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 strontium90-yttrium90 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 C13 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. 0 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 •0 ^-£ 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 .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 0 0 ? 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 STRONTIUM90-YTTRIUM90 IN TELEOST FISH1'2 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 strontium90-yttriumt)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 Sr90-Y90 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 Sr90, 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 Sr90-Y90 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"0-Yso 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, SrR5 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 106/mm3 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 106 X 106 X 106 X 106 X 10« X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 10" X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 106 X 106 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 106 X 106 X 106 X 106 X 10" X 106 X 106 X 10« 106 106 X 10« X 10« X 106 X 106 X 106 X 10" X X 1.102 X 106 1.396 X 106 1.598 X 106 .199 X 106 .298 X 106 .362 X 106 .273 X 106 .175 X 106 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^-Y110 IN TELEOST FISH BLOOD 67 Q. O 140 120 100 80 60 40 20 0 O 120 o 100 80 60 40 20 0 Whole Blood Plasma a. o o o o 120 100 80 60 20 0 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^-Y90 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 Srs3 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 Sr90-Y90 from the blood Figure 1 shows the rate of disappearance of Sr90-Y9u 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 Sr90-Y90. Strontium110 has a half-life of about 28 years and a maximum beta energy of 0.61 Mev. It decays to form radioactive Y90 which has a half-life of 2.54 days and a maximum beta energy of 2.18 Mev. Secular equilibrium exists when the Y90 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^-Y90 IN TELEOST FISH BLOOD 69 observed if no fractionation has occurred. If the rate increases, Y90 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 Y90 which is decaying, and the counts will decrease until a level is reached which is a function of the amount of Sr90 present. The role of the blood fractions in the transport of Sr90-Y90 The increase in the counts/minute of the whole blood and plasma in Figure 2 is due to the build up of Y90. 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 Y90. 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 Sr90-Y90 present in the syringe used for injections. It can be seen that over a period of time, the cpm increased, indicating that some Y90 was lost from the equilibrium mixture. This Y90 was lost to the glassware. The curve for whole blood and plasma, however, increased to a much higher value, indicating that additional Y90 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 Sr90 and Y90 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 Sr90 is readily removable either from or through the cell wall. The activity remaining in the washed cells and ghosts indicates it to be Y90, because the decay rates are very similar to the rate for pure Y90. 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 0 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 Y90 rather than Sr80. 90 Retention and distribution of Srgo-Y Figure 4 shows the retention by the fish of the injected Sr90-Y90 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^-Y90 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 Sr90-Y°° Three fish were injected with Sr90-Y90 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 5V90-]'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 Y90 more than they did Sr90, 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/mm3. 3. The mixing time of Sr90-Y90 injected in the ventricle of Tilapia mossambica, a teleost fish, is approximately 30 minutes. 4. Sr90-Y90 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 Sr90 in whole blood is carried by the plasma. 7 '. Very little Sr1'0 is found either in the cells or on the cell walls. 8. Yttrium90, on the other hand, is present in the stroma. Sr^-Y90 IN TELEOST FISH BLOOD 9. The pattern of internal distribution of intravascularly injected Sr90-Y90 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 Ym) 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 strontiums9 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 EXTRACTS1 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 4659s) 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 wrater 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 CO2 tensions were maintained essentially constant through EXOGENOUS BIOLOGICAL RHYTHMICITY 83 use of the principle of continuous CX-replacement together with CO2 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 O2 and the CO2 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 O2-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, O2-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 O2-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 O2-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 0 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 5-6—7 AM mean deviations (without sign) in O2-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- 0 +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 0 B O -I 0 +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 O2-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 O2-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 2—6 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 0 -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 Q10 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 0 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 107 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 fewrer 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 0 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 0 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 0 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 (T8f,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 ARTHROPODS1 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 /Ar 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 wrere 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 wrere 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 20—40 /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 O2 (95% CO/Oo), after flushing out the air. Xinety-five per cent XL,/O2 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 IfT5 M 99 \ 99jP°la 49 r bodies 30 20 1 1 1 5-10-6 M 2 1 4 46 4ft 1 0 3 96 1 O.KOH and O,Ca(OH)o were controls to compare the CO2-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 CO2 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% N2/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/O2 and the control labelled N2/O2, it is equally clear that, in the light, CO also stimulates the gas-uptake of these eggs. Illumination had no inhibitory effect on the O2- 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% N2/O2 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 O2 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% N2 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 N2 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% O2 rv ' 1 1 Irt1^ ' resp. in 95% N2-5% Oz 95% CO-5% 02 95% N2-5% 02 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/O2 is consistently lower than in 95% N2/O2. 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/O2 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/O2 over that of the parallel controls in 95^ N2/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/O2 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) DHL, + O, -> H,O, + D. if written in the form DH2 + 2 cyt.c3* -> 2 cyt.c2- + 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/O2 in the dark, and this increased (to ca. 100%) upon illumination. With decrease in oxygen tension to 5% (+ 15% N2 and 75% CO) the stimulation decreased. For freshly fertilized eggs in the dark he obtained a slight stimulation in 75% CO/O2 and a marked inhibition in 95% CO/O2. In the light the fertilized eggs showed marked stimulation by 75% CO/O2 and this effect decreased as the O2 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/O2 with 95% N2/O2 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/O2 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/O2 in the dark there was an average of 55% increase in respiration above that in 80% N,/O2, 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/O2 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 CO2 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 CO2. 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 O2-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 mouse4 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 O2. 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. 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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 Vrersuchen 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 (Z2) 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 (Z2) 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 (Z3) at the sphere stage were cut off 220 large buds (Z2) 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 0 1.4 ± .1 A5g A5h 13 13 0 0 1.6 ± .1 1.7 ± .1 A6a 7 0 1.8 ± .1 A6b 11 0 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 Z3 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 (Z2), and the hemispherical buds (Z3) . 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 C13 LABEL1 ROBERT E. BLACK,2 SAMUEL EPSTEIN3 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% OL,) in the light was greatly increased above that of the con- trols (95% N2:5% O2). 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 CO2. 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 C13-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 CO2 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 H2SO4. 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 K2CO3 is largely insoluble, and diluted with CO2-free double-distilled water under CO2-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 C13 offers some advantages over C14 for these experiments. Use of C1 1 would involve precipitating and weighing very small quantities ( less than 4 mg. as BaCO3) of the carbonate derived from the respired CO2 in the alkali well of the usual manometer vessels. This is unnecessary for the mass spectrometric measurement of C13 which provides the required quantitative data in the form of the ratio of C13 to O~ in the sample. It also avoids such uncertainties as are en- tailed by the self-absorption of radiation in the measurement of C14. In addition, use of C13 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% C13. 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 CO2 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 C13-enriched BaCO3 wras 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 CO2 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 CO2 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 C13 content of 2.14%. Relative to a C13 content of 1.17% found for the CO2 from Urechis eggs respiring in air, this gives 82.9% for the atom percentage excess C13 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 C13- 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-O2 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 C13 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 CI3-to-C12 ratios can be readily detected. For introduction of the respired CO2 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 CO2-free water and with precautions to avoid contamination with atmospheric CO2, to a reaction vessel wherein the CO2 could be liberated by tipping-in concentrated H3PO4 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 CO2 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 NaHCO3 was added to the reaction vessel in order to decrease the C13 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-O2 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-O2 mixture could (and, as later shown, does) represent disappearance of CO. However, calcula- tions using the solubility of CO instead of O2 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 mm3., respectively, for the excess gas uptake. Assuming that this is due to the oxidation of CO ( 2 CO + O2 — » 2 CO2), then % of these quantities represent the amounts of CO oxidized and the corresponding amounts of CO2 produced therefrom; namely, 102 and 87 mm3., respectively. The corresponding control vessels yield 318 and 305 mm3, of CO2, respectively, on the basis of an R.Q. of unity (Horowitz, 1940). The percentage of the CO2 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 Cn-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/O2 (mm.3) Total gas-uptake In air (mm.3) In 80 "c CO/O2 (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 C13 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 C13 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 C13 in respired COs Percentage of CC>2 de- rived from oxidation of CO With reference to standard CO2 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 0 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 C13 abundance in the respired CO, of the above three experiments are presented in Table II. The atom percentage excess C13 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 C13 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 NaHCO3 added. The respective percentages of excess C13, 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 C13 relative to the standard source. This simply reflects variation in C13/C12 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 C13 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 CO2 from the air-control vessel was not sub- jected to C13 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% O2 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 CO2 and the labelled CO. For this purpose about 300 mm3, of CO2 were introduced into the Warburg flask. The analysis of the CO2 in the alkali of this flask showed no difference in C13 con- tent from that of the standard. This indicates that no significant exchange of carbon atoms between the CO and CO2 occurs in this system. The determined value for atom percentage excess C13 in the CO2 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 C13. A correction of the same order as in experiment No. 1 would lower very little the calculated percentage of CO2 derived from oxidation of CO (column 6). The principal source of uncertainty in these two experiments is CO2-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 CO2 collected in the alkali for analysis would then have been diluted with the unlabelled CO2 present in the egg suspension at the start of the experiment. Also, some of the labelled CO2 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 C13 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 CO2 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/O2 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 C13 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 CO2 originally present. The retained, as well as the respired, CO2 was determined before transfer to the reaction vessel of the mass spectrometer, as described in Materials and Methods. The total amounts of CO2 (375 mm3, in experimental and 384 mm3, in control flask) were diluted with 0.5 ml. of carrier 0.04 M NaHCO3 (480 mm3, of CO2). Initial bicarbonate content of the CO/O2 blank amounted to 160 mm3. 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 CO2 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-O2 mixture. In the dark the percentage of CO2 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 CO2 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-O2 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/O2 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 CO2 both in the light and in the dark. This has been shown by the use of C13-labelled CO. In the light there is a previously described increase in gas-uptake in 80% CO/O2 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 CO2 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/O2. LITERATURE CITED BERNSTEIN, R. B., and T. I. TAYLOR, 1947. Conversion of isotopically enriched CO2 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 CO2 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 GULL1 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. 2Present 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 twro 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 1O2, 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 0 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. The significance of some ion, however dilute, to the survival of crabs in 0 mM. Cl/L. SALT AND WATER REGULATION IN CRABS 193 fluids is shown by the doubling of survival time by the retention in the blood of 7 mM. SCN/L. for 162 mM. Cl/L. lost in land crabs, 25 for 129 in ghost crabs and 9 for 72 in mangrove crabs during the first 24 hours of exposure. There was no such increase in the survival time of crabs in 720 mM. Cl/L. fluids to which similar amounts of Na SCN had been added. None of the animals showed any signs of depression. Urine chloride of ghost crabs exposed to distilled water for 24 hours was 48% of blood concentration, indicating that some, though obviously not all, filtered chloride is re-absorbed. This is also indicated by comparable SCN data. The high urine chloride is not high enough to suggest chloride secretion by the antennal gland. The impression that these animals formed very little urine is supported by the fact that only 10% of the injected inulin is cleared during this 24-hour period. The less-than-blood concentration and the small volume of urine and the loss of one-third of blood chloride suggest a removal of chloride, and SCN, from the blood by a pathway other than the antennal gland. The urine chloride concentration in mangrove crabs, 62% of blood level, after a similar exposure indicates partial chloride recovery by the antennal gland. This re- absorption continues through 48 hours, but fails to repair a falling blood chloride concentration. Comparison between a 35-gram ghost crab and a similar mangrove crab, cal- culated from data presented in Table I, serves to demonstrate the possible differ- ence in pathways of chloride loss in animals exposed to distilled water. From Figures 1 and 2, it may be seen that 88 mgm. NaCl were lost from the blood of such a ghost crab during 24 hours in distilled water and that the concurrently formed urine contained 11 mgm. NaCl per cubic centimeter. For the chloride lost from the blood to have been cleared by only the antennal gland, 8.0 cc. of urine would have had to be formed. As calculated by inulin clearance, only 0.8 cc. of fluid was filtered during this period. Urine inulin concentrations were roughly equal to blood levels, indicating little or no water re-absorption or secretion after filtration. Ninety per cent of the chloride loss must have been by another route in the ghost crab. From Figures 1 and 2 it appears that 45 mgm. NaCl were lost from a comparable mangrove crab during a similar exposure and that the urine formed contained 15 mgm. NaCl per cubic centimeter. The filtration and excretion of 3.0 cc. of this urine would account for the blood chloride loss. Accord- ing to inulin clearance, 3.5 cc. fluid were filtered, and according to urine inulin concentrations there was no appreciable re-absorption of water. In spite of the clearance of proportionately less chloride than water by the antennal gland, sug- gesting re-absorption of chloride, this is the only pathway necessary to account for the observed failure in chloride ion regulation in mangrove crabs exposed to distilled water. Re-absorption of chloride occurred in both species in distilled water. Since 0.8 cc. blood was filtered in the ghost crab, 18.0 mgm. NaCl crossed over into the lumen of the antennal gland. Since urine contained 48% blood chloride concentration, 8.6 mgm. NaCl were lost, and the remaining 9.4 mgm. must have been re-absorbed. The 3.5 cc. blood filtered in the mangrove crab carried 86.4 mgm. NaCl into the antennal gland. The urine, containing 62% blood chloride concentration, removed 53.6 mgm. NaCl, leaving 32.8 mgm. to be re-absorbed. This is in agreement with the observed chloride loss. The removal of more water than electrolyte from the blood of ghost crabs and mangrove crabs, 194 LAUNCH J. FLEMISTER and the decrease in blood chloride concentrations of all three species exposed to distilled water for 24 hours, make it apparent that the water entering the animals is flushing chloride out through the antennal gland. Moreover, loss of chloride through another pathway is indicated in land crabs and ghost crabs, but not necessarily in mangrove crabs. Although complete extraction of electrolytes in one passage through the gill chamber can not be assumed, comparisons of absorption rates can be made. The absorption of SCN added to 0 mM. Cl/L. environmental fluids indicates a with- drawal from a volume of environmental fluid equal to the SCN space, about 11 cc. for a 35-gram animal, in 2 hours for ghost crabs, 6 hours for mangrove crabs and 15 hours for land crabs. Leveling off of the concentration curves in time suggests that if absorption rates hold, the rate of diffusion outward increases with increasing concentration. This suggests a far more rapid turnover at the gill membrane and, perhaps, branchial epithelium than clearance rates in the antennal gland would indicate. The gill and, perhaps, branchial epithelium appear to be the site of this absorption activity since animals whose digestive tracts were closed at both ends with grafting wax did not differ in absorption rates from those animals not blocked. Similar blocking prior to electrolyte and inulin loss determinations indi- cated that the digestive tract has no significant role in the clearances observed. The appreciable, W% rise in blood chloride concentrations in land crabs ex- posed to 120, 240, and 360 mM. Cl/L. fluids indicates that the rate of absorption of chloride from these environments exceeds the rate of loss until a new steady-state is reached. The steadily maintained higher blood level, failing only in 120 mM. Cl/L. at 72 hours, shows that the regulation is effective, if not compensating. The much more elevated, 20% higher, yet steady concentrations found in animals exposed to 480 mM. Cl/L. for 72 hours, 600 for 48 and 720 for 24, indicate that this regulation persists and has some flexibility and upper limits in situations hyper- tonic to the blood. Absorption rates are greater than indicated by the concen- tration curves, for it must be assumed that during absorption the ions are being lost at rates suggested by the SCN loss curves. The slower rate of SCN loss from injected animals, the slower rate of SCN absorption by uninjected ones and the slower rate of filtration in 600 and 720 mM. Cl/L. fluids indicate that there is reduced exchange with the environmental fluids perhaps due to reduced exposure which in turn may be due to a partial restriction of gill chamber volume or flow as suggested by the work of Gross (1957) on the brachyuran shore crab (Pachy- gmpsus crassipes} exposed to hypertonic fluids. The loss of ions across the gill membrane and, possibly, the