by its weighted character. The shorter period, three-hour, means were found necessary, however, to expose the relationship between day-by-day 6 AM deviations in CX-consumption from daily linear trends and the concurrent day-by-day mean rates of barometric pressure change for the 2-6 AM interval. Although some clearly significant short-period fluctuations were obscured, therefore, by the seven-hour weighted "moving means," these were considered superior to the shorter periods for the accurate description of the general characters of the longer-period, daily and annual cycles to be described herein. The records for the five completely independent, respirometer-recording systems were first dealt with individually and three-hour and weighted seven-hour "moving means" were prepared month by month for the period of study. From the latter values were calculated the mean daily rates of O 2 -consumption and the data were then converted into hourly deviations from the solar daily means. The number of uninterrupted days of data from the 24 months of study ranged from 93 to 129 each month. The hourly deviations for all the respirometers operating were aver- aged for each calendar day, and these average daily cycles then converted to hourly deviations from a 1 AM to 12 midnight linear trend-line. This will be referred to as the deviations from linear dailv trend. From these data the forms of the mean 84 FRANK A. BROWN, JR. daily cycles for each month were obtained. The slope of this linear trend-line it- self shows apparent monthly and annual periodisms which have been treated else- where (Brown, 1957c; Brown, Bennett and Webb, 1958). The trend involved a mean daily increase during the two-year study of 6.7%, and included, as a large o\ *< LJ cr +3 O cc LJ Q +1 o\0 Z LJ O LJ Q + 1 6PM o o HOUR 12 OF DAY 18 24 FIGURE 1. A. The mean solar-day cycle of Oo-consumption in the potato (solid line) with standard errors for selected hours. This is expressed as % deviations from linear daily trend. The dashed curve is the cycle for the first year of study, the dotted curve, for the second. B. The mean apparent sidereal-day cycle of the potato for the two-year period of study. component, the apparent smoothly gradual recovery over a 3- to 5-day period, from the inhibitory influence of the room-illumination intensity. The mechanical re- cording system, itself, departed from linearity over its total range by 10%, departing in such a direction that there would be expected on this basis an average of about 2% increase per day. EXOGENOUS BIOLOGICAL RHYTHMICITY 85 An entirely independent and parallel study was made of Go-consumption of the sections of the carrots, for the 8-month period Oct. 1, 1956 through May 31, 1957. Two respirometer-barostat ensembles were employed for the first three months, and four for the remaining five months. These were maintained in darkness in a photo- graphic darkroom about 60 feet away from the place of the potato study, but simi- larly on the ground floor of Cresap Biological Laboratory, a three-story steel and mortar building. The respirometers were maintained and the data processed by a person not involved until the termination of the carrot study in the paralleling and continuing potato study. M A 1956 M A S N D J F M A M MONTHS 1957 O N D J F 1958 FIGURE 2. The relationship between average % noon deviation in O 2 -consumption in the potato from linear daily trend and month of year during the 25-month study. Standard errors of means are shown. RESULTS During the period, Feb. 1, 1956 through Feb. the only days omitted were May 25, the month July, 1956, and October 4, 1957. The form of the mean daily deviation from the daily mean rate, is shown, with the standard values, in the solid-line curve of Figure 1, A. quite comparable in size. Superimposed on this two years separately: Feb. 1, 1956 through Jan 28, 1958, in the study of the potato, of June and the first three days of trend, expressed as percentage of errors of arbitrarily selected mean The errors of the other values are are the mean cycles for each of the . 31, 1957 (the dashed curve) and 86 FRANK A. BROWN, JR. FEB MAR APR JUNE JUL SEPT OCT DEC JAN 6AM 12 HOUR OF DAY 6PM FIGURE 3. The forms of the average daily cycles for each month of the year obtained in the two- year study. The ordinate values are deviations in comparable arbitrary units. EXOGENOUS BIOLOGICAL RHYTHMICITY 87 Feb. 1, 1957 through Jan. 31, 1958 (the dotted curve). The average amplitude of the daily cycle was clearly quite reproducible for the two years at about 3.7%. There was also clear suggestion, in the skewed cycle form, of a bimodality with morning and afternoon maxima, a condition more conspicuous for the second than for the first year of study. The mean sidereal-day cycle (23 hours, 56.07 minutes) for the two-year period is shown in Figure 1, B. This was obtained by displacing M M MONTHS FIGURE 4. The relationship between the noon deviations in Da-consumption from linear daily trend and calendar month in the carrot during an 8-month study. The standard errors are depicted. the consecutive mean monthly solar-day cycles each by two hours to the right dur- ing the two-year period, to bring into reasonably close synchrony (1 hour) the hours of the sidereal day. The numbered hours are fixed by the solar-day hours of the first month, February, 1956. This process also randomizes daily trend. The form and amplitude of the solar daily cycle showed differences from month to month which revealed that it was undergoing a modulation of an annual fre- quency. This was quite evident when one used, for example, the parameter of average monthly noon deviation, in percentage, from 1 AM to midnight daily 88 FRANK A. BROWN, JR. linear trend. The deviations, month-by-month, for the period Feb. 1, 1956 through Feb. 28, 1958, together with their standard errors, are depicted in Figure 2. These indicate minimum annual values, involving often even apparent cycle inver- sion, during the coldest months of the year and a major maximum in the month of October. A lesser, or incipient, maximum occurred in April-May. The maxi- mum range is seen to extend from -- 3.4% to + 14.2%. An annual cycle in over-all form of the mean daily cycles for the months of the year is evident in Figure 3, where twelve average cycles, the means for two years, have been plotted in terms of average deviation in arbitrary units from linear-trend. 6AM 12 HOUR OF DAY 6PM 6AM 12 HOUR OF DAY 6 PM FIGURE 5. A comparison, for the same 8-month period of study, of the forms of the mean daily cycles for carrots (A) and potatoes (B). Solid curves show the mean cycles for the whole 8-month period. The dashed curves show the average cycles for October, November, April, and May. The dotted curves show the average cycles for December, January, February, and March. Standard errors of selected times of day are shown. Although these data are not expressed as percentage deviations, they do illustrate the gradually-changing form of the cycles from unimodality with essential inversion in February, but with a 7 PM maximum, through a period of bimodality with the two daily maxima gradually converging towards noon to reach unimodality \vith a maximum at 11 AM in October. Thereafter, bimodality reappears and continues, becoming only feebly evident as an apparent residual in the essentially unimodal in- verted cycle of January which like the succeeding month, February, has a 7 PM maximum. The study of the carrot revealed striking similarity of its major mean cycles with those of the potato. Figure 4 shows the mean % noon deviation from linear trend for each of the eight months. Like the results obtained with the potato for EXOGENOUS BIOLOGICAL RHYTHMICITY 89 the same calendar period, this passed from an early-fall higher value, through a winter minimum and back to a higher spring value. Fewer data were available dur- ing the first three months, hence the errors were larger. The range was less than for the potatoes. Figure 5, A and B solid curves, compares the mean 8-month daily cycles for the carrot and potato, and the average cycles for the two fall and two spring months (dashed curves) as compared with those for the four intervening colder months (dotted curves), The similarities of these two widely different kinds of plants and plant portions (roots vs. stems) for the same periods, in the % z o H Q_ 10 Z O o Icvj id I- < ct 5 J M M J J MONTHS A O N D FIGURE 6. The relationship between mean rates of Oo-consumption and each of the 24 months studied during 1956, 1957, and 1958, and time of year. Standard errors of the means are indicated. amplitudes of the fluctuations, in the times and the changing times with time of year of the primary maxima, and in the times of secondary, or incipient, maxima, are strikingly apparent from the figures. A second kind of annual cycle appears also present in the data. This is in the mean daily metabolic rates. In Figure 6, are to be found the mean monthly rates of Oo-consumption, in arbitrary units, for each of the 24 months of study, together with their standard errors. Two conclusions are evident from the figure : ( 1 ) The maximum rate of Oo-consumption occurs in the April-May period of the year and minimum rate in October-November. The rate for the former period ap- 90 FRANK A. BROWN, JR. TABLE I Signs of the average monthly correlations of the 6 A M deviations from linear trend with the mean 2-6 AM rate of barometric pressure change Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Year 1 + + + + + Year 2 + + + + + -f + + Year 3 preaches twice that of the latter. (2) The mean rates for corresponding months of the two consecutive years may be quite significantly different from one another, sug- gestive of a specific, time-environmental factor involved in an exogenous regulation. There is nothing in these data to suggest other than that the mean form of this annual cycle will ultimately be found essentially sinusoidal. In view of the correlations highly significantly different from zero earlier re- ported (Brown, 1957a) to exist between the 5-67 AM mean deviations (without sign) in O 2 -consumption from the daily means and the mean 2-6 AAI rates of baro- metric pressure change, this relationship was examined for the two-year period involved here. Three-hour values of (X-consumption centered on 6 AM were re- corded as deviations from linear daily trend, and three-day moving means calcu- lated. These were correlated with comparable three-day moving means of the av- +1 +2 2-6 A.M. B. R C. o\o-8 -16 85 FIGURE 7. A. Solid line : An outline of the general form of the scatterplot between mean 2-6 AM rate of barometric pressure change and the 5-6-7 AM mean deviation of rate CL-con- sutnption from daily trend for the same day during the "colder" months (see text). Broken line : The same for the "warmer" months. Data for both involve three-day moving means. The two patterns together include 98% of all points. B. An outline of the form of the scatter- plot (97% of all points) between % noon deviations from linear trend in potatoes in constant conditions and concurrent outdoor air temperature, taken from data of 149 non-overlapping three-day averages. EXOGENOUS BIOLOGICAL RHYTHMICITY 91 erage 2-6 AM rate of barometric pressure change, for the corresponding days. It should be emphasized that only a single value was used for each day for each phe- nomenon ; hence, this did not involve a correlation of parallel daily cyclic trends. A positive coefficient, highly significantly different from zero, was obtained. This correlation, as one would anticipate in view of the essentially aperiodic, large cli- matic barometric pressure changes, rapidly drops to insignificance as one correlates .6 Q U 4 cr h- Q 5. i CD +.4 +.2 B O -I +1 + 2 +3 2-6 A.M. BAR. PRESS. CHANGE FIGURE 8. The regressional relationship of the average three-hour rate of O^-consumption of the potato centered on 6 AM, and expressed as deviation from linear daily trend, on the average rate of barometric pressure change during the 2-6 AM interval f jr the same morning for the colder months cf the year (see Table I ). P < 0.005. B. The relationship comparable to that in A. but for the warmer months of the year (see Table I). P< 0.001. 92 FRANK A. BROWN, JR. in increasing lag on lead relationships up to two to three days (Brown, 1957a) indi- cating a direct response of the organisms to some pressure-change-correlated ex- ternal variable. But this relationship was found to contain a characteristic sign- change twice a year as seen in Table I. In this table, a dash indicates those months in which there was a negative correlation between the rate of the 2-6 AM baro- metric pressure change and the 6 AM deviation, without sign, from linear trend. The form of the scatterplot relationship for the 299 days of this negative period is outlined by the solid curve (encloses 91 % of the points) in Figure 7, A. The re- gressional relationship of the deviation in CX-consumption, without sign, upon pres- sure change is seen in Figure 8, A. During the + months, on the other hand, there was a positive correlation between the rate of 2-6 AM barometric pressure change and the 6 AM deviation in O 2 -consumption from linear trend. Ninety-one per cent of the 389 days in a scatterplot of the relationship for these months fell within the broken curve of Figure 7, A (98% of all 688 daily points + or months fell in the areas prescribed by both the solid or broken lines). The regressional relationship of O 2 -consumption on pressure for the + months, with sign, is seen in Figure 8, B. TABLE II Signs of the average monthly 2-6 PM change in barometric pressure Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Year 1 + + + + + Year 2 + + + + Year 3 + Since in the warmer, positive, months of the year, the overwhelming mass of the deviations was +, it was not possible to find any real difference between the corre- lations whether the deviations were treated with, or without, sign. In the colder, negative, months, on the other hand, about half of the deviations were negative, and the range of the latter as great as for positive deviations. In view of the earlier report (Brown, 1957a) of comparable correlations be- tween the 6 PM deviation in CX-consumption from daily mean values in potatoes, and the afternoon rate of barometric pressure change, and also correlations with the mean daily pressure of the second day thereafter (Brown, Webb and Macey, 1957), the former relationship including signs, it is of interest to compare the an- nual cycle in the sign of the average 26 PM barometric pressure change. These are seen in Table II. The similarities between Tables I and II suggest that this aspect of organismic annual cyclicity, involving the mean forms of daily cycles, might in some manner be caused by a factor whose daily fluctuation reflects the annual cycle in form of the well-known mean daily tidal atmospheric pressure cycles. In these daily pressure cycles, the time of the morning maximum remains relatively fixed throughout the year at 9-10 AM, but the afternoon, major minimum of the day gradually shifts from about 2 PM in winter to about 7 PM in summer. This last is the basis for the sign changes in Table II. Thus, any pressure-correlated effective external physical factor could provide such an annual cycle in the daily cycles as that described herein. Another clearly evident correlation is seen in the relationship of the % noon de- EXOGENOUS BIOLOGICAL RHYTHMICITY 93 viation from linear trend in the potato to the concurrent outside mean daily air temperatures. 3 A two-year study of the comparison of non-overlapping three-day periods of air-temperature and of noon deviation of the daily cycles in constant con- ditions yields a scatter plot relationship as illustrated in Figure 7, B. The line in- cludes 97% of the 149 values. The regressional relationship of noon-deviation of the potatoes on temperature (using 5 F. class intervals) is illustrated in Figure 9. The relationship seems adequately described as a linear one, but with a sign change near 57.5 F. Calculation of the coefficient of correlation for noon deviation in Q Z 8 LJ CC. r- 4 o O cr LJ Q .-4 O o -8 o -7.5 2.5 12.5 22.5 32.5 42.5 52.5 62.5 725 82.5 OUTSIDE AIR TEMP. E FIGURE 9. The regressional relationship of the noon deviation in O^-consumption in the potato, expressed as % deviation from daily linear trend on simultaneous outdoor air temperature. O, -consumption with the deviation in temperature from 57.5 F., yielded a value of - 0.51 0.049. This clearly indicates that the external factor responsible for the 24.0-hour cycles of metabolism is correlated in its fluctuations with air tempera- ture, resulting in a condition where a spurious organismic Q 10 of cycle amplitude of more than 3 could be apparent (e.g., in range 32.5 to 52.5 F.). This provides another piece of information which will probably lead eventually to identification of the still unknown external factor responsible for the organismic basic periodisms. That the relationship to temperature is rather substantial, is given further sup- port in that the regressional relationship of noon deviation on temperature exhibited a sign change about 57 F. in the first year, 1956-57, just as it did again in the sec- ond. 1957-58, despite the fact that in 1956 there was no clear absolute summer de- 3 These data were generously provided to me by the Chicago Office of the U. S. Weather Bureau. 94 FRANK A. BROWN, JR. cline in amplitude of the mean monthly cycles (Fig. 2) as was found in 1957. Also, during the winter months the correlations are observed (Fig. 9) to continue in the same linear relationship even at temperatures lower than the lowest mean monthly ones in the two years involved in this correlation (29.4, 28.7, 18.8, 27.3, and 26.3 F.) which averaged about 26 F. These last facts suggested intra-month significant temperature correlations which were borne out by investigation of the correlations using the data of the five coldest months now expressed as deviations from monthly means. The correlations continued in temperature ranges well ex- ceeding any mean month-to-month difference. Again, using the data for both of the two years, the transitional months, April, May, October, with a mean temperature of 54.8 F. (47.2, 60.5, 60.8, 49.5. 58.8, 52.0), expressed as deviations from monthly means, the critical temperature for sign change was again quite apparent. Finally, employing the warmest months of the year, June, July, August, and September with a mean temperature of 71.1 F., (72.9, 74.1, 65.0, 71.2, 76.4, 73.4, 64.2), there was a suggestion of the existence of a second sign change with again a positive correlation at the higher temperature. The number of high-temperature days was insufficient, however, to enable resolu- tion of this last point. DISCUSSION From the foregoing results it is evident that potatoes, and apparently the car- rots too, display a quite reproducible mean solar daily cycle provided adequately long periods of time are used to render random the influences of such modulating longer cycles as a lunar day (Brown, Freeland and Ralph, 1955), a synodic month (Brown, Freeland and Ralph, 1955 ; Brown, Bennett and Webb, 1958), and, in this report, a low amplitude apparent sidereal day, and an annual cycle. These described mean solar-day cycles are obviously of quite precise 24.0-hour frequency, and ade- quate evidence is at hand to be assured, beyond all reasonable doubt, that these have their frequency exogenously determined. This last conclusion is assured in part through the well-known knowledge that there are solar-day tidal rhythms of atmospheric pressure, together with the fact that the living organism has access to information of them through its responses to the day-to-day, essentially random, weather-induced, disturbances in their regularity. That the factor influencing the organism is not pressure itself, is evident from the fact that these and other experi- ments have involved organisms maintained for long periods in constant pressure. The external factor which is involved appears to have its primary action upon the organism at the times correlated with the early-morning rise in barometric pressure and the afternoon fall. These would presumably be the times of most rapid change in physical factors fluctuating with the day-night cycle, and hence be the times of their maximal stimulative effectiveness. As pointed out earlier in this report, the presence of the well-known annual change in the form of the daily, tidal, barometric pressure cycles, and the de- scribed response of the organism in the late afternoon to a pressure-correlated ex- ternal variable would have led to the prediction of the occurrence of an annual cycle in the form of the daily cycles. Such a prediction has been fulfilled in this study. This adds still further, therefore, to the assurance that the forms of the daily basic metabolic oscillations in living organisms are exogenous. EXOGENOUS BIOLOGICAL RHYTHMICITY 95 Since background radiation, too, possesses good mean solar-day cyclicity, and the organism follows the essentially random fluctuations in its cycle amplitudes from day-to-day (Brown, Shriner and Webb, 1957) very safely beyond what would be expected through chance, when and only when contemporary data are correlated, this constitutes a third line of evidence for exogenous origin of mean daily metabolic cycles. The existence of an annual cycle in the potato in constant illumination, tempera- ture, pressure, etc., was reported earlier for fluctuations in linear daily trend (Brown, 1957c), as were also synodic monthly cycles of this parameter (Brown, Bennett and Webb, 1958). In this paper there is described an apparent annual cycle in basic metabolic rate, a cycle which appears to be of simple sinusoidal char- acter with maximum in April-May and minimum in October-November. This cycle involves an approximate doubling of rate in passing from minimum to maxi- mum values in the annual cycle. Comparable, synodic monthly, cycles in metabolic rate in potatoes (Brown, Bennett and Webb, 1958) involved, as the average during a year of study, about a 15% increase from minimum (new moon) to maximum (third quarter) values. By further comparison, the amplitude of the daily cycles, though undoubtedly artificially depressed through the use of the seven-hour weighted moving means, displayed about a 3.7% increase from midnight minimum to 6 PM maximum values. The regressional relationship of amplitude of the daily cycles on mean daily temperature for three-day periods (Fig. 9), with its coefficient of determination of about 0.26, and its critical temperature for sign-reversal, together with the earlier barometric-pressure-change reversing correlation, suggests again the exogenous origin of this daily cycle period, and, at least in large measure, also cycle form. This is especially true, since the relationship to temperature seems to persist into the weather-correlated, intra-month, temperature fluctuations. In examining Figure 9 and noting the relationship of cycle amplitude to tem- perature, and recalling that the mean daily temperature range is about 16 F., with not very uncommonly single days with ranges up to 25 to 30 F., one is tempted to postulate that the factor that is responsible for transmitting to the organism in "constant conditions" information on outside air-temperature, is, through its tempera- ture-correlated fluctuations alone, contributing importantly to the 24-hour periodic metabolic fluctuations themselves. In support of this hypothesis is the rough simi- larity in the average forms of the annual fluctuations in the amplitudes of ground- level daily temperature change and metabolic cycles. Both, as average for the two years, showed lowest values in the coldest winter months and highest values in late spring and late summer to early fall, with a summer amplitude reduction. The re- lationship between these two phenomena is seen in Figure 10. The October peak, so conspicuous for the metabolic cycles, is much less evident for the temperature changes. For each year this relationship between these two phenomena appeared to trace out general ovoid form. The two-year mean month-by-month relationship is shown by the numerals 1 (January) through 12 (December), and the dotted ovoid curve roughly traces their course. It is interesting to speculate that this difference be- tween the organismic and temperature annual cycles may find its explanation in the changing natural smog content of the atmosphere. The terpenes, volatilized from 96 FRANK A. BROWN, JR. plants, polymerized by the ultra violet light from sun, reach a maximum in October (personal communication from Professor F. W. Went). This smog may, through influencing the amount of heat absorption from sunlight, produce in October the highest amplitude daily temperature changes of the year at levels in the atmosphere where temperature changes produce greatest influence upon the factor directly af- fecting the organism. One process, known to be temperature-dependent, is the rate of spontaneous decay of cosmic-ray-derived mesons. The larger the atmospheric depth involved in the temperature change, in this instance, the larger would be ex- pected the temperature influence. O 16 Z LJ 8 o: > 4 LJ Q Z O o Z 4 60 no inhibitor >60 p-chloromercuribenzoate 12 p-chloromercuribenzoate 14 (0.0005 M) 16 (0.0005 Af) 17 NaCX (0.001 J/) 10 12 iodoacetate (0.002 M) 2 1 RESULTS Several experiments indicate that the acid is within the cells, yet not free in the cytoplasm. Tissue extracts were capable of reducing methylene blue with a variety of substrates, under nitrogen, only if buffered near neutrality. No activity was noted in preparations in which the pH of the extract was less than five or if the tissues were homogenized in unbuffered sea water. Rates of dye reduction were somewhat greater in the presence of ribose and aspartate than with other substrates. Some activity was also present in buffered extracts with no substrate added, but quantitative studies were not made. In other experiments, discs of the thalli, cut out with a two-cm, cork borer, were tested for acid loss in sea water in the presence and absence of inhibitors. Samples of five or ten discs were placed in ten ml. of sea water containing a drop of methyl orange, and the time required for a color change of the indicator was recorded. The rate of acid loss was much greater in neutralized sea water in the presence of 0.0005 M p-chloromercuribenzoate, 0.001 M sodium cyanide, and 0.002 M iodo- acetate than in sea water alone (Table I). Rates of acid loss in dinitrophenol (0.005 M) were determined by measuring the pH of the solution. The curve resulting from a plot of pH against time (Fig. 1) suggests an autocatalytic reaction. This autocatalytic injury is implicit in Blinks' (1951) description of the rates of carotenoid color change in Desmarestia as the alga dies. SULFURIC ACID IN DESMARESTIA 103 8 PH Control DNP 20 40 MINUTES 60 FIGURE 1. Acid release by Desmarestia munda tissues in sea water (control) and in sea water containing 0.0005 M dinitrophenol (DNP). On accumulation of the dye, brilliant cresyl blue, the vacuoles of D. munda and D. herbacea are stained purple in confirmation of Kylin's results (1938). How- ever, we feel the color to be indicative of the change at pH 1.0-0.7, rather than 7.0 7.5. If the acid is localized within the vacuoles, one might expect the cations normally found in the vacuoles of brown algae to be replaced by hydrogen ions. In Egregia laevigata and Dictyoneurum californicum (Table II) potassium is the most abun- dant cellular cation measured. It occurs at a concentration approximately isotonic with sea water. In D. munda about 75 per cent of the potassium is replaced by hy- TABLE II Potassium, sodium, and hydrogen ion contents of Desmarestia munda, D. herbacea, and two non-acid- accumulating species of brown algae. Values are corrected for the ion contents of killed tissues and represent averages of four determinations. Units milli-equivalents /liter estimated cell osmotic volume Alga est. cell. H K Na Sum osm. vol. Desmarestia munda 84% 438 148 586 Desmarestia herbacea 69% 254 234 13 501 Dictyoneurum californicum 63% 523 21 544 Egregia laevigata 71% 542 45 587 104 RICHARD W. EPPLEY AND CARLTON R. BOVELL drogen, and about 50 per cent is replaced in D. hcrbacea (Fig. 2). The reciprocity of potassium and hydrogen ion concentrations agrees with the above mentioned ex- pectation. The approximation of the total cation concentration among the four brown algal species to that of sea water suggests that most of the cation content is accounted for, although magnesium and calcium were not measured and may be present. 