branchial epithelium and the persisting, though re- duced, filtration through the antennal gland are not sufficiently rapid to prevent an accumulation of ions from the environment resulting in the elevated blood chloride level observed. Although the lack of urine data precludes further analy- sis and appraisal of this regulation, it appears that there is a correlation between the dry habitat of land crabs and their relatively slow electrolyte clearance resulting in elevated blood chloride levels even in hypotonic environmental fluids. The regulation of blood chloride concentration in ghost crabs is more rigid from 120 to 600 mM. Cl/L. than in land crabs. The similarity of the SCN loss curves for 360 to 720 mM. Cl/L. fluids, faster than inulin clearance, indicates that the antennal gland is of only secondary importance in electrolyte loss in near-isotonic and hypertonic environments. Since in all fluids the injected SCN is cleared in SALT AND WATER REGULATION IN CRABS 195 about 24 hours, the constancy of the blood chloride concentration would appear to depend on the net absorption, or retention of the same quantity of electrolyte irrespective of environmental concentration. This can be concluded to happen from the net absorption curves. Proportional to chloride ions present in environ- mental fluids, there is about three times as much SCN in 120 mM. Cl/L. fluids as in 360 and almost twice as much in 360 as in 600. This is approximately the ratio of net absorption concentration of SCN accumulating in the blood during exposure to the various environments. The greater volume of environmental water involved in this extraction process in 120 mM. Cl/L. fluids is reflected in the more rapid filtration through the antennal gland. Blood chloride is held constant over a wider range, 120 to 720 mM. Cl/L., for 72 hours in mangrove crabs than in either of the other two species. The close approximation of urine chloride concentrations to those of environmental fluids suggests that the regulation is closely held and yet flexible in that absorbed ions are apparently retained in hypotonic situations and cleared in hypertonic ones. The fact that filtration continues at the same rate for all environmental fluids, even distilled water, shows that constant blood chloride levels must be maintained by prompt re-absorption of ions and water by the antennal gland and by absorption and loss by any other route of exchange involved. The more rapid loss of SCN in hypertonic environments than in near-isotonic ones, and these more rapid than in hypotonic ones, at a time when blood chlorides are constant, shows that the turnover, absorption and loss, of electrolytes is more rapid in the more concen- trated environments. This may account for the fact that the accumulated SCN curves fail to level off at points which suggest the ratios of proportionate SCN and chloride concentrations, as was found in ghost crabs. The outbound passage of the same amount of water through the antennal gland in all environmental con- centrations, indicated by inulin clearance, fails to account for electrolyte clearance, except from crabs in distilled water. The fact that urine chloride concentrations approach environmental fluid levels and not blood levels during exposure to 120 to 720 mM. Cl/L. environments for up to 48 hours in ghost crabs and 72 hours in mangrove crabs suggests that the antennal gland re-absorbs some chloride in hypotonic and some water in hypertonic situations after filtration. It is apparent that the reabsorption of chloride is not completely adequate in hypotonic environments in either species and begins to fail earlier in ghost crabs than in mangrove crabs. The re-absorption of water in hypertonic environments is more effective in both species. Urine electrolyte and inulin concentrations indicate that the high urine chloride in hypertonic and near- isotonic environments is due to re-absorption of water. Inasmuch as blood chlo- ride levels continue to be maintained, and inulin data indicate only a moderate increase in filtration and only a moderate decrease in water re-absorption by the antennal gland, the markedly reduced level of urine chloride in hypertonic environ- ments at 72 hours implies a closing of a portal of entry of chloride in the ghost crab. This might be due in part to restricted gill chamber exposure suggested in the shore crab in hypertonic fluids by Gross (1957). The marked increase in urine chloride concentration in the ghost crab in 120 mM. Cl/L. fluid in 72 hours, when blood chloride level remains constant and urine inulin concentrations indi- cate no re-absorption of water, suggests accelerated chloride absorption from the environment. It is interesting that urine chloride concentrations in the two species 196 LAUNCH J. FLEMISTER are near normal when the mangrove crab is in 600 mM. Cl/L. fluid, its normal habitat, and when the ghost crab is in 360 mM. Cl/L. fluid, near isotonicity with its blood. It is also interesting that urine and blood chloride concentrations are equal when the animals are exposed to environmental chlorides 100 mM. Cl/L. less concentrated than the blood. This gives a rough estimate of the re-absorption gradient in the antennal gland and indicates that similar mechanisms and thresholds are involved in the two species. When animals of these three species are exposed to environmental fluids rang- ing from 120 to 600 mM. Cl/L., the rate of turnover, absorption and loss, of elec- trolytes and the rate of filtration are less in the land crabs than in the others. The difficulty in getting urine samples suggests re-absorption of most of the filtered water, which might be expected in view of this crab's adaptation to a dry habitat. The electrolyte turnover and filtration rates are most rapid in the ghost crab in hypotonic and in the mangrove crab in hypertonic environmental fluids. There is an apparent correlation between the almost constant exposure of the mangrove crab to sea water hypertonic to its own blood and a rapid turnover and clearance rate. It appears that the defense in the ghost crab is against the inbound move- ment of hypotonic fluids and that this is a poor defense at best in view of the in- efficient re-absorption of chloride by its antennal gland. It is interesting that when animals of these species are exposed to environmental fluids which are near iso- tonic to their own blood concentrations, the filtration rates through the antennal glands are similar. This indicates that the hydrostatic and osmotic factors in filtra- tion are similar in all three of the species. This augments the interpretation based on the uniformity of re-absorption gradients that similar mechanisms and thresholds are involved in antennal gland function in the three species. In the early intervals of SCN absorption and loss determinations, before blood levels are much altered, absorption rates exceed loss rates in ghost crabs and man- grove crabs and are about equal in land crabs in hypotonic, 120 mM. Cl/L., fluids. Land crabs and ghost crabs hold about equal in near-isotonic, 360 mM. Cl/L., fluids, but mangrove crabs show an absorption advantage in the same fluid, which is hypotonic to their blood. Early loss rates exceed early absorption rates in all three species in hypertonic, 600 mM. Cl/L., sea water. These absorption and loss rate differences are parallel to the leveling-off points in the SCN accumulation curves, \vhich are interpreted to arise from the equating of outbound and inbound passage of ions across gills and, perhaps, branchial epithelium as blood concentra- tions are increased as a result of absorption exceeding loss earlier. Comparison of these leveling-off concentrations of net absorbed, accumulated, SCN and the ratio of the concentration of chloride maintained in the blood and that imposed by the environmental fluid shows close agreement for near-isotonic and for hypertonic situations. In both the ghost crab and the mangrove crab, the plateau has not been reached in hypotonic fluid, but in the land crab there is evidence of both a leveling-off and a breakdown in blood chloride regulation at 72 hours. In crabs of all three species exposed to distilled water, the plateau is so remote and the breakdown so severe that no conclusions can be drawn. Leveling-off of loss curves in time is interpreted to reflect rates markedly reduced by the falling blood concentration. After ten days no SCN or inulin could be found in injected ani- mals kept in the laboratory under normal conditions. The evidence from the net absorption curves is that electrolyte movement is rapid and precise. In hypotonic SALT AND WATER REGULATION IN CRABS 197 environments an appreciably longer time is required to reach a plateau than is required to clear the ion once it is injected, which suggests that there is a choke on the rate of absorption of ions from hypotonic fluids. This is altogether reasonable when the handling of the required amount of fluid is considered. The loss of injected SCN to a level well above a concentration in equilibrium with SCN added to 120 mM. Cl/L. environmental fluid coincides with and sup- ports the evidence from urine chlorides of animals in such environments that electrolyte loss continues even in situations where ions must be acquired to main- tain constancy. The fact that injected SCN falls to approximate equilibrium with environmental SCN in animals exposed to near-isotonic fluids also supports this evidence. The loss of injected SCN to a concentration less than environmental in animals in 600 mM. Cl/L. fluids, in which absorbed SCN is held to less than equilibrium concentration, shows that the capacity to lose electrolyte is not satu- rated by this degree of hypertonicity. It is clear that these loss rates are much greater than can be accounted for by passage through the antennal gland. On the basis of early clearance rates, before blood concentrations are greatly decreased, fluid equal to the inulin space volume is filtered by the antennal gland of land crabs in their normal habitat on sand in about 60 hours, and in mangrove crabs in 600 mM. Cl/L. sea water, their normal habitat, in 24 hours. In ghost crabs on sand no appreciable filtration was found. However, in sea water between 360 and 600 mM. Cl/L., to which ghost crabs normally have access, the filtration rates are similar to those of mangrove crabs. This indicates the importance of the ghost crab's brief nightly exposure to the surf. It is assumed that in their normal habitat, ghost crabs filter somewhat slower than do mangrove crabs, but that they do filter is apparent from the fact that fresh-caught crabs have urine. Therefore, an obvious correlation exists between filtration rate and type of habitat in these three species. The turnover rates indicate the activity in electrolyte and water movement which goes on during the maintenance of constancy of volumes and concentrations in the water compartments measured. The persistence of normal values for these quantities in the variety of devised and imposed environmental stress situations is as remarkable as the rate of continuous change which underlies it. It must be concluded that in submerged crabs of these three species, the gills and, possibly, the branchial epithelium provide the principal pathway for this rapid and precise absorption and loss of electrolytes and water, and that the antennal gland plays only a limited role in this turnover. However, the urine chloride, thiocyanate and inulin concentrations indicate that clearance through the antennal gland may provide the all-important fine adjustment in blood concentration of electrolytes and water. Among these three species, found in different degrees of exposure to seas of different salinity, the mangrove crab, most constantly and continuously exposed intimately to the stable environment of the sea, is the one showing the greatest capacity to regulate the concentration of blood chloride when subjected to environ- ments of widely differing salinities. The crab most independent of the sea, the land crab, has the most definitively regulated volume of total, inulin space and SCN space water, and an adequate electrolyte regulation when exposed to a limited hypotonic range or to food containing proportionately more water than salt, but little or none when environmental chloride exceeds that of blood as does the sea water accessible in its habitat. In the ghost crab, intermediate between them. 198 LAUNCE J. FLEMISTER there appear to be the mechanisms for effective regulation with diminished chloride absorption in hypertonic fluids and increased absorption in hypotonic ones, but with the threat of an extravagantly wasteful chloride loss through the antennal gland. The independence of the land crab from the sea depends on the mainte- nance of a gradient and not on effective regulation. The land habitat of the ghost crab is critically dependent on access to the surf, albeit for a short nightly exposure. The sea habitat of the mangrove crab is a complete commitment despite a wide range of effective regulation. There emerges a picture of independence which depends on the constancy of a normal gradient, and the capacity to tolerate a changing gradient which depends on effective regulation. They afford the mecha- nisms of adaptation to totally different habitats. SUMMARY 1. Exposure to environmental salinities ranging from 120 to 720 mM. Cl/L. for 72 hours did not produce changes in fresh weights of the land crab (Gccarcinus lateralis), the ghost crab (Ocypode albicans) or the mangrove crab (Goniopsis cruentatus). There was an increase in weight of questionable significance after 24 hours in crabs exposed to distilled water. Only in distilled water was there any change in the blood specific gravity of ghost crabs. Even this change was of questionable significance. 2. The total body water content of ghost crabs is significantly larger than those of land crabs and mangrove crabs, which are similar. The fractions of total water content which are available for the dilution of thiocyanate and inulin are similar in the three species. The volumes available for the dilution of inulin are about two-third the volumes in which SCN appears to be diluted. This suggests the interesting possibility of a functionally closed, lumen flow, circulation. 3. The blood chloride concentration of mangrove crabs, although less than that of their environment, is significantly greater than those of the more terrestrial ghost crabs and land crabs, which are similar. The urine chloride concentration of mangrove crabs is identical to that of its environment and is more concentrated than that of ghost crabs. 4. Exposed to environmental fluids of 120 to 600 mM. Cl/L. sea water for 72 hours, land crabs show adequate regulation of blood chloride concentration over a limited hypotonic range, but little or no regulation in fluids hypertonic to its blood chloride. Blood chloride regulation in ghost crabs is adequate over this range, but with the production of a urine which wastes chloride in hypotonic fluids. Mangrove crabs show an adequate and closely held regulation of blood chloride concentration in this range and the production of a urine with chloride levels similar to those of the environment, but with some chloride leakage in hypotonic fluids. Blood chloride regulation failed in all three species wrhen exposed to dis- tilled water for 24 hours, and in land crabs and ghost crabs exposed to 720 mM. Cl/L. for about 24 hours. Mangrove crabs survived 72 hours in 720 mM. Cl/L. fluid with regulation intact, but could not survive 24 hours in air. 5. On dry sand, land crabs filter across the antennal gland a volume equal to their inulin space in 60 hours. It also re-absorbs most of the water of the urine thus formed. This is not true of ghost crabs in which the formation of urine ap- pears to depend on water gained during brief nightly exposures to the surf. When SALT AND WATER REGULATION IN CRABS 199 0 exposed to 600 mM. Cl/L. sea water, their normal habitat, mangrove crabs filter their inulin volume in 24 hours. There is an apparent correlation between these filtration rates and the availability of water in the habitat. 6. Antennal gland filtration and re-absorption rates are adequate to account for the rate of chloride loss in mangrove crabs in distilled water. This is not true for ghost crabs and land crabs in which filtration rates are not much faster than those on sand. Electrolytes are escaping across some other membrane, sup- posedly gills and, perhaps, branchial epithelium. The loss of electrolyte by a route other than the antennal gland is also apparent in animals of all three species ex- posed to environmental fluids from 120 to 720 mM. Cl/L. 7. Re-absorption of chloride by the antennal gland of ghost crabs and man- grove crabs exposed to hypotonic fluids and of water in animals exposed to hyper- tonic fluids is apparent from the similarity between urine and environmental chlo- ride concentrations. Similar re-absorptions can be inferred from data presented on land crabs. 8. The similarity of the mechanisms and thresholds involved in antennal gland function is indicated by (1) the approach of urine chloride concentrations to the blood chloride levels when ghost crabs and mangrove crabs are exposed to environ- mental fluid chloride levels 100 mM. Cl/L. less concentrated than the blood, and (2) the similarity in filtration rates in all three species when animals are exposed to environmental fluids which are near isotonic to their own blood chloride concentrations. 9. The blood concentrations of SCN absorbed from 120 to 720 mM. Cl/L. environmental fluids tend to plateau, due to equating of inbound and outbound ion passage, at a point roughly equal to the ratio between blood chloride and environmental chloride levels. The point of plateau is reached more slowly in hypo- tonic situations indicating the difficulty of handling the required volume of environ- mental fluid. The persistence of electrolyte loss, even in situations where ions must be rapidly absorbed to maintain constancy, is indicated by the SCN loss rate curves for the various environments. 10. The rates of turnover of water and electrolyte are as remarkable as the con- stancy of the regulation from which they result and for which they are responsible. The effectiveness of this regulation in mangrove crabs and the maintenance of a concentration gradient in land crabs can be related to the successful adaptation of these two species to totally different habitats. LITERATURE CITED BURGER, J. W., 1955. Excretion in the lobster, Homanis. Anat. Rec., 122: 460-461. BURGER, J. W., 1957. The general form of excretion in the lobster, Homarus. Biol. Bull., 113: 207-223. CRANDALL, L. A., AND M. X. ANDERSON, 1934. Estimate of the state of hydration of the body by the amount of water available for the solution of sodium thiocyanate. Amer. J. Digest. Dis. and Nutr., 1 : 126-131. CUNNINGHAM, B., P. L. KIRK AND S. C. BROOKS, 1941. Quantitative drop analysis: XIV. Po- tentiometric determination of chloride. /. Biol. Chem., 139: 11-19. FLEMISTER, L. J., AND S. C. FLEMISTER, 1951. Chloride ion regulation and oxygen consumption in the crab Ocypodc albicans (Bosq). Biol. Bull., 101: 259-273. FORSTER, R. P., AND P. ZiA-\VALRATH, 1941. The absence of active secretion as a factor in the elimination of inulin and other substances by the green gland of the lobster, Homanis americanus. Anat. Rcc., 81 : siippl. 128. 200 LAUNCE J. FLEMISTER GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea. Biol. Bull., 112: 43-62. JACOBSEN, C. F., AND K. LINDERSTROM-LANG, 1940. Method for rapid determination of specific gravity. Acta. Physiol. Scand., 1 :_ 149-152. MALUF, N. S. R., 1941. Secretion of inulin, xylose and dyes and its bearing on the manner of urine formation by the kidney of the crayfish. Biol. Bull, 81 : 235-260. MARTIN, A. W., 1957. Recent advances in knowledge of invertebrate renal function. Recent Advances in Invertebrate Physiology. Univ. of Oregon Publications. PICKEN, L. E. R., 1936. The mechanism of urine formation in invertebrates. I. The excretion mechanism in certain Arthropoda. /. Exp. Biol., 13 : 309-328. SCHOLANDER, P. F., G. A. EDWARDS AND L. IRVING, 1943. Improved micrometer burette. /. Biol. Chan., 148 : 495-500. SOMOGYI, M., 1930. A method for the preparation of blood filtrates for the determination of sugar. J. Biol. Chcm., 86: 655-663. YOUNG, M. K., AND L. G. RAISZ, 1952. An anthrone procedure for determination of inulin in biological fluids. Proc. Soc. Exp. Biol. Mcd., 80: 771-774. REGIONAL LOCALIZATION OF NEURAL AND LENS ANTIGENS IN THE FROG EMBRYO IN RELATION TO INDUCTION REED A. FLICKINGER Department of Zoology, State University of loiva, lo-iva City, Iowa A number of embryologists recently have attempted to characterize embryonic cells by their constituent proteins. This approach is of particular interest when this characterization is attempted before, or at the time of, embryonic determination since it might be expected that a protein, or proteins, usually associated with a given tissue would increase in amount once the differentiation and growth of that tissue has already begun. The serological experiments of Ebert et al. (1955) in localizing cardiac myosin and actin in the early chick blastoderm, and those of Ten Cate and Van Doorenmaalen (1950) in determining the time of appearance and location of the lens antigen in frog and chick embryos are examples of this approach. In relation to embryonic induction it would appear to be of some theoretical significance to be able to map or localize the protein that may characterize the re- acting tissue in an induction system. In the induction of the medullary plate by the underlying chorda mesoderm, where is the greater amount of neural antigen localized just before this induction occurs? Is there more specific neural protein in the inductor (chorda mesoderm) or in the reacting tissue (gastrula ectoderm) ? If the inductor has more neural protein, then this may imply the passage of spe- cific protein from the inducing to the reacting tissue and subsequent synthesis of this protein in the reacting tissue. If, on the other hand, more of the neural antigen is present in the gastrula ectoderm, this implies that the induction stimulus is of a less specific nature and may merely be an activator for the synthesis of more neural protein in the reacting tissue. MATERIALS AND METHODS In order to examine this question, antisera were developed against adult male frog brains (Rana pipiens) and antisera developed against adult frog lenses and cattle lenses for a previous investigation (Flickinger et al., 1955) were also utilized. The method of preparing the anti-lens sera was presented in the above paper. Two antisera against adult male frog brains were prepared by cutting open the brains and freeing them of all visible blood, washing them several times in cold 0.65% saline, homogenizing them in a glass tissue grinder in the cold, and injection of the supernate obtained from centrifugation at 3000 g. This supernate was about 1% protein as shown by nitrogen determinations. Intravenous injections of 0.5, 1.0 and 2.0 ml. and an intraperitoneal injection of 4.0 ml. were given on alternate days and constituted an injection series. Three such series of injections were administered a week apart with the modification that the whole uncentrifuged homogenate was injected intraperitoneally in the third series of injections. The rabbits were bled 7 days after completion of injections. One of the antisera 201 202 REED A. FLICKINGER reacted with a 0.1 % protein supernate from adults' brains at an antiserum dilution of 1/128. Obviously extracts of adult frog brains are not highly antigenic. In the preparation of test antigens, early gastrulae (Shumway stage 10) were operated upon under sterile conditions and cut in four regions ; ectoderm, dorsal mesoderm, ventral mesoderm and endoderm, as seen in Figure 1. Most of the large white yolky cells were removed from the ventral and dorsal mesoderm tissues. Recently hatched larvae (Shumway stage 19-20) were cut into three parts; head, trunk and gut (Fig. 1). The heads were removed by cutting verti- ECTODERM HEAD TRUNK VENTRAL MESODERM DORSAL MESODERM ENDODERM GUT FIGURE 1. See text for explanation. cally just posterior to the gill plate; the trunks were separated from the guts by cutting horizontally just ventral to the somites. No attempt was made to count the number of gastrulae and larvae that were operated upon, but for each prepara- tion of test antigen from an embryo fraction, several hundred of the appropriate tissues were homogenized with an equal volume of 0.9% NaCl in micro-tissue grinders and then centrifuged in micro-centrifuge tubes (3 mm. inside diameter ( 55 mm. length) in adaptors designed for the 7-ml. high speed head of the Inter- national centrifuge. After removal of the pigment, yolk, and lipid cap by re- peated centrifugation at 15,000 g, some 0.2-0.3 ml. of a centrifugal supernate is obtained which can be used as the test antigen. The precipitin reactions were carried out in the same type of small tubes used for the centrifugations so as to conserve the antigen preparations. In some cases more concentrated embryo anti- gen preparations wrere obtained by homogenizing the embryo parts with an equal volume of supernate from a previous fractionation of the same tissues. A number of nitrogen determinations were made upon the test antigen preparations by the nesslerization method. The protein concentrations ranged from .35-1.5% pro- tein, but for any given fractionation the protein concentrations were usually fairly similar for the four parts of the gastrula or the three regions of the larva. The test antigen preparations were adjusted to the same protein concentration before any given set of serological tests. RESULTS The anti-brain serum was not organ-specific and cross-reacted with frog serum, muscle, heart and kidney. It was found that serial absorption of 1.0 ml. of anti- brain serum with a total of 1.15 ml. of female frog serum, 1.0 ml. of a centrifugal supernate from a frog heart homogenate and 0.95 ml. of a frog kidney supernate ANTIGEN LOCALIZATION AND INDUCTION 203 was sufficient to render the anti-brain serum specific. If the frog serum was not used in the absorptions then it required 2.4 ml. each of the heart supernate and the kidney supernate to render 1.0 ml. of the anti-brain serum organ specific. After absorption the antiserum did not react with centrifugal supernates obtained from adult frog heart, kidney, muscle, liver, spleen, ovary and frog serum. In four separate experiments where the anti-adult male frog brain serum had been absorbed to completion with male frog serum, the antiserum showed a pre- cipitin reaction with female frog serum. This was also found to hold true for similar absorption of an anti-adult male muscle serum. Upon further dilution with male frog serum the reactivity towards female frog serum disappeared. If the anti-brain serum was diluted 1/4 with normal rabbit serum and reacted against male and female frog sera, the latter reaction occurred at once while a faint pre- cipitate did not appear in the male serum reaction for a period of twenty minutes. At the end of an hour the female serum-antiserum precipitate was much heavier than the male serum-antiserum reaction. It is known from agar-plate serology experiments of Flickinger and Rounds (1956) that an anti-embryonic yolk frac- tion serum gives 5 precipitate bands with female frog serum, and only two with male frog serum, so that the female frog serum apparently contains some proteins in higher concentration, or with different determinate groups, than those found in serum of the male frog. It is more difficult to understand the situation in this work where the antibodies have been formed in response to injections of an adult organ from a male frog. It might be expected that absorption to completion with male frog serum would not only remove activity to the male serum but also to female serum, especially since the serum contaminating the injection antigen was male serum. The most likely explanation is that the common frog serum proteins in male and female are in higher concentration in the female serum and therefore the female serum is a better antigen and can still react with anti-adult male organ sera absorbed to completion with male frog serum. This explanation seems more plausible than trying to invoke any type of pangenesis for adult organ antigens. Anti-brain serum, rendered specific by absorption, reacted positively with all test antigen preparations from the four parts of the gastrula and the three regions of the larva (Fig. 1). The precipitates in every case were distinct after twenty minutes at room temperature; after twenty minutes at 37° C. and after twenty minutes at 6° C. the precipitates were still of equal intensity. Normal rabbit serum-antigen controls were negative. No attempt was made to titer the anti- serum against the various test antigen fractions since it was apparent that the antigens reacting with the brain-specific antiserum were to be found in all regions of the gastrula and larva in approximately equal amounts. Absorption of pre- viously unabsorbed anti-brain serum with the antigen fraction from the larval gut ( where no elements of the nervous system are present) also removed activity against all other antigen fractions, thus indicating in another way that proteins 1 tearing the determinate groups that react with the anti-adult brain serum are located in all regions of the frog gastrula and larva. In order to see if different proteins may be given off by different tissues, as some of the work of Niu (1956) suggests, a series of forty explantation experiments was carried out in which 20-30 pieces of larval brain (stage 19) or larval trunk (Fig. 1) were stripped of their epidermis and cultured in the bottoms of deep well depression slides in Niu-Twitty solution (1953). After a week of explantation 204 REED A. FLICKINGER the culture fluids were collected from above the tissues, centrifuged, and the super- nates from the various cultures of denuded heads were pooled, as were the trunk culture supernates. There was some slight cytolysis in these cultures which would account for the release of some soluble protein into the medium, but it is felt that some of the protein probably was released into the medium by "natural means" other than cytolysis. These solutions were dialyzed against distilled water and then evaporated in dialysis bags suspended in front of a fan in a cold room (2-4° C.). An individual dialysis bag usually contained about 8 ml. of the culture supernate and this volume was reduced to about 0.2 ml. Nitrogen determinations in one case revealed the protein level of the concentrated supernate to be 0.06% protein. The absorbed brain-specific antiserum gave positive ring tests of equal intensity with both head and trunk supernates twenty minutes after the test anti- gens were layered over the antiserum. It would appear that the proteins given off from the denuded heads and trunks bear similar determinate groups that react with the brain-specific antibodies. These results are certainly not definite enough to state that proteins or nucleoproteins (inducing substances?) given off by differ- ent inductors are similar, even though much of the protein from the trunk cultures would be derived from the exposed myotomes and that from the head cultures would come from the brain, since the antiserum lacks the desired degree of specificity. However, the data tend to support the many biological examples of non-specificity of the inducing agent (Holtfreter, 1951). The obvious disadvantage to the use of anti-brain serum is its lack of speci- ficity. Hence it was decided to use anti-lens sera where it is known that this organ has a greater degree of organ specificity. This organ is of course also an induced organ and the localization of lens antigen in the embryo would be of value in relation to the induction problem. In a previous investigation (Flickinger ct al., 1955) anti-frog lens serum was used to demonstrate the presence of lens anti- gen in the anterior half, but not in the posterior half, of feeding frog larvae. How- ever, the negative results do not necessarily mean an absence of the lens antigen but might imply only a reduced amount of the antigen in the posterior region of the embryo. As a first attempt to improve the means of localizing lens antigen, flank ecto- derm, which is known to possess the ability to respond to an induction stimulus and form lens, was stripped from several hundred hatched larvae (Shumway stage 19), homogenized in 0.9% NaCl, and a supernate fraction obtained by centrifuga- tion. This extract did not react in the ring tests with an anti-cattle lens serum, or an antiserum to cattle lens a- crystalline (previously provided to me by Dr. Ten Gate of the University of Amsterdam). The anti-a-crystalline was also negative against the gastrula ectoderm and chorda mesoderm test antigens. It is believed that the epidermis alone did not provide sufficient soluble protein to give a pre- cipitin reaction. Therefore, in order to concentrate the test antigen preparations, it was decided to use test antigens from the head and trunk regions of hatched larvae in which the tissues were homogenized with an equal volume of supernate of heads or trunks from a previous fractionation. This has an advantage over the use of posterior halves of larvae (Flickinger et al., 1955) in that the less metaboli- cally active gut region, containing more storage protein, is not included with the trunk tissues. The anti-frog lens serum gave immediate strong ring tests with both the head ANTIGEN LOCALIZATION AND INDUCTION 205 and trunk antigen preparations, but this antiserum also reacted with adult frog serum and therefore was not organ-specific. After absorption of 1 volume of anti- frog lens serum with l/2 volume of female frog serum the antiserum was negative to serum, and still gave definite ring tests after 20 minutes with head and trunk supernates. This antiserum was negative against test antigen preparations from adult muscle, heart, kidney and ovarian supernate, but it did give a weak positive re- action with an adult brain supernate after one hour. Therefore three volumes of the anti-frog lens serum were absorbed with two volumes of a centrifugal supernate obtained from homogenizing adult frog brains with an equal volume of frog serum. This absorption left the anti-frog lens serum negative to serum and brain and the antiserum gave a reaction with the test antigen preparation from larval trunks. The use of antiserum against adult frog lens in localizing the presence of lens protein, or a protein bearing lens determinate groups, has the disadvantage that the antibodies are directed against determinate groups characteristic of frog pro- teins as well as those characteristic of lens protein. It would be preferable to use an organ-specific anti-cattle lens serum where, if the antibodies did react with frog embryo test antigens, the reaction would most likely be due to the lens deter- minate groups of the frog antigens reacting with the antibodies directed against cattle lens proteins. The anti-cattle lens serum was tested against frog serum, brain and kidney and found to give no reaction. However, this antiserum gave immediate strong posi- tive reactions with the concentrated head and trunk antigens from the hatched larva. The appropriate normal rabbit serum-test antigen controls were negative. The anti-cattle lens serum did not react with test antigen preparations from the four parts of the early gastrula. This negative result might be explained by the fact the gastrula supernates were not prepared as concentrated antigens, as in the case of the larval heads and trunks, but it is also likely that the lens antigen is present in lower concentration at earlier stages. In looking back at the previous work with this antiserum (Flickinger ct a!., 1955) it was noted that the antiserum reacted with supernates from both the anterior and posterior halves of 69-hour chick embryos as well as with the ovarian supernate and hatching larva supernate from frog embryos. DISCUSSION If antigens with neural determinate groups are localized in all parts of the gastrula and tailbud larva, and particularly in the more critical case where antigens with lens determinate groups are situated in the trunk region (somites, neural tube, notochord, and dorsal epidermis) of the early larva, this indicates that organ antigens may be more disperse for a certain time than the organ-forming areas in the embryos. Ebert et al., (1955) have shown this to be the case for cardiac myosin in the chick blastoderm, although cardiac myosin did become localized in the heart-forming areas after a period of time. Cardiac actin was always confined to these heart-forming regions. It seems that the presence of organ antigens outside their organ-forming dis- tricts does not invalidate the idea that one may in part characterize a cell, tissue or organ by the structural and functional (enzymes) proteins they contain. The wide distribution of organ-specific proteins is an indication of the totipotency of 206 REED A. FLICKINGER various regions of the embryo which has been demonstrated by numerous trans- plantation experiments. It would also tend to support the idea that embryonic induction could be any one of a number of stimuli which might evoke protein synthesis at a particular region in the embryo. For example : any specific protein, which the genetic machinery of the cells would allow to be synthesized, might be stimulated (induced) to this synthesis by a number of factors. Ribonucleic acid or ribonucleo-protein is a critical component for protein synthesis (Brachet, 1950; Gale and Folkes, 1954) and for embryonic induction (Niu and Twitty, 1953). It is known that protein synthesis is an •endergonic process demanding energy (Fruton and Simmonds, 1953) and Miller (1939) has demonstrated the reversal of the anterior-posterior polarity of a section •of stem of a regenerating hydroid by raising the temperature (and therefore the level of energy-yielding reactions) at the posterior end of the stem. The presence of free amino acids is known to be necessary for adaptive enzyme formation (Halvorson and Spiegelman, 1953) and it is known from the work of Earth (1941), Holtfreter (1945), Yamada (1950) and Flickinger (1958) that competent tissues can be stimulated to differentiate independently of an induction stimulus from another tissue by pH shock treatments wdiich can dissolve yolk (Holtfreter, 1946). Flickinger (1957) has emphasized that the solubilization of yolk protein, which provides the material from which cytoplasmic proteins are synthesized, can be a causal step in embryonic induction. Even after specific cytoplasmic proteins have been synthesized the provision of an enzymatic substrate, as in Wilde's (1955) conversion of gastrula ectoderm cells into melanophores by giving them phenylala- nine, could be considered an embryonic induction. Activators or inhibitors of an enzymatic reaction might then also act in an induction system. Viewed in this manner there may be multiple aspects of embryonic induction with any factor, or combination of factors, that would facilitate the synthesis, or activity, of specific proteins being considered an inductor. This is somewhat similar to the case shown by Spiegelman and Reiner (1947) where, under conditions optimal for growth and protein synthesis, adaptive enzymes may be formed without the substrate or inducer being present. If a sub-differentiation threshold level of any given specific protein, or proteins, exists throughout the embryo, it may be that a preferential hierarchy of cell and tissue specialization exists such that when conditions become optimal for protein synthesis in a given region of the embryo then a specific cell type or tissue will be formed. That there is some kind of preferential hierarchy can be seen from the tendency for gastrula ectoderm activated by sub-lethal cytolysis to form forebrain structures (Holtfreter, 1944). This tendency to form head structures first can be seen in Sabella regeneration (Berrill, 1931) where the most anterior part forms first and then fills in the missing parts. From studies of regeneration and embry- onic development it is evident that differentiation occurs time-wise along anterior- posterior and dorsal-ventral axes with the anterior and dorsal differentiations usually preceding the posterior and ventral ones. Possibly these anterior-posterior and dorsal-ventral patterns of specialization are due to gradients of factors which favor protein synthesis, and that certain types of cell or tissue specialization are favored when conditions for protein synthesis become optimal. For example, Flickinger (1957) has hypothesized that the primary organizer area forms where the first and most active conversion of yolk to cytoplasm occurs. The biological ANTIGEN LOCALIZATION AND INDUCTION 207 totipotency of most parts of the embryo, and the serological evidence tend to indi- cate that some organ-specific proteins may be more widely distributed than the corresponding specific organ-forming areas. Possibly the postulated sequential protein synthesis, and cell specializations and growth that may depend upon these syntheses, are of a self-limiting type as postulated by Rose (1952, 1957) and Weiss (1952). The question why the synthesis of a given type of protein may be favored when conditions become optimal for protein synthesis is indeed puzzling. It is apparently not due to a purely qualitative distribution of protein, or to unequal nuclei (Briggs and King, 1952). It might be ascribed to a quantitative distribu- tion of various organ-specific proteins, or nucleoproteins, but although there are gradient-wise distributions of soluble proteins and ribonucleoproteins, there is as yet no evidence concerning the specificity of these compounds. Another possi- bility might be a preferential sequence of activity of different specific genes. It may be well at this time to review the idea of Driesch that the fate of a cell or tissue is a function of its position. It is well known that undetermined embryonic cells and tissues tend to "fit in" to the particular locale in which they find them- selves. The fact that the nuclei of the cells of the determined neural plate are apparently undifferentiated and still able to promote complete development when injected into the enucleated egg (King and Briggs, 1954) and the apparent deter- mination of the whole mouse embryonic shield before the determination of its individual constituent cells (Grobstein, 1952) argues for some sort of "supra- cellular patterning," perhaps of a polar or axial type (Child, 1941; Rose, 1957), which precedes cell specialization. This is a question which deserves a good deal of attention from embryologists. SUMMARY 1. Anti-adult male frog brain and muscle sera absorbed to completion with male frog serum still react with female frog serum. It is believed that serum proteins common to the male and female may be in higher concentration in the female serum and hence account for this reaction. 2. Anti-brain serum, rendered organ-specific by absorption, reacted positively with test antigen preparations from four regions of the early frog gastrula (ecto- derm, dorsal mesoderm, ventral mesoderm, and endoderm) and three regions of the hatched frog larva (head, trunk and gut). The proteins bearing brain deter- minate groups are apparently situated throughout the embryo at these stages. 3. The organ-specific anti-brain serum gave positive precipitin reactions with culture supernates from both larval heads and trunks which had been denuded of their epidermis and explanted for a period of a week. The proteins given off by these cultured heads and trunks bear similar determinate groups that react with the brain antibodies. 4. An absorbed organ-specific anti-frog lens serum, and an organ-specific anti-cattle lens serum, reacted with concentrated test antigen preparations from both the heads and trunks of hatched frog larvae. It seems that lens antigen, or protein bearing lens determinate groups, is localized in areas other than the lens- forming region. 5. The significance of these results is discussed. 