100 UJ CD O cr Q 50 D. mundo Ov D. herbaceo other browns 50 100 POTASSIUM FIGURE 2. Hydrogen and potassium ion contents of Desmarestia inunda, D. herbacea, and two other brown algae : Egregia laevigata and Dictyoneumm califoniicum. Units : per cent of total cation content determined. The binding of large amounts of sodium by dead tissues was detected. This may represent adsorption of the cation to the carboxyl groups of alginic acid, a structural polysaccharide of the brown algae (Wasserman, 1949). DISCUSSION The vacuoles of Desmarestia contain sulfuric acid in amounts up to 0.44 N, in D. inunda. Direct evidence for this view is the purple color of brilliant cresyl blue accumulated by the vacuoles of D. inunda and D. herbacea. Indirect supporting evidence includes the following : 1 ) The acid is lost more rapidly on exposure of tissues to inhibitors which abolish selective membrane permeability than it is in the absence of such inhibitors. In this group are sodium cyanide, iodoacetate, p-chloro- SULFURIC ACID IN DESMARESTIA 105 mercuribenzoate, and dinitrophenol. 2) The autocatalytic release of acid in the presence of dinitrophenol suggests that extra-vacuolar acid injures the cells, caus- ing an increasing rate of acid release. 3) Oxidative metabolism is sensitive to high hydrogen ion concentrations as evidenced by the inability of tissue extracts to re- duce methylene blue in unbuffered suspensions. 4) The reciprocity of potassium and hydrogen ion concentrations among the brown algae tested suggests that hy- drogen replaces potassium as the most abundant cellular cation in D. munda, and that about one-half of the potassium is replaced in D. herbacea. The tonoplasts of Desmarestia cells must be quite unique in their resistance to acid injury, and in their permeability characteristics. A hydrogen ion concentration gradient of about 10 7 is apparently maintained between the vacuolar sap and sea water. However, the sea water is probably not the "substrate" for hydrogen ion ac- cumulation. Metabolically produced hydrogen in the cytoplasm may well be the source for vacuolar accumulation. Efforts to leach the acid from the cells so that the progress of acid reaccumulation could be studied have not been successful. The cells are killed as the acid is released. The production of hydrogen ion due to anaerobic conditions in the interior cells of massive species of Desmarestia may explain Blinks' (1951) observation of a cor- relation between tissue massiveness and acid content. The interior cells of D. munda are much larger, contain few r er plastids, and show a greater percentage of purple vacuoles, on staining with brilliant cresyl blue, than the peripheral cells or the cells of D. herbacea. The high acidity of Desmarestia cells may limit the vertical distribution of the alga in the intertidal zone. Because injury spreads so rapidly when water circula- tion is poor, it seems reasonable that the acid-accumulating species must be confined to regions of constant water circulation. Desmarestia herbacea occurs below the lowest-lower-low-water tide mark (Doty, 1946) and D. munda is limited to the lower portion of the intertidal zone (Smith, 1944). SUMMARY 1. Brilliant cresyl blue accumulates in the vacuoles of Desmarestia munda and D. herbacea and the accumulated dye appears purple, indicating that the pH of the vacuolar sap is less than 1.0 or greater than 7.5. However, the expressed saps of these two brown algae have pH 1.0 or less and about 2.0, respectively. The outer cell membranes are injured by the low pH of the sap and methylene blue is not re- duced by tissue homogenates at such low pH values. 2. Sodium cyanide, dinitrophenol, iodoacetate, and p-chloromercuribenzoate in- duce the release of acid from the cells, in which potassium, normally the cation most abundant in brown algal cells, is largely replaced by hydrogen. In D. munda hy- drogen accounts for 75 per cent of the intracellular cation content. Tissue sodium is largely bound and contributes little to the cellular cation content. 3. The simplest interpretation of these data is that the acid is localized within the vacuoles of Desmarestia cells. LITERATURE CITED BLINKS, L. R., 1951. Physiology and biochemistry of algae. In: Manual of Phycology (G. M. Smith, editor). Chronica Botanica Co., Waltham. Mass.; pp. 263-91. 106 RICHARD W. EPPLEY AND CARLTON R. BOVELL BRIGGS, G. E., AND R. N. ROBERTSON, 1957. Apparent free space. Ann. Rev. Plant Physio!., 8: 11-30. DOTY, MAXWELL, 1946. Critical tide factors that are correlated with the vertical distribution of marine algae and other organisms along the Pacific Coast. Ecology, 27: 315-328. EPPLEY, R. W., AND L. R. BLINKS, 1957. Cell space and apparent free space in the red alga, Porphyra pcrforata. Plant PhysioL, 32 : 63-64. KYLIN, HARALD, 1938. t)ber die Konzentration der Wasserstofinonen in den Vakuolen einiger Meeresalgan. I'drh. Kgl. Fysiograf. Sallsk. Lund, 8 : 194-204. MEEUSE, B. J. D., 1956. Free sulfuric acid in the brown alga, Dcsmarcstia. Biochhn. Biophys. A eta, 19: 372-374. SMITH, G. M., 1944. Marine Algae of the Monterey Peninsula. Stanford Univ. Press, Stan- ford, Calif. ; 622 pp. WASSERMAN, A., 1949. Cation adsorption by brown algae. The mode of occurrence of alginic acid. Annals Bot., 13 : 79-88. WIRTH, H. E., AND G. B. RIGG, 1937. The acidity of the juice of Dcsmarcstia. .hncr. J. Bot., 24 : 68-70. THE SENSITIVITY OF ECHOLOCATION IN THE FRUIT BAT, ROUSETTUS D. R. GRIFFIN, A. NOVICK 1 AND M. KORNFIELD 2 Biological Laboratories, Harvard University, Cambridge 38, Massachusetts Moehres and Kulzer (1956b) have reported that among the Megachiroptera (Old World fruit bats and flying foxes) the genus Pier opus orient visually while Rousettus aegypticus orient visually but also echolocate. Six additional mega- chiropteran genera, Eidolon, Cynopterus, Ptenochirus, Lissonycteris, Eonycteris, and Macroglossus, have all proved, like Pteropus, to orient visually and not acousti- cally. Observations of two additional species of Rousettus, R. amplexicaudatus and R. seminudus as well as R. aegypticus, have confirmed Moehres and Kulzer's con- clusions (Novick, 1958). Rousettus generate clicks by movements of the tongue and emit these through the open corners of the mouth (Kulzer, 1956) rather than producing sounds laryngeally as do the Microchiroptera (Griffin, 1946, 1952; No- vick, 1955; Griffin, 1958). As far as is known at present all of the Megachiroptera except Rousettus are helpless in total darkness. Rousettus apparently make use of vision and/or echolo- cation depending upon the light conditions, the difficulty of their flight path, and the type of flight required (take-offs and landings, for example). The echolocation system used by Rousettus has almost surely evolved independently of the system employed by the Microchiroptera. Furthermore, it resembles in design the system serving much the same purpose in the cave-dwelling birds, Steatornis and Collocalia. The isolation of these three natural sonars in single genera, their simple designs, and their facultative employment (all three genera orient visually in adequate light) make it seem likely that they are recent developments compared with undoubtedly ancient microchiropteran echolocation systems. There is, therefore, considerable interest in comparing the effectiveness of the echolocation system of Rousettus in the detection of small objects with that achieved by the Microchiroptera, especially some carefully studied species of the families Vespertilionidae and Phyllostomatidae (Curtis, 1952; Griffin and Novick, 1955; Grinnell and Griffin, 1958). Since the orientation clicks of Rousettus, Steatornis, and Collocalia are clearly audible to man, they obviously contain more energy at frequencies below 20 kc than do the orientation pulses of most of the Microchiroptera. The principal compo- nent in Rousettus clicks is between 12 and 18 kc, depending upon the species and the individual, but overtones and harmonics are present to a considerable degree (Novick, 1958). Saccopteryx and Taphosous (Emballonuridae) and some species of Tadarida (Molossidae) emit partly audible orientation cries. Rhino poma also emit orientation pulses with audible components (Moehres and Kulzer, 1956a). Rousettus, Steatornis, and Collocalia, though, unlike all of the Microchiroptera, 1 Present address: Osborn Zoological Laboratory, Yale University, New Haven 11, Connecticut. - Present address : New York University-Bellevue Medical Center, New York, N. Y. 107 108 D. R. GRIFFIN, A. NOVICK AND M. KORNFIELD produce clicks with relatively little energy above 20 kc. Thus, it appeared that only relatively long wave-lengths would be available for echolocation and that Rouscttus and the two cave-dwelling birds might be unable to detect obstacles as small as the wires that had been used as standardized test objects for the Microchiroptera (Hahn, 1908; Griffin and Galambos, 1941 ; Griffin and Novick, 1955; and Grinnell and Griffin, 1958). A single male Rouscttus acgypticus, captured in a dimly lighted cave at Eaux Chaudes, Katana, Kivu Province, Belgian Congo in July, 1956, was brought to Harvard University in good health in August, 1956. This bat survived for nine months on a diet of bananas and, after a short period of recuperation from its jour- ney and its restriction to a small cage, flew skillfully in an experimental flight room 32' long, 12' wide, and 8' high. Its ability to avoid a variety of cylindrical test ob- stacles arranged in a row across the center of this room was tested by methods di- rectly comparable with those previously used to measure obstacle-avoiding skill in the Microchiroptera. This Rouscttus proved able to avoid surprisingly small wires even in total darkness. Its skill is here compared with that, measured previously, of the vespertilionid, Myotis I. liicifiigus (Curtis, 1952). This work was partly sponsored by the Office of Naval Research, the United States Public Health Service, and the Belgian American Education Foundation. During this time, Novick held a Post-doctoral Fellowship of the National Institute of Neurological Diseases and Blindness. We are grateful to the personnel of the Institut pour la Recherche Scientifique en Afriquc Ccntrale, Lwiro, Belgian Congo for their help in capturing the experimental subject. Reproduction of this paper in whole or in part is permitted for any purpose of the United States government. METHODS After the bat had become accustomed to the problems of flight both in light and in total darkness in the flight room, and to the task of dodging between vertical ob- stacles suspended from the ceiling across the middle of the room, we tested its ability to avoid cylindrical obstacles, spaced 53 cm. apart, varying in size from cardboard tubes 5 cm. in diameter to bare metal wires 0.28 mm. in diameter. In each case these obstacles were suspended in a movable frame in a plane parallel to the end walls of the room. This plane had to be crossed by the bat in flying from its roost at one end to its roost at the other end. We forced such flights by agitating the roost which was a loosely suspended horizontal bar of wood. The bat would take off and fly the length of the room to the opposite roost or would, on occasion, make sev- eral flights back and forth before landing. In each of the tests considered below, the frame holding the obstacles was shifted horizontally in the dark just before each flight so that the absolute position of the obstacles and their location relative to the walls were unknown to the bat, though their position relative to one another was constant. Thus, the bat could not learn the location of the open spaces nor could it depend on following the walls because the space adjacent to the walls was fre- quently and randomly too narrow to permit passage. The room was totally dark during all these observations, but we often noticed by listening to the bat's audible clicks or to its wingbeats that it hesitated in front of the obstacles and executed dodging maneuvers to pass between them. ECHOLOCATION IN ROUSETTUS 109 The Rousettus was thus required to fly through an obstacle plane and its ac- curacy of echolocation was evaluated in terms of its ability to avoid the obstacles. One must consider whether it was constantly and equally motivated to avoid col- lisions and whether its physical agility was sufficient for it to make the maximum use of its orientation system. The flights were scored simply as hits or misses by means of the sound of hits or in doubtful cases by inspecting the obstacles in light switched on immediately after the bat's passage. A hit always caused a clearly visi- ble, sustained vibration of the obstacles as they were suspended from rubber bands. All hits were considered equal although some undoubtedly represented the bat's TABLE I Comparison of the obstacle avoidance scores of a Rousettus aegypticus with those of Myotis I. lucifugus (Curtis, 1952}. The wires or other cylindrical obstacles were arranged vertically and spaced 53 cm. apart for Rousettus and 30 cm. apart for Myotis Diameter of obstacle (mm.) Myotis I. lucifugus Rousettus aegypticus No. trials % misses No. trials % misses Cardboard tubes 25 109 76% Rubber tubing 19 161 78% Rubber tubing 13 100 77% Rubber tubing 6 50 80% Metal rods 4.76 140 85% Insulated metal wires 3 442 85% Bare metal wire 1.5 200 77% Bare metal wire 1.21 3820 82% Bare metal wire 1.07 280 68% Bare metal wire 0.68 480 77% Bare metal wire 0.65 225 58% Bare metal wire 0.46 134 45% Bare metal wire 0.35 660 72% Bare metal wire 0.28 50 18% Bare metal wire 0.26 600 52% Bare metal wire 0.12 530 38% Bare metal wire 0.07 460 36% inability to maneuver successfully even though it had detected the obstacle, and some represented light touches by the wingtips which may have been sufficiently painless to call for no great effort to avoid their occurrence. Unlike the Micro- chiroptera, this Rousettus rarely turned back from the obstacles. Its position and attitude in passing through the obstacle plane were recorded on about 40 flights with a camera and electronic flash. All wing positions from completely spread to considerably folded were photographed both just before and just after passage through the barrier, but we could not determine whether the bat was reducing its potential collision diameter just at the obstacle plane. Its maximum wingspread was about 75 cm., and while we cannot accurately estimate its mean wingspread this must have been at least 45 cm. or very little below the spacing between the wires. Finally, the possibility that the bat would detect the presence of the obstacles by their fastenings to the ceiling and/or floor and learn that they were suspended ver- 110 D. R. GRIFFIN, A. NOVICK AND M. KORNFIELD tically between these two points was excluded by framing the obstacle plane with uniform fiberboard so that only the obstacles themselves and not their fastenings were exposed to acoustic or visual inspection. As a last precaution, lest the bat learn to recognize the position of the obstacles by listening to the movement of the frame between flights, the readjustment was covered with loud noise. The nature and size of the obstacles used are shown in Table I. TABLE II Experiments with a captive Rousettus exposed to thermal noise while flying through a row of vertical wires, 3 mm. in diameter spaced 53 cm. apart. All flights in total darkness except as noted. The noise was filtered with high pass (HP) or low pass (LP) electronic filters as noted Date Conditions of test No. misses/No, trials Per cent misses Remarks Apr. 23 Quiet 30/40 75% Noise, 25 kc HP 0/10 Totally disoriented Quiet 17/20 85% Noise, 25 kc HP with lights on 9/10 90% Flew normally Noise, 15 kc LP 4/10 40% Somewhat disoriented Quiet 7/10 70% Noise, 15 kc LP 3/10 30% Disoriented, but less so than at 25 kc HP Quiet 10/10 100% Apr. 26 Quiet 10/10 100% Reluctant to fly- Noise, 25 kc HP 0/8 Badly disoriented Quiet 6/6 100% Very tired Apr. 28 Quiet 8/10 80% Noise, 15 kc LP 1/10 10% Badly disoriented Quiet 4/10 40% Tired May 3 Died Averages Quiet 93/116 79% of all Noise, 25 kc HP 0/18 tests Noise, 25 kc HP with lights on 9/10 90% Noise, 15 kc LP 8/30 27% RESULTS The results are presented in tabular form. The only data excluded from con- sideration are those which were obtained when the bat was clearly fatigued or in poor condition near the end of a long series of trials or after many days of inac- tivity. The data are compared directly in Table I with similar data obtained by Curtis (1952) with Myotis I. lucifugus. A short series of experiments was carried out to compare the resistance of Rousettus to interference with its echolocation by thermal noise but before further studies could be completed the bat died, possibly of injuries sustained in these ex- perimental flights. The data are shown in Table II, because they indicate a mark- ECHOLOCATION IN ROUSETTUS 111 edly greater vulnerability to interference by noise than occurs with the Vespertilioni- dae (Griffin, 1958). Thermal noise was generated in 20 electrostatic loudspeakers adjacent to the plane of obstacles. This noise was limited in frequency band, by electronic niters, in one of two ways. Either the filter was set at 15 kc high pass so that frequencies above 15 kc were generated at a high level while lower frequencies were attenuated progressively at 24 db per octave, or else a 25 kc low pass filter was used to transmit audio frequencies while attenuating ultrasonic components of the noise, also at 24 db per octave. Without noise, the bat avoided 3 mm. wires 79% of the time in the dark. In the light, and with the noise, in a very short series, it avoided the wires 90% of the time. But in the dark the bat was incapable of avoid- ing these wires at all in intense noise above 25 kc. In noise below 15 kc, it scored 27% misses. The bat's total inability to avoid large wires in noise above 25 kc and its very poor performance in noise below 15 kc suggest several hypotheses. If we assume that the poor performance was due to unfavorable signal-noise ratio at the same frequencies, then we have evidence that Rouscttns depends upon a wide range (from less than 15 kc to more than 25 kc) of frequencies in echolocation. But al- ternatively the analytical ability of Rouscttus' ears may not suffice for distinguish- ing a 14 kc echo from either type of noise tested, that is, we may simply have shown that the accuracy of acoustic orientation in Rouscttus can be reduced (even totally) by noise. The results may also have been complicated by the bat's panic, discomfort, loss of motivation, or confusion in an unusual situation aside from its ability to perceive echoes in a noisy environment. DISCUSSION In these experiments, the wires were less widely spaced relative to the wing- spread of Rouscttus than in Curtis' experiments with M \otis, but Roiiscttns almost always approached the plane of the obstacles perpendicularly while Myotis often approached obliquely. Our flight room was also considerably larger than the 15' X 9' X ()' room used by Curtis. The percentage of misses for relatively large obstacles was, nevertheless, almost exactly the same 85.0% for Myotis with 4.76- mm. rods and 84.5% for Rouscttus dodging 3 -mm. wires. Rouscttus was slightly less successful at avoiding even larger obstacles (cardboard and rubber tubes) but these tests were conducted early in the bat's experience in the exacting task of flying in a dark room (with its multiplicity of echoing surfaces). This Rouscttus was able to detect and avoid, with a considerable degree of suc- cess, wires as small as 1.07 mm. in diameter. Only when confronted with wires of less than 1 mm. did its skill fall seriously below its own standards as well as those of Myotis. Rouscttus' score decreased rather gradually. If we consider its poor performance (T8 f ,Y misses) against 0.28-mm. wires as due to chance, then Rouscttus was clearly detecting 0.46-mm. wires against which it scored 45% misses. Even 18%' misses against 0.28-mm. wires may have represented some degree of echolocation for. when flying in a noise field, this bat did even more poorly (100% hits) against 3-nim. wires. It seems reasonable that the ease with which a small object is echolocated depends upon its position relative to the angle of sound emis- sion and its beaming and the angle of sound reception. Thus there is likely to be an optimal angle of approach (probably, but not necessarily, straight ahead) where the maximum echo will be received and less easilv detected obstacles will be echo- 112 D. R. GRIFFIN, A. NOVICK AND M. KORXFIFLD located. Obstacles which lie less optimally relative to the bat will have to have more effectively echoing surfaces to be detectable. Thus the bat might well succeed in avoiding a 0.46-mm. or 0.65-mm. wire only if it chanced to approach it favorably and so its score when working against obstacles of marginal size would be an aver- age of chance misses, active misses, and "blind" hits. One of the limiting factors in exploring the threshold of echolocation is the danger of serious injury to the bat every time it collides with an obstacle. Such collisions may be major accidents or simply touches. Collisions with small wires tend to be more serious than those with large obstacles. Roiiscttns' performance varied considerably from trial to trial. Whenever possible we ran long series of tests and interspersed tests with 3-mm. wire between those with smaller sizes. The results were consistent with the average scores listed in Table I. The design of Myotis orientation pulses is very different from that of Rouse ft its clicks. Mvotis pulses are produced laryngeally and emitted through the open mouth. They have a frequency modulated pattern with a gradually fall- ing frequency starting on the average at about 80 kc and ending at about 40 kc but with beginnings ranging from at least 60 to 120 kc. Similar variety among terminal frequencies also occurs. Thus Myofis in single pulses and in consecutive pulses produce prominent frequencies covering about two octaves (Griffin, 1958; Xovick. 