208 REED A. FLICKINGER LITERATURE CITED EARTH, L. G., 1941. Neural differentiation without organizer. /. Exp. Zoo}., 87: 371-382. BERRILL, N. J., 1931. Regeneration in Sabclla pavonia (Sav.), and other sabellid worms. /. Exp. Zool., 58: 495-523. BRACKET, J., 1950. Chemical Embryology. Interscience Publishers, New York. BRIGGS, R., AND T. J. KING, 1952. Transplantation of living nuclei from blastula cells into enucleated frog's eggs. Proc. Nat. Acad. Sci., 38: 455-463. CHILD, C. M., 1941. Patterns and Problems of Development. Univ. of Chicago Press, Chicago, Illinois. EBERT, J. D., R. A. TOLMAN, A. M. MUN AND J. F. ALBRIGHT, 1955. The molecular basis of the first heart beats. Ann. N. Y. Acad. Sci.. 60: 968-985. FLICKINGER, R. A., 1957. The relation between yolk utilization and differentiation in the frog embryo. Amcr. Nat., 91 : 373-380. FLICKINGER, R. A., 1958. Induction of neural tissue in ventral explants from frog gastrulae by carbon dioxide shock. Science, 127 : 145-146. FLICKINGER, R. A., E. LEVI AND A. E. SMITH, 1955. Some serological experiments relating to the embryonic development of the lens. Physiol. Zool., 28 : 79-85. FLICKINGER, R. A., AND D. E. ROUNDS, 1956. The maternal synthesis of egg yolk proteins as demonstrated by isotopic and serological means. Biochim. ct Biophys. Acta, 22: 38-42. FRUTON, J. S., AND S. SIMMONDS, 1953. General Biochemistry. Page 624. John Wiley and Sons, New York. GALE, E. F., AND J. P. FOLKES, 1954. Effect of nucleic acids on protein synthesis and amino- acid incorporation in disrupted staphylococcal cells. Nature, 173: 1223-1227. GROBSTEIN, C., 1952. Effect of fragmentation of mouse embryonic shields on their differen- tiative behavior after culturing. /. E.vp. Zool., 120: 437-456. HALVORSON, H. O., AND S. SPIEGELMAN, 1953. Net utilization of free amino acids during the induced synthesis of maltozymase in yeast. /. Bact., 65 : 601-608. HOLTFRETER, J., 1944. Neural differentiation of ectoderm through exposure to saline solution. /. Exp. Zool., 95 : 307-340. HOLTFRETER, J., 1945. Neuralization and epidermization of gastrula ectoderm. /. E.rp. Zool., 98: 161-209. HOLTFRETER, J., 1946. Experiments on the formed inclusions of the amphibian egg. I. The effect of pH and electrolytes on yolk and lipochondria. /. Exp. Zool., 101 : 355-405. HOLTFRETER, J., 1951. Some aspects of embryonic induction. Tenth Growth Symposium, pp. 117-152. KING, T. J., AND R. BRIGGS, 1954. Transplantation of living nuclei of late gastrulae into enucleated eggs of Rana pipiens. J. Embryol. Exp. Morph., 2 : 73-80. MILLER, J. A., 1939. Experiments on polarity determination in Tubularia regenerates. (Abst. Amer. Soc. Zool.) . Anat. Rcc., 75: 4 Suppl. Niu, M. C., 1956. New approaches to the problems of embryonic induction. (In: Cellular Mechanisms in Differentiation and Growth, edit, by D. Rudnick, pp. 155-171, Prince- ton University Press, Princeton, N. J.) Niu, M. C., AND V. C. TWITTY, 1953. The differentiation of gastrula ectoderm in medium conditioned by axial mesoderm. Proc. Nat. Acad. Sci., 39 : 985-989. ROSE, S. M., 1952. A hierarchy 'of self-limiting reactions as the basis of cellular differentia- tion and growth control. Amcr. Nat., 86 : 337-354. ROSE, S. M., 1957. Cellular interaction during differentiation. Biol. Rev., 32: 351-382. SPIEGELMAN, S., AND J. M. REINER, 1947. The formation and stabilization of an adaptive enzyme in the absence of its substrate. /. Gen. Physiol., 31 : 175-193. TEN GATE, G., AND J. VAN DOORENMAALEN, 1950. Analysis of the development of the eye-lens in chicken and frog embryos by means of the precipitin reaction. Proc. Kon. Nedcrl. Akad. Wetensch., 53: 3-18. WEISS, P., 1952. Self-regulation of organ growth by its own products. Science, 115: 487-488. WILDE, C. E., JR., 1955. The role of phenylalanine in the differentiation of neural crest cells. Ann. N. Y. Acad. Sci., 60: 1015-1025. YAMADA, T., 1950. Dorsalization of the ventral marginal zone of the Triturus gastrula. I. Ammonia-treatment of the medio-ventral marginal zone. Biol. Bull., 98: 98-121. STUDIES ON NEUROMUSCULAR TRANSMISSION IN LIMULUS GRAHAM HOYLE * Marine Biological Laboratory, Woods Hole, Mass.2 Among invertebrates only certain crustaceans and insects have been the sub- jects of detailed study in regard to neuromuscular mechanisms. There have proved to be very considerable differences between the various arthropod mecha- nisms encountered on the one hand (Wiersma, 1957 — Crustacea; Hoyle, 1957 — insects) and those of vertebrates on the other (Fatt, 1954). The differences con- tribute to the difficulty in arriving at a general concept of the way in which coupling between excitation of the surface membrane of the muscle fiber, which is achieved by nervous action, and shortening of the contractile material, is brought about. But they also show that certain favored hypotheses in regard to vertebrate muscle are either of only limited applicability for muscle as a whole, or are wide of the mark. There is a strong difference of opinion regarding the relevance of the electrical activity of the muscle fiber membrane in the process. Most recent authors (cf. Sten-Knudsen, 1954; Huxley, 1956) have regarded the contractile machinery as being in some way connected with the membrane potential. Some (e.g., Csapo and Suzuki, 1957) believe that contraction is initiated by current flow resulting from membrane action potentials. For the Crustacea, it has been found necessary to postulate a separate coupling mechanism within the muscle fiber which may be activated differently by neuromuscular transmitter action in different cases. In some (Hoyle and Wiersma, 1958b) there may be a direct action by the transmitter substance on the coupling mechanism, the electrical intermediate (or propagation) stage having been by-passed. In others, electrical changes, or the ionic fluxes associated with them, affect the coupling mechanism. From this it seems likely that in the elucidation of the general problems of excitation-contraction coupling, the arthropods will provide favorable material. In them single muscle fibers are innervated by more than one motor axon, each having different motor effects, and in the Crustacea there are also inhibitory axons which uncouple the excitatory action. In many arthropod systems the unit of contraction is not an all-or-nothing twitch, and contractions are minutely graded. This difference between arthropod muscle and ordinary skeletal muscle of verte- brates is probably attributable to the absence of propagated muscle action poten- tials in the former. In spite of their potential interest, and the variety of their mechanisms, several major subdivisions of the phylum remain unexplored, no arachnid, for example, having been examined in regard to its detailed neuro- muscular mechanisms. It seems desirable, therefore, to have information regard- ing the motor mechanisms of the particularly interesting primitive arachnids, the Xiphosura. Accordingly a preliminary study has been made on Limuhis polyphe- mus Latr. and has revealed several interesting features which are reported here. 1 Fellow of the Rockefeller Foundation. - Permanent address : Department of Zoology, University of Glasgow, Scotland. 209 210 GRAHAM HOYLE METHODS The walking legs, except the specialized fifth pair, have been examined from specimens 16"-22" long, obtained at Woods Hole, with a view to finding suitable nerve-muscle preparations. The legs were severed by a quick snip of the coxo- trochanteral joint. A few of the leg muscles can be used, in particular the closer of the claw (adductor or depressor of the tarsus) and the flexor (levator) tibiae (situated in the patella). The present studies were carried out entirely on the claw closer. This muscle exhibits in the freshly-excised leg a remarkable pseudo- reflex. If the inside of the pollex (fixed extension of the tibia) is gently stroked, the clawr closes sharply. This reflex can be obtained repeatedly for up to 15 minutes or so after removal of the leg. It seems highly improbable that any nervous machinery of true synaptic type can be present in the isolated leg to account for this curious phenomenon. Similar phenomena have been described Electrodes for stimulating axon bundle FIGURE 1. Drawing of the preparation seen from above. The leg is placed in a trough cut in a wax block. The opener of the claw has been removed, exposing the closer muscle. in excised crustacean legs in which stretching of the chela, for example, can lead to its opening. Wiersma (unpublished) has suggested as an explanation of the crustacean responses that following excision the excitability of the cut ends of the motor nerves is raised to such an extent that an ephaptic transmission occurs from adjacent sensory axons. To make a preparation, the excised leg was laid in a sculptured trough of wax and stapled into position with the tarsus uppermost. The main leg nerve can then be easily exposed in the femur by cutting away the shell and removing part of the extensor patella muscle. The nerve has no surrounding sheath and very little connective tissue so that it can easily be split into bundles. These may be stimulated in turn and any having an effect on the tarsus retained, the rest being cut away. The retained bundles can then be split again until either very small bundles, or eventually single axons, remain. In this way it was ascertained that the closers of the claws of legs I-IV are innervated by two motor axons. No inhibitory axons were found. In this re- spect Linuihis resembles the insects rather than the crustaceans. There is no tested physiological saline for Linnthts so filtered sea water was LIMULUS NEUROMUSCULAR TRANSMISSION 211 used to bathe the preparation. Cole (1940) has analyzed the haemolymph and found that the mineral composition approximates very closely indeed that of the local sea water in two different localities, one of which was Woods Hole. Since the present work was done, a physiological saline has been developed for the Japanese horseshoe crab, Tachypleus tridentatiis (Kikuchi and Tanaka, 1957). At this stage a strip of shell was carefully snipped away from the margin of the tibia in order to expose the outer edge of the opener muscle (abductor tarsi). The opener apodeme was then cut close to the tarsus, grasped with forceps, lifted and stretched until the whole muscle came away. This leaves the V-shaped closer muscle exposed, with its innervation intact. The pollex was fixed in a hole in the wax block and the tip of the tarsus was attached by a thread to an electromechanical transducer. Pairs of fine silver wires were micromanipulated onto the exposed nerve bundles. A drawing of the prepa- ration, seen from above, is presented in Figure 1. The muscle fibers are of fairly uniform diameter but only 25-40 ^ thick, i.e., they are appreciably thinner than many insect muscle fibers and very much thinner than those of the larger decapod crustaceans. Glass capillary micro-electrodes, filled with 3 M KC1, were used to record trans-membrane potentials from muscle fibers of the claw closer. The nerve bundles were stimulated with brief rectangu- lar pulses isolated by radiofrequency coupling units. Display was conventional. RESULTS In the more vigorous preparations a single stimulus applied to either of the two nerve fibers evokes in each case a small twitch. Repetitive stimuli lead to partial and complete tetani. The mechanical response to one of the two axons is, however, always larger than the other, and at a given frequency of stimulation also appears slower. Hence the two axons may be referred to as "fast" and "slow" according to the nature of the contraction evoked, as is customary in deal- lOOrnsec J^ i* i* r^ r FIGURE 2. "Spontaneous" potentials. Four records from claw-closer muscle fibers of a fresh-excised Limidus leg, showing recurring potentials. The deflections marked + are attrib- uted to discharges in the fast axon ; those marked • to the slow axon. The single spike response in b was associated with a twitch which must have caused the electrode to be jerked out of the fiber. The upper trace in each record marks the zero baseline and the lower one the internal potential recorded with a 3 M KCl-filled glass capillary micro-electrode. 212 GRAHAM HOYLE ing with crustacean motor nerve fibers (Wiersma, 1941). The corresponding responses are then called fast and slow, respectively. "Spontaneous" responses. When the preparation is very fresh, discharges originating in the hypersensitive cut ends of the axons lead to spontaneous "tone" in the closer muscle and contractions which cause small movements of the tarsus. If a micro-electrode is inserted at random into a muscle fiber of the closer at this time, recurring electrical potentials of small size are seen (Fig. 2). The resting potentials of the muscle fibers are of small magnitude, ranging from 35-55 mV. The peak amplitudes of the recurring potentials are from 0.5 mV to a maximum of 25 mV in different fibers. In any one fiber they are clearly of two distinct sizes, the smaller being due to the slow axon and the larger to the fast. Single small potentials are not usually associated with visible twitches although in the more vigorous preparations, when they occurred singly, this was the case, and twitches were seen. A small proportion of muscle fibers gave "fast" potentials which were compound, i.e., they had an initial component resembling an ordinary end-plate- potential (e.p.p.) giving rise to a small spike response (Fig. 2b). The slozv responses. Responses attributable to the "slow" axon could be observed in about 60% of those muscle fibers in which any appreciable electrical o • mV SLOW 40 | i *" I i FAST lOOmsec lOO-xsec FIGURE 3. Potentials and tension due to single excitations applied to : a, the slow axon and b, the fast axon. Left hand side : electrical responses from the same single muscle fiber. Right hand side : mechanical responses of whole muscle recorded at tarsal tip. change could be obtained during stimulation of both fast and slow axons (usually the bundles containing them) together. The single electrical response was always a very small one resembling a small e.p.p. It will be referred to as a junctional potential (j.p.) rather than an e.p.p. since nothing is known of the nature of the nerve terminals in Limulus muscle. To distinguish it from the corresponding response to the "fast" axon it will be called a slow junctional potential (s.j.p.) The long latency following the stimulus artifact, which is apparent in the records, is clue largely to the conduction time of the nerve impulse along the nerve in the femur and patella into the tibia. The s.j.p.'s rise to a peak in 12-18 msec, and decay in about 60 msec. The largest one found had a peak amplitude of 5 mV. Although no tension was usually recorded at the tip of the tarsus during stimulation of the slow axon with a single shock, some preparations did show a small twitch, giving not more than LIMULUS NEUROMUSCULAR TRANSMISSION 213 o- 4O lOOmsec II III FIGURE 4. The responses to short trains of stimuli in three different muscle fibers (i-iii). (i) Slow axon. Three responses from the same fiber showing the summation of s.j.p.'s and the small degree of facilitation. A small spike arises in c from the plateau of depolarization, (ii and iii) Fast axon. a, low frequency; b-d, higher frequency. Successive steps (f.j.p.'s) are progressively larger (facilitation). Summation is evident; occasional spikes arise from the depolarization plateau. 0.5 gm. tension at the tip of the tibia (Fig. 3). On repetitive stimulation appre- ciable tension developed, increasing with increasing frequency of stimulation up to a maximum of just over 50 gm. at 200 per second. Thus the tetanus/twitch ratio was more than 100: 1. The s.j.p.'s initially increased in magnitude by two or three times during a train of stimulation, a phenomenon usually referred to as facilitation, but later diminished as they also summated to give a plateau of de- polarization. From the plateau occasionally a small spike arises (Fig. 4i, c). The fast response. The fast axon evoked electrical responses in most of the muscle fibers penetrated. They were often very small, but they were always larger OT mV 40-L lOOmsec FIGURE 5. Electrical response of one fiber and total mechanical response at tip of tarsus to: single (a) and paired (b) stimulation of the fast axon. Note that the f.j.p. is followed by a small spike in each case. 214 GRAHAM HOYLE than the corresponding slow responses, when these were seen, in the same fibers. There was no overlap of s.j.p. and f.j.p. magnitudes in individual libers, such as was found in several muscles of decapod crustaceans (Hoyle and Wiersma, 1958a). The typical response to a single shock is shown in Figure 2. The response, like the s.j.p., is of end-plate-potential type and will be referred to as the fast junctional potential (f.j.p.). The rise-time of the f.j.p.'s was usually about the same as that of the s.j.p.'s, i.e., 12-18 msec, and the decay likewise about 60 msec. But occa- sionally an f.j.p. had a faster rise-time of only 5-6 msec, and/or a faster decay of about 40 msec. In some fibers the f.j.p. leads to a small spike of 10-15 mV. The larger f.j.p.'s reached a peak amplitude of 11 mV. so OT 100 \OO.,„ corresponding to approximately physiological pH's. That value of p for which y equals 50% is the />.|0. The "sigmoid coefficient," n, is a measure of the heme-heme interactions. Hence, />-„ is an inverse measure of the oxygen affinity, and n determines the shape of the oxygen dissociation curve. If the slope of the transformation, log [v/(100 -- y)] as a function of log p, is one, then the hemes are totally independent — i.e., there is no heme-heme interaction. As can be seen from Figures 1 and 3, where the solid lines are drawn with a slope of 1.00, this is true of hagfish hemoglobin inside and outside the erythrocyte, and at high and low temperatures. 230 CLYDE MANWELL Between pH 6.7 and 9.0 hagfish hemoglobin in solution shows no detectable Bohr effect. Outside that pH range a significant decrease in oxygen affinity occurs ; however, this effect appears to be a prelude to more drastic changes (methemoglobin formation and decrease in solubility), which become apparent several hours after equilibrium measurements. This is in contrast to the solutions at intermediate pH which are stable for days and display identical oxygen equi- libria when re-analyzed one or two days after the original measurements (see Figure 1 ) . No Bohr effect was observed for erythrocyte suspensions at pH's above neutrality ; however, paralleling the behavior of hemoglobin in solution, a slight oxygen affinity decrease occurs at acid pH's. The effect was shown not only 100 0 50 X o Polistotrenta. stoi/ti PH 7.21 6.17 0 10 20 Partial Pressure of 30 I (mm. FIGURE 2. Oxygen dissociation curves of erythrocyte suspensions of the California hagfish, Polistotrema stouti, at two different pH's, showing the possible very slight Bohr effect. Erythrocytes in phosphate-buffered saline; no Karo present. Temperature = 20-21° C. by the partially clarified suspensions (Fig. 1), but also when no Karo was present (Fig. 2). In contrast to hemoglobin solutions such acidic erythrocyte suspensions were stable, possibly because of the presence of cellular reducing systems able to reduce any methemoglobin. The observed decrease in oxygen affinity could rep- resent a very small Bohr effect ; however, until it is shown that the decrease in oxygen affinity is rapid and entirely reversible, the possibility of slight denaturative changes in the protein cannot be overlooked, especially in view of the results obtained for hemoglobin solutions. The presence of CCX specifically decreases the oxygen affinity, in addition to its effect resulting from the increase in acidity, for hemoglobin of the horse (Mar- garia and Milla, 1955) and the teleost Sebastodcs ruberrinms (Manwell, unpub- HEMOGLOBIN EVOLUTION 231 lished data). That CO2 does not cause any special Bohr effect for hagfish hemo- globin is shown in Figure 3. Because the possible Bohr effect of hagfish hemoglobin is so small and occurs at almost one pH unit below the normal pH of hagfish blood (7.5-7.7; Prosser ct ol, 1950; David Jensen, personal communication), it is reasonable to assume that it is of no physiological significance, especially as CO2 does not have any specific effect. Knowledge of the heat of oxygenation (AH°) of hagfish hemoglobin enables one to predict the position of the oxygen equilibrium at any particular physiological 10 -y PoliS to tr etna sfov ft «/» II V* prj • 11.5 7.50 o 29.5 7.3* 0 Ig.O 7.30 Pco2 0 0 10 p FIGURE 3. Oxygen equilibria under a variety of conditions of hemoglobin solutions prepared from the blood of the California hagfish, Polistotrema stouti. Dashed lines are drawn to ap- proximate the oxygen equilibrium at 11.5 and 29.5° C. The solid line is drawn on the basis of data presented in Figure 1 in order to facilitate the comparison of hemoglobin solutions in the presence and in the virtual absence of CO-,. temperature. In addition, absence of a Bohr effect facilitates the evaluation of AH°, for corrections representing the effects of ionizations of heme-linked groups do not need to be applied. Using data presented in Figure 3, a value of AH° -9.3 kcal. (one atmosphere of dissolved oxygen gas at the standard state) was obtained as in a previous study (Manwell, 1958a). This value of AH° is similar to those observed for hemoglobin of sheep (— 8.2 kcal. ; Paul and Roughton, 1951), the holothurian Cucumaria miniata (—8.4 kcal.; Manwell, 1958f), and adult and fetal spiny dogfish, Sqnalns siicklcyi (-8.7 to -9.5 kcal.; Manwell, 1958d). This group of values for the heat of oxygenation of various hemoglobins is not CLYDE MANWELL characterized by the extensive variation seen in older data (reviewed by Paul and Roughton, 1951) ; theoretical considerations imply that there be relatively little variation in these values for a particular respiratory pigment, although significant differences occur between hemoglobin, hemocyanin, and hemerythrin (Klotz and Klotz, 1955; see, also, Manwell, 1958a). DISCUSSION Biochemical and Physiological Hagfish hemoglobin has a hyperbolic oxygen dissociation curve. Beyond that point, however, resemblance to sea lamprey hemoglobin (Wald and Riggs, 1951 ) ceases. Hagfish hemoglobin lacks a Bohr effect over a pH range well in excess of pH's to be expected in living hagfish. The hagfish is, accordingly, the first adult vertebrate whose blood is known to lack a Bohr effect. In addition, the oxygen affinity of hagfish blood is very high — at physiological conditions as high, if not higher than that of any known vertebrate blood ; the />-„ is 2-4 mm. Hg - over the temperature range of 5-15° C. By way of comparison, />-0 for human blood is 26 mm. Hg at pH == 7.3-7.4 and a temperature of 37° C. (Prosser ct al, 1950). Arenicola hemoglobin has an especially high oxygen affinity, />r>0 = 2—2.5 mm. Hg at 20° C. (Allen and Wyman, 1952) ; yet, by virtue of its extremely sigmoid oxy- gen dissociation curve (n -- 6) Arenicola hemoglobin appears to be more suitable for oxygen transport than hagfish hemoglobin. The comparison between hemoglobin solutions and erythrocyte suspensions prepared from the blood of the hagfish indicates that no specific interaction occurs between the hemes of adjacent hemoglobin molecules inside the cell. Hence, these data are consistent with — but do not necessarily establish — the idea that the molecu- lar weight of hagfish hemoglobin in situ corresponds to but one heme per molecule— i.e., approximately 17,000-18,000. Comparison of the results with the introduction to this study shows that hagfish hemoglobin possesses all three of the features of the oxygenation reaction con- sidered to be primitive by Wald (1952). However, the properties of hagfish hemoglobin, considered to be characteristic of storage hemoglobin by Wald, are displayed by a vascular hemoglobin, which one might accordingly assume to be involved in oxygen transport. Living hagfish have been examined in an attempt to see whether there may be a significant difference in the color of blood entering and leaving the tissues and the gills, a condition which would indicate participation of the hemoglobin in oxy- gen transport. Unanesthetized hagfish were pinned at the extreme caudal and cranial ends (but not unnaturally stretched out) to a board immersed in oxy- genated sea water at 10-12° C. The animals struggled violently until both ends were pinned down ; they then remained quiescent for the duration of the experi- ment. A median ventral incision was made in the vicinity of the liver and the heart, care being taken to avoid cutting any blood vessels. The slime secretions were periodically removed. When slime production ceased, blood in the dorsal aorta (leaving the gills) and in various veins (leaving the tissues) was compared - This approximate range of />.-„ for hagfish blood at physiological temperatures has been calculated (Manwell, 1958a) on the basis of the />.