1955). Furthermore, harmonics also occur in Myotis pulses and represent a sec- ond octave sweep within the pulses in which they occur. The importance of the harmonics as components of the outgoing pulses and the returning echoes and in the carrying of information about the environment to the bats has not been evaluated. In Ronsettns. the pulses are produced by tongue clicks and are impure in frequency and irregular in frequency pattern. The bulk of the energy, however, appears to be in the range of about 12 to 18 kc. Additional energy is scattered from 6.5 to over 100 kc with a second maximum at about 20 to 40 kc ( Moehres and Kulzer, 1956a; Kulzer, 1956; Novick. 1958). SUMMARY 1. The ability of a single specimen of the fruit bat, Roiiscttns aegypticus, to avoid test obstacles of various sizes by echolocation in total darkness was tested. This bat avoided vertically placed 3-mm. metallic wires 85% of the time. Its success de- clined gradually as the wires were reduced in size but the bat displayed considerable success (68% misses) against 1.07-mm. wire and did significantly better than chance (45% misses) against wires 0.46 mm. in diameter. 2. These results have been compared with those of Curtis (1952) who studied the vespertilionid, Al \otis I. In din;/ its. 3. Roiiscttns' success at echolocation was considerably reduced when it was forced to fly in a field of intense thermal noise. LITERATURE CITED CURTIS, W. E., 1952. Quantitative studies of echolocation in bats (Myotis I. lucifugus) ; stud- ies of vision in bats (Myotis I. Iucifn(jus and Eptcsicus f. fuse us) ; and quantitative studies of vision of owls (Tyto alba pratincola) . Thesis deposited in the library of Cornell University, Ithaca, New York. GRIFFIN, D. R., 1946. The mechanism by which bats produce supersonic sounds. Anat. Rec., 96: 519. ECHOLOCATION IN ROUSKTTUS 113 GRIFFIN, D. R., 1952. Mechanisms in the bat larynx for production of ultrasonic sounds. l ; cd. Proc.. 11: 59. GRIFFIN, D. R., 1958. Listening in the Dark. New Haven, Yale University Press. GRIFFIN, D. R., AND R. GALAMBOS, 1941. The sensory basis of obstacle avoidance by flying bats. /. .r/>. Zooi, 86: 481-506. GRIFFIN, D. R., AND A. NOVICK, 1955. Acoustic orientation of neotropical bats. /. E.rp. Zoo/.. 130: 251-300. GRINNELL, A. D., AND D. R. GRIFFIN, 1958. The sensitivity of echolocation in bats. Biol. Bull., 114: 10-22. HAHN, W. L., 1908. Some habits and sensory adaptations of cave-inhabiting bats. I and II. Biol. Bull., 15: 135-193. KULXER, E., 1956. Flughunde erzeugen Orientierungslaute durch Zungenschlag. Naturzviss., 43: 117-118. MOKIIKKS, I". P., AND K. KuLZEK, 1956a. Untersuchungen iiber die Ultraschallorientierung von vier afrikanischen Fledermausfamilien. I'crli. dtsch. zool. Gcs. in Erlangcn, Zool. .Inzciticr Siifplemcntlnind. 19: 59-65. MOEHRES, F. P., AND F. KULZER, 1956b. tjber die Orientierung der Flughunde ( Chiroptera- Pteropodidae). Zcitschr. f. rcrt/l. Pliysiol., 38: 1-29. NOVICK, A., 1955. Laryngeal muscles of the bat and production of ultrasonic sounds. Aincr. J. Physiol.. 183': 648. N'uvicK. A., 1958. Orientation in palaeotropical bats. II. Megachiroptera. /. E.\-p. Zool., 137 ( in press). ELECTROPHYSIOLOGICAL STUDIES OF ARTHROPOD CHEMO- RECEPTION. III. CHEMORECEPTORS OF TERRESTRIAL AND FRESH-WATER ARTHROPODS 1 EDWARD S. HODGSON Department of Zoology, Columbia University, New York 27, A'. )'.. and Mountain Lake Bio- logical Station, r While an extensive literature documents the role of chemoreceptors in the be- havior of invertebrates (Hodgson, 1955), the small size of chemoreceptor cells is a major handicap in any attempt to study their functions using conventional electro- physiological procedures ( Chapman and Craig, 1953 ; Roys, 1954). Barber ( 1956) recorded afferent impulses from neurons which supply the gnathobase chemore- ceptors of Lunnlns and noted an increase in nerve activity when aqueous extracts of marine bivalves were applied to the gnathobase. Use of microelectrodes enabled Schneider (1957) to record afferent impulses from groups of antennal chemore- ceptors in male silkmoths (Bomb\.\-) during stimulation with extracts of the scent glands from female moths. Possible synaptic effects between receptor cells and nerves supplying them, or the unpredictable numbers of cells represented in most recordings, make it difficult, however, to interpret the results in terms of single unit activity of the actual chemoreceptor cells. A relatively simpler technique is that of recording the afferent impulses from primary chemoreceptor cells through the same fluid which is applied as a stimulus (Hodgson, Lettvin and Roeder, 1955). This method has thus far been applied only in studying contact chemoreceptors of two animals: labellar chemoreceptors of the blowfly Phornria (Hodgson and Roeder, 1956; Wolbarsht. 1957) and tarsal chemoreceptors of the butterfly J \iucssa ( Morita ct al., 1957). The conclusions from studies of these two preparations point to a number of unexpected properties of primary chemoreceptor cells. With both Phonnia and 1'ancssa, it was found that different chemoreceptor cells were specialized to respond, not to the different modalities of stimuli generally held to be effective for contact chemoreceptors of vertebrates (e.g. Beidler, 1952), but either to sugars or to various non-sugars, with the presence of a water-specific re- ceptor also strongly indicated in 1'ancssa (Morita ct al., 1957). Seemingly at vari- ance with the usual concept of single specificities of receptor cells (Granit, 1955), a single primary receptor cell of Phonnia may respond to chemical, tactile, and thermal stimuli within normal physiological ranges (Hodgson and Roeder, 1956). Unfortunately, information on this point is not available for 1'ancssa. In view of these unexpected results, and the lack of any comparable electrophysi- ological data on primary chemoreceptors of other invertebrates, it seemed desirable that the method of recording through fluid-filled, externally applied electrodes 1 This investigation was aided by Public Health Service Grant No. E-1010, and by the Higgins Fund of Columbia University. The field work was aided by a National Science Foundation Grant to the Mountain Lake Biological Station. 114 ARTHROPOD CHEMORECEPTION 115 should be tried on chemoreceptors of a wider variety of animals, in order to de- termine how generally the characteristics found in Plwnnia and Vanessa receptors may apply to the functions of other primary chemoreceptor cells. For technical reasons, this method is best adapted to recording from chemoreceptors in arthropods (Hodgson, Lettvin and Roecler, 1955). The object of the present paper is to report the results of tests conducted using this method upon the chemoreceptors of some terrestrial and fresh-water arthropods. In each case where the method could be successfully applied, answers to the following questions were sought : ( 1 ) Does the same receptor cell respond to chemical, tactile, and thermal stimuli within normal physiological ranges? (2) What modalities of chemical stimuli excite the indi- vidual primary chemoreceptor cells? (3) Does the relationship between the reac- tion of the animal to chemicals and the range of sensitivity of its chemoreceptors indicate a peripheral discrimination mechanism, such as found in PJwrmia? METHODS Thirty-seven species, representing the major classes of arthropods and eight or- ders of insects, were tested. These species are arranged according to taxonomic status below. All specimens were collected in the field and tested within 12 hours after capture. The animals were allowed to drink water to repletion, but no attempt was made to control their diet prior to testing. At least three individuals, usually more, belonging to each species were studied. The technique of recording action potentials from chemoreceptors using ex- ternally applied, fluid-filled electrodes has been described in detail elsewhere (Hodg- son, Lettvin and Roeder, 1955; Hodgson and Roeder, 1956). This technique was used with only such minor modifications as were necessary to manipulate the va- riety of receptor-bearing appendages tested. All experiments were tape recorded and photographs made from the tape recordings, beginning one-half second after the stimulus was applied, thus avoiding the base-line fluctuations which commonly accompany the stimulus artifact. The species tested were as follows, with each group and each species yielding potentials from chemoreceptors designated by an asterisk. (Except as otherwise noted, identifications were checked through the courtesy of Dr. R. E. Blackwelder of the U. S. National Museum.) Class: Crustacea* Cambarus bartomi scioten- sis* (Det. H. H. Hobbs, Jr.) ; Class: Arachnida Latrodectus mactans (black widow spider). Theridion tepidariorum (house spider) ; Class: Diphpoda* -Pseudotremis sp. (Det. H. F. Loomis), Pseudopolydesmus serratus* (Det. M. Walton) ; Class: Insccta; Order: Odonata Aeschna constricta. Libellula pulchella, Progomphus obscurus ; Order: Ortlwptera* Acheta assimilis (common field cricket), Ceu- thophilus gracilipes* (cave cricket), Crytocercus punctulatus (wood-eating roach) (Det. L. R. Cleveland). Hadenoecus putaneus* (cave cricket). Scudderia furcata (katydid) ; Order: Heiuiptera Arilus cristatus, Oncopeltus fasciatus (large milk- weed bug) ; Order: Coleoptera Cicindela sexguttata (six-spotted tiger beetle), Dineutes americanus (whirligig beetle), Dytiscus fasciventris (large diving beetle), Laccophilus maculosus (common pond beetle), Nicrophorus tomentosus (carrion- beetle), Phymatodes dimidiatus ( longhorn beetle), Saperda Candida (apple tree borer), Silpha americana (carrion beetle), Tropisternus lateralis (keeled water beetle) ; Order: Megaloptera Carydalus cornutus (dobsonfly) ; Order: Nenrop- 116 EDWARD S. HODGSON tcra Chrysopa sp. (golden eyed lacewing) ; Order: Diptera* -Amoebaleria de- fessa* (cave fly) (Det. C. H. Curron). Tipula trivittata (crane fly) ; Order: Lef>i- doptera* Atlides halesus (purple hairstreak) ; Epargyreus clarus* (silver spotted .skipper), Limenitis arthemis astyanax* (red spotted purple), Papilio marcellus* ( zebra swallowtail), Papilio philenor* (pipe vine swallowtail), Protoparce quin- quemaculata ( five-spotted hawk moth), Speyeria cybele* (great spangled britillary), Tropaea luna (luna moth), Vanessa atalanta* (red admiral). The chemicals tested were sodium chloride, sucrose, d-levulose, glycine. DL glutamic acid, citric acid, oil of citronella and oil of wintergreen. Sodium chloride was tested as a 0.25 molar aqueous solution. Oils of citronella and wintergreen were tested by bringing swabs soaked in these chemicals to within an inch of the sensory structure. Although quantitative control of stimulus concentration was not obtained by this method, the results obtained with these two oils were quite reproducible. All of the other chemicals were mixed with sodium chloride so that the final test solution was an unbuffered aqueous solution containing 0.1 molar XaCl and a 0.25 molar concentration of the test chemical. Results were compared with activity re- corded when 0.1 molar XaCl was applied alone. Temperatures were measured with a thermistor implanted just under the cuticle near the receptor being studied. The temperature was changed by bringing a warm glass rod or small ice-pack near the preparation. Spike potentials from mechano- receptors were recorded by bending sensilla or whole appendages with needles. Certain departures from the usual tests are described at appropriate points below. RESULTS All of the preparations yielded numerous spike potentials originating from tac- tile receptors, thus providing assurance that the preparations were alive when studied. In only five orders of the arthropods tested, however, was it possible to obtain unequivocal recordings from chemoreceptors. These five groups are desig- nated by asterisks above. The several factors believed to be responsible for failure to record action potentials in all of the tested species are considered in the discussion, and a complete description of the results will be presented only for those forms in which chemoreceptors could be studied using fluid-filled electrodes. 1. DECAPODA Cainbants bartonii sciotensis (16 individuals) This large crayfish proved to be an exceptionally interesting experimental ani- mal. Recordings could be made with the usual 0.1 molar NaCl conducting solution in the electrode, or else by using distilled water or pond water as a solvent for the chemicals. Although the results showed few differences whichever solvent was used, all of the tests were run with chemicals dissolved in distilled water, thus avoid- ing any possible complications of the sodium chloride. The antennae and the lateral branches of the antennules were alike in yielding only records of mechanoreceptors at low amplitudes (30 /iV). From the entire medial branch of the antennule, however, it was possible to record a variety of spike potentials ranging in amplitude from 30 /A r to 500 /tY . The large-amplitude spikes (200 fjiV to 500 p.\ 7 ) were recorded only when the antennule was bent. Conse- quently, the cells giving rise to these potentials, which are relatively few in this ARTHROPOD CHEMORECEPTION 117 A FIGURE 1. Typical spike potentials from arthropod chemoreceptors. A, response of medial branch of Cainhanis antennule to glutamic acid; Bl, single sensillum on Cambarus walk- ing leg, tested with distilled water ; B2, same as Bl, except glycine test solution ; Cl, Pscudo- polydcsmus tarsus, NaCl control: C"_', same as Cl, except sucrose test solution; Dl, spontaneous activity, Hadcnoccits tibia; I~>2, same as Dl, except exposed to citronella vapor; E, single tarsal sensillum of Eparyyreus, control NaCl solution ; /", same as E, except sucrose test solution ; G, antenna of Amocbalcria, distilled water in electrode; H, same as G, except exposure of antenna to oil of citronella vapor. Time bases for all records, 100 cycles per second. Consult text for additional details. 118 EDWARD S. HODGSON branch of the antennule, are mechanoreceptors. The majority of the spikes have amplitudes of 30 ^V to 50 ^V. These respond to the application of glycine and glutamic acid, of the test series of chemicals used. Because a number of different amplitudes of spikes were recorded even with the smallest practicable areas of elec- trode contact, it was not possible to determine whether identical cells were respond- ing to both chemical and tactile stimuli. Record A of Figure 1 is taken from an experiment in which a test solution of glutamic acid was allowed to flow around the medial branch of an antennule without changing electrode contact. Activity re- corded when the antennule is in distilled water (on the left of the large stimulus artifact) is negligible, but many small-amplitude spike potentials follow the intro- duction of the glutamic acid. The frequency of firing during chemical stimulation was not influenced by temperature changes within the range tested -five degrees (C.) above or below the room temperature of 25 degrees. Chemoreceptors were also found on the first two pairs of walking legs. The chemoreceptors w r ere located on the chelae and, to a lesser extent, elsewhere on the protopodites of those legs. The external chemosensory structures can be recog- nized in C. barton ii as tufts of setae, numbering ten to twenty setae per tuft, each such tuft arising from a circular depression in the cuticle. Contact of the electrodes with other parts of the cuticle failed to terminate the open circuit condition between the indifferent electrode inside and the recording electrode outside the cuticle. The best records were obtained after the claw had been allowed to dry at room temperature for thirty minutes following its removal. This prevented short circuits between the recording and indifferent electrodes. By teasing apart the hairs of a single tuft, the tip of an electrode could then be positioned over a single sensory hair. In this way the firing of a single chemoreceptor cell could be studied. The spike potentials recorded from different sensilla ranged from 30 to 60 /A /r in ampli- tude. It was found that these receptors resemble chemoreceptors on the antennule in being insensitive to test chemicals other than amino acids of the series used. (Records Bl and B2 of Figure 1 illustrate typical results during applications of a control NaCl solution, and the test mixture of NaCl and glycine, respectively.) The chemoreceptors on the first two walking legs were never observed to respond to mechanical movement of the sensory hairs during recordings. The small size of the hairs (about 20 microns in length) and their position surrounded by rigid cuticle would appear to render their usefulness as tactile receptors unlikely. The insensi- tivity of these receptors to temperature changes within the range tested resembles that of the receptors on the antennule. Impulses from chemoreceptors were not detected from the chelipeds, third maxillae, or elsewhere on the body of the crayfish using the present method. Behavioral experiments were run to check the possibility of a peripheral dis- crimination for amino acids. Ablations of antennae, antennules, or the first two pairs of walking legs, and combinations of these operations, were performed on thirty crayfish. The results were difficult to interpret in many cases because of abnormal behavior of operated animals. It was easy to demonstate, however, that the animals can locate food using the first two pairs of walking legs, even when antennae, anten- nules, and maxillipeds are removed. Activity resembles that during normal feed- ing and can be initiated by injecting 0.25 molar solutions of glycine and glutamic acid into the water, while even intact animals fail to give clear-cut responses to the other test solutions. Thus there seems to be a clear correlation between the elec- ARTHROPOD CHEMORECEPTION 119 trophysiological data and the behavioral results. Attempts to determine by be- havioral tests whether the antennae and lateral branches of the antennules bear chemoreceptors yielded results which could not be unequivocally interpreted. Doflein (1910), on the basis of behavioral tests, has reported that the antennules of decapods contained chemoreceptors, and Luther (1930), using similar methods, reported chemoreceptors on mouth parts, walking legs, and pincers of brachyurans. 2. DIPLOPODA Pseudotremis sp. (4 individuals) ; Pseudopolydesmus scrratus (4 individuals) In both Pseudotremis and Pseudopolydesmus many action potentials could be recorded from the tips of the antennae and from the tips of the legs when an elec- trode filled with 0.1 molar NaCl was applied to those parts. In Pseudotremis. the smaller species, the action potentials were never more than 40 /zV in amplitude, and all clearly responded to mechanical bending of the antenna or leg. In Pseudopoly- desmus the largest spikes from the antenna were about 60 //.V in amplitude, and those from the tarsus were about 80 //.V. All of the larger spikes increased in frequency during bending of the appendages being tested, and it was therefore assumed that these spikes represented the afferent impulses from mechanoreceptors. Spike po- tentials of smaller amplitude (30-50 ju,V) from tarsi of Pseudopolydesmus occurred with increased frequency when the tarsi were bent, or sugars applied. (See Fig. 1C.) They did not change during application of other test solutions or during tem- perature changes within five degrees (C.) of the room temperature of 25 degrees. No significant changes in the frequency or pattern of impulses were noted in re- cordings from the antennae of the two species when chemical stimuli were applied. The small trichoid sensilla which probably enclose the actual chemosensory cells on the tarsi of Pseudopolydesmus are too closely spaced to make possible a re- striction of the area of electrode contact to a single sensillum. Attempts to record activity using electrodes filled with distilled water were likewise unsuccessful. In view of the smaller size of the mechanoreceptor spikes recorded from Pseudotremis, and the generally smaller size of action potentials from chemoreceptors as compared with mechanoreceptors, it would hardly be expected that chemoreceptor spikes from Pseudotremis would be detectable above the inherent "noise level" of the apparatus Behavioral test showed that sucrose or levulose, placed in contact with the tarsi, initiated feeding responses even after the antennae w r ere removed. Tarsal contact with citric acid caused the animals to move away from the test solution, but this was the only test solution, other than the sugars, which elicited a behavioral re- sponse. With the exception of citric acid, receptors for which could not be de- tected electrophysiologically, the behavioral and electrophysiological results suggest the existence of a peripheral discrimination mechanism. 3. ORTHOPTERA Ceuthophilns c/racilipcs (7 individuals) ; Hadcnoccus pn- tancus (3 individuals) The orthopterans tested showed considerable variation, some of which appears to be related to habitat. Cryptocercus, a wood-eating roach, was completely refrac- tory to the recording method, except for a few mechanoreceptors in the antennae and palpi. A larger number of tactile receptors were recorded from the antennae and 120 EDWARD S. HODGSON palpi of the katydid, Scuddcria, and the field cricket. Acheta. Hest results, how- ever, were obtained with the cave crickets Ceuthophilus and Hadenoecus, which have antennae elongated to many times the length of the body and also have un- usually long legs and palpi. The data support the generally expressed assumption that these anatomical modifications are associated with hypertrophy of tactile and chemical senses which would presumably be of selective value in dark subterranean environments. In tests of seven adult specimens of Ceuthophilus and three of Hadenoecus, the antennae were found to contain spontaneously active and quick-adapting mechano- receptors (spike amplitudes 50-80 yuV) along with spontaneously active, relatively non-adapting chemoreceptors (spike amplitude 2040 /tV). The latter were seen in one antennal preparation of Ceuthophilus and all three preparations of Hade- noecus. The frequency of the small spikes did not change during application of any of the test chemicals in solution, or during temperature changes between 20 and 30 degrees C.. but did increase when swabs soaked in citronella or wintergreen were brought near the region of the antenna in contact with the electrode. Essentially similar results were obtained from recordings of the receptor activity in both the maxillary and labial palpi and the tarsi of Ceuthophilus and Hadenoecus. In addi- tion, small spikes (30-50 ^V) were recorded from the trochanter and tibia of the prothoracic and mesothoracic legs of Hadenoecus, in six out of eight preparations when the legs were exposed to vapors of wintergreen or citronella. Mechanical bending of sensilla on the trochanter and tibia also increased the frequency of these same spike potentials. Record Dl of Figure 1 shows the spontaneous activity of receptors in the tibia of a prothoracic leg of Hadenoecus, and record D2 shows the increase in frequency of spikes during application of citronella vapor. Xo effects of the test chemicals in solution could be detected in either Ceuthophilus or Hade- noecus, and chemoreceptor activity could not be recorded from the cerci, ovipositor, general body surface, or the larger spines on the legs of either species. Ceuthophilus did not give any clear-cut behavioral response to citronella or wintergreen in tests of the intact animals, but Hadenoecus gave intense avoidance reactions, moving quickly away from these stimuli. Removal of the antennae and palpi did not abolish this reaction in Hadenoecus, which always responded most strongly when stimuli were near the legs. 4. LEPIDOPTERA Nine species of Lepidoptera were tested. Only a few impulses associated with tactile stimulation could lie recorded from the antennae of any of these species, even when vapors were applied. In all six species of butterflies tested, records were ob- tained from the tarsal receptors (described by Minnich, 1921). Tests upon the tarsal sensilla of Eparyyrcus and Limcnitis revealed that each sensillum had a few- receptor systems functioning similarly to that in the labellar hairs of Phonnia. (Compare the records E and F of Figure 1. taken from tests of a single tarsal sensillum of Epargyreus, and note that the small spike potentials predominate only in record F when sugar is present in the electrode.) The maximum number of re- ceptors represented in recordings from single sensilla of these two species is four, and the minimum two. Variations within these limits were commonly encountered in comparisons of the records from several hairs, even on the same tarsus. The ARTHROPOD CHEMORKCKPTION 121 variations characteristically occurred in the smaller spike potentials, but under the conditions of these tests all of the smaller spikes increased in frequency during stimulation with sugars, and the largest spike responded with increased frequencies during application of any of the non-sugar solutions. These receptors were not observed to respond to vapors of citronella or wintergreen. With the other species of butterflies tested, there appeared to be as many as 12 different receptors associated with each tarsal sensillum and the records were too complex for analysis of the functions of any single receptor cells. Responses to tactile stimulation were obtained in tests with tarsal hairs of all the butterflies used ; in those preparations involving only a few fibers it was clear that all fibers responded to bending of the tarsal hair, and probably this was the case with the many-fiber preparations also, but this could not be determined with certainty because of the complexity of the records. The frequency of impulses recorded during continuous stimulation of single sensory hairs of Eparc/yrcns and Li men it is was increased by temperature rises of as little as 1.2 degrees C. These particular tarsal receptors,, then, bear a greater resemblance to the labellar chemoreceptors of flies than do any of the other preparations (excluding the labellar chemoreceptors of Amoebalcria] encountered in this survey. Feeding responses (proboscis extensions) in butter- flies are known to be elicited by sugars, with negative responses being elicited by other types of chemicals (Dethier, 1953 ; Minnich, 1921 ). A peripheral mechanism for discrimination of acceptable and unacceptable chemicals is thus indicated by both the behavioral and electrophysiological results with butterflies. Tarsal chemoreceptors were not detected in any of the three species of moths. No impulses could be recorded from the trichoid sensilla described by Frings and Frings (1949) on the proboscis of lepidopterans. The characteristics of the records obtained from such tests indicated, however, that a short-circuit between the record- ing and indifferent electrodes, established through the fluids in the proboscis, prob- ably accounted for the lack of any spike potentials detected through an active electrode near the tip of the proboscis. 