-,„ for erythrocyte suspensions at 18° C. (see Fig. 1) and the heat of oxygenation (AH°) of hagfish hemoglobin in solution. HEMOGLOBIN EVOLUTION 233 visually with "reduced" and oxygenated standards in hagfish blood vessels. Blood in the veins appeared to be almost de-oxygenated ; blood in the arteries was ap- proximately 50% oxygenated. This condition did not change over several hours of continuous observation. By reference to the oxygen dissociation curves, it can be seen that the internal oxygen tensions were extremely low, although the hemo- globin was functional in oxygen transport. An improved physiological experi- mental approach would be highly desirable ; however, the hagfish — considering its small size, its surprisingly violent activity when handled, and its copious slime- producing abilities — is not an especially suitable form in which to determine arterial-venous oxygen concentrations. Wald (1952) comments (p. 367) : "The business of a circulatory hemoglobin, having combined with oxygen at the body surface, is to release it in the tissues at high tensions. . . ." (Italics are those of Wald.) Clearly, hagfish hemoglobin is biochemically unable to function in such a way ; and, the observations made on living specimens tend to strengthen the idea of oxygen transport at lou> internal oxygen tensions in Polistotrcma stouti. Redmond (1955) has found extensive evidence for oxygen transport at lowr internal oxygen tensions in several decapod crustaceans. Several studies indicate that such a condition also exists in some but not all annelids (reviewed by Eliassen, 1953; see also, Jones, 1954; Eliassen, 1955; Manwell, 1958e). Adult spiny dogfish, S quoins suckleyi, have a hemo- globin with a hyperbolic oxygen dissociation curve inside and outside the erythro- cyte (Manwell, 1958d) ; yet, polarographically determined oxygen tensions of blood leaving the heart were never above 5 mm. Hg in 15 resting dogfish. Very low venous oxygen tensions have been observed in some teleosts — but not the mackerel (Black, 1951). Especially interesting in this regard is the marked suppression of heme-heme interaction by the erythrocytes of some teleosts and a species of holocephalian (Manwell, unpublished data) ; although n for clingfish Gobicsox hemoglobin in solution is 2.5-2.6 and thus approaches values of n for mammalian hemoglobins (2.6-3.0), inside the red blood cell the oxygen equi- librium of Gobieso.r hemoglobin is almost devoid of heme-heme interaction (n -• 1.2-1.4) ; this trend is exactly the opposite of what would be expected were the sigmoid oxygen dissociation curve always so vital for oxygen transport. Under conditions where the tissues tolerate — or require — low oxygen tensions the properties usually associated with a transport hemoglobin would be of little selective advantage. In addition, if a large diffusion gradient were necessary to account for movement of sufficient oxygen across the epithelium of the gills or skin, then such properties as low oxygen affinity and large Bohr effect would pre- vent loading of the respiratory pigment with sufficient oxygen in the organ of external respiration. ("Sufficient" does not imply complete saturation; see Red- mond, 1955.) Partial use of anaerobic metabolism could free tissues from de- pendence on large internal oxygen tension gradients. At the same time as such a rigorous dependence on oxygen were reduced, however, so would the metabolic efficiency decline (aerobic metabolism yielding several times more energy per unit weight of substrate than anaerobic metabolism). Consequently, one might expect large, very active animals (e.g., cephalopods, some fishes, birds, and mammals) to have evolved increasing dependence on the more efficient aerobic metabolic path- ways— and at the same time oxygen transport at high internal oxygen tensions. In such cases the sigmoid oxygen dissociation curve, the low oxygen affinity, and 234 CLYDE MANWELL the large Bohr effect would be of the greatest selective advantage in increasing the efficiency of the respiratory pigment. It is well known that squid hemocyanin, mammalian and avian hemoglobins, and mackerel and trout hemoglobins possess all of these characteristics (reviewed by Florkin, 1949; Prosser et al., 1950). Phylogenetic In terms of Wald's (1952) previously mentioned theory on the origin and evolution of hemoglobin one might be tempted to infer that the primitive hagfish has retained in a hemoglobin used in oxygen transport all three oxygenation prop- erties to be expected of hemoglobin in an earlier stage of evolution — that repre- sented by an oxygen storage hemoglobin. However, some or all of the properties of hagfish hemoglobin may represent specialization to a particular mode of life far different from that of known fossil Agnatha. The hagfishes are, in spite of some primitive characteristics, well-adapted, biologically successful animals. Over sev- eral types of ocean bottom in temperate seas the hagfishes are among the dominant scavengers — or parasites — feeding on dead and dying fishes ; they are often present in such numbers as to restrict or prevent several types of fishing operations (Young, 1950). Certain characteristics of the hagfish, such as the rasping tongue, com- plete absence of scales and bone, and the habit of feeding on teleost fishes, are not properties of fossil Agnatha (Ostracoderms). These features must have evolved independently of other aspects of early vertebrate phylogeny. The differences in the properties of sea lamprey (Wald and Riggs, 1951) and hagfish hemoglobin may be correlated with the well-known ecological observation: the hagfish enters, often in large numbers, the body of its prey and thus is often exposed to low O._, and high CO2 tensions ; the lamprey remains attached to the surface of its host, thereby having well-oxygenated water of low carbon dioxide tension available for its respiration at all tim^s. In addition, so far as is known, the hagfish does not make any sustained active movement comparable to the anadromous migration of the sea lamprey. Several other objections to Wald's (1952) theory in its present form can be raised : ( 1 ) Cytochrome oxidase has been considered the phylogenetic precursor of hemoglobin because: (a) it combines reversibly with CO and reacts with O2; and, (b) beef heart cytochrome oxidase has an extremely high oxygen affinity, no Bohr effect, and an almost hyperbolic equilibrium curve with CO — all properties that a "primitive" hemoglobin ought to possess (Wald and Allen, 1957). Unfortu- nately, neither the prosthetic group (Paul, 1951; Stotz, Morrison and Marinetti. 1956) nor the protein moiety (Lemberg and Legge, 1949) of this respiratory enzyme (or enzyme complex) resembles the corresponding parts of hemoglobin as closely as might be desired. Cytochrome r would be a better, although not entirely satisfactory, hemoglobin phylogenetic precursor. At least its prosthetic group is the same as that of hemoglobin, although linked to the protein differently; and, its protein moiety is readily water-soluble, although of lower molecular weight (one heme per 13,000-15,000) and higher isoelectric point (pi - : 10) (Paleus, 1955) than any known hemoglobin. When the heme of cytochrome c is not com- pletely protected by coordination of the iron with the imidazole groups of two HEMOGLOBIN EVOLUTION 235 histidine residues, the enzyme combines with CO and is oxidized by CX (Lemberg and Legge, 1949; Theorell, 1956). Bartsch and Kamen (1958) isolated a bac- terial heme protein — originally called a "pseudohemoglobin"- —which resembles cytochrome c in many respects, although its isoelectric point (pi = 5) is com- parable to that of invertebrate and cyclostome hemoglobins (Prosser et al., 1950) and it is readily oxidized by O2 and combines reversibly with CO. The carbon monoxide reaction of this bacterial heme protein is not invariant to pH change- in contrast to cytochrome oxidase (Wald and Allen, 1957). In support of sonic connection between the syntheses of cytochrome and hemoglobin is the finding of Yeas (1956) that aerobically grown yeast in the presence of antimycin produces less cytochrome a and more hemoglobin than controls ; however, as Yeas suggests, this relation may be explained by assuming that the heme of hemoglobin is a precursor to the modified heme of cytochrome a. At present there is so little comparative biochemical information on the cytochromes and other heme-containing enzymes that one cannot rule out the possibility that the proteins of various hemo- globins have arisen from apoenzymes of quite unrelated biocatalysts ; certainly, the protoheme prosthetic group is always phylogenetically available. Several proteins besides globin will combine with heme, although none are yet known that will enable this heme to combine reversibly with molecular oxygen (Lemberg and« Legge, 1949). (2) Wald (1952) states (p. 369): "The hemoglobins that have arisen so sporadically among invertebrates of various orders are all storage hemoglobins." However, oxygen transport by hemoglobin occurs in several annelids (Johnson, 1942; Eliassen, 1955; reviewed by Eliassen, 1953; Manwell. 1958e), the brine shrimp Artcmia (Gilchrist. 1954), and even such small arthropods as daphnids (Hoshi, 1957). As the experiments of Redmond (1955) show, the presence of a respiratory pigment in the blood of invertebrates in low concentration does not rule out significant oxygen transport by that pigment. Coelomic hemoglobins, such as those of Urechis (Redfield and Florkin, 1931) and Citcumaria winiata (Manwell, 1958f), are usually assumed to function in oxygen storage; however, the movement of coelomic fluid, either by muscular contraction or cilia, presents the possibility of oxygen transport by the coelomic hemoglobin from cloacal diver- ticula (Urechis) or respiratory trees (Cucumaria) to tissues in or adjacent to the coelom. (3) That a hyperbolic oxygen dissociation curve, high oxygen affinity, and no Bohr effect should represent primitive conditions (Wald, 1952) requires comment. The properties of the oxygen equilibrium of the vertebrate storage hemoglobin (myoglobin) rest on studies of crude extracts or purified preparations prepared from the muscles of five species of mammals (reviewed by Lemberg and Legge, 1949; see also, Rossi-Fanelli and Antonini. 1958). As Lemberg and Legge point out, the oxygen equilibrium of myoglobin in situ in the muscle remains to be evaluated. The findings, that n could be as high as 1.6 for oxygen equilibria of extractions of Cryptochiton myoglobin (Manwell, 1958c) and that n could be as high as 2.8 in the reaction of horse metmyoglobin with various ligands (Kiese and Kaeske, 1942), indicate that heme-heme interactions can exist under certain con- ditions in tissue hemoglobins. One would expect tissue hemoglobins to have a high oxygen affinity because of limitations on the intracellular oxygen tensions imposed by the combination of passive diffusion of oxygen and aerobic cellular 236 CLYDE MANWELL metabolism. In the case of Cryptochiton even when the oxygen dissociation curve of the radular myoglobin is sigmoid, it lies far to the left of the corresponding curve for the vascular hemocyanin ; hence, the presence of heme-heme interactions in the myoglobin does not interfere with the functional oxygen transfer system (Manwell, 1958c). Interactions between oxygen-affine centers have evolved in all four major classes of respiratory pigments (hemoglobin, hemocyanin, chlorocruorin, and heme- rythrin) ; Bohr effects are found in all these classes except hemerythrin (reviewed by Prosser ct al., 1950). 3 There is reason to believe that heme-heme interactions and the Bohr effect are not necessarily specialized acquisitions restricted to respiratory pigments in an advanced state of evolution but are expressions of very basic properties found in many unrelated proteins. The frequently observed variation of enzyme kinetics as a function of pH often involves interaction between proton-affine centers on the protein moiety and the active center (Alberty, 1956). Heme-heme interaction, likewise, has its parallel in the interaction between centers having similar reactivi- ties in proteins possessing two or more such sites per molecule. Such interactions occur in the binding of dyes and ions to some multivalent proteins (Klotz, 1954) and in the kinetics of some enzymes (Botts and Morales, 1953). Finally, the ease with which certain reagents (various mercurials, formaldehyde, and glutathione) will remove the heme-heme interactions, partially restore those interactions, greatly increase the oxygen affinity, and/or modify the Bohr effect (Guthe, 1954; Riggs and Wolbach, 1956) implies that these properties are not invariant for a particular hemoglobin molecule. In addition, the differences in the oxygen equilibrium of some hemoglobins inside and outside the red blood cell (Root, Irving and Black, 1939; Manwell, unpublished data) indicate also that a considerable lability exists with regard to the properties of the oxygen equilibrium. It seems reasonable to assume that the phylogenetic order of first appearances was : heme-containing respiratory enzymes, tissue hemoglobins, vascular hemo- globins. However, the present discussion indicates the difficulty of knowing (a) if a certain set of characteristics of the oxygen-hemoglobin equilibrium — e.g., high oxygen affinity, no heme-heme interactions, and no Bohr effect — is basically primitive, and (b) if any particular component of the cytochrome system or any other heme-containing enzyme is evolutionally the forerunner of hemoglobin. SUMMARY 1. Oxygen equilibria of hagfish hemoglobin inside and outside the red blood cell have been obtained under a variety of conditions. The oxygen affinity of the hemoglobin in the erythrocyte suspensions is high (/>50 = 3-4 mm. Hg at 18°), although it is even higher in hemoglobin solutions (/>r>0 -- 1.8 mm. Hg at 18° C.). There is no interaction between hemes (n-- 1.00) and virtually no Bohr effect. The effect of temperature on the oxygen equilibrium of hagfish hemoglobin is 3 Absence of the Bohr effect has been confirmed for various sipunculid coelomic hemeryth- rins (Manwell, 1958a, and unpublished studies on Dendrostomutn zostericolum and Siphonosoma ing ens) ; however, the coelomic hemerythrin of the brachiopod Linc/nla, a form that is morpho- logically essentially unchanged since the Cambrian period, has a Bohr effect that is two-thirds the magnitude of that observed for human adult hemoglobin (Manwell, 1958, unpublished experiments) ! HEMOGLOBIN EVOLUTION 237 similar to that observed in recent experiments on other hemoglobins (AH° -9.3 kcal. for hagfish hemoglobin). 2. Several aspects of Wald's (1952; see, also, Wald and Allen, 1957) theories on the. evolution and function of hemoglobin are criticized in view of these data on hagfish hemoglobin and on the basis of information in the literature. It is concluded that: (1) At present there is no reason to favor cytochrome oxidase as the phylo- genetic precursor of hemoglobin. (2) Many invertebrate hemoglobins function in oxygen transport. (3) If the internal oxygen tensions are sufficiently low, a respiratory pigment participating in oxygen transport does not need to possess a low oxygen affinity, a sigmoid oxygen dissociation curve, and a marked Bohr effect. (4) It is impossible to say if a particular set of properties of the oxygen equilibrium is basically "primitive." (5) Physiological conclusions on hemoglobin should be made upon studies of the pigment in the natural condition — i.e., myoglobin in the muscle, or intracellular vascular hemoglobin in the erythrocyte. LITERATURE CITED ALBERTY, R. A., 1956. Kinetic effects of the ionization of groups in the enzyme molecule. /. Cell. Comp. Physio!,, 47, Sup. 1 : 245-281. ALLEN, D. W., AND J. WYMAN, 1952. The oxygen equilibrium of hemerythrin of Arcnicola cristata. J. Cell. Comp. Phys'wl, 39: 383-389. BARTSCH, R. G., AND M. D. KAMEN, 1958. On the new heme protein of facultative photo- heterotrophs. /. Biol. Chcm., 230: 41-63. BLACK, E. C, 1951. Respiration in fishes. Univ. Toronto Studies Biol. No. 59: 91-111. BOTTS, J., AND M. MORALES, 1953. Analytical description of the effects of modifiers and of enzyme multi valency upon the steady state catalyzed reaction rate. Trans. Faraday Soc., 49: 1-12. ELIASSEN, E., 1953. The physiology of the vascular system of invertebrates. I. A monography on the blood pigments. Univ. Bergen Arbok, Naturvit. rekkc, 11 : 1-65. ELIASSEN, E., 1955. The oxygen supply during ebb of Arenicola marina in the Danish Wad- densea. Univ. Bergen Arbok, Naturvit. rekkc, 12: 1-9. FLORKIN, M., 1949. Biochemical Evolution. Edited and translated by S. Morgulis. Academic Press, New York. GILCHRIST, B. M., 1954. Haemoglobin in Artcmia. Proc. Rov. Soc. London, Ser. B, 143 : 136-146. GUTHE, K. F., 1954. The effect of formaldehyde on the oxygen equilibrium of hemoglobin. /. Gen. Physio!., 37 : 775-780. HOSHI, T., 1957. Studies on physiology and ecology of plankton. XIII. Haemoglobin and its role in the respiration of the daphnid, Simoccplialiis retains. Sci. Rep. Tohoku Univ., 4th Ser. (Biology), 23: 35-58. JOHNSON, M. L., 1942. The respiratory function of the haemoglobin of the earthworm. /. Exp. Biol., 18: 266-277. JONES, J. D., 1954. Observations on the respiratory physiology and on the haemoglobin of the polychaete genus Ncphthys, with special reference to N. homberqii (Aud. et M.-Edw.). /. Exp. Biol., 32: 110-125. KIESE, M., AND H. KAESKE, 1942. Verbindungen des Muskelhamoglobins. Biochem. Zeitschr., 312: 121-149. KLOTZ, L, 1954. Protein interactions. In: The Proteins, H. Neurath and K. Bailey, eds. Academic Press, New York, Vol. 1, Part A, 727-806. KLOTZ, L, AND T. A. KLOTZ, 1955. Oxygen-carrying proteins: a comparison of the oxygena- tion reaction in hemocyanin and hemerythrin with that in hemoglobin. Science, 121 : 477^80. LEMBERG, R., AND J. W. LEGGE, 1949. Hematin Compounds and Bile Pigments. Interscience Publishers, Inc., New York. 238 CLYDE MANWELL LENHERT, P. G., W. E. LOWE AND F. D. CARLSON, 1956. The molecular weight of hemoglobin from Petromyzon iiuiriuiis. Biol. Bull., Ill: 293-294. MANWELL, C., 1958a. Oxygen equilibrium of Phascolosonia agassizii hemerythrin. Science, 127: 592-593. MANWELL, C., 1958b. Respiratory properties of the hemoglobin of two species of diving birds. Science, 127 : 705-706. MANWELL, C., 1958c. Oxygea equilibrium of myoglobin and hemocyanin from the amphineuran mollusc Cryptochiton stcllcri. J. Cell. Conip. Physiol., in press. MANWELL, C., 1958d. A "Fetal-Maternal Shift" in the ovoviviparous spiny dogfish Squalus sncklcyi. Physiol. ZooL, in press. MANWELL, C., 1958e. Alkaline denaturation and oxygen equilibrium of annelid hemoglobins. J. Cell. Coinp. Physiol., in press. MANWELL, C., 1958f. Oxygen equilibrium of Cucumaria ininiata hemoglobin and the absence of the Bohr effect. /. Cell. Coinp. Physiol., in press. MARGARIA, R., AND E. AliLLA, 1955. Effetti acidificanti dell'ossigenazione dell'Hb in condizioni fisiologiche. Boll. Soc. ital. biol. spcr., 31 : 1250-1253. PALEUS, S., 1955. Studies on cytochrome c . Svcnsk. Kern. Tidskr., 67 : 273-290. PAUL, K. G., 1951. The iron-containing enzymes. A. Cytochromes. In: The Enzymes, J. B. Sumner and K. Myrback, eds. Academic Press, New York, Vol. 2, Part 1 : 357-396. PAUL, W., AND F. J. W. ROUGHTON, 1951. The equilibrium between oxygen and sheep haemo- globin at very low percentage saturations. /. Physiol., 113: 23-35. PROSSER, C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. J. WULFF, 1950. Com- parative Animal Physiology. Saunders, Philadelphia. REDFIELD, A. C., AND M. FLORKIN, 1931. The respiratory function of the blood of Urechis caupo. Biol. Bull., 61 : 185-210. REDMOND, J. R., 1955. The respiratory function of hemocyanin in Crustacea. /. Cell. Conip. Physiol, 46: 209-247. RIGGS, A. F., AND R. A. WOLBACH, 1956. Sulfhydryl groups and the structure of hemoglobin. /. Gen. Physiol., 39: 585-605. ROOT, R. W., L. IRVING AND E. C. BLACK, 1939. The effect of hemolysis on the combination of oxygen with the blood of some marine fishes. /. Cell. Conip. Physiol., 13: 303-313. ROSSI-FANELLI, A., AND E. ANTONINI, 1958. Reversible splitting of human myoglobin. Physico- chemical properties and oxygen equilibrium of reconstituted proto- and deutero-myo- globin. Arch. Biochem. Biophys., 72: 243-244. STOTZ, E. H., M. MORRISON AND G. MARINETTI, 1956. Components of the cytochrome system. /;;: Enzymes: Units of Biological Structure and Function, O. H. Gaebler, ed. Academic Press, New York, 401-416. SVEDBERG, T., 1933. Sedimentation constants, molecular weights, and isoelectric points of the respiratory proteins. J. Biol. Chan., 103: 311-325. THEORELL, H., 1956. Nature and mode of action of oxidizing enzymes. Science, 124 : 467-470. WALD, G., 1952. Biochemical evolution. hi: Modern Trends in Physiology and Biochemistry, E. S. G. Barren, ed. Academic Press, New York, 337-376. WALD, G., AND D. W. AL/LEN, 1957. The equilibrium between cytochrome oxidase and carbon monoxide. /. Gen. Physiol., 40: 593-608. WALD, G., AND A. RIGGS, 1951. The hemoglobin of the sea lamprey, Petromyzon marinus. J. Gen. Physiol., 35 : 45-53. YCAS, M., 1956. Formation of Hb and the cytochromes by yeast in the presence of antimycin "A." Ex p. Cell. Res. 11: 1-6. YOUNG, J. Z., 1950. Life of the Vertebrates. Oxford University Press. TOXIC EFFECTS OF NORMAL SERA AND HOMOLOGOUS ANTISERA ON THE CHICK EMBRYO1 ALTON M. MUN Department of Zoology, Indiana University, Bloomington, Indiana -> 3 The possibility of identifying embryonic antigens of unique function, of localiz- ing their sites of origin and action, and of manipulating them experimentally in order to analyze their developmental significance by the use of specific toxic sera at lethal or sub-lethal doses was advanced by Nace (1955). By modifying the normal function of an antigen with sub-lethal doses of a toxic antiserum, a specific anomaly may be produced, affording a key to the localization and the time and nature of the action of the antigen. Similar arguments have been advanced by those who have sought to block the growth of tumors with specific antisera (re- viewed by Ross, 1957; Wissler and Flax, 1957). However, before this approach can be employed critically in studying the synthesis of specific antigens and their role in development, the following questions must be considered : ( 1 ) Are the proteins and other macromolecules of the embryo antigenic ? Or does the embryo contain a population of molecules capable of reacting with antibody produced against adult antigens but incapable of eliciting antibody production? The dis- tinction must be made between the occurrence in embryos of combining groups identical with those of adult antigens and the occurrence of embryonic antigens (Ebert, 1958a). (2) What are the effects of antisera on the embryo? Does the reaction between antigen and antibody, in vivo, result in measurable modifications of, or interference with, biological function? As a general rule, tissue-specific molecules exhibit species-specificity to some degree, making analysis by immuno- chemical techniques possible ; the principal advantage of these methods is their exquisite sensitivity, which makes possible the analysis of the rate of synthesis and accumulation and site of localization of proteins or other macromolecules present in embryos in trace amounts. The principal difficulty, one which is often not appreciated, is that antigenic specificity depends upon relatively small determinant groups rather than on the complete structure of the molecule, and that the mole- cule may contain more than one kind of determinant group. Little is known of the kind, number, and size of determinant groups of natural proteins. The anti- genically active groups and physiologically active groups of a molecule may not be 1 This investigation was supported in part by grants to Professor James D. Ebert from the National Science Foundation, the American Heart Association, the National Heart Institute of the National Institutes of Health of the United States Public Health Service (grant No. H-1709), and the Indiana University Foundation Research Division. 2 From work reported in a thesis submitted to the Faculty of the Graduate School in partial fulfillment of the requirements for the degree, Doctor of Philosophy, in the Department of Zoology, Indiana University, July, 1956. Eigenmann Fellow in Zoology, 1954-1955. Con- tribution number 648 from the Department of Zoology. 3 Present address : Department of Poultry Science, State College of Washington, Pullman, Washington. 