5. DIPTERA Amocbalcria dcfcssa (7 individuals) ; Tipnla tririttata (3 individu- als) Studies on four genera of Diptera having been previously reported (Hodgson and Roeder, 1956), the present work was confined to two types in which the chemo- receptors might be expected to be of special interest. The helomyzid fly Amoe- balcria was tested because of its occurrence in caves, a habitat often associated with hypertrophy of chemical or tactile senses (Hodgson, 1955), and the crane fly Tipitla was tested because the branching structure of its antennae suggested that recordings might be made from one or a few antennal receptors in a single antennal branch. Only Ainoebalcria yielded results of interest, however. The labellar chemoreceptors and chemoreceptors within the tarsal hairs of Amocbolcria proved to function similarly to those in Plionnict, in that they exhibited L and S spikes when stimulated by sugars or non-sugars, and showed comparable responses to tactile and temperature stimulation. Some data on olfactory receptors were obtained in recordings from the antennae of Amocbalcria. A typical result, obtained by placing a fluid-filled electrode on the antenna, is shown in record G of Figure 1 . Distilled water is adequate in the electrode, and the results are essentially 122 EDWARD S. HODGSON the same whether contact is made with the distal tip of the antenna or the enlarged third segment near the hase of the antenna. Ablation experiments show that most of the activity recorded originates in the third segment of the antenna in either case. The abundant spikes which seem to represent the basal level of receptor ac- tivity in the absence of externally applied stimulation are not affected by any of the test solutions applied, but are decreased in frequency by vapors of wintergreen, or citronella (see record H of Figure 1). This result was so contrary to anticipated findings that tests were run with benzene, toluene, and carbon tetrachloride vapors, all of which produced similar reversible decreases in amount of receptor activity. Unfortunately, so little is known of the natural historv of this fly that it is im- possible to say what might constitute the normal olfactory stimuli. Tactile effects upon the antennal receptors were observed only when the surface of the antenna was prodded or bent in excess of any amount of stimulation which the antenna would encounter in flight. Blowing upon the antenna during a recording or varying the temperature from 20 to 28 degrees C. produced no discernible effect upon the frequencv or pattern of the impulses recorded. Attempts to make similar antennal recordings using other species of flies have yielded only negative results. DISCUSSION In view of the considerable differences in chemoreceptors which have already been reported from electrophysiological studies of mammals (Beidler. Fishman and Hardiman, 1955) it is not surprising that much greater differences should be found among members of such a heterogeneous group as the arthropods. It seems clear that sensitivities to tactile and temperature stimuli within the normal physiological range are not essential characteristics of primary chemoreceptor cells, even among the arthropods, because several exceptions to this situation were found as soon as tests were made of chemoreceptors other than those on the fly labellum. Yet it would probably be incorrect to regard the labellar receptors as primitive or unspecial- ized receptor cells. Their similarity to receptors in the tarsal sensilla of at least two of the butterflies tested suggests that a sensitivity of the same cell to more than one type of energy in the environment may have a high selective value in cases where only a relatively small number of receptors contact a substrate, man}' fea- tures of which are significant for the animal's behavior. This certainly would be the case with receptors on the tarsus or proboscis of a fly or butterfly, or on the tips of the tarsi of a millipede. The demonstrated multiple sensitivities of single receptor cells in those locations may. therefore, be one of the solutions which evolution has produced for the problem of obtaining a variety of information about the environ- ment when only very small areas of the body are actually in contact with the en- vironment. Whether the several types of stimuli all eventually affect the same excitatory process within a single receptor cell will have to be determined by further investigations. Cases of double specificities of receptors in vertebrates, such as the temperature-touch receptors of the rattlesnake facial pit (Bullock and Diecke, 1956), have been reported but it is very doubtful that more than one type of stimulation normally acts upon the same receptor units, and even if this were true these would have to be considered exceptions to the general rule of single specificities for single receptors (Granit, 1955). Several correlations might be noted between receptor distribution or function ARTHROPOD CHEMORECEPTION 123 and the natural history of the particular animals concerned. Of the two cave crickets providing favorable receptor preparations, Hadenoecits, with the more ex- tensively distributed chemoreceptors on the legs, is reported to be more strictly lim- ited to caves than CcutJiophilus (Giovannoli. 1933). The selective advantage of highly developed chemical senses in a totally dark environment is obvious. The sensitivity of the chemoreceptors of Cainbarus to amino acids is undoubtedly related to a diet of decaying meat, and the absence of any response of its receptors to sugars can be correlated with the lack of any behavioral response to sugars by this species. The results with butterflies likewise indicate the existence of a peripheral discrimina- tion mechanism for the chemicals constituting the normal food in this case, sugars. All of the spike potentials recorded from chemoreceptors were smaller in am- plitude than the spikes from mechanoreceptors of the same animal, unless the same receptor cell responded to both types of stimuli. This is in accord with the usual assumption that chemoreceptor fibers are smaller than mechanoreceptor fibers (Dethier, 1953; Hodgson, 1955). The fact that many receptors in Cainbarus, Hadcnoccus, and Aiuoebaleria showed spontaneous activity supports another idea believed to be of some general applicability that spontaneous activity is widespread among sensory cells, and that anv changes in the frequency or pattern of the spon- taneous activity ( rather than the mere presence of impulses ) may constitute the af- ferent ''message" from the sense organs (Roeder, 1955). The antennal receptors of Amoebaleria, showing decreased numbers of impulses during administration of vapors, may illustrate a less common direction of change in spontaneous activity which serves as the afferent message. The present experiments resolve a discrepancy between the earlier work on the labellar chemoreceptors of the blowfly (Hodgson, Lettvin and Roeder, 1955) and the results obtained by Morita ct al. (1957, and personal communication) using the butterfly. I'ancssa. The polarity of the spike potentials recorded from Phonnia was previously reported as negative, using the present recording method, but posi- tive under similar conditions in Vanessa. All the spike potentials recorded from chemoreceptors in the present studv resulted from an increase in positivity at the distal tip of the sensory hairs (position of the recording electrode) relative to the base of the same hairs (position of the indifferent electrode), and the contrary po- larity reported in Phonnia was subsequently traced to an error in instrumentation. A precise explanation for the positive spike potentials obtained by this method can- not be given at the present time, but might possibly be explained by generation of the main negative spike potential at the cell body region of the receptor, which would leave the actual chemosensory area with a relatively positive charge. Ex- periments to localize the main impulse generating area within the receptor are now underway. The failure to record potentials from chemoreceptors in a large majority of the arthropods tested could result from a real absence of these receptors in the ap- pendages tested or from limitations of the technique. The latter is the more probable explanation in most cases. Particularly unfortunate is the apparent inap- plicability of the technique to recordings from the antennae of most insects. Unavoid- able short circuits between indifferent and recording electrodes explain some nega- tive results, as noted above, but inability to position the recording electrode over one or a few receptor sensilla and the small size of the spike potentials from the chemo- 124 EDWARD S. HODGSON receptors undoubtedly account for most of the failures. The optimum preparation for use with this technique appears to he an elongated sensillum, well isolated from surrounding sensilla, and containing very few receptor cells an ideal approached more conveniently in the lahellar chemoreceptors of flies than with any other ar- thropod preparations }et tested. A similar survey of the chemnreceptors of marine arthropods is planned. It is a pleasure to acknowledge the courtesy of Dr. Horton H. Hobhs, Jr., Di- rector of the Mountain Lake Biological Station, who facilitated the held work in many ways. Mr. David Bardack assisted in collecting the animals. Drs. V. G. Dethier and K. D. Roeder have been most helpful in critically reading the manuscript. SUMMARY 1. Electrophysiological tests with externally applied, fluid-filled electrodes were performed upon thirty-seven species representing four classes of arthropods. Af- ferent chemoreceptor impulses were recorded in animals of five types: a crayfish ( Caiubants ) , a millipede (Pseudopolydesmus}, two orthopterans (Ceuthophilus and Hadenoccus) , a helomyzid fly (Ainocbalcria), and six species of butterflies. 2. Receptors sensitive to chemical, tactile, and temperature stimuli within nor- mal physiological ranges are found in certain Lepidoptera (Epargyreus and Linic- nitis) and Diptera (Aiuocbaleria] . Receptors with a dual sensitivity to at least two of the above types of stimulation are found in Pscitdopolydesinus. Ccitthophilns, and Hadenoccus. It is concluded that multiple sensitivities of receptors are not exceptional in arthropods. 3. Chemoreceptors sensitive to amino acids, but insensitive to tactile and tem- perature stimuli, are found on the chelae and protopodites of the first two walking legs of Caiubants bartonii sciotcnsis. 4. \Yith the present recording method, spike potentials from chemoreceptors represent increases in positivity at the distal tip of the receptor cell, relative to the cell body. 5. Relationships between functional characteristics of chemoreceptors and the natural history of the animals are discussed. LITERATURE CITED BARBER, S. B., 1956. Chemoreception and proprioception in Limulus. /. P..rp. Zonl.. 131 : 51-73. BEIDLER, L. M., 1952. Our taste receptors. Sci. Monthly, 75: 343-349. BEIDLER, L. M., I. Y. FISHMAX AND C. \Y. HARDIMAN, 1955. Species differences in taste re- sponses. Amer. J. Physiol.. 181 : 235-239. BULLOCK, T. H., AND F. P. J. DIECKE, 1956. Properties of an infra-red receptor. /. Phvsiol.. 134 : 47-87. CHAPMAN, J. A., AND R. CRAIG, 1953. An electrophysiological approach to the study of chemical sensory reception in certain insects. Canad. Ent.. 85: 182-189. DETHIER, V. G., 1953. Chemoreception. Chap. 21 in "Insect Physiology," edited by K. D. Roeder. Wiley, New York. DOFLEIN, F., 1910. Lebensgewohnheiten und Anpassungen bei dekapoden Krebsen. Fcstschr. R. Hertivig, 3 : 1-76. FRINGS, H., AND M. FRINGS, 1949. The loci of contact chemoreceptors in insects. Amer. Midi. Nat.. 41: 602-658. ARTHROPOD CHEMORECEPTION 125 GIOVANNOLI, L., 1933. Invertebrate life of Mammoth and other neighboring caves. Ainer. Midi. Nat.. 14: 600-623. GRANIT, R., 1955. Receptors and Sensory Perception. Yale University Press, New Haven. HODGSON, E. S., 1955. Problems in invertebrate chemoreception. Quart. Rev. Biol.. 30: 331- 347. HODGSOX, E. S., 1957. Electrophysiological studies of arthropod chemoreception. II. Re- sponses of labellar chemoreceptors of the blowfly to stimulation by carbohydrates. /. Insect Physiol., 1 : 240-247. HODGSOX, E. S., J. Y. LETTVIN AND K. D. ROEDER, 1955. The physiology of a primary chemo- receptor unit. Science, 122: 417-418. HODGSON, E. S., AND K. D. ROEDER, 1956. Electrophysiological studies of arthropod chemo- reception. I. General properties of the labellar chemoreceptors of Diptera. /. Cell. Comp. Physiol.. 48: 51-76. LUTHER, \V., 1930. Versuche iiber die Chemorezeption der Brachyuren. Zcitschr. rcryl. Physiol., 12: 177-205. MORITA, H., S. DOIRA, K. TAKEDA AND M. KUWABARA, 1957. Electrical response of contact chemoreceptor on tarsus of the butterfly, J'anessa indica. Mem. Pac. Sci.. Kyushu Univ., Scries E. 2: 119-139. MINNICH, D. E., 1921. An experimental study of the tarsal chemoreceptors of two nymphalid butterflies. /. 7:.r/>. ZooL, 33: 173-203. ROEDER, K. D., 1955. Spontaneous activity and behavior. Sci. Monthly, 80: 362-370. ROYS, C. C., 1954. Olfactory nerve potentials a direct measure of chemoreception in insects. Ami. N. Y. Acad. Sci.. 58: 250-255. SCHNEIDER, D., 1957. Electrophysiologische Untersuchungen von Chemo- und Merchanorezept- oren der Anteene des Seidenspinners Bomb\.\- uiori L. Zcitschr. vcrgl. Physiol., 40: 8-41. WOLBARSHT, M. L., 1957. Water taste in Phormia. Science. 125: 1248. MORPHOLOGY OF MAIN AND ACCESSORY ELECTRIC ORGANS OF NARCINE BRASILIENSIS (OLFERS) AND SOME CORRELATIONS WITH THEIR ELECTRO- PHYSIOLOGICAL PROPERTIES ROBERT MATHEWSON, 1 ALEXANDER MAURO,- ERNEST AMATNIEK AND HARRY GRUNDFEST 3 Department of Neurology, (- ollajc of Physicians and Surgeons, Columbia University, Ncv.' }'ork, and Marineland Research Laboratory, St. Aitf/iistine, Like other Torpedinidae ( Bigelow and Schroeder, 1953). Xarcine brasiliensis (Olfers) possesses electric organs. While they have previously been studied physiologically (Chagas ct 5X10 10-' 5X1Q-" Molarity of Ca(CN) 2 in 10% Ca(OH) 2 (U8 0.046 0.023 0.0054 0.0028 138 LORD ROTHSCHILD AND ALBERT TYLER In some experiments an appropriate quantity of XaCX was added to the egg-sus- pensions in the manometer flasks just before the beginning of the experiment. In the latter case the equilibration-time was reduced from thirty to fifteen minutes. Manometer flasks and other vessels containing cyanide solutions were kept stoppered at all times except when eggs were added and the flasks were put on the manometers. CO experiments. The gas phase of the manometers was filled with 95% CO in O 2 (95% CO/Oo), after flushing out the air. Xinety-five per cent X L ,/O 2 and air controls were run at the same time. The center wells contained 0.3 ml. X/l KOH and filter papers. Equilibration was in the dark for ten minutes. RESULTS Cyanide experiments. The results of three sets of experiments were clear-cut in the sense that, even at low concentrations, cyanide inhibited the respiration of fer- tilized eggs. Data from one of these are plotted in Figure 1. The lines labelled TABLK 1 The effect of cyanide, added 20 to 25 minutes after fertilization, on the percentage development of eggs of Urechis caitpo, examined at 3 hours. The sen water contained 0.0 1 M glycyl glycine, pH 8.0, Cone. HCN 1 "ncleaved 2-cell 4-cell 8-cell 16-32 cell Unfertilized 10~ 4 M 5-10- 5 M IfT 5 M 99 \ 99jP la 49 r bodies 30 20 1 1 1 5-10- 6 M 2 1 4 46 4ft 1 3 96 1 O.KOH and O,Ca(OH)o were controls to compare the CO 2 -absorptive powers of 10% KOH and Ca(OH), in the center-wells of the manometer flasks. As this and other tests showed, the Ca(OH) 2 proved as effective as the KOH in absorbing CO 2 under the conditions of these experiments. Table I shows the effects of the different concentrations of cyanide on the de- velopment of the eggs when examined at the end of the experiment. Carbon monoxide experiments. The results of an experiment in which just- fertilized eggs were subjected to 95% CO/Oo and 95% N 2 /O, are shown in Figure 2, in which periods of illumination and darkness are indicated by black and white blocks along the time axis. If the rate of Oo-consumption " in the curve labelled CO/Oo is examined by itself, it is clear that it rises upon illumination and falls in darkness in the manner considered characteristic of cytochrome-catalyzed respira- tion. When, however, comparison is made between the curve labelled CO/O 2 and the control labelled N 2 /O 2 , it is equally clear that, in the light, CO also stimulates the gas-uptake of these eggs. Illumination had no inhibitory effect on the O 2 - uptake of eggs in equilibrium with air. Table II shows the effect of CO in this experiment on egg development. The 3 The use of the terms Oo-consumption, O.-uptake, and respiration in the description and discussion of the CO-experiments is subject to the qualification that there is the possibility (see Discussion) that some of the gas consumed might be CO. METABOLISM OF URECHIS EGGS 139 inhibition is not so marked as in the cyanide experiments, but it might, of course, be more dramatic if higher CO tensions were used. The results of six sets of experiments with 95% CO/CX and 95% N 2 /O 2 are presented in Table III. The last two columns of the table give a measure of the effect of CO on the respiratory rate, in the dark and in the light, based on lateral 60 time, minutes 90 FIGURE 1. The respiration of eggs of Urcchis caiipo in the presence of HCN. For further details see text. 140 LORD ROTHSCHILD AND ALBERT TYLER 20 40 80 120 time, minutes 200 FIGURE 2. The oxygen uptake of eggs of Vrcchis canpo in the presence of 95% CO in O 2 and of 95% N in O,. The black and white blocks along the time axis correspond to periods of darkness and illumination. For further details see text. METABOLISM OF URECHIS EGGS 141 TABLE 1 1 The effect of 95% CO in O* and 95% N 2 in Oi on the percentage development of eggs of Urechis caupo, exposed at j hour and examined at 5 hours after fertilization. The sea water contained 0.01 M glycyl glycine, pH 8.0, T C. 20 Gas I'ncleaved 64-cell 128-cell Air 15 50 35 N 2 15 50 35 CO 15 85 TABLE 1 1 1 Effect of carbon monoxide on the respiration of eggs of L'rechis caupo in the light and in the dark (All experiments started about 40 minutes after fertilization. Temp. 20 C.) Experiment Respiration period Cu.mm. O? per hr. per 10-' eggs Resp. in 95% CO-5% O 2 rv ' 1 1 Irt 1 ^ ' resp. in 95% N 2 -5% Oz 95% CO-5% 2 95% N 2 -5% 2 Dark Light 1 0'-15' dark 7.6 9.7 0.78 15'-30' light 14.6 9.7 1.59 30'-45' light 13.5 8.2 1.65 45'-60' dark 5.9 7.7 0.77 60'-75' dark 4.9 6.7 0.73 2 0'-20' light 15.7 9.8 1.60 20'-60' dark 7.1 9.1 0.78 60'~100' light 19.5 9.3 1.88 100'- 140' dark 7.8 8.8 0.89 140'-160' light 15.7 9.8 1.60 3 0'-21' dark- 7.5, 7.6 9.4, 8.7 0.83 20'-40' light 17.1, 18.3 8.0, 10.1 1.95 40'-80' dark 6.8, 6.8 7.2, 6.1 1.02 80'-120' light 13.5, 15.1 6.9, 8.4 1.87 120' 140' dark 6.9, 6.6 8.0, 7.5 0.87 4 0'-30' dark 8.7 11.8, 10.8 0.77 30'-60' dark 9.6 11.1, 14.7 0.75 60'-90' light 18.4 8.2, 9.3 2.10 90'-120' light 17.6 8.8, 8.5 2.02 120'-240' light 15.2 8.9, 8.9 1.71 240'-270' light 14.4 8.9, 8.5 1.66 270'-300' light 15.2 10.4, 10.0 1.49 5 0'-90' light 15.8 6.4 2.47 90'- 150' light 13.0 5.7 2.28 150'-180' light 14.1 5.7 2.47 180'-240' light 13.8 6.5 2.12 6 0'-60' light 13.5 9.4 1.44 60'-210' light 15.4 10.9 1.41 210'-240' light 13.5 7.4 1.82 240'-270' light 11.8 5.7 2.07 270'-300' light 15.2 9.9 1.53 142 LORD ROTHSCHILD AND ALBERT TYLER comparisons (i.e., of different vessels run in parallel with aliquots of the same egg- suspension). In the dark the respiratory rate in 95% CO/O 2 is consistently lower than in 95% N 2 /O 2 . Rigid statistical treatment would be complicated because of the differences in times of readings, magnitude of respiration, etc., in the different experiments. However, a simple averaging of the percentage decrease (with double and quadruple weights for experiments 4 and 3, respectively) gives a 15 per cent inhibition of respiratory rate in 95% CO/O 2 in the dark. Similarly calculated, there is in these experiments, in the light, an 85 per cent average increase in respiratory rate of the eggs in 95% CO/O 2 over that of the parallel controls in 95^ N 2 /O,. The figures in Table III also show, for individual manometer vessels, the great effect of alternate light and dark periods on the respira- tion of the eggs in 95% CO/O 2 and the lack of significant effect of light and dark periods on the respiration of the eggs in 95% N../O.,. Spectroscopic examination of eggs. We have examined the unfertilized eggs of Urechis with a narrow-dispersion hand spectroscope (Keilin, 1925) at the tem- perature of liquid nitrogen, the eggs being suspended in 5Q% glycerol (v/v) with sodium dithionite added (Keilin and Hartree, 1939, 1949, 1955). A double ab- sorption band at 551 m^, which is in the region of the a-band of cytochrome c, could be clearly seen. A further, faint, absorption band at 580-590 m/x (cyto- chrome a) was also seen. The presence of these absorption bands was confirmed by Professor D. Keilin and Dr. R. Hill. Reduced cytochrome c was rapidly oxidized by egg brei in phosphate buffer. A peculiar phenomenon was observed during examination of the oxidation of cyto- chrome r by egg brei. When the oxidized cytochrome c and egg brei was kept in comparative darkness and then illuminated through the microscope sub-stage con- denser (which automatically occurs during spectroscopic examination), the absorp- tion bands of reduced cytochrome c gradually reappeared. This also was con- firmed by Professor D. Keilin and Dr. R. Hill. The most probable interpretation is that in the presence of light, some reducing substance is produced by the eggs, caus- ing the reduction of cytochrome c. This phenomenon may have some connection with the inhibitory action of light on the respiration of sea urchin eggs (Rothschild, 1949), though, as mentioned above, we have not observed any comparable light- inhibition of respiration in Urechis eggs. Certain dyes are affected by light in ways which would be consistent with the observed reduction of cytochrome c in light, which raises the possibility that urechrome may be concerned in the phenomenon. For example, Equ. (3) in Clare's article in Hollaender's Radiation Biology, Vol. Ill (1956) DH L , + O, -> H,O, + D. if written in the form DH 2 + 2 cyt.c 3 * -> 2 cyt.c 2 - + D + 2H + is suggestive in this connection. DISCUSSION In the introduction to this paper, reference was made to Horowitz's (1940a) view that urechrome and not cytochrome catalyzed the respiration of Urechis eggs ; METABOLISM OF URECHIS EGGS 143 this opinion was based on the facts that urechrome is reversibly autoxidizable and that no absorption bands of cytochrome were observed. We have now shown that the absorption bands of cytochrome are present in these eggs and that an egg brei can oxidize reduced cytochrome c. Moreover, the inhibition studies with cyanide and carbon monoxide support the view that the respiration of these eggs is cyto- chrome-catalyzed. Just where urechrome fits into the picture is, at present, un- certain. The effects of CO and cyanide on this pigment have not, as yet, been studied. The stimulating effect of carbon monoxide on respiration has been noted in many experiments with eggs and other tissues. The following citations from the litera- ture on this subject will serve to illustrate the widespread occurrence of the phenomenon. Runnstrom ( 1930) found that the respiration of unfertilized eggs of Paracentro- tus and Arbacia was either not inhibited or somewhat higher in carbon monoxide- oxygen mixtures than in air, while that of the fertilized eggs was greatly inhibited. Presumably, although not explicitly stated, these experiments were run in the dark. Lindahl (1939) obtained a 44% stimulation of the respiration of unfertilized eggs of Paracentrotus by 75% CO/O 2 in the dark, and this increased (to ca. 100%) upon illumination. With decrease in oxygen tension to 5% (+ 15% N 2 and 75% CO) the stimulation decreased. For freshly fertilized eggs in the dark he obtained a slight stimulation in 75% CO/O 2 and a marked inhibition in 95% CO/O 2 . In the light the fertilized eggs showed marked stimulation by 75% CO/O 2 and this effect decreased as the O 2 concentration was dropped to 5% at constant CO. Rothschild (1949) measured the respiration of unfertilized eggs of Psam- mechinus miliaris in various CO-O, mixtures. In 14 comparisons of the effect of 95% CO/O 2 with 95% N 2 /O 2 in the dark there was no difference in two, an 11% decrease in three and a 14% increase in nine. Twenty-four comparisons of the effect of 95% CO/O 2 in dark with that in light showed a 44% increase in the light. At the same time he found an inhibitory effect of light on the respiration of the un- fertilized eggs in air. This averaged 38% in 44 experiments. With 80% CO/O 2 in the dark there was an average of 55% increase in respiration above that in 80% N,/O 2 , and no significant change upon illumination. In the ascidian Phalhisia mamniillata Minganti (1957) found an increase in respiration of the unfertilized eggs in 95% CO/O 2 in the dark and a further in- crease in the light. The fertilized eggs showed a 14% to 20% decrease in the dark, which is about the same degree of inhibition as in the present experiments, and an increase (up to 40%) in the light. Bodine and Boell (1934) obtained CO-stimulation of respiration of diapause embryos of the grasshopper Mclanoplus differentialis and no significant effect of light. A similar stimulation by CO was found by Wolsky (1941) in a bivoltine race of the silkworm Bombyx inori, but not (Wolsky, 1938) in pupae of Drosophila inclanogaster. Wolsky (1938) attributes this difference to the pupal stage being one of great activity as compared with diapause. Schneiderman and Williams (1954) found that the respiration of diapausing pupae of the Cecropia silkworm was but slightly affected by high concentrations of carbon monoxide ; further experiments (Harvey and Williams, 1958) demonstrated that a cytochrome system functioned in this material, the resistance to CO being accounted for by cytochrome oxiclase being present in great excess relative to cytochrome c. 144 LORD ROTHSCHILD AND ALBERT TYLER In non-embryonic tissue the most extensively studied examples of CO-stimula- tion of respiration were those first reported by Fenn and Cobb (1932a, 1932b) in skeletal and heart muscle of frog and rat. This stimulation occurs in the dark or diffuse daylight and, as shown by Schmitt and Scott (1934), is increased by strong illumination. Fenn and Cobb (1932b) adduced evidence to show that the CO was oxidized to CO 2 and this has been further substantiated by Clark, Stannard and Fenn (1950) by the use of isotopically labelled CO. The latter investigators (1949) also reported such oxidation of CO by the intact animal (turtles and mice). In plants Daly (1954) obtained increases of about 20% to 30%, in 95% to 97% CO, with leaf tissue of the wild plum, Primus americana, in the dark. From the re- sults of experiments with labelled CO he concluded that the increased gas-uptake by the tissue represents a real stimulation of respiration rather than oxidation of CO to CO 2 . He also found a rather high R.Q. (up to 1.33) for the extra gas con- sumed and therefore suggested that aerobic glycolysis was increased by CO to a greater degree than O 2 -uptake. He cited cases of such stimulation of aerobic gly- colysis by CO which have been reported in spinach (Ducet and Rosenberg, 1952 4 ), carrot (Marsh and Goddard, 1939), and rat retina and mouse 4 chorion (Laser, 1937). The above-mentioned investigations indicate that the stimulating action of CO on respiration is of wide incidence in cells and tissues of animals and plants. In some cases (skeletal and heart muscle of frog and rat) there is strong evidence that the extra gas-uptake is due to the oxidation of CO. In others (plum leaves) it ap- pears to be due to the stimulation of endogenous respiration. In the case of the fertilized Urechis eggs, and the other cases that have been cited above, the mecha- nism of the stimulating action of CO is, as yet, unknown and would constitute an in- teresting area of further investigation. For the present purpose the demonstration of a light-sensitive action of CO on the gas-uptake of the Urechis eggs serves to sup- port the other evidence presented that a cytochrome system is operative in this material. One of us (R.) is indebted to the Biology Division, the California Institute of Technology, for their hospitality during the course of these experiments. We are indebted to Miss Mary Jones for technical assistance. SUMMARY 1. The respiration and normal development of fertilized eggs of Urechis caupo are inhibited by low concentrtaions of HCN, 5 X 10~ 6 M. Known concentrations of HCN were established within the manometer flasks by the use of Ca(CN) 2 - Ca(OH) 2 mixtures in the center- wells, with and without the appropriate amounts of NaCN in the egg suspensions. 2. The respiration of fertilized eggs was photo-reversibly inhibited by 95% CO in O 2 . The inhibition of development was not so marked at this tension as in the cyanide experiments. 3. CO markedly stimulated the respiration of the eggs in the light. The oc- currence of a similar action in the dark is presumed to account for the moderate de- gree of depression of respiration by CO in the dark. * Daly (1954) cited a 1951 paper instead of the 1952 paper listed here; also he referred to chicken chorion whereas Laser (1937) refers to mouse chorion. METABOLISM OF URECHIS EGGS 145 4. Spectroscopic examination of the eggs at the temperature of liquid nitrogen revealed absorption bands at 551 m/i and 580-590 niju. Absorption bands at these wave-lengths are associated with the presence of cytochromes c and a. 5. An egg brei rapidly oxidized reduced cytochrome c, but intense illumination of the system reversed the process. 6. It is concluded that the respiration of Urechis eggs is cytochrome-catalyzed. LITERATURE CITED BALL, E. G., AND B. MEYERHOF, 1940. On the occurrence of iron-porphyrin compounds and succinic dehydrogenase in marine organisms possessing the copper blood pigment hemocyanin. /. Biol. Chew., 134: 483-493. BODINE, J. H., AND E. J. BOELL, 1934. Carbon monoxide and respiration. Action of carbon monoxide on respiration of normal and blocked embryonic cells (Orthoptera). /. Cell. Comp. Physiol., 4 : 475^182. BOREI, H., 1951. Cytochrome c in sea urchin eggs. Ada Chem. Scand., 4: 1607-1608. BOREI, H., AND S. LYBING, 1949. Temperature coefficients of respiration in Psammechimis eggs. Biol. Bull., 96: 93-116. BRACKET, J., 1934. fitude du metabolisme de 1'oeuf de Grenouille (Rana fusca) au cours du developpement. I. La respiration et la glycolyse, de la segmentation a 1'eclosion. Arch, dc Biol. 45: 611-727. CLARE, N. T., 1956. Photodynamic action and its pathological effects. Chapter 15, pp. 693- 723 in Radiation Biology, Vol. Ill, ed. by A. Hollaender. McGraw Hill Book Co., New York. CLARK, R. T., J. N. STANNARD AND W. O. FENN, 1949. Evidence for the conversion of carbon monoxide to carbon dioxide by the intact animal. Science, 109: 615-616. CLARK, R. T., J. N. STANNARD AND W. O. FENN, 1950. The burning of CO to CO, by iso- lated tissues as shown by the use of radioactive carbon. Amer. J. Physio!., 161 : 40-46. DALY, J. M., 1954. Stimulation of respiration by carbon monoxide. Arch. Biochcm. Biophys., 51 : 24-29. DUCET, G., AND A. J. ROSENBERG, 1952. Action d'oxyde de carbone sur la respiration des feuilles vertes. C. R. Acad. Sci. (Paris), 234: 549-551. FENN, W. O., AND D. M. COBB, 1932a. The stimulation of muscle respiration by carbon monox- ide. Amer. J. Physiol., 102 : 379-392. FENN, W. O., AND D. M. COBB, 1932b. The burning of carbon monoxide by heart and skeletal muscle. Amer. J. Physiol., 102: 393-401. HARVEY, W. R., AND C. M. WILLIAMS, 1958. Physiology of insect diapause. XII. The mecha- nism of carbon monoxide-sensitivity and -insensitivity during the pupal diapause of the Cecropia silkworm. Biol. Bull., 114: 36-53. HOROWITZ, N. H., 1940a. A respiratory pigment from the eggs of a marine worm. Proc. Nat. Acad. Sci., 26: 161-163. HOROWITZ, N. H., 1940b. The respiratory metabolism of the developing eggs of Urechis caupo. J. Cell. Comp. Physiol., 16 : 299-308. HOROWITZ, N. H., AND J. P. BAUMBERGER, 1941. Studies on the respiratory pigment of Urechis eggs. /. Biol. Chem., 141 : 407^15. KEILIN, D., 1925. On cytochrome, a respiratory pigment, common to animals, yeasts, and higher plants. Proc. Roy. Soc. (London], Scr. B, 98: 312-338. KEILIN, D., AND E. F. HARTREE, 1939. Cytochrome and cytochrome oxidase. Proc. Roy. Soc. (London}, Ser. B, 127: 167-191. KEILIN, D., AND E. F. HARTREE, 1949. Effect of low temperature on the absorption spectra of haemoproteins ; with observations on the absorption spectrum of oxygen. Nature, 164 : 254-259. KEILIN, D., AND E. F. HARTREE, 1955. Relationship between certain components of the cyto- chrome system. Nature, 176: 200-206. KORR, I. M., 1937. Respiratory mechanisms in the unfertilized and fertilized sea urchin egg : a temperature analysis. /. Cell. Comp. Physiol., 10: 461-485. KORR, I. M., 1939. Oxidation-reductions in heterogeneous systems. Cold Spring Harbor Sym- posia Quant. Biol., 7 : 74-93. 146 LORD ROTHSCHILD AND ALBERT TYLER KRAHL, M. E., A. K. KELTCH AND G. H. A. CLOWES, 1939. Oxygen consumption and cell di- vision of fertilized Arbacia eggs in the presence of respiratory inhibitors. Biol. Dull., 77: 318-319. LASER, H., 1937. Tissue metabolism under the influence of carbon monoxide. Biochem. J., 31: 1677-1682. LINDAHL, P. E., 1936. Zrr Kenntnis der physiologischen Grundlagen der Determination im Seeigelkeim. Acta Zool., 17: 179-366. LINDAHL, P. E., 1939. t)ber die biologische Sauerstoffaktivierung nach V r ersuchen mit Kohlen- monoxyd an Seeigeleiern und Keimen. Zeitschr. vergl. Physiologic, 27 : 136-168. MARSH, P. B., AND D. R. GODDARD, 1939. Respiration and fermentation in the carrot, Dancus carota. II. Fermentatiun and the Pasteur effect. Amcr. J. Botany, 26: 767-772. MINGANTI, A., 1957. Experiments on the respiration of Phallusia eggs and embryos (ascidians). Acta Embryol. et Murflwl. Exp., 1 : 150-163. ROBBIE, W. A., 1946a. The quantitative control of cyanide in manometric experimentation. /. Cell. Comp. Physiol, 27 : 181-209. ROBBIE, W. A., 1946b. The effect of cyanide on the oxygen consumption and cleavage of the sea urchin egg. /. Cell. Comp. Physiol., 28: 305-324. ROBBIE, W. A., AND P. J. LEINFELDER, 1945. Calcium cyanide solutions as constant sources of hydrogen cyanide gas for animal experiments. /. Ind. Hyg. To.vicol., 27 : 269-274. ROTHSCHILD, LORD, 1949. The metabolism of fertilized and unfertilized sea-urchin eggs. The action of light and carbon monoxide. /. Exp. Biol., 26: 100-111. RUBENSTEIN, B. B., AND R. W. GERARD, 1934. Fertilization and the temperature coefficients of oxygen consumption in the eggs of Arbacia punctulata. J. Gen. Physiol., 17: 677-686. RUNNSTROM, J., 1930. Atmungsmechanismus und Entwicklungserregung bei dem Seeigelei. Protoplasma, 10: 106-173. Sen MITT, F. O., AND M. G. SCOTT, 1934. The effect of carbon monoxide on tissue respiration. Amer. J. Physiol., 107 : 85-93. SCHNEIDERMAN, H. A., AND C. M. WILLIAMS, 1954. The physiology of insect diapause. VIII. Qualitative changes in the metabolism of the Cecropia silkworm during diapause and development. Biol. Bull., 106: 210-229. TYLER, A., 1936. On the energetics of differentiation. IV. Comparison of the rates of oxygen consumption and of development at different temperatures of eggs of some marine animals. Biol. Bull, 71: 82-100. TYLER, A., 1937. Influence of temperature and other agents on the respiration and develop- ment of marine eggs. Coll. \et, 12 : 38-39. TYLER, A., AND N. H. HOROWITZ, 1937. Glycylglycine as a sea water buffer. S'cicncc, 86: 85-86. TYLER, A., AND N. H. HOROWITZ, 1938. On the energetics of differentiation. VII. Comparison of the respiratory rates of parthenogenetic and fertilized Urechis eggs. Biol. Bull., 74 : 99-107. TYLER, A., AND W. D. HUMASON, 1937. On the energetics of differentiation. VI. Comparison of the temperature coefficients of the respiratory rates of unfertilized and fertilized eggs. Biol. Bull., 73 : 261-279. WOLSKY, A., 1938. The effect of carbon monoxide on the oxygen consumption of Drosophila melanogastcr pupae. /. Exp. Biol., 15 : 225-234. WOLSKY, A., 1941. The respiration of silk-worm eggs. I. Respiratory activity in various stages of development with special regard to the effect of carbon monoxide. Math. naturiv. Anz. unyar. Akad. Wiss., 59: 893-901 (from Chew. Abstr., 35: 5571). YCAS, M., 1954. The respiratory and glycolytic enzymes of sea-urchin eggs. /. Exp. Biol., 31 : 208-217. REGENERATION OF BUDS IN BOTRYLLUS x MARGARET J. WATKINS Department of Zoology, University of Minnesota, Minneapolis, Minnesota The process of budding in the colonial ascidian, Botryllus schlosseri, has been carefully analyzed by N. J. Berrill (1941a) and recently by Sabbadin (1955). The new buds (Z.,) arise from the atrial epithelium and epidermis of large buds (Z 2 ) in which internal structure is nearly complete but which are still attached to the parent (ZJ. The disc-like thickening of the atrial epithelium increases in cell number and area until a certain size, called the maximum disc, is reached. It then folds out into a hemisphere and finally to a closed sphere attached to the large bud by a stalk. Three generations are thus present and connected together at one time. The sphere then goes through a process of expansion, folding, and evagination to form the internal structure of the new zooid. The bud continues to grow until it reaches a size nearly equal to the parent, at which time the latter degenerates and the bud becomes functional. There is considerable variation among colonies in the number of buds formed and the number which reach maturity. Berrill (1941a, 1941b, 1945) has shown that in young colonies the diameters of the maximum disc and sphere stages are less than half those of older colonies and that they gradually increase with each successive generation. The size of the adult zooid is closely related to the size of the bud and hence to the number of cells initially present. A sphere with a diameter of 0.035 mm., for example, becomes a zooid with a length of 1.1 mm., while a sphere of 0.080 mm. becomes a zooid of 2.6 mm. Sabbadin (1956a, 1956b) showed that the growth of the bud is conditioned not only by its initial dimensions, but also by the quantity of food made available to it by the regression of the parent zooid and the duration of its growth period. In his ex- periments all but one bud was removed from each zooid in the experimental colo- nies. These buds and the zooids from them attained greater maximum length than corresponding buds and zooids in control colonies. Sabbadin concludes that the buds on one zooid compete for food made available by the parent as it regresses. This does not explain, however, the gradual increase in size of the zooids with each generation. It was deemed of interest to determine whether the size of the zooid depends directly on the number of cells present in the early bud or is determined in some other manner by the parent. In order to study this problem, buds were damaged at an early stage and the amount of regeneration as shown by the final size was noted. This degeneration of the parent zooid has generally been thought to be due to the increasing need of the bud for space and nourishment (Berrill, 1935). To test 1 This work was done while the author held an Anderson Summer Fellowship from the University of Minnesota Graduate School as a part of the Embryology Course given at the Marine Biological Laboratory, Woods Hole, Massachusetts. The author wishes to thank Dr. Mac V. Edds, Jr. for his help with both the research and the manuscript. Dr. N. J. Berrill read the manuscript and offered valuable advice. 147 148 MARGARET J. WATKINS whether this is true the buds were removed in an attempt to prolong the life of the zooid. MATERIALS AND METHODS Adult colonies of Botryllus were collected from the dock in Eel Pond at Woods Hole, Massachusetts, and placed in finger bowls on a table of running sea water. Each day a few of the tadpole larvae were released from the colonies and these were collected and placed in Syracuse dishes for approximately 12 hours. After the tad- poles had attached to the glass, the Syracuse dishes were inverted in wooden racks placed in tanks of running sea water. The tadpoles metamorphosed in less than one day, forming oozoids with large right buds which became the first blastozooids. Under these conditions at 22.5 0.5 C. the adult zooid persisted for from 4 to 6 days, with as much as 24 hours' variation between two colonies in the same dish. The experimental colonies were examined every day or two with a binocular dis- secting microscope and rough sketches were made to follow the fate of individual zooids and buds. Buds were removed by cutting through the stalk with a needle sharpened to a blade. With care this could be done with very little damage to the parent, but sometimes the latter was damaged severely and disappeared. To determine whether the new buds originated from the same area as the destroyed buds or from bud pri- mordia posterior to it, all the small buds (Z,) were cut off 220 large buds (Z,) in 31 colonies. The site of formation of the new buds was then observed. In 5 col- onies an attempt was made to keep the parent zooids from degenerating by con- stantly removing new buds as they appeared. Buds were damaged with sharpened steel needles inserted through the tunic. An effort was made to destroy half or more of the forming bud. Although the amount of actual damage varied from bud to bud, in most cases at least half of the bud was destroyed. Frequently, part of the bud was torn away and could be seen sticking to the needle. In preliminary experiments on 21 colonies, buds ranging from the sphere stage to those with some internal structure present were damaged. These were watched to see whether they reached maturity, but no measurements were made to determine if they were full size. In order to examine the effect of destroy- ing approximately half of the cells at a stage before the closed sphere, both right and left buds were damaged when the atrial epithelium had begun to fold out into a hemisphere (between stages 2 and 3 of Berrill, stage 2 Sabbadin). The length of the zooid which formed these buds was then measured with an ocular micrometer and compared with the length of undamaged zooids. The width of the zooids varied in different colonies of the same age and seemed to decrease as the number of zooids around the cloaca increased ; therefore, no measurements of width were made. RESULTS a) Degeneration of parent sooids In no case observed did an adult zooid persist beyond 24 hours of the time of degeneration of other zooids of the same age. When all the buds were removed from a zooid, that zooid degenerated at the same time as the rest of the zooids in that colonv or in other colonies, whether new buds formed or not. The five col- REGENERATION OF BUDS IN BOTRYLLUS 149 onies in which new buds were constantly removed as they appeared degenerated and disappeared within 24 hours of the time of degeneration of control colonies. b) Formation of new buds If the large bud which normally occurred on the right side of an oozoid was re- moved, a bud then appeared in four cases out of five on the left side of the oozoid and became a normal blastozooid. In later generations, if all the large buds (Z 2 ) with complete internal structure were cut off (4 colonies), the colony degenerated. If, however, only approximately half of the larger buds were cut off (5 colonies), those remaining proceeded to maturity, and in addition a few new buds appeared. When all the small buds (Z 3 ) at the sphere stage were cut off 220 large buds (Z 2 ) in 31 colonies, a total of 72 new buds appeared, an average of one bud for every three parents. There was a great deal of variation among the colonies, with any- where from zero to seven buds produced by the six or seven parents. Of the new buds, 26 appeared on the left side of the blastozooids and 29 on the right side. Of the latter 6 definitely were from the same area as the destroyed bud, 18 were prob- ably from this area, and 5 appeared posterior to the destroyed bud. The origin of the other 17 buds was impossible to determine. Most of these were first seen in the midst of a degenerating colony quite separate from any blastozooid. c) Bud regeneration after damage In preliminary experiments in which 107 buds ranging from the sphere stage to those with some internal structure present were damaged, 55 reached maturity. The rest of the buds became progressively smaller and eventually disappeared. In 36 colonies in which 361 hemispheres were damaged, 40% reached maturity as com- pared to 78% in 8 control colonies with 124 hemisphere stages. TABLE I Size regulation of zooids in partially damaged colonies Colony number No. of zooids in colony No. of zooids damaged Average length of all zooids in colony, in mm.zfcS.D. Average length of damaged zooids in colony, in mm. S.D. Average length of undamaged zooids in mm. S.D. A12c 12 5 1.5 .1 1.6 .1 1.5 .1 A12e 1 3 1.8 .1 1.8 .2 1.9 .1 A12h 21 8 1.9 .1 2.0 .1 1.8 .1 A12k 13 6 1.6 .1 1.6 .1 1.6 .1 A121 17 4 1.4 .1 1.5 .1 1.4 .1 B2d 25 4 1.8 .2 1.8 .1 1.8 .2 A5a 8 3 1.8 .1 1.8 .2 1.9 .1 A5e 15 13 1.6 .1 1.6 .1 1.6 .1 A5i 7 6 1.8 .1 1.8 .1 1.9 Ala 16 16 1.8 .2 A5b 6 1.4 .1 A5g A5h 13 13 1.6 .1 1.7 .1 A6a 7 1.8 .1 A6b 11 1.7 .1 150 MARGARET J. W ATKINS TABLE II Size regulation of zooids in experimental and control colonies Colony number No. of Zi Length of Zi in mm.iS.D. at time of experi- ment No. of Zi Length of Zi in mm.iS.D. 1-2 days after reach- ing maturity No. of Z 3 Length of Zs in mm.iS.D. 1-2 days after reach- ing maturity Length of Z* in mm.iS.D. 3-4 days after reach- ing maturity Experimental Colonies A3e 9 1.7 .1 8 2.0 .2 12 2.0 .2 2.4 .3 A7a 5 1.8 .1 8 2.0 .1 12 2.2 .1 2.8 .1 A8a 7 1.7 .1 4 1.7 .1 4 1.9 .1 2.4 .1 AlOa 4 1.6 .1 4 8 2.2 .1 AlOe 6 1.7 .1 10 1.8 .1 Total 25 1.7 .1 30 1.9 .2 46 2.1 .2 2.6 .3 Control Colonies A3d 6 1.8 .1 8 1.8 .2 13 1.8 .1 A7b 5 1.6 .2 9 1.8 .1 15 1.9 .1 2.3 .2 A8a 7 1.8 .1 10 2.0 .1 17 2.0 .1 2.6 .1 AlOb 5 1.8 .1 5 1.8 .1 11 2.1 .1 AlOf 5 1.7 .1 9 1.8 .2 Total 23 1.7 .1 37 1.8 .2 65 1.9 .2 2.4 .2 d) Size regulation in damaged buds In the first experiments, only some of the hemispherical buds in each colony were damaged with the idea of using the others as controls. The data for 15 such colonies are given in Table I. In colonies A12h and A121 the damaged left buds did not survive, so the right buds measured received the full food supply from the parents. In all other colonies as many damaged left buds survived as undamaged, so the supply of food did not affect the results. The colonies are not all of the same size or age at the time of the experiments, so the average length for different col- onies cannot be directly compared, but the average lengths of damaged and un- damaged zooids in the same colony show no significant difference. In later experiments, all the hemisphere stages in a colony were damaged and these colonies were compared with control colonies. Three generations were pres- ent at the time of the experiment: the parent zooids (ZJ, the large buds (Z 2 ), and the hemispherical buds (Z 3 ) . Each of these was measured as it in turn reached ma- turity. In both experimental and control colonies, most of the left buds reached maturity, so the food supply was about the same for all buds. The data for these experiments are given in Table II. No significant difference can be seen between the experimental and control colonies. DISCUSSION If the degeneration of the adult zooid is due only to the increasing need of the growing buds for space and nourishment, removal of all the buds in a colony ought REGENERATION OF BUDS IN BOTRYLLUS 151 to have prolonged the life of the zooids. In this study, any attempt