239 240 ALTON M. MUN identical. Similarity in immunochemical properties of natural molecules of diverse origin may result from ( 1 ) identity of one or more antigenic determinant groups, (2) a degree of structural similarity, or (3) the presence of one as an undetected trace contaminant in the other, for, inasmuch as traces of antigen may evoke large amounts of antibody, such contaminants tend to be revealed (Ebert, 1958b). The antigenicity of embryonic tissues has been demonstrated in several species, for example in amphibians (Cooper, 1948, 1950; Flickinger and Nace, 1952; Clayton, 1953) and in the sea urchin (Perlmann, 1954; Perlmann and Perlmann, 1957). Much of the earlier literature has been reviewed by Tyler (1955, 1957). The ability of the chick embryo to react with antisera against tissue-specific components of adult chickens was shown by Burke et al. (1944), Nace and Schecht- man (1948), Ebert (1950, 1951), and Ebert et al. (1955). For example, spe- cific effects of antibodies against adult antigens on the development of chick embryos were reported by Ebert (1950). Saline extracts of heart, brain, and spleen from adult chickens were used as antigens. Rabbit antiserum against chicken brain affected chiefly nervous tissue, and anti-heart and anti-spleen sera affected primarily mesodermal elements in early chick blastoderms cultivated in vitro. Moreover, comparing the effects of anti-heart and anti-spleen preparations, it was clear that the former antisera affected the development of the heart, whereas the latter did not. A striking extension of this approach is found in the report by Langman et al. (1957) who have demonstrated specific effects of antisera devel- oped in rabbits against antigens of chick lens, purified alpha crystallin, and myosin. Anti-lens and anti-alpha crystallin sera prevented the formation of the lens from ectoderm-optic cup combinations in vitro, w^hereas anti-myosin sera permitted nor- mal lens development but inhibited mesenchyme formation. These studies clearly demonstrate the existence of reactive or combining groups in the embryo capable of reacting with antibodies to adult tissue antigens. However, the antigenicity of the chick embryo, i.e., its ability to elicit the production of precipitating anti- bodies in the rabbit, cannot be inferred from these studies. After complete absorption with adult laying hen serum, antisera against the serum of the 10-day chick embryo showed positive precipitating activity with the homologous antigens (Schechtman ct al., 1954). Moreover, Levi and Schecht- man (1954) concluded from similar studies that the 12-day embryo contained distinct embryonic red blood cell antigens. Nettleship (1953) injected 1-day, 2-day, and 6-day chick embryos (p. 325), "emulsified in normal saline by syringe suction and expulsion or grinding in a mortar without abrasive," into hamsters. The hamster anti-chick sera were dropped or injected into or near the "embryo site" of unincubated eggs. The titers of the antisera used were not determined, nor were the antisera against the various stages tested with the homologous em- bryos. The hamster anti-chick embryo serum (p. 326) "placed in proximity to the preincubated chick embryo stops the development of these embryos at a time which corresponds to the time the embryo antigen was obtained." The results were interpreted as pointing strongly (p. 327) "to the development of qualitatively different protein complexes in the embryo concurrent with the embryo's growth." These results are consistent with the studies of Cooper (1950), Spar (1953), Nace (1953), and Flickinger and Nace (1952) showing changes in the antigenic constitution of the developing embryo. However, critical absorption techniques, such as those of Cooper (1950) and Spar (1953) in which antisera to later stages TOXIC EFFECTS OF ANTISERA 241 were absorbed with antigens of the earlier stage, thus separating those antibodies peculiar to the later stages, were not reported. Tyler (1957) remarked (p. 356) that "For an unabsorbed antiserum of this type to produce a highly specific effect does seem surprising, and one wonders whether or not this might be due to for- tuitous variation in the antibody content of the various antisera." The present experiments were undertaken with two-fold objectives: (1) To establish whether or not the chick embryo contains specific antigens (as opposed to combining groups capable of reacting with antibodies to adult tissues) by in- jecting whole chick embryos or embryo extracts into rabbits, and (2) to examine the effects of the antisera thus produced on the development of the homologous" embryo. However, because fresh rabbit serum was found to be highly toxic to the chick, it became necessary first to study the known heat-labile and heat-stable factors in fresh rabbit serum in order to devise means of reducing or removing false reactions, after which the action of antisera could be explored. MATERIALS AND METHODS Preparation of antigens The 72-hour chick embryo (stages 16 to 18, Hamburger and Hamilton, 1951) was used for the preparation of antigens because of the significant number of well defined histogenetic and morphogenetic processes wyhich occur during this period, e.g., morphogenesis and the growth of the limb buds, the deposition of the pigment in the eyes. Another factor in selecting the 72-hour embryo was its larger size and higher content of protein nitrogen as compared with that of the 48-hour embryo. Even in using the 72-hour embryo, a substantial number of embryos was required for the preparation of the antigens. For example, it required approxi- mately twenty 72-hour embryos to furnish material for one injection into a single rabbit. The embryos were cut from the yolk and transferred immediately into a dish containing ice cold 0.15 M NaCl. The adhering yolk was trimmed off with sharp- ened steel needles, and all membranes were removed with sharpened jewelers forceps. The embryos were rinsed several times in ice cold saline and stored in the freezer (—20° C.) until used. Saline extracts of the 72-hour embryo were prepared by permitting the frozen embryos to thaw at room temperature, after which they were homogenized with ice cold saline in a chilled Ten Broeck grinder. Approximately 1 ml. of saline was added for each 5 embryos. The cloudy suspension was refrigerated for 10 to 12 hours, after which it was centrifuged at 3000 RPM (1200 RCF) for 30 minutes at 0 to 4° C. The protein nitrogen content of the resultant translucent extract was approximately 0.2 mgN/ml., as determined by semi-micro Kjeldahl method. Preparation of antibodies In preliminary experiments, 6 white rabbits weighing 2 to 3 kilograms were injected intravenously with the saline extract and intraperitoneally with homoge- nized 72-hour chick embryos. Although injections and booster shots were given repeatedly, antisera with workable titers were not obtained. In subsequent experi- 242 ALTON M. MUN ments, 10 rabbits were injected with antigens in adjuvant; 72-hour chick embryos and equal amounts of Falba, paraffin oil and heat-killed tubercle bacilli were ground together in a mortar without abrasive and emulsified by syringe suction and expul- sion. Each rabbit received approximately 10 to 12 embryos (at least 10 mg. of protein) in each of three injections administered subcutaneously in the region of the neck, one week aj art. The rabbits were bled from the marginal vein of the ear one month after the first injection and one week after an intravenous booster shot of 10 to 20 embryos homogenized in a small amount of saline. This method yielded antibodies with titers of 1 : 32 to 1 : 128. By standards conventionally employed for antibodies against purified antigens, these titers are low. As will be made clear, however, they proved to be valuable tools. Tests of antibody content The presence of antibodies was detected by the use of interfacial "ring" tests in which 0.1 ml. of the test serum was overlaid with 0.1 ml. of the serially diluted chick embryo extract. The tests were performed in 6 X 50 mm. culture tubes and test materials were delivered with measuring pipettes ground and fitted with hypo- dermic needles at the tip. After the appearance of the rings was noted, the tubes were mixed and placed in the refrigerator overnight. The next morning the tubes were tapped gently and the presence of precipitate was detected as a thin white spiral rising from the bottom of the tube. The titer of the antiserum used was determined by precipitin tests in which 0.1 ml. of the embryo extract was added to 1 ml. of the serially diluted antiserum. The tubes were then incubated in a water bath at 37° C. for 30 minutes. The tubes were read after incubation and again after being refrigerated overnight. Operative procedures A technique for the study of the effects of antisera on the chick embryo was described by Witebsky and Neter (1935), who added serum drop by drop to the embryo. A modification of this technique was adopted in this study. New Hamp- shire eggs obtained from a local hatchery were incubated for 72 hours at 37.5 to 38° C. Preparatory to operation, the egg was swabbed with 70% alcohol. Then a square window 1 cm. X 1 cm. was cut in the shell with a sharpened hack saw blade. The cut piece of shell was removed, and the shell membrane was cut off. After the embryo was in position immediately under the window, a small hole was made in the vitelline membrane just anterior to the heart, after which 0.05 to 0.1 ml. of the test serum was inserted. The material, especially if colored and dense, could be seen to envelop the embryo and remain in position for several hours. The window was sealed with cellophane tape, and the egg was returned to the incubator. All test materials were sterilized by autoclaving or by Seitz filtration. After the latter procedure the titer of each serum was checked because of the report (Dilks and Wolfe, 1949) that significant decreases in titer result from Seitz filtration. In the present study, decreases in titer were minimized by filtering large volumes of undiluted serum. More than 50 experiments were conducted. Each experiment consisted of at least 25 to 35 embryos, including sham-operated or saline controls, and normal or TOXIC EFFECTS OF ANTISERA 243 absorbed serum controls. Each embryo was numbered; the time of operation, the stage of the embryo, and the amount of the test substance administered recorded. All observations were recorded following examination of the specimens under a binocular dissecting microscope, after which the embryos were removed and dissected or fixed in calcium formol for further histological studies. In the interest of objectivity, frequently the assistance of a second person was enlisted to code the randomly numbered treated eggs and to record the observa- tions made by the experimenter. THE EFFECTS OF NORMAL RABBIT SERUM ON THE 72-HouR CHICK EMBRYO In preliminary experiments, it was found that sera from both uninjected and injected rabbits were toxic to the embryo. Within a few minutes after the appli- cation of fresh rabbit serum the blastoderm begins to shrivel, the embryo gradually sinks and the heart stops beating. The toxicity of fresh rabbit serum has been encountered by others. Witebsky and Neter (1935) described its effects on the chick embryo. Bernheimer and Harrison (1940) observed the ability of normal rabbit serum to immobilize Paramecium. Green (1946) observed that normal TABLE I The effect of heating on the toxicity of fresh normal rabbit serum (NRS) Serum Effects on 72-hour chick embryos No. embryos treated Normal Abnormal Dead Unheated NRS 117 3 3 111 NRS heated at 37° C. 30 minutes 8 0 0 8 NRS heated at 42° C. 30 minutes 8 1 0 7 NRS heated at 50° C. 30 minutes 10 5 0 5 XRS heated at 56° C. 30 minutes 17 17 0 0 rabbit serum interfered with the rapid growth of cancer cells, and Imagawa et al. (1954) observed that normal rabbit serum inhibited the proliferative capacity of mouse mammary cancer cells. A spermicidal factor in fresh human, bovine, rabbit, and rat sera was reported by Chang (1947). Nace has described normal rabbit sera which were toxic to the Rana piplens embryo (1955; see also Nace and Inoue, 1957). Witebsky and Neter (1935) reported that the toxic effects of fresh rabbit serum were removed by heating at 56° C. for 30 minutes, an observation confirmed in the present study. Partial inactivation was obtained by heating at 50° C. for 30 minutes, but below 50° C. inactivation did not occur (Table I). What is the nature of the heat-labile substance? Is it complement which is defined in part on the basis of its destruction by heating at 56° C. for 30 minutes? Is it propcrdin, the heat-labile substance recently found in normal serum of a number of animals (Pillemer et al., 1954), or is it another heat-labile substance as yet undescribed? Other questions may be asked, among them : does the heat-labile factor act inde- pendently, or does it require the presence of heat-stable and/or other heat-labile factors for its action? What is the mechanism of its action? 244 ALTON M. MUN TABLE II The toxicity of heated normal rabbit serum (HNRS) coupled with unheated chicken serum (CS) or unheated guinea pig serum (GPS) Serum Effects on embryos No. embryos treated Normal Abnormal Dead HNRS 43 43 0 0 CS 29 26 0 3 HNRS + CS (10 to 50%) 11 11 0 0 GPS 40 38 2 0 HNRS + GPS (10 to 50%) 27 26 1 0 The role of complement in the toxicity of fresh rabbit serum was examined first. The sufficiency of complement was tested by adding complement in the form of fresh guinea pig serum or fresh chicken serum to heated normal rabbit serum. The toxicity which wras characteristic of fresh rabbit serum was not restored to heated rabbit serum by the addition of either fresh chicken serum pooled from 4 to 6 chickens, or fresh guinea pig serum pooled from 4 to 6 guinea pigs (Table II). Next, complement or components of complement were removed from rabbit serum by absorbing unheated fresh rabbit serum with a nonspecific precipitate which had been prepared by combining beef serum albumin (BSA) with heated homolo- gous rabbit antiserum (anti-BSA). The precipitate was washed three times with cold saline, after which 10 ml. of fresh normal rabbit serum were added to 0.5 ml. of packed precipitate. The mixture was refrigerated (0 to 4° C.) for 8 to 12 hours with frequent stirring. The mixture was centrifuged ; next the supernatant was poured into another tube containing 0.5 ml. of packed beef serum albumin pre- cipitate. After three absorptions, the rabbit serum was unable to lyse chicken red blood cells. The fresh rabbit serum absorbed in this manner was still toxic to the 72-hour chick embryo. The toxicity was lost only after heating for 30 minutes at 56° C. (Table III). These experiments clearly demonstrate that the heat- labile substance, complement, which can be absorbed by a nonspecific precipitate, is neither sufficient nor necessary for the toxic action of fresh rabbit serum. TABLE III The toxicity of normal rabbit serum (NRS) absorbed in the. cold with beef serum albumin (BSA) precipitate and chicken red blood cells (RBC) Effects on embryos Serum No. embryos treated Normal Abnormal Dead NRS BSA 70 37 5 28 NRS BSA then heated 33 32 0 1 NRS RBC 36 6 3 27 NRS RBC then heated 30 28 2 0 NRS BSA and RBC 16 6 4 6 NRS BSA and RBC then heated 10 10 0 0 TOXIC EFFECTS OF ANTISERA 245 To determine whether or not heat-labile factors were capable of acting inde- pendently, heat-stable substances found in normal rabbit serum were removed by absorption. Several substances can be used for absorption purposes, among them, chicken red blood cells which were selected because they contain Forssman anti- gens (Boyd, 1956). In this manner, Forssman antibodies, as well as other sub- stances absorbable by chicken red blood cells, can be removed. To minimize the destruction of heat-labile substances during the absorption process, the procedure was conducted in the cold (0 to 4° C.). Red blood cells were obtained from the pooled blood of 4 to 6 adult New Hampshire chickens. Approximately 2 ml. of packed red blood cells were used in the absorption of each 10 ml. of serum. The red blood cells and serum were thoroughly mixed by frequent stirring. After 8 to 12 hours, the cells were removed by centrifugation at 1200 RCF for 30 minutes. The serum was poured into another tube containing 2 ml. of packed red blood cells. The process was repeated until no further agglutination of red blood cells was observed under the microscope. The cold-absorbed rabbit serum was still highly toxic to the 72-hour chick embryo. Since lysis frequently occurred during the long course of absorption in the cold, it was necessary to inactivate complement by removing cations before absorbing with red blood cells either by filtering the serum through a column of cation ex- change resin (IRC-50, Rohm and Haas Company, Philadelphia, Pa.), as described by Levine ct al. (1953), or by adding Versene (sodium ethylene diamine tetra- acetate) to the serum. The latter method was found to be more successful. Upon completion of absorption, calcium and magnesium ions were reconstituted to a final concentration of 0.00015 M and 0.0005 M, respectively (Mayer and Levine, 1954). The cold-absorbed serum which contained Versene and an insufficient amount of calcium and magnesium ions was found to be toxic to the 72-hour chick embryo. The picture of toxicity, however, differed from that produced by fresh rabbit serum. Upon the injection of absorbed serum containing Versene (a 6 millimolar solution of Versene in serum), the embryo dies within a few minutes. The heart is engorged writh blood and becomes bright red in appearance. However, the puckering of the blastoderm and the sinking of the embryo, which is characteristic of the effect of fresh rabbit serum, is not observed. The toxic effects of Versene are observed only when insufficient calcium and magnesium ions are present. To determine whether or not complement was still present in the cold-absorbed rabbit serum, its ability to lyse chicken red blood cells was tested. The cold- absorbed serum was unable to lyse chicken red blood cells. However, when a sufficient amount of heated but unabsorbed rabbit serum was added ( 1 : 1 ) , lysis occurred readily. Thus, complement, which is dependent on the presence of heat- stable substances, was not destroyed in the process of absorption in the cold. Again, the toxic effects of cold-absorbed rabbit serum can be removed by heating (Table III). This result demonstrates the presence of a toxic heat-labile sub- stance in fresh rabbit serum, a substance not absorbed by chicken red blood cells. Absorption of fresh rabbit serum with beef serum albumin precipitates followed by chicken red blood cells in the cold also failed to remove the toxicity of the serum (Table III). It thus may be concluded that complement is neither sufficient nor necessary for the toxic action of fresh normal rabbit serum which is evoked in the absence of substances absorbable with chicken red blood cells. Moreover, since PLATE I Photographs were taken in ovo through the cut window 20-24 hours after treatment. 246 TOXIC EFFECTS OF ANTISERA 247 the action of properdin requires both complement and magnesium ions (Pillemer et al., 1954), it is suggested that this heat-labile substance is not properdin. THE EFFECTS OF ANTISERA AGAINST THE 72-HouR CHICK EMBRYO ON THE HOMOLOGOUS EMBRYO It is clear from the foregoing experiments that to remove nonspecific toxic factors, normal rabbit sera and antisera must be heated for 30 minutes at 56° C. When heated rabbit antiserum against the 72-hour chick embryo was placed on the embryo, the immediate puckering of the blastoderm, together with its sinking, which was characteristic of fresh rabbit serum, was not observed. However, a number of the embryos died after 6 to 8 hours ; in some cases the embryos did not show any visible effects until 15 to 18 hours after the operation, at which time slight abnormalities were detected. Usually no further changes appeared in the surviving embryos after 18 to 20 hours. Occasionally, some of the embryos with TABLE IV The effects of heated rabbit antiserum against the 72-hour chick embryo (HA72) coupled with guinea pig serum (GPS) Graded effects on 72-hour chick embryos Serum No. embryos treated 1 2 3 4 5 HNRS 60 59 1 0 0 0 A72 51 1 0 0 0 50 HA72 57 28 7 6 16 0 HNRS + GPS 27 25 1 1 0 0 HA72 + GPS 69 19 3 8 6 33 slight visible abnormalities appeared to recover completely. The toxic effects of the sera on the chick embryo arbitrarily are divided into five different groups (Table IV; Plate I). PLATE I Croup 1. The embryos appear essentially normal with good cole r, as compared with unoperated embryos of the same stage (Fig. 1). Group 2. The embryos appear essentially normal in stage and color but show slight morphological abnormalities, e.g., the trunk may be turned ventrad, instead of to the left in embryos in stage 22. These abnormalities may be detected 15 to 18 hours after the operation (Fig. 2). FIGURE 1. A group 1 embryo which is alive and appears normal (6X). FIGURE 2. A group 2 embryo with accumulation of blood in trunk region (6 X). FIG. 3. A group 3 embryo which is alive with its head beneath the puckered portion of the blastoderm (6 X). FIGURE 4. A group 4 embryo which is dead. The embryo lies on top of the blastoderm which is smooth in appearance (6X). FIGURE 5. A group 5 embryo which is dead and partially hidden by the puckered blasto- derm (6X). 