to postpone de- generation of the zooid in this way met with failure. No zooid was observed to per- sist more than 24 hours longer than other zooids of the same age even if its buds were continually removed. Sabbadin (1956b), however, found that when all but one bud was removed from each zooid, that zooid had a prolonged stage of func- tional maturity. Perhaps removal of all buds was a shock to the zooid and partially caused its regression ; however, it seems likely that adult regression will occur with- out the presence of buds. At the same time the buds may play an important part in the process by their increasing need for nourishment. There is some question as to the origin of new buds after removal of these already growing. Blastozooids have two potential budding areas, one on the right side and one on the left, although frequently only the bud on the right side reaches ma- turity. At times, a third bud may be formed posterior to the bud normally found on the right side (see Watterson, 1945, and Sabbadin, 1956a, for a discussion of the number of buds usually formed). It would appear that a certain amount of atrial epithelium is set aside for bud formation ; after that is used no more buds can be formed. Frequently in these experiments, when buds were removed from the blastozooid, new buds were formed at the same area as the buds were destroyed. Sabbadin (1956a) reports that he never observed buds arising "de novo" after re- moval of buds present. Sometimes, however, after he had removed buds in the hemisphere stage, he saw fragments adhering to the atrial side of the parent zooid, and these fragments formed new buds. This is a possible explanation of the pres- ent results although every effort was made to remove the entire bud intact. In these experiments all the buds removed were at least in the closed sphere stage and many were quite large and visibly separated from the parent though still attached by the stalk. It would take considerable powers of regeneration for fragments of such buds to form a whole new zooid. The 17 buds whose origin it was impossible to determine might possibly be cases of vascular budding (Oka and Watanabe, 1957). They arose during or after the regression of the adult zooids, so a vascular origin seems likely. They were not observed, however, until they were large enough to obscure their point of origin. Although development of ascidians from egg to tadpole is determinate (Conk- lin, 1905), the adults have remarkable powers of regeneration (Berrill, 1951). Zhinken (1939) has shown that while tadpoles have little ability to replace lost parts, the oozooid has acquired considerable regulative powers. The present study would indicate that buds also have the ability to regenerate lost tissues from the earliest stages onward. Berrill has shown (1941b, 1941c, 1945) that the size of the maximum disc and sphere stages increases with succeeding generations and that the size of the adult zooid is clearly related to the number of cells or the diameter of the maximum disc and sphere. This might suggest that the parent determines the size of the new zooid by the number of cells which are initially incorporated into the early stages of the bud. If this were true, then destroying some of these cells would have resulted in smaller adult zooids. However, using length as an index of zooid size, it was found that there was no decrease in size of the zooids damaged at the hemisphere stage. After a bud was damaged it either disappeared completely or reached the predeter- mined size. Thus the size of the adult does not appear to depend directly on the number of cells originally present since these cells can be replaced. Either the 152 MARGARET J. WATKINS parent zooid retains control over the growth of the bud or the bud has "received instructions" as to the size it should attain and follows them by regenerating lost tissue and then continuing to grow. Sabbadin (1956c) has shown that zooids with the position of the digestive tube reversed may appear if the growth of the bud is delayed at an early stage. The buds on these abnormal zooids showed a marked tendency to be the same as their parents unless the parent has started to regress be- fore organogenesis is complete. This would indicate that the parents do retain con- trol over the growth and organogenesis of their buds. SUMMARY 1. The degeneration of the adult zooid of Botryllus schlosscri, which normally occurs when the buds become functional, occurred even after all buds were removed. 2. All stages of the growing buds of Botryllus have considerable regenerative ability. 3. Buds damaged in the hemisphere stage became adult zooids with the same length as undamaged zooids of the same age. Control over the size of the adult zooid appears to be maintained during the growth of the bud. LITERATURE CITED BERRILL, N. J., 1935. Studies in tunicate development. IV. Asexual reproduction. Phil. Trans. Roy. Soc. London, Ser. B, 225 : 327-379. BERRILL, N. J., 1941a. The development of the bud in Botryllus. Biol. Bull., 80 : 169-184. BERRILL, N. J., 1941b. Size and morphogenesis in the bud of Botryllus. Biol. Bull., 80 : 185-193. BERRILL, N. J., 1941c. Spatial and temporal growth patterns in colonial organisms. Growth, 51 (Supplement) : 89-111. BERRILL, N. J., 1945. Size and organization in the development of ascidians. In: Essays on Growth and Form. Ed. by W. E. Le Gros Clark and P. B. Medawar. Oxford Univ. Press ; pp. 231-263. BERRILL, N. J., 1951. Regeneration and budding in tunicates. Biol. Rev., 26: 456-475. CONKLIN, E. G., 1905. Mosaic development in ascidian eggs. /. E.rp. Zoo/., 2 : 145-223. OKA, HIDEMITI, AND HIROSHI WATANABE, 1957. Vascular budding, a new type of budding in Botryllus. Biol. Bull, 112: 225-240. SABBADIN, ARMANDO, 1955. Osservazioni sullo suiluppo, 1'accrescimento e la riproduzione d. Botryllus schlosseri (Pallas), in condizioni di laboratorio. Boll. d. Zoo/., 22: 243-263. SABBADIN, ARMANDO, 1956a. Studio sperimentale della gemmazione in "Botryllus schlosscri" (Pallas). Rend. d'Accademia Nasionalc dei Lincei, 20: 380-385. SABBADIN, ARMANDO, 1956b. Osservazioni suH'accrescimento delle gemme e degli zooidi di "Botryllus schlosscri" (Pallas) (Ascidiacea), in condizioni normal! e sperimentali. Rend. d'Accademia Nasionale dei Lincei, 20: 485-491. SABBADIN, ARMANDO, 1956c. "Situs inversus viscerum" provocato sperimentalmente in "Botryllus schlosscri" (Pallas) (Ascidiacea). Rend. d'Accademia Nasionale dei Lined, 20 : 659-666. WATTERSON, RAY L., 1945. Asexual reproduction in the colonial tunicate, Botryllus schlosseri (Pallus) Savigny, with special reference to the developmental history of inter siphonal bands of pigment cells. Biol. Bull., 88 : 71-103. ZHINKEN, L., 1939. Alteration of regenerative power of the larvae of ascidea during meta- morphoses. C. R. Acad. Sci. Moscozv (N.S.), 24: 623-625. Vol. 115, No. 2 \AV MA* /CV/ October, 1958 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE OXIDATION OF CARBON MONOXIDE BY FERTILIZED EGGS OF URECHIS CAUPO SHOWN BY USE OF A C 13 LABEL 1 ROBERT E. BLACK, 2 SAMUEL EPSTEIN 3 AND ALBERT TYLER Division of Biology, California Institute of Technology, Pasadena, California In some previous experiments (Rothschild and Tyler, 1958) with eggs of Urecliis, it was found that the rate of respiration in the presence of carbon monoxide (95% CO: 5% O L ,) in the light was greatly increased above that of the con- trols (95% N 2 :5% O 2 ). The average increase amounted to 85 per cent. In the dark there was a slight decrease, averaging about 15 per cent. In many earlier investigations on eggs and other tissues of various animals and plants there have been reports of a stimulating action of CO on respiratory rate. Examples of this are found in experiments on eggs of sea urchins by Runnstrom (1930), Lindahl (1939) and Rothschild (1949); on ascidian eggs by Minganti (1957) ; on diapausing grasshopper- and silkworm-embryos by Bodine and Boell (1934) and by Wolsky (1941) ; on skeletal and heart muscle of frog and rat by Fenn and Cob'b (1932a, 1932b), Schmitt and Scott (1934), and Clark. Stannard and Fenn (1950) ; on leaf tissue of the wild plum by Daly (1954). In the experiments on vertebrate muscle tissues, Fenn and Cobb (1932b) and Clark, Stannard and Fenn (1950) obtained evidence that CO is oxidized to CO 2 . Clark ct al. (1949) also reported that intact whole turtles and mice could effect such oxidation of CO when this was administered at very low tensions. In the experiments on plum-leaves, on the other hand, Daly (1954) found that the in- creased gas-uptake in the presence of CO represents a stimulation of ordinary respiration rather than an oxidation of the CO. The question of whether or not the stimulation of respiration in eggs of sea urchins and ascidians is due to oxida- tion of the CO was considered by Lindahl (1939), Minganti (1957) and Roths- child (1949). The former two investigators rejected this view while the latter considered it to be the most probable explanation of the increased respiration. In a review of various experiments Runnstrom (1956) concludes that the evidence is against the possibility of oxidation of CO by sea urchin eggs. However, there has as yet been no direct test of this proposition, such as would be provided by the use of isotopically labelled CO. 1 This investigation was supported by a research grant (C-2302) from the National Cancer Institute of the National Institutes of Health, Public Health Service. - Postdoctoral Research Fellow of the U. S. Public Health Service. 3 Division of Geological Sciences, California Institute of Technology. 153 Copyright 1958, by the Marine Biological Laboratory 154 R. E. BLACK, S. EPSTEIN AND A. TYLER In the present experiments C 13 -labelled CO was employed in an investigation of the possibility of its oxidation by eggs of UrccJiis. The results show that such oxidation occurs and that it accounts for all of the extra gas-uptake of the eggs in the light. The data also show that an oxidation of CO occurs in darkness, but at a lower rate. MATERIAL AND METHODS Eggs of the gephyrean worm Urcchis canpo were employed in these experi- ments. They were inseminated in sea water and washed in sea water buffered at pH 8 with 0.01 M glycylglycine (Tyler and Horowitz, 1937). Gas-uptake was measured with Warburg-Barcroft manometers using vessels whose calibration volumes ranged around 25 ml. The vessels generally contained 3 ml. of egg suspension and 0.3 ml. of M/\ KOH (low in CO,). In some experiments in which CO 2 was to be released from the egg suspension as well as from the alkali, magnetically held cups were employed, one for the alkali and one containing 0.3 ml. of 6 M H 2 SO 4 . The contents of these could be separately tipped into the egg suspension at the desired time by removal of an externally supported magnet. The KOH used in the alkali-wells of the manometer vessels was prepared from a saturated solution, in which K 2 CO 3 is largely insoluble, and diluted with CO 2 -free double-distilled water under CO 2 -free air. An analysis of the alkali prepared in this manner gave 0.8 X 10~ G mole of total carbonate per 0.3 ml. In filling the manometer vessels the alkali was introduced last. The use of C 13 offers some advantages over C 14 for these experiments. Use of C 1 1 would involve precipitating and weighing very small quantities ( less than 4 mg. as BaCO 3 ) of the carbonate derived from the respired CO 2 in the alkali well of the usual manometer vessels. This is unnecessary for the mass spectrometric measurement of C 13 which provides the required quantitative data in the form of the ratio of C 13 to O~ in the sample. It also avoids such uncertainties as are en- tailed by the self-absorption of radiation in the measurement of C 14 . In addition, use of C 13 eliminates possible health-hazards and possible effects of radiation on the system under investigation. The labelled carbon monoxide was prepared from barium carbonate containing 3.85% C 13 . This was obtained from the Stable Isotopes Division of the Oak Ridge National Laboratories. The method employed was essentially similar to the continuous flow technique described by Bernstein and Taylor (1947). The apparatus consisted of a CO 2 generator connected to a Pyrex combustion tube (8 mm. i.d.), containing about 50 grams of zinc-dust-asbestos fiber (95: 5), within a combustion-furnace of 18 cm. length, and leading through a three-way stopcock to the top of a storage bulb. The latter was provided also with a bottom stop- cock leading to a levelling bottle containing N/10 NaOH. At the start of the preparation the storage bulb was filled with the alkali up to the three-way stop- cock. A weighed amount of the C 13 -enriched BaCO 3 w r as placed in the generator, and the generator and combustion tube, up to the three-way stopcock, were flushed with unlabelled CO. The furnace was set at 520 C. Hydrochloric acid was introduced into the generator at a rate producing about 25 to 50 cc. of CO 2 per minute. Measurements of the volume of fluid displaced in the storage bulb showed OXIDATION OF CO BY EGGS OF URECHIS 155 that the amount of CO obtained in this system was close to that expected. After CO 2 generation had stopped, the gas remaining in the generator and combustion tube was flushed into the storage bulb with enough unlabelled CO to make a final volume of one liter. The relative volumes of labelled and unlabelled CO were 362 to 638 for the preparation, giving a C 13 content of 2.14%. Relative to a C 13 content of 1.17% found for the CO 2 from Urechis eggs respiring in air, this gives 82.9% for the atom percentage excess C 13 of the preparation. The labelled CO was stored over alkali for at least one day prior to use. Storage over alkali for several weeks showed no change in gas volume, indicating no significant contamination by acidic gases. After attachment of the Warburg vessels to their manometers they were flushed with one liter or more of oxygen. They were then attached to a Toepler pump and evacuated to one-fifth of the original pressure, precautions being taken, by stopper- ing the open end of the manometers and closing-ofr" the bottom rubber well with a clamp, to avoid drawing the Brodie's fluid out of the manometers. The C 13 - labelled CO was then introduced through the three-way stopcock at the top of the manometers, after a preliminary flushing of connecting tubes. By this procedure the CO-O 2 ratio could be fixed with considerable accuracy to the desired value, which was 4:1 in the present experiments. About 15 minutes were required for these procedures and 10 minutes were allowed for equilibration in the temperature bath. The control vessels were left open to air during the gassing of the experi- mental vessels. The experiments were run at 20 C. Shaker speed was 95 c.p.m. at 3-cm. stroke. Illumination was provided by a bank of 30-watt reflector-type G-E incandescent lamps located below a glass shelf of the water bath. This sup- plied 1100 to 1200 foot-candles at the level of the egg suspensions in the Warburg vessels. The C 13 determinations were made with a Nier mass spectrometer (Nier, 1947) modified for detection of relatively small enrichments by McKinney at al. (1950). The sensitivity of the instrument is such that differences of two parts in ten thou- sand in the C I3 -to-C 12 ratios can be readily detected. For introduction of the respired CO 2 into the mass spectrometer, the procedure followed in two of the experiments (No. 1 and No. 2) was to transfer the alkali from the center well of the Warburg vessel quantitatively, with CO 2 -free water and with precautions to avoid contamination with atmospheric CO 2 , to a reaction vessel wherein the CO 2 could be liberated by tipping-in concentrated H 3 PO 4 from a side-arm (McCrea, 1950). This was attached to the vacuum-line of the mass spectrometer. In one of the experiments (No. 3), the CO 2 was liberated within the Warburg vessels by tipping acid from one of the contained insert-wells into the egg suspension and the alkali. After measurement of their amounts the CO, samples were transferred to the reaction vessels by means of the Toepler pump. In two of the experiments (No. 2 and No. 3) a measured amount of NaHCO 3 was added to the reaction vessel in order to decrease the C 13 enrichment to values within the range best suited for the mass spectrometer. The measurements are corrected for the dilution factor. 156 R. E. BLACK, S. EPSTEIN AND A. TYLER RESULTS Effect of CO on gas-uptake of eggs of Ureehis The relevant respiration-data for three experiments are presented in Table I. The first two are for eggs run in the light starting shortly after fertilization, and the third is a dark-experiment with eggs at a similar period of development. The increase in gas-uptake reported by Rothschild and Tyler (1958) for freshly fertilized eggs of Ureehis in the light in 95% CO/O, is shown also in the present experiments (No. 1 and No. 2) with 80% CO/CX. Likewise, the lack of appre- ciable inhibition in the dark is shown in the results of experiment No. 3. Exami- nation of the eggs at the end of the respiration runs in experiments No. 1 and No. 2 showed no significant difference in rate of development between those in 80% CO/CX and those in air. The eggs from experiment No. 3 were not available for examination because of the acidification, but separate experiments on eggs run in the dark in CO-O 2 mixtures show only a small amount of inhibition of develop- ment, as reported previously (Rothschild and Tyler, 1958). The data in Table I present amounts of gas-uptake calculated as if the total gas were oxygen. Part of the gas-uptake of the eggs in the CO-O 2 mixture could (and, as later shown, does) represent disappearance of CO. However, calcula- tions using the solubility of CO instead of O 2 in the usual formula for converting the manometric pressure difference into volume of gas would change these figures by only 0.1%, since the solubility coefficients of the two gases are of the same order of magnitude and this factor contributes relatively little to the vessel constant. This difference is negligible here. Experiments No. 1 and No. 2 give values of 154 and 130 mm 3 ., respectively, for the excess gas uptake. Assuming that this is due to the oxidation of CO ( 2 CO + O 2 2 CO 2 ), then % of these quantities represent the amounts of CO oxidized and the corresponding amounts of CO 2 produced therefrom; namely, 102 and 87 mm 3 ., respectively. The corresponding control vessels yield 318 and 305 mm 3 , of CO 2 , respectively, on the basis of an R.Q. of unity (Horowitz, 1940). The percentage of the CO 2 derived from oxidation of CO would therefore be 24.3 for experiment No. 1 and 23.4 for experiment No. 2. These are entered in the last column of Table II as expected values, and involve also the assumption that in the light there is no inhibition of the ordinary respiration. TABLE I Respiration-data for eggs of Ureehis used in C n -labelled CO experiments (1) Experiment (2) Number of eggs per flask (3) Time interval of experiment in hours after fertilization (4) (5) (6) Excess gas- uptake in 80% CO/O 2 (mm. 3 ) Total gas-uptake In air (mm. 3 ) In 80 " c CO/O 2 (mm. 3 ) 1 (light) 2 (light) 3 (dark) 389,000 622,000 421,000 l*-8* ii-6* 1-10 318 305 408 472 435 394 + 154 + 130 -14 OXIDATION OF CO BY EGGS OF URECHIS 157 TABLE II Percentage of respired COz derived from oxidation of CO, as determined from 'measurements of C 13 in mass spectrometer and as calculated on the assumption that such oxidation accounts for all excess gas-uptake in CO-Oz mixtures in the light (1) Experiment No. (2) Atom % excess C 13 in CO used in gas space of manometer vessels (3) (4) (5) (6) (7) Expected percentage of total CO* derived from CO as calculated from excess gas-uptake in light Mass spectrometer data Atom % excess C 13 in respired COs Percentage of CC>2 de- rived from oxidation of CO With reference to standard CO 2 With reference to control Experimental vessel Control vessel Experimental vessel 1 (light) 2 (light) 3 (dark) 82.9 82.9 82.9 19.92 20.28 16.23 0.61 1.35 19.31 20.38 14.88 23.3 24.5 18.0 24.3 23.4 A calculation of expected CO-oxidation cannot be made in this way for experi- ment No. 3 which was run in the dark, wherein both inhibition of ordinary respira- tion and oxidation of the CO might well take place. Moss spectrometer data relating to oxidation of CO The results of determinations of C 13 abundance in the respired CO, of the above three experiments are presented in Table II. The atom percentage excess C 13 in the CO used in these experiments is listed in the second column of the table. These figures also represent the excess that would be expected if all of the respired CO, were derived from oxidation of CO. The values obtained from the mass spectrometer measurements for the excess C 13 in the CO, from experimental, rela- tive to that from control vessels, are given in the fifth column of the table. Division of these figures by the corresponding ones of column two gives the percentages (column 6) of the CO, derived from CO-oxidation in these three experiments. Comparison with the expected percentages (column 7) calculated from the manometrically determined extra gas-uptake, on the assumption that all of this surplus in the light is derived from CO-oxidation, shows close agreement in experiments No. 1 and No. 2. This closeness of agreement may, however, be largely fortuitous as the follow- ing considerations of further details of the experiments indicate. In experiment No. 1 the control was an aliquot of the same egg suspension respiring in air. The alkali from both experimental and control flasks was transferred quantitatively to the reaction vessels and no carrier NaHCO 3 added. The respective percentages of excess C 13 , relative to the standard used in the instrument, are given in columns 3 and 4 of the table. The air-control shows a small excess of C 13 relative to the standard source. This simply reflects variation in C 13 /C 12 ratios of living and non-living materials from various sources (rf. Craig, 1953). Since the carbon of the respired CO, of the air-control is all derived from the eggs this indicates 158 R. E. BLACK, S. EPSTEIN AND A. TYLER a higher C 13 content in the eggs than in the standard. In the absence of other information the best method of applying a correction for the control is uncertain, but it seemed most reasonable to us simply to subtract it from the value for the experimental flask. In any case this correction has relatively little effect on the calculations of CO-oxidation. In experiment No. 2 the respired CO 2 from the air-control vessel was not sub- jected to C 13 analysis. Instead, a second type of control was investigated. This consisted of a preparation of lyophilized eggs that was run along with the experi- mental flask in the 80% labelled CO-20% O 2 atmosphere in the light. This preparation showed a negligible amount of gas-uptake, and was employed to test for possible exchange of carbon atoms between CO 2 and the labelled CO. For this purpose about 300 mm 3 , of CO 2 were introduced into the Warburg flask. The analysis of the CO 2 in the alkali of this flask showed no difference in C 13 con- tent from that of the standard. This indicates that no significant exchange of carbon atoms between the CO and CO 2 occurs in this system. The determined value for atom percentage excess C 13 in the CO 2 of the experi- mental flask of experiment No. 2 was not corrected for any possible contribution from ordinary respiration since the air control in this experiment was not analyzed for C 13 . A correction of the same order as in experiment No. 1 would lower very little the calculated percentage of CO 2 derived from oxidation of CO (column 6). The principal source of uncertainty in these two experiments is CO 2 -retention in the egg suspensions of the Warburg flasks. As shown in later experiments the egg suspensions may contain considerable amounts of bicarbonate at the beginning of the experiments, despite the normal precautions to keep this at a low value. This unlabelled bicarbonate would presumably form a common pool during the run with bicarbonate derived both from ordinary respiration and from the oxida- tion of labelled CO. The CO 2 collected in the alkali for analysis would then have been diluted with the unlabelled CO 2 present in the egg suspension at the start of the experiment. Also, some of the labelled CO 2 produced during the experiment would be retained in the suspension at the end of the run. If corrections were made for the above effects, the values calculated in column 6 for CO-oxidation in experiments No. 1 and No. 2 would be higher than those presented. In other words, the value used for atom percentage excess C 13 to be expected if only CO- oxidation took place would be lower than those listed in column 2. Therefore, the calculated percentages of COo derived from CO-oxidation in these two experi- ments represent minimum values. It should be noted that the expected percentages of CO 2 produced from CO by the eggs, as calculated from excess gas-uptake (column 7), also represent minimum values, since they depend on the assumptions that the R.Q. is 1.0, and that there is no inhibition of ordinary respiration by CO in the light. Lindahl (1939) has shown that in 75% CO/O 2 in the light, the eggs of the sea urchin have a lower R.Q. than one would expect, even if one were to account for all the excess gas- uptake as CO oxidation. This could be due to an inhibition of ordinary respiration t>y CO in the light, which is masked by the utilization of CO. In the present experiments if an R.Q. of 0.67 instead of 1.0 were assumed for the ordinary respiration, as well as the CO-oxidation, then the expected percentages of CO, derived from CO-oxidation (column 7) would be 32 and 30 for experiments No. 1 and No. 2, respectively. OXIDATION OF CO BY EGGS OF URECHIS 159 In experiment No. 3 the bicarbonate in the egg suspension, as well as that in the alkali well, was collected for analysis of C 13 content in the mass spectrometer. A control flask of egg suspension, into which acid was tipped at the time of the first reading of the manometers, provided a measure of unlabelled CO 2 originally present. The retained, as well as the respired, CO 2 was determined before transfer to the reaction vessel of the mass spectrometer, as described in Materials and Methods. The total amounts of CO 2 (375 mm 3 , in experimental and 384 mm 3 , in control flask) were diluted with 0.5 ml. of carrier 0.04 M NaHCO 3 (480 mm 3 , of CO 2 ). Initial bicarbonate content of the CO/O 2 blank amounted to 160 mm 3 . The corresponding dilution factors applied to the mass spectrometer data were therefore (375 + 480) / (375 -- 160) and (384 + 480)/384 for experimental and control flasks, respectively. The figures entered in columns 3 and 4 of Table II are corrected for the dilution factor. The value of 18 per cent for the CO 2 derived from CO-oxidation in this experi- ment is then not subject to uncertainties of retention and can be considered to represent reasonably closely the extent of CO-oxidation occurring in the dark. Since there is about 3% inhibition of gas-uptake (Table I) in this experiment and since 27% (% of 18%) of the gas-uptake represents CO-oxidation, then there is 29% inhibition (100-97(0.73)) of the ordinary respiration by the CO in the dark. DISCUSSION The results show that eggs of Urcchis can oxidize carbon monoxide. This occurs both in the light and in the dark. The amount of carbon monoxide that is oxidized in the light can account for all of the excess gas-uptake that occurs in a CO-O 2 mixture. In the dark the percentage of CO 2 derived from CO-oxidation is somewhat less than in the light, according to the present data. It should be noted again that the values obtained for oxidation of CO in the light are probably minimal. In other words, there may be a small amount of inhibition of the "ordinary" respiration in the light which is obscured by the oxidation of CO. It is possible that in the dark CO may be inhibiting, to some extent, its own oxidation. Clark, Stannard and Fenn (1950) found that sodium azide and hydroxylamine completely blocked the oxidation of CO by skeletal muscle, as measured both by manometric and isotope techniques. Information available from the literature and from the present experiments does not permit identification of the enzymatic system(s) involved in the oxidation of CO. It seems likely that a haem compound is involved because of the known affinity of CO for the Fe ++ of such substances. Also, it may well go through cytochrome oxidase. However, tests of cytochrome oxidase preparations from Urechis and sea urchin eggs (to be reported later) gave no oxidation of CO. In certain bacteria CO can serve as the sole carbon source (cf. van Niel, 1954). Fixation of CO has been demonstrated in barley leaves (Krall and Tolbert, 1957), in which the labelled carbon appears initially in serine and choline. This fixation occurs in both light and dark but the rate is much higher in the light. The possi- bility of fixation of CO has not, as yet, been examined in animals, but it does seem likely that some of the CO 2 produced by its oxidation would be assimilated. As previously reported (Rothschild and Tyler, 1958) and as noted here, the development of the eggs was not significantly accelerated or retarded in the CO-O 2 160 R. E. BLACK, S. EPSTEIN AND A. TYLER mixtures in the light. It might appear, then, that the energy released by the oxidation of the CO is not put to useful developmental work in this system. However, it should be noted that the CO-oxidation would provide much less energy per mole of carbon than the oxidation of the ordinary substrates of the cell. So, even if the energy were utilized, the increase in developmental rate might be too small to be readily detected under the present conditions in which roughly 25 per cent of the respiration is attributed to oxidation of CO. Furthermore, as indicated above, the figure of 25 per cent is a minimum value. Some inhibition of ordinary respiration could be occurring in the light. If, for example, the inhibition amounted to 25 per cent and if it is assumed that oxidation of CO supplies half as much energy per mole of carbon as does the ordinary respiration, then the total rate of energy supply would be the same for eggs in 80% CO/O 2 in the light as for eggs respiring in air. It is then possible that the energy released by oxidation of CO is utilized by the cell for developmental work. SUMMARY 1. The fertilized eggs of Urechis canpo have been found to oxidize CO to CO 2 both in the light and in the dark. This has been shown by the use of C 13 -labelled CO. In the light there is a previously described increase in gas-uptake in 80% CO/O 2 as compared with air. All of this excess gas-uptake can be attributed to the oxidation of CO. 2. In the dark the percentage of respiratory CO 2 derived from CO is less than in the light. If the oxidation of CO is subtracted from the total gas uptake, the "ordinary" respiration is shown to be inhibited about 29% in the dark by 80% CO/O 2 . LITERATURE CITED BERNSTEIN, R. B., and T. I. TAYLOR, 1947. Conversion of isotopically enriched CO 2 to CO. Science, 106: 498-499. BODINE, J. H., and E. J. BOELL, 1Q34. Carbon monoxide and respiration. Action of carbon monoxide on respiration of normal and blocked embryonic cells (Orthoptera). /. Cell. Comp. Physiol, 4: 475^82. CLARK, R. T., J. N. STANNARD and W. O. FENN, 1949. Evidence for the conversion of carbon monoxide to carbon dioxide by the intact animal. Science, 109: 615-616. CLARK, R. T., J. N. STANNARD and W. O. FENN, 1950. The burning of CO to CO 2 by isolated tissues as shown by the use of radioactive carbon. Amer. J. Physiol., 161 : 40-46. CRAIG, H., 1953. The geochemistry of the stable carbon isotopes. Gcochimica et Cosino- chimica Acta, 3 : 53-92. DALY, J. M., 1954. Stimulation of respiration by carbon monoxide. Arch. Biochcm. Biophys., 51: 24-29. FENN, W. O., and D. M. COBB, 1932a. The stimulation of muscle respiration by carbon monoxide. Amer. J. Physiol., 102 : 379-392. FENN, W. O., and D. M. COBB, 1932b. The burning of carbon monoxide by heart and skeletal muscle. Amer. J. Physiol, 102: 393-401. HOROWITZ, N. H., 1940. The respiratory metabolism of the developing eggs of Urechis caupo. J. Cell. Comp. Physiol., 15: 299-308. KRALL, A. R., and N. E. TOLBERT, 1957. A comparison of the light dependent metabolism of carbon monoxide by barley leaves with that of formaldehyde, formate and carbon dioxide. Plant Physiol., 32: 321-326. LINDAHL, P. E., 1939. t)ber die biologische Sauerstoffaktivierung nach Versuchen mit Kohlenmonoxyd an Seeigeleiern und Keimen. Zeitschr. I'crgl. Physiol., 27 : 136-168. OXIDATION OF CO BY EGGS OF URECHIS 161 McCREA, J. M., 1950. On the isotopic chemisty of carbonates and a paleotemperature scale. /. Chem. Phys., 18: 849-857. McKiNNEY, C. R., J. M. McCREA, S. EPSTEIN, H. A. ALLEN and H. C. UREY, 1950. Improve- ments in mass spectrometers for the measurement of small differences in isotope abundance ratios. Rev. Sci. Instr., 21 : 724-730. MINGANTI, A., 1957. Experiments on the respiration of Phallusia eggs and embryos (ascidi- ans). Acta Embryologiac et Morphologiae Experimentalis, 1: 150-163. NIER, A. O., 1947. Mass spectrometer for isotope and gas analysis. Rev. Sci. Instr., 18 : 398-411. ROTHSCHILD, LORD, 1949. The metabolism of fertilized and unfertilized sea-urchin eggs. The action of light and carbon monoxide. /. E.rp. Biol., 26: 100-111. ROTHSCHILD, LORD, and A. TYLER, 1958. The oxidative metabolism of eggs of Urechis caupo. Biol. Bull, 115: 136-146. RUNNSTROM, J., 1930. Atmungsmechanismus und Entwicklungserregung bei dem Seeigelei. Protoplasma, 10: 106-173. RUNNSTROM, J., 1956. Some considerations on metabolic changes occurring at fertilization and during early development of the sea urchin egg. Pubbl. Staz. Zoo/. Napoli, 28 : 315-340. SCHMITT, F. O., and M. G. SCOTT, 1934. The effect of carbon monoxide on tissue respiration. Amer. J. Physiol., 107: 85-93. TYLER, A., and N. H. HOROWITZ, 1937. Glycylglycine as a sea water buffer. Science, 86 : 85-86. VAN NIEL, C. B., 1954. The chemoautotrophic and photosynthetic bacteria. Ann. Rev. Micro- biol., 8: 105-132. WOLSKY, A., 1941. The respiration of silk-worm eggs. I. Respiratory activity in various stages of development with special regard to the effect of carbon monoxide. Math. naturw. Anz. ungar. Akad. Wiss., 59: 893-901. THE SALT GLAND OF THE HERRING GULL 1 R. FANGE, 2 K. SCHMIDT-NIELSEN AND H. OSAKI Department of Zooloi/y, Duke University, Durham, North Carolina, and Mount Desert Island Biological Laboratory, Salisbury Cove, Maine The long known fact that the nasal gland is conspicuously larger in marine birds than in terrestrial species has recently been given a functional explanation. It has been found that in birds taking their food from the ocean the nasal gland is developed into an organ whose main function is the secretion of salt. We have, therefore, in our publications on the function of this gland, referred to it as the "salt gland." After large salt intake, due to ingestion of sea water or marine invertebrate organisms, the salt gland assists the kidney in the excretion of excess of sodium chloride. In some marine birds the gland is more important than the kidney in the elimination of salt from the organism (Schmidt-Nielsen and Sladen, 1958; Schmidt-Nielsen and Fange. 1958b). The anatomy of the avian nasal gland in a large number of birds, both terres- trial and marine, was described in a monograph by Technau (1936). Although Technau mainly dealt with the gross anatomy of the gland he also made histo- logical observations. Other microscopical observations have been made by Mar- pies (1932) and Mihalik (1932). and the embryology has been studied by Grewe (1951). The discovery of the osmoregulatory importance of the salt gland of marine birds made it necessary to re-investigate its histology in the light of the present knowledge of its function. MATERIALS AND METHODS The material consisted of young specimens of the herring gull (Larns argen- tatus) caught at the Atlantic coast at Beaufort, North Carolina, and at Mount Desert Island, Maine. For histological examination glands were fixed in Bouin's fluid, and paraffin sections were stained in azan (Romeis, 1924) or haematoxylin-eosin. The main structure of the arterial supply to the gland was studied by injection of methacrylate plastic into the carotid arteries, followed by maceration of the tissues with KOH. The detailed vascularization was studied in preparations injected with India ink through the carotids, fixed in Bouin's fluid, and subse- quently cleared in benzyl benzoate. The glandular duct system was studied by injection of India ink or methacrylate plastic into the lateral duct opening. Paraf- fin sections were prepared of some of the India ink-injected specimens. 1 Supported by National Institutes of Health, Grant No. H-2228. 2 Present address : Department of Zoophysiology, University of Lund, Lund, Sweden. 162 SALT GLAND OF THE GULL 163 Gross anatomy In the gull the large, paired salt gland is situated on the top of the skull in the supraorbital grooves of the frontal bone (Fig. 1). Strictly speaking each gland consists of two parts, as seen from the fact that there are two ducts on each side of the head leading forwards into the beak (Technau, 1936). However, the two parts of the gland have a similar structure and are joined so closely together that they can be considered as one functional unit and may be regarded as one gland. Thus, the glands are flat and crescent shaped, and two ducts pass from the anterior end of each to the anterior nasal cavity (vestibulum). On the upper side the gland is covered by a thin, tough connective tissue membrane. The anterior part of it extends somewhat laterally from the margin of the frontal bone and forms part of the roof of the orbit. Blood vessels and nerves pass from the orbit into the gland through holes in the frontal bone. FIGURE 1. Skull of the herring gull from above, showing the position of the salt gland. The two ducts on each side of the head take their origin from the lower side of the anterior part of the gland and run close together into the beak, where they open at the posterior end of the vestibular concha (Marples, 1932; Technau 1936). The lateral duct opens on the lower median side of the vestibular concha (pre-concha) while the median duct has its opening on the nasal septum close to the transverse fold separating the anterior nasal cavity (vestibulum) from the upper nasal cavity. The openings of the ducts can be found if a longitudinal incision is made in the palate somewhat lateral to the midline, and it is then pos- sible to cannulate the lateral duct opening for the collection of secretion in living birds (Fange, Schmidt-Nielsen and Robinson, 1958), or for injection of fluids into the duct. For some reason our attempts to cannulate the median duct were unsuccessful. Marples (1932) found in Lams ridibimdiis (black-headed gull) that the ducts are formed at an early embryonic stage as outgrowths from the nasal cavity. Later the ducts branch above the frontal bone, forming the glandular tissue. Corresponding to the branches of the embryonic ducts, the gland of the adult is composed of tubes or lobes, giving it a characteristic surface structure (Fig. 2). Most of the gland consists of long lobes, some of which stretch along the whole length of the gland. In the gland of Lams argentatus about 15 such longitudinal 164 R. FANGE, K. SCHMIDT-NIELSEN AND H. OSAKI lobes can be seen in a transversal section. In addition to these longitudinal lobes there are shorter lobes oriented in various directions. In our material the combined weight of the two salt glands varied from 700 to 900 mg. The weight of the animals was 700-1000 grams (young specimens). Technau (1936) found in the herring gull a gland weight (probably unilateral), of 555 mg., but in the related common gull. L. canus, 150 mg., and in the black- headed gull. L. ridibnndns, only 50 mg. Of these three gulls, the herring gull is the most salt water-bound species while the black-headed gull is, to a large extent, associated with fresh water. Thus, there is a good correlation between the size of the salt gland and the habitat of the different gull species (Schildmacher, 1932). GLANDULAR DUCTS CENTRAL CANAL ! i It! Ml. I II / I" I " I ^ ANTERIOR LOBE POSTERIOR FIGURE 2. Diagram showing the gross structure of the salt gland (left side). Microscopic structure In each lobe there is a central canal (Fig. 2) which connects with the lumen of one of the two main ducts from the gland. Branching tubular glands radiate out from this central canal which is surrounded by a rather voluminous connective tissue mass (Figs. 3, 4). Close to the central canal, where the gland tubules have not yet branched extensively, the tubules are round in transverse section and separated by the connective tissue. In the periphery of the lobe the tubules are closely packed together and run parallel to each other, separated by very delicate connective tissue membranes and blood capillaries. In tangential sections through SALT GLAND OF THE GULL 165 FIGURE 3. Longitudinal section through a lobe. Note the difference in stainability between the outer and the inner zone of tubules. The blue-stained connective tissue is dark, due to use of a yellow filter when taking the microphotograph. (Bourn's fluid, azan.) FIGURE 4. Transverse section through the central part of a lobe. An artery (vertical in the figure) passes into the connective tissue around the central canal. (Bouin's fluid, azan, yellow filter. ) FIGURE 5. Tangential section through a lobe half-way between the surface of the lobe and the central canal. The capillaries between the tubules are partly filled with blood. (Bouin's fluid, azan, yellow filter.) FIGURE 6. Transverse section through a lobe halfway between the surface and the central canal. India ink was injected into the lateral duct of the gland before fixation. (Bouin's fluid, azan.) 166 R. FANGE, K. SCHMIDT-NIELSEN AND H. OSAKI the peripheral parts of a lobe the cross-sectioned tubules have a polygonal outline and form a honeycomb-like pattern (Fig. 5). The tubules branch 4-6 times forming different "generations" or "orders" of tubules. In the center of the lobe, close to the central canal, the tubules are thick and consist of cylindrical epithelial cells with the approximate dimensions 6 p X 15- 20 /z.. In the periphery of the lobe the diameter of the tubules is smaller, and here the size of the cell is 6-9 X 6-9 . The cell nuclei are about the same size in ARTERY TO THE BEAK ANASTOMOSIS A.OPHTH. INTERNA A.OPHTH. EXTERNA FIGURE 7. The arterial supply of the salt gland. The sketch shows the left gland from below. Drawn from a methacrylate plastic cast of the vascular system. the central and the peripheral tubules. Thus, the amount of cytoplasm in relation to the nuclear volume is largest in the central portion of the tubules, possibly indicating that these gland cells carry out more work than those of the peripheral portions. The cytoplasm of the cells has a lamellated or striated appearance. The stria- tion is not limited to a striated border, but extends through Lhe cells from the lumen to the periphery, where the cells are in contact with blood capillaries. In sections from specimens in which India ink had been injected into the lateral duct, the lumen of the tubules had an irregular shape, indicating the presence of secretory intra- or intercellular canaliculi (Fig. 6). SALT GLAND OF THE GULL 167 The cytoplasm takes a reddish colour in azan stain. In the most peripheral part of the tubules the cytoplasm is less heavily stained than in the central tubules (Fig. 3). The central canal consists of 2-4 layers of cuboidal epithelium. The two main ducts passing to the anterior nasal cavity also consist of a multi-layered epithelium. In some preparations the boundaries between the epithelial cells, especially those of the central canal, had a vacuolated appearance which gave the illusion of a system of intercellular canals. This, however, could be a fixation artefact due to shrinkage of the cells. The two main ducts are surrounded by the same con- nective tissue which surrounds the accompanying blood vessels and nerve and have no connective tissue of their own. No smooth muscle cells could be found in the walls of the ducts. Neither was it possible to detect any smooth muscle in the gland except that of the arteries supplying the lobes. Neither the connective tissue of the upper side of the gland, the interlobular connective tissue mass, the con- nective tissue membranes around the tubules, nor the central connective tissue mass around the central canal contains any smooth muscle. Vascularization The blood supply of the nasal glands of the duck has been described by Mar- pies (1932) and earlier authors (Gadow, 1891). According to our observations in Lams argentatus the main arterial supply comes from the arteria ophthalmica interna. The vessel penetrates the wall of the orbit above the optic nerve and, passing upwards along the median wall of the orbit, it divides into two branches to the salt gland. The anterior branch gives off several small arteries to the gland and then continues into the beak (Fig. 7). The posterior branch supplies the posterior part of the glands. Anastomosing with this branch another artery from the posterior wall of the orbit also gives blood to the gland. This artery probably corresponds to the arteria ophthalmica externa described by previous authors (Gadow, 1891; Slonaker, 1918). Both the arteria ophthalmica interna and the arteria ophthalmica externa are branches of the arteria carotis interna. The arteries reaching the salt glands are among the largest arteries in the head of the gull. The arrangement of the arteries is such that, in spite of the rich blood supply, the blood could probably bypass the gland via the arterial arch formed by the anastomosis between the anterior and posterior branch of the arteria ophthalmica interna (Fig. 7). This arrangement may permit a large reduction in glandular blood flow without reducing the blood flow to the upper beak when the glands are not functioning. The control of the blood flow through the glands may be exerted by contractile arterioles in the glands. The veins from the salt glands follow the arteries in their main courses (Marples, 1932). Microscopic distribution of blood vessels The connective tissue between the individual gland lobes contains a large num- ber of branching arteries and veins. At intervals the arteries give off branches which pass into the lobes. These arteries pass straight through the gland tissue between the tubules towards the central canal without branching (Figs. 4, 8), but after reaching the central connective tissue mass they break up into numerous 168 R. FANGE, K. SCHMIDT-NIELSEN AND H. OSAKI capillaries. These capillaries, which have frequent branchings and anastomoses, run radially out towards the surface of the lobes. In their main course the capil- laries are parallel to the tubules. Tubules and capillaries form a regular pattern in sections cut tangentially through a lobe (see Fig. 5). The tubules are polygonal in shape and the capillaries are situated at the corners of the polygons, each tubule being surrounded by 5-7 capillaries. The regularity of the arrangement reminds of the rete mirabile of the fish swimbladder, or the regular arrangement of tubules and blood vessels in the medulla of the mammalian kidney. At the surface of the ARTERY VEIN 1 central connective tissue interlobular connective tissue FIGURE 8. Diagram of a transverse section through a lobe of the salt gland. lobe the capillaries leave the tubules and pass over into a venous plexus drained by veins in the interlobular connective tissue (Fig. 8). No veins were observed within the lobes. No lymph vessels could be observed in the glands, but as they may be difficult to detect in histological sections, we hesitate to claim that there are none in the salt gland. A diagrammatic picture of the blood flow in the gland is shown in Figure 9. Innervation The nasal gland of birds has been reported to be innervated from a para- sympathetic ganglion in the anterior part of the orbit (Cords, 1904; Webb, 1957). The ganglion has connections with different cranial nerves and with the sympathetic system (Cords, 1904). The nerve supply of the salt gland in the herring gull will be described in another publication which will also deal with the physiological responses of the gland to various kinds of stimulation (Fange, Schmidt-Nielsen and Robinson, 1958). Other bird species The presence of salt glands has been demonstrated in birds of five different orders (Schmidt-Nielsen and Fange, 1958a). We have undertaken some pre- SALT GLAND OF THE GULL 169 ARTERY VEIN INTERLOBULAR CONNECTIVE TISSUE SECRETORY TUBULES CENTRAL CONNECTIVE TISSUE CENTRAL CANAL FIGURE 9. Diagram of the circulation showing the opposing directions of the flow in the gland tubules and in the capillaries. The tubules branch repeatedly, but for simplicity only t\vo ramifications are pictured. liminary histological studies of the salt glands of pelican (Pclecanus*), cormorant (Phalacrocora.v) , eider duck (Somateria), petrel (Occanodroma), etc. In these birds the glands have essentially the same histological structure as in the gull, and consists of lobes with tubular glands radially arranged around a central canal. In the pelican and the cormorant the lobes are not tubiform as in the gull, but rather short and of a rounded shape. In the connective tissue of the salt glands of many birds black pigment cells occur. 