248 ALTON M. MUN Group 3. The embryos are alive but show distinct abnormalities, e.g., the trunk may be turned to the left or even doubled back upon itself. The embryos are usually pale in color. These abnormalities may be detected 10 to 12 hours after operation (Fig. 3). Group 4. The embryos are dead and appear quite small and shrunken ; blood vessels are not distinct. These embryos usually die 5 to 8 hours after the operation (Fig. 4). Group 5. The embryos are dead. The blastoderm appears puckered or pursed. The blastoderm may be seen to begin to shrivel 3 to 5 minutes after the operation. The red blood cells may be seen to clump in the blood vessels in a few minutes and then cease to flow in the smaller vessels. The heart may stop beating as soon as 5 minutes after the operation (Fig. 5). This classification of the extent of the toxic action on the embryo does not imply the expression of basically different mechanisms or functions in each of the five groups, nor does it indicate distinct and separate stages or steps of a single mechanism or function. The embryos earlier described as "normal" fall into either group 1 or 2. Embryos described as "abnormal" are similar to those in group 3, whereas embryos described as "dead" fall into either group 4 or 5. The low sensitivity of the system, probably owing in part to the low titer of the anti- sera employed (1 : 32 to 1 : 128), as well as to the heating of the antisera, increased the possibility of introducing false negative reactions. Methods were sought, therefore, to increase the sensitivity of the system. From the foregoing dis- cussion, it is apparent that methods to achieve this end are available ; vis., the expedient of adding back those substances which are destroyed by heating, such as complement and properdin, but which do not contribute to the toxicity of normal rabbit serum. THE EFFECTS OF GUINEA PIG AND RAT SERUM ON THE ACTIVITY OF RABBIT ANTISERUM AGAINST THE 72-HouR CHICK EMBRYO The role of complement in vivo is not fully understood. It is needed in addi- tion to antibody for bactericidal and hemolytic reactions of immune sera, as well as for other toxic effects (Boyd, 1956). Witebsky and Neter (1935) found that adding fresh guinea pig serum to heated rabbit antiserum against sheep red blood cells restored the toxic activity of the antiserum but not that of normal rabbit j serum. Imagawa ct al. (1954) showed that antisera produced in guinea pigs against mouse cancer cells when heated lost the ability to inactivate mammary cancer cells but that this activity could be restored by the addition of fresh guinea pig complement. Therefore, because previous experiments showed that comple- ment was neither sufficient nor necessary for the toxicity of fresh rabbit serum, in an attempt to increase the effectiveness of the heated antiserum, complement was returned to the heated rabbit antiserum in the form of fresh guinea pig serum. Fresh unheated guinea pig serum, obtained from the pooled blood of 4 to 6 guinea pigs, had no visible effects on the 72-hour chick embryo. Fresh guinea pig serum, when mixed with heated normal rabbit serum in varying proportions, also showed no visible effects. However, a mixture of guinea pig serum and heated antiserum against the 72-hour chick embryo was quite toxic to the 72-hour chick embryo, resulting in embryos in the group 5 condition (Table IV). TOXIC EFFECTS OF ANTISERA 249 The effects of the addition of a second heat-labile substance, properdin, were examined next. Pillemer et al. (1954) found properdin in high concentration in the rat (25-50 units properdin/ml. serum), in intermediate concentration in the rabbit (4-8 units properdin/ml. serum), and in low concentration in guinea pig serum (1-2 units properdin/ml. serum). Therefore, rat serum was chosen as the source of properdin. Fresh rat serum was obtained from the pooled blood obtained by cardiac punctures from 4 to 6 large white rats. Fresh rat serum alone was highly toxic to the 72-hour chick embryo, producing the striking vascular phe- nomena described previously at all concentrations above 4%. Heating for 30 minutes at 56° C. removed all observable toxic effects. Preliminary experiments in which the embryos were examined 5 minutes to 4 hours after treatment showed that 4% fresh rat serum mixed with heated normal rabbit serum was extremely toxic to the 72-hour chick embryo. In the case of rat serum, the titer of complement is low but the concentration of properdin was shown to be high, whereas, in the case of the guinea pig serum, the titer of complement is high but the concentration of properdin is low ; there- fore a study of the combination of rat and guinea pig serum, together with rabbit serum, was conducted. A mixture of fresh rat serum and guinea pig serum, at a dilution in which neither was capable of eliciting toxic effects alone, was quite toxic to the chick embryo. The toxicity of this mixture was also increased when heated normal rabbit serum was added to this mixture. The toxicity was lessened when the concentration of the heated normal rabbit serum was reduced by dilution with saline (1:2 to 1:4). These experiments suggest the possible interaction of heat- labile substances in guinea pig and rat sera with heat-stable substances in guinea pig, rat. and normal rabbit serum. Thus, the following absorption studies were conducted to remove nonspecific heat-stable substances. ABSORPTION STUDIES Forssman antigen is reported to be present in the tissues of the chick embryo from the beginning of its development. Heated rabbit antiserum against sheep red blood cells mixed with guinea pig serum evoked the characteristic vascular phenomenon in the early chick embryo, whereas heated normal rabbit serum mixed with guinea pig serum would not (Witebsky and Neter, 1935). Therefore, it appeared imperative that Forssman type antibodies formed as a result of the injection of chick embryos into the rabbit be removed by absorption with chicken red blood cells (RBC). This procedure increased the specificity of the reaction but. owing to the concomitant dilution, decreased the sensitivity. When heated antiserum against 72-hour chick embryos was absorbed with chicken red blood cells at 37° C., the proportion of embryos showing the group 4 condition was de- creased (Table V). Adding fresh guinea pig serum increased the toxicity of the heated and ab- sorbed antiserum. Several embryos in the group 5 condition were observed. Absorption of the fresh guinea pig serum with chicken red blood cells in the cold in the presence of Versene decreased the action of the heated and absorbed anti- serum and absorbed guinea pig serum combination (Table V). This result may have been due to some inactivation of complement during the process of absorption. As shown previously, fresh rat serum was extremely toxic to the 72-hour chick 250 ALTON M. MUN embryo at concentrations above 4%. After absorption with chicken red blood cells in the presence of Versene in the cold (0 to 4° C.), the rat serum was no longer toxic to the embryo at concentrations below 10%. A mixture of 6% absorbed rat serum and 94% heated and absorbed rabbit antiserum was without effect on the embryo, as was a mixture of 6% rat serum and 94% heated and absorbed rabbit antiserum (Table VI). This is in strong contrast to the boosting effect of the addition of guinea pig serum to the heated rabbit antiserum. However, this find- ing is not unexpected, because properdin acts only in conjunction with complement and magnesium ions (Pillemer et a/., 1954) and the concentration of comple- ment in rat serum is low (Hegediis and Greiner, 1938). A mixture of guinea pig serum and rat serum at a dilution in which neither could elicit toxic effects was shown to be extremely toxic to the 72-hour chick embryo. After absorption of the rat serum in the cold with RBC following filtra- tion through a cation exchange (IRC-50) column, the toxic activity of the rat and guinea pig serum mixture was decreased. TABLE V The effects of absorption on the toxicity of heated rabbit antiserum against 72-hour chick embryos (PIA72) and unheated guinea pig serum (GPS) combinations TVT/\ Graded effects on embryos Serum Absorbed with Combined with IN O. embryos Vv 1 L 1 1 treated 1 2 3 4 5 HNRS RBC None 52 40 9 3 0 0 HA72 None None 57 28 7 6 16 0 HA72 RBC None 45 30 2 6 7 0 HNRS RBC GPS 19 18 1 0 0 0 HA72 RBC GPS 63 5 7 8 3 40 HA72 RBC GPS, heated 23 10 6 2 5 0 | GPS RBC None 7 7 0 0 0 0 HNRS RBC Absorbed GPS 14 13 1 0 0 0 HA72 RBC Absorbed GPS 17 3 3 1 4 6 The toxicity of a mixture of 6% fresh rat serum, 10% guinea pig serum, and 84% heated normal rabbit serum mixture was also reduced or removed altogether by the absorption of the guinea pig serum and rat serum at 0 to 4° C. with chicken red blood cells in the presence of Versene, and by the absorption of the heated normal rabbit serum at 37° C. with chicken red blood cells. On the other hand, a similar mixture of 10% absorbed guinea pig serum, 6% absorbed rat serum, with 84% heated and absorbed rabbit antiserum was toxic to the embryo. Imme- diate vascular effects were observed, followed by the cessation of heart contractions within 30 minutes (Table VI). The proportion of embryos exhibiting toxic effects was greater in this rabbit antiserum mixture containing both guinea pig serum and rat serum than that in rabbit antiserum mixtures containing either guinea pig serum or rat serum alone. Hence, a method is available to increase the effectiveness of antisera. Since rat serum enhanced the effect of the antiserum only in the presence of complement, it is suggested that a factor or factors analo- gous to properdin may be involved. Need for further experiments using purified properdin is indicated. TOXIC EFFECTS OF ANTISERA 251 Properdin can participate in such diverse activities as the destruction of bac- teria, the neutralization of viruses, and the lysis of certain red blood cells (Pillemer et al., 1955). Although the presently reported experiments suggest the interaction of "the properdin system" with this specific antibody in the serum (A72), it is possible that the rat serum acts by supplementing the components of complement which are low in both rabbit and guinea pig sera, e.g., the C'l component (HegediAs and Greiner, 1938). The application of quantitative techniques for handling com- plement and the use of purified components of complement, C'l, C'2, C'3, and C'4, may elucidate this aspect of the problem. The absorption of the antiserum against 72-hour chick embryos with the hom- ologous antigen removed most of the toxic effects of the antiserum on the 72-hour chick embryo. The antiserum was first heated and absorbed with chicken red blood cells in the manner described previously, and then mixed with a slight excess of minced and homogenized 72-hour chick embryos. The suspension was placed TABLE VI The effects of the addition of absorbed rat scrum (RAT-RBC) and absorbed guinea pig serum (GPS-RBC) to heated and absorbed antiserum against the 72-hour chick embryo (HA72-RBC) No. Graded effects on embryos Serum embryos treated 1 2 3 4 5 RAT-RBC (100%) 10 0 1 1 2 6 RAT-RBC (6 to 10%) 8 8 0 0 0 0 HNRS-RBC (04%) + RAT-RBC (6%) 9 9 0 0 0 0 HNRS-RBC (84%) + GPS-RBC (10%) + RAT-RBC (6%) 16 14 2 0 0 0 HA72-RBC (100%) 45 30 2 6 7 0 HA72-RBC (94%) + RAT-RBC (6%) 18 9 8 0 1 0 HA72-RBC (84%) + GPS-RBC (10%) + RAT-RBC (6%) 32 5 2 2 5 18 in the water bath for two to three hours at 37.5° C. A dense white precipitate was usually observed after 15 to 30 minutes. The tube was placed in the refrigerator at 0 to 4° C. for 12 hours and later centrifuged at 1200 RCF for 30 minutes. The supernatant was poured off into another tube containing a slight excess of homoge- nized 72-hour chick embryos. A smaller amount of precipitate was observed after the second absorption. The process was repeated until a negative interfacial "ring" test was obtained. When guinea pig and rat sera were added to this heated antiserum which had been absorbed with both chicken red blood cells and 72-hour chick embryos, the toxic effects of the antiserum were not found to be completely removed (Table VII) . The failure of the absorption of the antiserum by the homologous antigen is sur- prising but not without precedent. Ebert (1950) reported the failure of absorp- tion by homologous antigen to remove the striking lethal and growth inhibitory powers of anti-organ sera. This non-absorption of one fraction of the antiserum was attributed to individual differences in the organ antigens used in injections 252 ALTON M. MUN TABLE VII The toxicity of heated antiserum against the 72-hour chick embryo (HA 72) absorbed with the homologous antigen, singly, and in combination with absorbed guinea pig serum (GPS) and absorbed rat serum (RA T) No. Graded effects on embryos Serum Absorbed with embryos treated 1 2 3 4 5 HA72 RBC 11 8 1 0 2 0 HA72 RBC, 72-hour chick embryos 23 21 2 0 0 0 HA72 (90%) + GPS (10%) RBC 57 3 5 7 3 39 HA72 (90%) + GPS (10%) RBC, 72-hour embryos 34 21 6 2 4 1 HA72 (94%) + RAT (6%) RBC 23 14 8 0 1 0 HA72 (94%) + RAT (6%) RBC, 72-hour embryos 12 8 0 1 3 0 HA72(84%)+GPS(10%) + RAT (6%) RBC 32 5 2 2 5 18 HA72 (84%) + GPS (10%) + RAT (6%) RBC, 72-hour embryos 14 7 1 1 1 4 and absorptions. Although large numbers of embryos were used in both injections and absorptions, a long course of injections was given. Such treatment often re- sults in antisera of reduced specificity. This result may be even more pronounced in animals receiving adjuvant. However, the injection of adjuvant with heterolo- gous antigen was insufficient to evoke a nonspecific response in the rabbit. Beef serum albumin (BSA) combined with adjuvant was injected into three rabbits. Tests of heated anti-BSA, and heated anti-BSA absorbed with BSA were nega- tive (Table VIII). The number of different kinds of antibodies may be so great as to be incompletely absorbed by the antigen, even though an excess of antigen was used in absorptions and negative interfacial "ring" tests were obtained after the final absorption. This is not to say, however, that antibodies with new and different specificities are formed. The in vivo system employed here may be so sensitive as to respond strongly to these weaker or less "avid" antibodies. That embryonic proteins may be unique in their behavior in precipitin reactions was reported by Schechtman (1952), who found an unusual result when antiserum against the plasma from the 10-day embryo was reacted with adult and 10-day TABLE VIII Effects of antiserum against beef serum albumin (A BSA) on the 72-hour chick embryo No. Effects on embryos Treatment of serum embryos treated 1 2 3 4 5 ABSA imheated 3 0 0 0 0 3 ABSA heated 7 6 1 0 0 0 ABSA heated and absorbed with RBC + GPS 15 15 0 0 0 0 ABSA heated and absorbed with RBC and BSA + GPS 6 6 0 0 0 0 TOXIC EFFECTS OF ANTISERA 253 serum. He wrote (p. 95), "This antiserum forms higher (antigen-antibody pre- cipitation) curves with the heterologous antigen, adult serum. The antiserum is obviously not lacking in antibody since it produces heavy precipitates with adult material." He concluded that the embryonic serum forms antigen-antibody com- plexes with inferior light-scattering properties or that it contains substances in- hibitory to the precipitin reaction. DISCUSSION The toxicity of fresh rabbit serum to the early chick embryo was destroyed by heating at 56° C. for 30 minutes. The above experiments show clearly that the toxic substance in fresh rabbit serum is not complement ; nor is it dependent on complement for its activity. In view of the latter observation, it is also probably not properdin. The following questions remain to be answered : ( 1 ) What are the physicochemical properties of this toxic heat-labile substance? (2) Is is com- posed of one or many substances? Can substances, other than complement, be separated or isolated from this heat-labile fraction which would further enhance the action of heat-stable fractions, as was shown above for complement and proper- din or properdin-like substances? (3) What is the mechanism of action of this heat-labile substance? Is it similar to that brought about by heat-stable fractions? It was observed that the toxic effects of fresh normal rabbit serum in general resembled those produced by the action of heated rabbit antiserum to the 72-hour chick embryo coupled with fresh guinea pig serum and rat serum. Witebsky and Neter (1935) also described similar toxic effects on the early chick embryo of heated rabbit anti-sheep red blood cell serum plus complement. Pomerat (1949) reported similar results with rabbit anti-chick spleen serum. However, although the final picture appears to be the same, the mechanisms involved may not be similar. The development and use of more specific antisera to embryonic anti- gens may reveal more definitive and specific morphological expressions than those elicited by toxic factors in fresh rabbit serum. The present study has demon- strated that the 72-hour chick embryo is antigenic, i.e., capable of eliciting the production of precipitating antibodies. The presently reported investigation also demonstrated the fact that comple- ment and properdin or properdin-like substances can play an active role in the action of the antiserum in vivo. The demonstrated ability of complement and properdin or properdin-like substances to increase the magnitude of the action of the antiserum will permit the observation of the effects of weaker but perhaps more specific antisera which otherwise would go unnoticed. Thus, the manner in which antisera act to block development or modify the normal function of reactive groups in the embryo may be studied more readily. The use of purified properdin or related substances, together with the components of complement, C'l, C'2, C'3, and C'4, may contribute to our understanding of the mechanism of action of the toxic antiserum in vivo. I wish to express my sincere appreciation to Professor James D. Ebert for his encouraging interest and valuable advice throughout the course of this investiga- tion, and to Dr. Royal F. Ruth for his many helpful suggestions. I also wish to thank Dr. Joseph F. Albright and Mr. Lowell M. Duffey for expert technical aid. 254 ALTON M. MUN SUMMARY 1. The specific objectives of the present investigation were at first two-fold: (1) to determine the antigenicity of the early chick embryo, and (2) to study the effects of homologous antisera on the chick embryo. However, because at the outset a profound toxic action of fresh normal rabbit serum was encountered, it became imperative to describe the toxic factor. 2. The toxic action of normal rabbit serum, characterized by the puckering of the blastoderm, the sinking of the embryo and its ultimate death, was removed In- heating at 56° C. for 30 minutes. The toxic action was not restored by adding fresh guinea pig serum to heated rabbit serum. The toxicity was not removed by absorption in the cold with nonspecific antigen-antibody precipitates and/or thicken red blood cells. These results are interpreted as indicating that comple- ment is neither necessary nor sufficient for the toxic action of fresh rabbit serum. The toxic heat-labile substance can also act independently of heat-stable substances which are removed by absorption with chicken red blood cells. 3. The antigenicity of the 72-hour chick embryo was demonstrated by its ability to elicit the production of precipitating antibodies in the rabbit. Heated rabbit antiserum against the 72-hour chick embryo evoked a weak but definite toxic response when placed on the homologous embryo. 4. In an attempt to decrease the probability of false negative reactions, methods were sought to increase the effectiveness of the antisera. Substances which may have been inactivated by heat were returned to the antiserum singly and in combination. 5. The toxic action of heated rabbit antiserum was partially enhanced by the addition of fresh guinea pig serum, rich in complement. 6. The toxic action of the heated rabbit antiserum was not increased by adding fresh rat serum, reported to contain large amounts of properdin, but was enhanced by a mixture of guinea pig serum and rat serum. 7. The results suggest the interaction of complement and properdin or a properdin-like factor in the action of the antiserum on the chick embryo. LITERATURE CITED BERNHEIMER, A. W., AND J. A. HARRISON, 1940. Antigen-antibody reactions in Parameciitm : the aurelia group. /. Immunol., 39: 73-83. BOYD, W. C., 1956. Fundamentals of Immunology. Third Edition. Interscience Publishers, Inc., New York. BURKE, V., N. P. SULLIVAN, H. PETERSEN AND R. WEED, 1944. Ontogenetic change in antigenic specificity of the organs of the chick. /. Inf. Disease, 74 : 225-233. CHANG, M. C., 1947. The effects of serum on spermatozoa. /. Gen. Physiol., 30 : 321-335. CLAYTON, R. M., 1953. Distribution of antigens in the developing newt embryo. /. Enibryol. and E.vp. Morph., 1 : 25-42. COOPER, R. S., 1948. A study of frog egg antigens with serum-like reactive groups. /. E.vp. Zool., 107: 397-438. COOPER, R. S., 1950. Antigens of frog embryos and of adult frog serum studied by diffusion of antigens into agar columns containing antisera. /. Exp. Zoo/., 114: 403-420. DILKS, E., AND H. R. WOLFE, 1949. The effect of Seitz filtration on the protein content and precipitin reaction of diluted antigen solutions. Physiol. Zoo/., 22 : 22-30. EBERT, J. D., 1950. An analysis of the effects of anti-organ sera on the development, hi vitro, of the early chick blastoderm. /. Exp. Zoo/., 115: 351-378. TOXIC EFFECTS OF ANTISERA 255 EBERT, J. D., 1951. Ontogenetic change in the antigenic specificity of the chick spleen. Physiol. Zool., 24: 20-41. EBERT, J. D., 1958a. Antigens as tracers of embryonic synthesis. In: Symposium on Embry- onic Nutritional Requirements, ed. by D. Rudnick. In press. University of Chicago Press, Chicago, Illinois. EBERT, J. D., 1958b. The acquisition of biological specificity. In: The Cell, in press. Aca- demic Press, New York. EBERT, J. D., R. A. TOLMAN, A. M. MUN AND J. F. ALBRIGHT, 1955. The molecular basis of the first heart beats. Ann. N. Y. A cad. Sci., 60 : 968-985. FLICKIKGER, R. A., AND G. W. NACE, 1952. An investigation of proteins during the develop- ment of the amphibian embryo. Exp. Cell Res., 3: 393-405. GREEN, R. G., 1946. Cytotoxic property of mouse cancer antiserum. Proc. Soc. E.rp. Biol. Med., 61: 113-114. HAMBURGER, V., AND H. L. HAMILTON, 1951. A series of normal stages in the development of the chick embryo. J. Morph., 88: 49-92. HEGEDUS, A., AND H. GREINER, 1938. Quantitative Bestimmung der Komplementbestandteile. Zcitschft. f. Immunitatsf., 92 : 1-9. IMAGAWA, D. T., J. T. SYVERTON AND J. J. BITTNER, 1954. The cytotoxicity of serum for mouse mammary cancer cells. I. The effects of admixture in vitro upon homoiotrans- plantability. Cancer Res., 14 : 1-7. LANGMAN, J., M. A. D. H. SCHALEKAMP, M. P. A. KUYKEN AND R. VEEN, 1957. Sero- immunological aspects of lens development in chick embryos. Acta Morphol. Nccr- land. Scand., 1 : 142. LEVI, E., AND A. M. SCHECHTMAN, 1954. Embryonic antigens in the red cells of the chick embryo. A not. Rec., 120: 764. LEVINE, L., K. M. COWAN, A. G. OSLER AND M. M. MAYER, 1953. Studies on the role of Ca++ and Mg++ in complement fixation and immune hemolysis. II. The essential role of calcium in complement fixation. /. Immunol., 71 : 367-373. MAYER, M. M., AND L. LEVINE, 1954. Kinetic studies on immune hemolysis. III. Description of a terminal process which follows the Ca++ and Mg++ reaction steps in the action of complement on sensitized erythrocytes. /. Immunol., 72: 511-515. NACE, G. W., 1953. Serological studies of the blood of the developing chick embryo. /. Exp. Zool., 122: 423-448. NACE, G. W., 1955. Development in the presence of antibodies. Ann. N. Y. Acad. Sci. 60- 1038-1055. NACE, G. W., AND K. INOUE, 1957. Cytolysis versus differentiation in anti-neurula serum. Science, 126: 259-261. NACE, G. W., AND A. M. SCHECHTMAN, 1948. Development of non-vitelloid substances in the blood of the chick embryo. /. Exp. Zool, 108: 217-233. NETTLESHIP, A., 1953. Growth and mortality effects produced in the early chick embryo by antiserum. Proc. Soc. Exp. Biol. Med., 84: 325-327. PERLMANN, P., 1954. Study on the effects of antisera on unfertilized sea urchin eggs. Exp. Cell Res., 6 : 485-490. PERLMANN, P., AND H. PERLMANN, 1957. Analysis of the surface structures of the sea urchin egg by means of antibodies. II. The J- and A- antigens. III. The C- and F- anti- gens. Exp. Cell Res., 13 : 454-474 and 475-487. PILLEMER, L., L. BLUM, I. H. LEPOW, O. A. Ross, E. W. TODD AND A. C. WARDLAW, 1954. The properdin system and immunity: I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science, 120 : 279-285. PILLEMER, L., M. D. SCHOENBERG, L. BLUM AND L. WURZ, 1955. Properdin system and immu- nity: II. Interaction of the properdin system with polysaccharides. Science, 122: 545-549. POMERAT, C. M., 1949. Morphogenetic effects of spleen antigen and antibody administrations to chick embryos. Exp. Cell Res., Suppl., 1 : 578-581. Ross, J. D., 1957. Cytotoxins and cytotoxic antibodies. Ann. N. Y. Acad. Sci., 69: 795-800. SCHECHTMAN, A. M., 1952. Physical and chemical changes in the circulating blood. Ann N. Y. Acad. Sci., 55: 85-98. 