170 R. FANGE, K. SCHMIDT-NIELSEN AND H. OSAKI DISCUSSION The salt gland of marine birds has a very characteristic structure consisting of closely packed secretory tubules with blood vessels between them. The tubules radiate from a central canal. In terrestrial birds, where the nasal glands have no salt excretory function, the glands contain only a few tubules or have sac-shaped diverticula instead of tubules (Marples, 1932). The strictly parallel arrangement of closely packed, glandular tubules may be necessary for the osmotic work per- formed by the gland. It is probable that the manner of distribution of the blood capillaries within the gland tissue is also of importance in this respect. It may be noted that the arrangement of blood vessels within the lobe is such that the capil- lary blood flows in a direction opposite to that of the secreted fluid. The func- tional significance of this counter-current flow in the salt gland is not clear. The counter-current principle, although manifested in a different way, seems to play an important role in the production of a concentrated urine in the kidney of mam- mals and birds (Hargitay and Kuhn, 1951). Although the structure of the salt gland in marine birds and of the mammalian kidney otherwise are entirely different, it is striking that a counter-current flow is found in both these organs, which in higher vertebrates are the only ones known to produce a highly hypertonic secretion. The counter-current flow in the salt gland cannot, as such, explain the large osmotic work performed by the gland. Active ionic transport can be assumed to be the fundamental cellular process responsible for the osmotic work. The striated or lamellated appearance of the cytoplasm of the gland cells and the presence of secretory canaliculi indicate a highly specialized transport function of the cyto- plasm. A more detailed study of the microscopic and electron microscopic struc- ture of the cytoplasm of the avian salt gland cells is in progress. SUMMARY 1. The salt gland of the herring gull (Lanis argcntatits) is a large, paired gland on top of the skull. On each side tw r o ducts lead to the anterior nasal cavity. When the gland is secreting, its discharge comes out through the nares and drips off from the tip of the beak. 2. The gland has long, tubular lobes, each with a central canal. Tubulous glands radiate from the central canal. The gland cells have a striated or lamellated cytoplasm, and seem to have secretory canaliculi. 3. The blood supply is mainly from arteria ophthalmica interna. Within the gland the capillary blood flow is in a direction opposite to that of the secreted fluid. The innervation of the gland is from a ganglion of predominantly para- sympathetic nature. 4. The salt glands of other marine birds have the same characteristic structure with the secreting tubules radiating out from a central canal. LITERATURE CITED CORDS, E., 1904. Beitrage zur Lehre vom Kopfnervensystem der Vogel. Anat. Heftc, 26: 49-100. FANGE, R., K. SCHMIDT-NIELSEN AND MARYANNE ROBINSON, 1958. The control of secretion from the avian salt gland. Amcr. J. Physiol. (in press). SALT GLAND OF THE GULL 171 GADOW, H., 1891. Dr. H. G. Bronn's Klassen und Ordnungen des Thier-Reichs. VI. 4 Abt. Vogel. I. Anatomischer Theil. 767-785. Das Arteriensystem. C. F. Winter'sche Verlagshandlung, Leipzig. GREWE, F. J., 1951. Nieuwe gegewens aangaaende die ontogenese van die neuskliere, die orgaan van Jacobson en die dekbene van die schedel by die benus Anas. Annals Univ. Stellenbosch, 27 : 69-99. HARGITAY, B., AND W. KUHN, 1951. Das Multiplikationsprinzip als Grundlage der Harn- konzentricrung in der Niere. Zcitschr. f. Elcktrochcm. 11. angeiv. physik. Cheinic., 55: 539-558. MARPLES, B. J., 1932. The structure and development of the nasal glands of birds. Proc. Zool. Soc. Land., 829-844. AimALiK, P. V., 1932. Uber die Glandula lateralis nasi der Vogel. Ergebn. d. Anat. u. Entw. Gesch., 29: 399-448. ROMEIS, B., 1924. Mikroskopische Technik. R. Oldenbourg, Mtinchen. SCHILDMACHER, H., 1932. Uber den Einfluss des Salswassers auf die Entwicklung der Nasendrusen. /. /. Ornitlwl., 80: 293-299. SCHMIDT-NIELSEN, K., AND R. FANGE, 1958a. Extrarenal salt excretion. Fed. Proc., 17: 142. SCHMIDT-NIELSEN, K., AND R. FANGE, 1958b. The function of the salt gland in the brown pelican. The Auk (in press). SCHMIDT-NIELSEN, K., AND W. J. L. SLADEN, 1958. Nasal salt secretion in the Humboldt Penguin. Nature, 181: 1217-1218. SLONAKER, J. R., 1918. A physiological study of the anatomy of the eye and its accessory parts of the English Sparrow (Passer domesticus). J. MorphoL, 31: 351-459. TECHNAU, G., 1936. Die Nasendriise der Vogel. /. /. Ornitlwl.. 84: 511-617. WEBB, M., 1957. The ontology of the cranial bones, cranial peripheral and cranial para- sympathetic nerves, together with a study of the visceral muscles of Struthio. Ada Zool, Stockh., 38: 1-203. THE SWIMBLADDER OF THE TOADFISH (OPSANUS TAU L.) RAGNAR FANGE 1 AND JONATHAN B. WITTENBERG 2 The Marine Biological Laboratory, Woods Hole. Massachusetts; The Department of Zoophysiology, University of Lund. Lund, Sweden; and The Departments of Physiology and Biochemistry, Albert Einstein College of Medicine of }'eshira University. AVtc- York 61. N. V. The swimbladder of the toadfish (Opsanus tail L.) offers a particularly favor- able object for the experimental study of gas secretion. To provide a basis for physiological studies we describe here the structure of the swimbladder, its gas gland and its vascular supply. In addition, some physiological observations are presented. Further physiological studies of this species are reported elsewhere (Wittenberg, 1958). Brief anatomical descriptions of the swimbladder of the toadfish are found in Tower (1908) and Rauther (1945). Greene (1924a) has studied a related species, Porichtliys. Tracy (1911) presents some embryological and histological data. Tracy observed that the posterior chamber of the embryonic toadfish de- velops from the pneumatic duct, which secondarily loses its connection with the gut. MATERIAL AND METHODS Animals: Toadfish caught at Woods Hole were maintained in a shallow live car for several months before they were used. Histological: After fixation in Bouin's fluid, histological sections were made and stained with azan (Romeis, 1948) or haematoxylin and eosin. The blood vessels were studied by injection of India ink into the coeliac artery. The injected specimens were fixed in Bouin's fluid and later cleared in benzyl benzoate. Gas analyses: These were by the method of Scholander ct al. (1955). RESULTS The swimbladder gases In contrast to the majority of shallow-living marine fishes, the toadfish nor- mally maintains a very high proportion of oxygen in the swimbladder gases. The oxygen ranges from 40 to 80 per cent and in most animals is about 50 per cent of the total gas. Similar high oxygen concentrations (maximum 88 per cent) have previously been observed in a related species, Porichthys (Greene, 1924b). When forced experimentally to renew repeatedly the gaseous contents of the bladder, the toadfish is able to maintain the secretion of gas undiminished in rate and oxygen content. Thus in one experimental series the swimbladders of three animals were emptied every 24 hours for six days. During this time, each animal 1 Present address : Department of Zoophysiology, University of Lund, Lund, Sweden. -This investigation was supported by a Senior Research Fellowship (S.F. 57) from the Public Health Service, and by a research grant from The National Science Foundation. 172 SWIMBLADDER OF THE TOADFISH 173 secreted a volume of gas equivalent to six times the volume of the swimbladder. At the end of the six-day period, the rate of secretion and the composition of the secreted gas remained unchanged. The newly secreted gas is characterized by an extraordinarily high proportion of oxygen which averages 90 per cent and may be as high as 96 per cent of the total gas. The proportion of carbon dioxide is low, about 4 per cent (Wittenberg, unpublished data). The ratio, argon to nitrogen, in the secreted gas is very high, 2.4 X 10~- to 2.6 X 1O 2 , and approaches the maximum which can be achieved by a mechanism of inert gas secretion proposed elsewhere (Wittenberg, 1958). These properties combine to indicate a very powerful development of oxygen transport in the gas gland of the toadfish, making this an animal of choice for experimental studies concerning oxygen transport. The principal layers of the sivimbladdcr wall The external appearance of the swimbladder is shown in Figure 1. It is of the euphysoclist type (Rauther, 1922; Fange, 1953). The wall may be described as formed of three layers, conveniently called tunica externa, submucosa and Coeliac artery Nerve : :: ' Nerve Sound producing striated muscles Swim bladder^ artery Swim bladder vein from posterior chamber DORSAL VIEW Portal vein ^^SlS^ VENTRAL VIEW FIGURE 1. External view of the swimbladder of the toadfish seen in dorsal and ventral view. The nerve shown in the picture is the motor nerve to the striated sound-producing muscle. According to Tracy (1911) it is a branch of the first spinal nerve. mucosa. The tunica externa is a tough, somewhat rigid external connective tissue capsule. Laterally this layer includes the sound-producing striated muscle masses (Figs. 1 and 2; compare with Rauther, 1945). The submucosa consists of very loose fibrous connective tissue which allows a limited movement of the mucosa relative to the tunica externa. In fresh specimens it is possible to take advantage of the loose consistency of the submucosa to dissect away the tunica externa, including the striated muscle masses. The mucosa is 174 RAGNAR FANGE AND JONATHAN B. WITTENBERG then revealed as a transparent, richly vascularized, sac composed of two chambers separated by a deep transverse constriction, the diaphragm (Fig. 2). The lumina of the two chambers communicate by a hole in the diaphragm (Fig. 2). Capillaries of the Resorbent Mucosa Posterior chamber Anterior chamber Gas Gland Capillaries Diaphragm Striated muscles Rete Mirabile FIGURE 2. The swimbladder opened dorsally. Portions of the secretory mucosa and the resorbent mucosa are shown in higher magnification, in order to demonstrate the typical appearance of the blood vessels. The anterior chamber, gas gland and retia mirabilia The gas gland forms the epithelial lining of the floor of the anterior chamber and to a lesser extent it is developed on the anterior face of the diaphragm. Periph- SWIMBLADDER OF THE TOADFISH 175 erally the gas gland is continuous with the cuboidal, apparently non-glandular, epithelium of the roof of the anterior chamber. The gas gland is most strongly developed and heavily folded within a few millimeters of the retia mirabilia (Fig, 3). At a distance from the retia the degree of folding dwindles rapidly and the glandular cells become smaller. The glandular epithelium is everywhere only one cell thick. The cells are columnar with a dense cytoplasm stained red by azan. An interesting feature of the gas gland cells is the position of the cell nuclei (Fig. 3). These are situated near the secretory lumen and not adjacent to the basal blood vessel as in most gland cells. This peculiar position of the nuclei has been noted by Woodland (1911) in the gas gland of the eel (Anguilla) and other species, but in the toadfish the nuclei are situated far more apically than in any of the fish studied by Woodland. Gas gland Vascular bed Dense connective tissue Muscularis mucosae Dense connective tissue Loose connective tissue FIGURE 3. Partly diagrammatic drawing of a section through the secretory mucosa. Blood vessels are found within the folds of the secretory epithelium. The structure of the retia mirabilia is essentially of the type described for the eel by Woodland (1911). There are 6-8 distinct retia ("red bodies") situated in the submucosa at the junction of the floor of the anterior chamber and the dia- phragm. The capillaries emanating from the retia mirabilia rejoin, to some ex- tent, forming arterioles and venules which go to the gas gland, where they break up into capillaries providing a very rich blood supply to the glandular membrane. Every fold of the membrane contains blood vessels (Fig. 3), and it is probable that each gland cell has access to a blood capillary at its base and is separated from the blood only by a very thin endothelium. Capillary connections are found between arterioles and venules emanating from the same rete as well as between blood vessels emanating from different retia (Fig. 2). The capillaries of a single rete mirabile were counted in a histological section. A very rough calculation indicated that the total number of capillaries of all the 176 RAGNAR FANGE AND JONATHAN B. WITTENBERG retia mirabilia is 200.000-300,000, which is of the order of magnitude found by Krogh (1929) in the eel. In the connective tissue surrounding the central parts of the retia mirabilia there are numerous nerves and ganglion cells. The ganglion cells probably give fibers to the gas gland or innervate the muscularis mucosae. /'//(' muscularis inncosac and the diaphragm In close connection with the inner epithelium of both the anterior and posterior chamber there is a smooth muscle layer, the muscularis mucosae. This is ex- tremely thin in the posterior chamber but well developed in the anterior chamber, especially ventrally in connection with the glandular portion of the epithelium. The muscularis mucosae also makes a large contribution to the diaphragm where it forms a sphincter around the hole. Tower (1908) observed that the position of the diaphragm varies from about one-third of the distance from the posterior end to less than one-sixth of the distance. We have observed the same variations. That these changes of the position of the diaphragm are due to reflex movements of the muscularis mucosae is shown by the following observations : ( 1 ) In a speci- men in which gas secretion had been stimulated by emptying the bladder three hours earlier, the diaphragm had a posterior position, by which consequence the anterior chamber was enlarged and the posterior chamber diminished. The hole in the diaphragm was closed. (2) In a specimen which suffered from asphyxia- tion and which in addition had received an injection of adrenaline (0.1 ml., 1:1000), the diaphragm was found in the anterior position and with its hole open. (Asphyxia and adrenaline each stimulate gas resorption.) (3) In indi- viduals, where the hole in the diaphragm was initially closed, application of a small drop of adrenaline solution to the margin of the hole caused this to open to a width of 2-3 mm. It is evident that movements of the muscularis mucosae are among the physiological regulatory mechanisms which control reflexly the function of the secretory chamber (the gas gland) and the resorbent chamber ("the posterior vascular organ"). The blood supply of the swimbladder The swimbladder receives its blood from a branch of the coeliac artery, the swimbladder artery (Fig. 1). The individual retia of the anterior chamber are supplied by branches from the swimbladder artery. Within each rete the arterial and venous capillaries form the typical counter-current exchange system studied by Woodland (1911), Haldane (1922), Krogh (1929) and Scholander (1954). All the blood to the anterior chamber passes through the retia. The entire venous return from the anterior chamber passes back through the retia and leaves the swimbladder by the swimbladder vein, which joins the portal vein (Fig. 1, ventral view). The blood supply to the resorbent capillary network (the "posterior vascular organ") of the posterior chamber resembles that of Fierasfcr (Emery, 1880) and the eel (Mott, 1950a, 1950b) in that the arterial blood is supplied from the swim- bladder artery instead of from the intercostal arteries as in most physoclists. The venous return is to the cardinal vein system (Fig. 1, dorsal view). SWIMBLADDER OF THE TOADFISH 177 DISCUSSION The swimbladder of the toadfish is of the typical euphysoclist type (Rauther, 1922; Fange, 1953). It shows many similarities, both physiologically and morpho- logically, with that of the eel. The swimbladder of the toadfish is apparently specialized for the production of sounds (Tower, 1908), and the tunica externa forms a thick capsule enclosing both the anterior and posterior chambers. Removal of this capsule reveals the homology of the two chambers with corresponding parts of the eel swimbladder (Fig. 4). (For previous descriptions of the swimbladder of the eel see Queckett 1. Pneumatic duct oesophagus 2. 3. swim bladder artery secretory part resorbent part (pneumatic duct) resorbent part (posterior chamber) OPSANUS ANGUILLA Anterior Posterior chamber chamber EMBRYOLOGICAL DEVELOPMENT FIGURE 4. The swimbladder of the toadfish (Opsanus tan) and the eel (Anguilla anguilla) illustrating the similarity in general structure. The embryological stages to the left in the figure are redrawn from Tracy (1911). Note the transformation of the embryonic pneumatic duct into the posterior chamber. (1844), Woodland (1911), Rauther (1922), Fange (1953).) The anterior chamber of the toadfish swimbladder corresponds to the swimbladder per sc in the eel and the posterior chamber corresponds to the pneumatic duct of the eel. The homology is further substantiated by the embryonic development of the toadfish swimbladder (Tracy, 1911) during which the posterior chamber develops from the embryonic pneumatic duct. The muscularis mucosae of the toadfish and the eel respond to adrenaline in a similar manner ; the anterior chamber of the toadfish swimbladder and the swimbladder of the eel both are contracted by adrenaline while the posterior chamber of the toadfish swimbladder and the pneumatic duct of the eel are relaxed (Fange, 1953). Woodland (1911), in his classic description of the gas gland, distinguishes three major types of gas glands : those in which the glandular epithelium is composed 178 RAGNAR FANGE AND JONATHAN B. WITTENBERG of a single layer of cells, those in which the gland is massive, and those in which a primitively single layer of cells is secondarily folded into a massive structure. The toadfish, in common with the eel, belongs to the first category (Woodland, 1911) in which (p. 193) "the glandular epithelium is composed of a single layer odc albicans}, bears a correlation to this crab's almost continuous exposure to sea water of high salinity. This correlation is also found by compar- SALT AND WATER REGULATION IN CRABS 191 ing mangrove crab and ghost crab urine chloride levels. It is interesting that urine taken from ghost crabs soon after capture on Nonesuch Island, Bermuda, did not differ appreciably in chloride content from that taken from the ones cap- tured on the Delaware beaches. The difference in salinity of the sea water avail- able to the two habitats does not impose a difference in urine chloride clearance. This might be expected in view of the brief nightly exposure to the surf during feeding. However, mangrove crabs, constantly exposed to 600 mM. Cl/L. sea water, did show the effect of high environmental salinity. During the first two hours after injection, inulin became diluted in a volume of fluid about two-thirds the indicated thiocyanate space. This suggests that either (1) the blood SCN after injection is less concentrated, indicating a larger dilution volume, due to absorption of SCN by cells, or (2) inulin more slowly penetrates the remote spaces invaded more rapidly by SCN. The similarity of the slope of the dilution curves for massive and light SCN injections and the similarity between simultaneous SCN and inulin curves suggest that only the mechanical factors of spreading are involved. Recovery determinations on ghost crabs, ac- counting for 87 to 97% of injected SCN under a variety of environmental condi- tions, indicate that little, if any, SCN is bound by cells. Whether or not inulin eventually invades all of the SCN volume can only be suggested on the basis of data presented here. The apparent cessation of antennal gland activity in ghost crabs on sand appears to offer some opportunity for an answer. So far, it appears that in the 70 hours following the first two, inulin still occupies only two-thirds of the SCN space. The suggestion of a functionally closed circulation, inulin space, within the larger extracellular compartment, SCN space, is an interesting one for which the mechanical factors of lumen flow and stream boundary diffusion seem reasonable. The breadth of the range within which two-thirds of the data are estimated to fall, presented in Table I, is taken to be a reliable indication of the effectiveness of regulation. Comparison of these ranges reveals that the three fluid compartments, total water, SCN space and inulin space, are more closely regulated in land crabs than in ghost crabs and mangrove crabs. A greater difference in the regulation of these volumes might be expected between ghost crabs and mangrove crabs in view of the difference in the stress imposed by their normal habitats. Chloride concentrations in the blood and urine of mangrove crabs are much more closely regulated than in land crabs and ghost crabs. This indicates that the land crab is farther along in the evolution of volume regulation and that the mangrove crab has a more definitive control of chloride concentration. Comparison of chloride and SCN loss from the blood of crabs exposed to the various environmental fluids shows that these ions move at about the same rate in each species and in each situation. Urine SCN concentrations stood in the same ratio and range to blood SCN levels as did these respective concentrations of chloride. The graphs summarizing data are not further complicated by adding these items, inasmuch as they duplicate the chloride data. These observations indicate that it is valid to use SCN as "tagged chloride" in an effort to determine the movement of chloride ions under conditions of electrolyte and water stress. The presence of inulin or SCN in the blood did not affect the clearance, rate or degree, of the other. Inulin was not absorbed from the environmental fluids. Its presence in the environment did not affect the rate or degree of absorption of 192 LAUNCH J. FLEMISTER SCN from the environmental fluids, or the rate or degree of inulin or SCN clear- ance from injected animals. This was true even when sufficient inulin was added to the environmental fluids to make them equal in concentration to the blood of animals injected for the determination of inulin clearance. Therefore, inulin was judged to exert no appreciable effect on the direction, rate or degree of electrolyte and water shifts in the concentrations used. The presence of SCN in the environ- ment in concentrations used did not affect the rate or degree of inulin clearance, but it did affect the rate of fall of SCN levels in injected animals. In injected animals placed in fluids to which SCN had been added, blood concentrations of SCN fell more slowly and only to a point well above equilibrium with environ- mental SCN in 120 mM. Cl/L., about equal in 360 and well below in 600, but not cleared. Of the three species, only the land crab and the ghost crab survived 24 hours out of water. This was to be expected from the differences in habitat and was one basis on which the three species were selected. The inulin and SCN clear- ance in land crabs and ghost crabs on sand for 72 hours, during which blood chlo- rides remained constant, indicate a difference in antennal gland activity. The indicated re-absorption of filtered water in land crabs could account for the lack of obtainable urine. If the re-absorption of electrolytes is obligatory, it could be a cause of the elevated chloride levels found in land crabs exposed to hypertonic environments. In ghost crabs, such a continuing nitration and re-absorption does not appear to exist. The dependence on contact with the sea for filtration and resulting urine formation is in agreement with the observations by Burger (1957) that haemoconcentration, from keeping lobsters in air, suppresses urine formation. His interpretation is that non-diffusible molecules in the blood draw in water principally through the gills, and that this water is bailed out as urine. The similarity between inulin clearance rates in land crabs on sand and for 24 hours in distilled water is interesting. The same is true of ghost crabs. The persistent high inulin concentrations in these latter animals suggest very little filtration in distilled water. The possibility is immediately obvious that cellular osmotic swelling in gill membranes and branchial epithelium may cause mechanical, if not metabolic, interference with absorption of water by crabs in such environ- ments. If this should be the case, why are mangrove crabs different? Chloride and SCN loss in mM. Cl/L. fluid, most rapid in land crabs and least so in mangrove crabs, appears to be compensated for by the absorption of available ion, SCN, from the environmental fluids. The rate and degree of net gain, blood concentration, of the absorbed ion is not proportional to the rate or degree of blood chloride, or SCN, loss. The three species clearly differ in their ability to retain normally present chloride ions and to absorb and hold SCN ions. The blood chloride level in land crabs seems to be least well held and the least well protected by absorption rates. The blood chloride of ghost crabs is somewhat better held and is better protected by a remarkably rapid absorption rate. Reten- tion of blood chloride in mangrove crabs, best of the three, is supported by an intermediate absorption rate. The apparent superiority of the holding and com- pensatory mechanisms in mangrove crabs is reflected by their longer survival, past 48 hours. It should be pointed out, however, that in all three of these species, the net absorptions are inadequate to compensate for a falling blood chloride. Th