256 ALTON M. MUN SCHECHTMAN, A. M., O. A. ScHjEiDE AND R. PicciRiLLO, 1954. Embryonic antigens in the serum of the chick embryo. Anat. Rcc., 120: 764. SPAR, I. L., 1953. Antigenic differences among early developmental stages of Rana pipiens. J. Exp. Zool, 123: 467-498. TYLER, A., 1955. Ontogeny of immunological properties. In: Analysis of Development, ed. by B. H. Willier, P. Weiss and V. Hamburger. W. B. Saunders Co., Philadelphia. TYLER, A., 1957. Immunolcgical studies of early development. In: The Beginnings of Embry- onic Development, ed. by A. Tyler, R. C. von Borstel and C. B. Metz. AAAS, Washington, D. C. WISSLER, R. W., AND M. H. FLAX, 1957. Cytotoxic effects of antitumor serum. Ann. N. Y. Acad. Sci., 69 : 773-794. WITEBSKY, E., AND E. NETER, 1935. Primary serum toxicity as demonstrated by the chicken embryo. /. Exp. Med., 61: 489-499. THE DYNAMICS OF A DIATOM BLOOM 1 1. H. RYTHER, C. S. YENTSCH, E. M. HULBURT AND R. F. VACCARO IVoods Hole Occanographic Institution, Woods Hole, Mass. Phytoplankton cells respond so rapidly to their environment that conventional methods of studying their populations fail to reveal many of the more subtle and more interesting aspects of their dynamic ecology. This is particularly true of surveys in which observations are made at intervals of weeks or months, where the very use of the term "the phytoplankton population," when carried over from one set of observations to the next, implies more knowledge than is available. It is also true of productivity measurements made over 24 hours or the daylight por- tion of a day, since Rodhe (personal communication). Doty and Oguri (1957), Yentsch and Ryther (1957) and others have shown that the plants vary in their composition and react differently to their environment at different times of the day. To study such phenomena it is obviously necessary to make intensive obser- vations at very frequent intervals throughout one or more days on single, isolated populations. This type of study presents obvious difficulties. The average natural population is sparse enough so that its properties can be measured only with rough accuracy, and the errors of such measurements may be larger than the changes in the organisms and their environment which are under investigation. Some insight into these problems may be had by studying cultures, but the difficulties of growing organisms under completely natural conditions need no elaboration here. A com- promise may be reached, however, by working with a dense phytoplankton bloom. Here natural populations may be studied under their natural growing conditions and very rapid responses of the organisms to changes in their physical or chemical environment may be detected and measured. The authors encountered such a diatom bloom in a small tidal creek on the south shore of Long Island, N. Y., in June, 1957. The following report will describe the studies of this bloom which were made over a 40-hour period including two days and one night. DESCRIPTION OF THE AREA Senix Creek is approximately one mile long, tapering from a width of about 300 meters at its mouth to less than 10 meters at its upper end in the town of Center Moriches. Our observations were made about halfway up its length where the water depth is approximately one meter. Underlying this shallow body of water is a thick deposit of black, organic muck which discharges H2S gas when disturbed. There is no river or other obvious source of fresh water to Senix Creek except for local runoff. The latter was negligible in the early summer of 1957 due to 1 Contribution No. 978 from the Woods Hole Oceanographic Institution. This study was made with the assistance of research grants from the National Science Foundation. 257 258 RYTHER, YENTSCH, HULBURT AND VACCARO abnormally low rainfall (none whatever during the month of June). This drought was undoubtedly an important contributory factor to the existence of the diatom bloom, since it helped to maintain the salinity at a moderately high level in the estuary and also reduced flushing action so that the latter did not greatly influence the population at the time and place of our observations. While many of the cributaries to Moriches Bay receive large quantities of pollu- tants from duck farms which line their shores (see Ryther, 1954), there is no such direct source of enrichment to Senix Creek. The origin of the nutrients which gave rise to the phytoplankton bloom in question was not investigated. They presumably resulted either from domestic pollution from Center Moriches and the residences located along the banks of the creek, or from an invasion of water from one of the other, heavily polluted estuaries via Moriches Bay (i.e., Forge River located just V FIGURE 2. The relation between salinity and total diatom concentration, showing least squares line (R = — .875). Data from Table I. The tidal influence in Senix Creek is extremely small. The range between high and low water on both days \vas approximately seven inches. Salinities ranged from 10-15 %0 and showed no obvious correlation with the stage of the tide. However, salinities at the bottom were slightly higher than those at the surface, and salinities were higher the second day of observations than on the first (Table I). Diatom counts were slightly lower at the bottom than at the surface and lower at both depths on the second day. Again there was no obvious relation to the tide, 262 RYTHER, YENTSCH, HULBURT AND VACCARO but there was a good inverse correlation (r — —0.875) between the diatom concen- tration and the salinity (Fig. 2). This correlation suggests that the diatom popu- lation did not change over the two-day period as a result of growth or death, but that the population was being diluted slowly with water from Moriches Bay, where the salinity ranged from 20 to 25 %0 and diatom concentrations were generally less than one million cells per liter. c. Dissolved oxygen and pH Both pH and dissolved oxygen behaved in essentially the same manner, as may be seen by comparing Figures IB and C. However, the high pH attained in the late afternoon of both days was maintained for several hours whereas the oxygen concentration reached its peak at the same time but then began to decline imme- diately. Water temperatures (which are not shown) ranged from 25° C. to 28° C. during the two-day period. Assuming a mean salinity of I2r/Cc, the water was approximately 90% saturated with oxygen at daybreak, about 270% saturated at 14 :00 on June 27. Despite this supersaturation, there did not appear to be a sig- nificant loss of oxygen to the air by diffusion since the decrease in oxygen concen- tration at night by respiration appears to have occurred at a constant rate. If appreciable loss by diffusion had occurred, this would have been dependent upon the oxygen concentration, and the decrease due to both causes (respiration and diffusion) would have been non-linear. The pH reached a minimum of about 8.5 early in the morning and a maximum of almost 9.5 in the afternoon. Presumably at its maximum, no free CO., was available and any further photosynthesis was dependent upon bicarbonate or car- bonate ions. Unfortunately no measurements were made of CO2 in any of its fractions, nor may these values be calculated from pH, salinity, and temperature for these estuarine conditions as they may for either fresh or sea water. Again the regular behavior of the pH curve with time, shown in Figure 1C, indicates that CO2 diffusion from air to water was negligible in comparison to the changes caused by photosynthesis and respiration. d. Plant pigments Figure ID shows the concentration of chlorophyll a in the composite samples taken during the two-day period. Since the cell counts \vere not made on the same samples, it is not possible to represent chlorophyll on a cellular basis. The chlorophyll concentration in the water ranged from 116 to 245 mg./m3.. about a two-fold variability. Although the highest concentrations coincided on both days with low water, the connection between these factors is probably fortuitous. Cer- tainly the variations in the pigment concentration are far greater than the observed differences in the diatom counts, caused by tidal fluctuations or otherwise. Despite the somewhat erratic distribution of the pigment concentration, it is still obvious that the chlorophyll increased gradually throughout the day, reaching its peak at about sunset, after which it decreased rapidly throughout the night until daybreak. Similarly the plant carotenoid pigments increased during the day and decreased at night (Fig. IE). Both chlorophyll and carotenoids increased to higher values on the second day, which differed from the first primarily in the amount of incident DYNAMICS OF A DIATOM BLOOM 263 o tr < o 01 _j X o -O I I I I I I I I I 1 I i .5 1.0 LIGHT INTENSITY (g CAL/CM2/MIN ) 1.5 FIGURE 3. The relation between incident radiation and the ratio chlorophyll a: carotenoid pigments in the diatom population. Open circles, June 26. Closed circles, June 27. radiation. The carotenoid pigments ranged on June 27 from a minimum of 30 SPU 3 at 05 :45 to 95 SPU at 17 :45, more than a three-fold variation. The ratio chlorophyll a: carotenoid pigments (Fig. IF) decreased throughout the daylight periods of both days from maximum values observed at sunrise, the more rapid decrease on June 27 again correlated with the greater incident radiation 3 The spectrophotometric analysis of carotenoid pigments has not been standardized in absolute units and they are reported in specific pigment units after Richards with Thompson (1952). One SPU, however, is closely equivalent to one milligram of pigment. 264 RYTHER, YENTSCH, HULBURT AND VACCARO on that day. Figure 3 shows the inverse relationship which was found between the intensity of solar radiation and the chlorophyll : carotenoid ratio. The varia- tions in this ratio are the result of a differential effect of light on pigment synthesis and decomposition where the chlorophyll changes are of much greater magnitude than are the carotenoid changes (Yentsch and Scagel, unpublished). The signifi- cance and interpretation of the magnitude and changes in this ratio will be discussed in the final section of this paper. c. Primary production Primary production was calculated by the following three methods : ( 1 ) the in situ changes of oxygen in the water, (2) the "light-and-dark-bottle" oxygen measurements, (3) the "chlorophyll-radiation" method of Ryther and Yentsch (1957). The inability to measure or calculate total CO2 prevented the use of pH changes or the C14 method for this purpose. On June 26, the dissolved oxygen in the integrated sample collected over the one-meter water column increased from a minimum of 4.5 ml. /liter at 07 :00 hours to a maximum of 11.8 ml./liter at 17:00 hours, a difference of 7.3 ml./liter. If an assimilatory quotient of 1.25 is used, this change in oxygen is equivalent to a carbon fixation of 3.15 grams/m2./day. As mentioned earlier, the decrease in oxygen at night appears to have been due almost entirely to respiration. This loss was equivalent to 0.5 ml. oxygen/liter/hour. During the 10 hours of daylight, if respiration occurred at the same rate, this wrould account for a total of 5.0 ml. O2/ liter or 2.15 grams carbon/m2./day. Adding this respiratory loss to the observed net production of 3.15 grams carbon/m2./day gives a total or gross production of 5.20 grams carbon/m2./day. In the same way production was calculated for June 27, the net change in oxygen being equivalent to assimilation of 3.8, the respiration loss 1.7 and the gross production 5.5 grams carbon/m2./day. The two-hour "light-and-dark-bottle" experiments which were described above were also used to calculate gross and net production. The differences between the oxygen concentration of the light bottles at 0, 0.5, and 1.0 meter and that of the accompanying dark bottle over the two-hour experimental periods, converted to carbon assimilation as above, are shown in Figure 1G. The carbon equivalent of respiration for the same two-hour periods was obtained from the difference be- tween the oxygen content of the water at the beginning of the two-hour period and that of the dark bottle. These curves were integrated to obtain daily photo- synthesis at each depth and daily respiration. These values in turn were plotted against depth and integrated to give daily photosynthesis and respiration beneath a square meter of surface. Gross photosynthesis for June 26 calculated by this method was 3.5, respiration was 1.7 and net photosynthesis 1.8 grams carbon/m2./ day. On June 27 the values were 6.2, 2.8 and 3.4 grams carbon/m2. /day for gross production, respiration and net production, respectively. The in situ oxygen changes at night appeared to indicate a constant respiration rate of 0.5 ml. O2/liter/hour, and this, as described above, was used to correct the net in situ change observed in daylight to give gross production. An examination of the two-hour bottle experiments during the day shows that, when measured in this way, respiration was by no means constant but varied roughly in proportion to the rate of photosynthesis. On June 27, for instance, the respiratory rate ranged DYNAMICS OF A DIATOM BLOOM 265 from 0.06 ml./liter/hour in the early morning to about 1.00 ml./liter/hour at mid- day. These measurements, though somewhat crude, emphasize the need for a reconsideration of the tacit assumption made by most ecologists that respiration measured at night, or for long periods in dark bottles, is the same as that which occurs in the light in conjunction with photosynthesis. The third method for estimating production is that developed by Ryther and Yentsch (1957). This method requires measurement of the concentration of chlorophyll a, the total daily incident radiation, and the extinction coefficient of visible light in the water. The latter was determined by the measurement of light penetration to one meter with a submarine photometer at 13:30 hours on June 26. The extinction coefficient (k) so determined was 4.0. Use of this method required an obvious over-simplification, since the chlorophyll a concentration, as has been pointed out, varied throughout the day. A mean value of 200 mg. chla/m3. was used for the calculation for both days, and this was assumed to be uniformly distributed over the one-meter water column. The resulting values for gross production were 3.2 and 5.1 grams carbon/m2./day f°r June 26 and 27, respectively. The results obtained by these three methods are summarized in Table II. They show rather good agreement except for the values obtained by in situ oxygen changes on June 25 which are almost twice as high as those obtained by the other TABLE II Primary production in Senix Creek on June 26 and June 27, as measured by three methods (grams carbon assimilated /m?/ day) Method Gross Net (day) Net (24 hrs.) Gross Net (day) Net (24 hrs.) In situ O2 5.3 3.15 0 5.5 3.8 0 L-D bottle O2 3.5 1.76 — 6.2 3.4 — Chlorophyll 3.2 — 5.1 1 two methods. It should be pointed out that the net production values which have been discussed refer to this process during the daylight hours only. The only estimates over a 24-hour period which can be made are based upon the in situ oxy- gen changes (and pH changes) which clearly reflect a net production for this period of zero. Finally, the net changes observed in situ and in vitro are acknowl- edged as representing changes brought about by the whole community including animals and bacteria, and do not characterize the plant population alone. The efficiency of production on the two days may be roughly estimated by taking the median of the values obtained by the three methods for daily gross produc- tion, 3.5 and 5.5 grams carbon/m2. on June 26 and 27, respectively. If the assump- tion is made that 50% of the photosynthetic production is carbon and has a heat of combustion of 5.5 k cal./gr. (see Krogh and Berg, 1931), and further that half the incident radiation may be used for photosynthesis, the efficiency may be cal- culated as : 3.5 X 2 X 5,500 a) June 26 • b) June 27 1,500,000 5.5 X 2 X 5,500 3,700,000 = 2. = 1-6% 266 RYTHER, YENTSCH, HULBURT AND VACCARO /. The physiology of the bloom There are several indications that the diatom population in Senix Creek was a non-growing one which had exhausted its supply of available nutrients and was able to subsist at a basal level, photosynthesizing just enough during the day to compensate for its metabolic requirement over a 24-hour period. This is best illustrated by the in situ oxygen and pH values, in which the net oxygen produced and CO2 assimilated during the day are exactly compensated by the reverse proc- esses at night. Further evidence of this is the fact that the concentration of diatoms remained unchanged over the 48 hours of observation except where such changes are attributable to tidal flushing. The evidence that the bloom was nutrient-limited is somewhat sparse and in- direct, but rather convincing. At 10 :30 hours on June 27 an integrated water sample was collected and frozen. This was later analyzed for nitrogen and phos- phorus fractions at the Woods Hole Oceanographic Institution. The results of these analyses are given below. /jg Atoms/liter NO2- + NO3- 3.40 NH3+ 1.49 PO4 3.80 Total P 16.0 A photosynthetic rate of 5.5 grams carbon/m2./day in a one-meter water column represents a requirement of 460 jugA carbon/liter/day. As Redfield (1934) and others have pointed out, marine phytoplankton assimilate carbon, nitrogen and phosphorus at an atomic ratio closely approaching 100: 15 : 1. This rate of carbon assimilation is therefore equivalent to a daily requirement of 71 /xg A/liter of nitro- gen and 4.6 /^gA/liter of phosphorus. Thus, the concentrations of these elements in the mid-morning of June 27 represented no more than a fraction of a day's supply of either nitrogen or phosphorus. These calculations were based upon the require- ment of normal cells. Photosynthesis may of course continue after nitrogen and phosphorus are exhausted with the storage of carbohydrates and lipids. This is presumedly what was happening in this population, the cells using these stored materials to satisfy their metabolic requirements at night. Further studies of this type of population, with emphasis placed upon the diurnal cycle of nutrients, would be particularly interesting. The behavior of the plant pigments is a further indication of the physiological condition of the population. The fact that both chlorophyll a and the carotenoids were synthesized during the day and decomposed at night signifies that the plants were drawing upon their cellular reserves to maintain themselves in the dark. When nutrients are available, this does not occur ; in fact, chlorophyll may be syn- thesized in the dark under favorable growing conditions if the cells have sufficient respiratory reserves (Harvey, 1953). Experiments in this laboratory (Ketchum ct a/., 1958) and elsewhere have shown that both chlorophyll a and the carotenoid pigments decrease in diatoms in response to nitrogen, phosphorus or iron deficiency or excessive illumination. This nutritional chlorosis results in a more rapid decomposition of chlorophyll than carotenoid pigments. As the day progressed the pigment ratio decreased, presum- ably in part because of nutrient exhaustion which was hastened by greater demands of photosynthesis at high light intensities (Fig. 3). DYNAMICS OF A DIATOM BLOOM 267 The picture which emerges from these various bits of evidence, then, is that of a static diatom bloom of great magnitude, its nutrient supply exhausted or at least re- duced to the level where growth could not occur. Yet it was not a dying population, except insofar as physical forces tended to disperse it. It was capable of carrying out organic synthesis at a rate some 10-100 times that of normal plankton commu- nities, drawing upon these materials for its metabolic requirements much the same as a mature animal maintains a balance between its assimilation and metabolism. It would appear, then, that populations of phytoplankton such as we have de- scribed here, though not actively growing, are not necessarily dying either. They are merely living in a different growth phase, a condition in which they may per- sist for long periods of time if they are not destroyed or dispersed by external factors. Perhaps in diatom populations, as elsewhere, the bloom of maturity may outlast the bloom of youth. SUMMARY 1. A dense population of planktonic diatoms was studied over a 40-hour period in a small tidal creek on the south shore of Long Island, New York. 2. Measurements were made at frequent intervals of incident radiation, light penetration, salinity, temperature, dissolved oxygen, pH, concentration of diatom cells and their pigments, and dissolved inorganic nutrients. Photosynthesis and respiration were measured by oxygen changes in bottle experiments and estimated from in situ oxygen changes and from chlorophyll a and radiation. 3. The plankton community appeared to be nutrient-limited and consisted of a static, non-growing diatom population which was being slowly diluted by tidal action. This was indicated by the diatom counts, the behavior of their pigments (which increased throughout the day and decreased during the night) and the concentration of available plant nutrients. 4. Rates of primary production measured by three methods showed good agree- ment, the values ranging from 3.2 to 5.3 grams carbon assimilated/m2./day on June 26, from 5.1-6.2 on June 27. Total incident radiation for the two days was 300 and 740 gram calories/cm2. /day, respectively, and the efficiency of the photo- synthetic utilization of visible radiation for the two days was estimated at 2.6% and 1.6%, respectively. LITERATURE CITED AMERICAN PUBLIC HEALTH ASSOCIATION, 1955. Standard methods for the analysis of water, sewage, and industrial wastes. Tenth Edition. American Public Health Assoc., Inc., New York. 259 pp. CREITZ, G. I., AND F. A. RICHARDS, 1955. The estimation and characterization of plankton populations by pigment analyses. III. A note on the use of "millipore" membrane niters in the estimation of plankton pigments. /. Mar. Res., 14: 211-216. DOTY, M. S., AND M. OGURI, 1957. Evidence for a photosynthetic daily periodicity. Limnol. and Occanogr., 2: 37-40. DUXBURY, A. C., AND C. S. YENTSCH, 1956. Plankton pigment nomographs. /. Mar. Res., 15 : 92-101. HARVEY, H. W., 1948. The estimation of phosphate and total phosphorus in sea waters. /. Mar. Biol. Assoc., 27: 337-359. HARVEY, H. W., 1953. Synthesis of organic nitrogen and chlorophyll by Nitsschia closterium. J. Mar. Biol. Assoc., 31 : 477-478. KETCHUM, B. H., J. R. RYTHER, C. S. YENTSCH AND N. CORWIN, 1958. Productivity in rela- tion to nutrients. Rapp. et Proc. Verb. Cons. Internal. Explor. Mcr., 144: 132-140. 268 RYTHER, YENTSCH, HULBURT AND VACCARO KROGH, A., AND K. BERG, 1931. Uber die chemische Zusammensetzung des Phytoplanktons aus dem Fredriksborg-Schlossee und ihre Bedeutung fiir die Maxima der Cladoceren. Int. Rev. Gesamt. Hydrobiol. u Hydrog., 25 : 205-218. MULLIN, J. B., AND J. P. RILEY, 1955. The spectrophotometric determination of nitrate in natural waters, with particular reference to sea water. Analyt. Chim. Acta, 12: 464-480. REDFIELD, A. C., 1934. On the proportions of organic derivatives in sea water and their rela- tion to the composition of plankton. James Johnstone Memorial Vol., The University Press, Liverpool. Pp. 176-192. RICHARDS, F. A., WITH T. G. THOMPSON, 1952. The estimation and characterization of plank- ton populations by pigment analysis. II. A spectrophotometric method for the esti- mation of plankton pigments. /. ]\Iar. Res., 11 : 156-172. RIDER, B. F., AND M. G. MELLON, 1946. Colorimetric determination of nitrites. Ind. Engin. Chem, Anal. Edit., 18 : 96-99. RILEY, J. P., 1953. The spectrophotometric determination of ammonia in natural waters with particular reference to sea water. Analyt. Chim. Acta, 9: 575-589. ROBINSON, R. J., AND T. G. THOMPSON, 1948. The determination of phosphates in sea water. /. Mar. Res., 7 : 33-41. RYTHER, J. H., 1954. The ecology of phytoplankton blooms in Moriches Bay and Great South Bay, Long Island, New York. Biol. Bull, 106: 198-209. RYTHER, J. H., AND C. S. YENTSCH, 1957. The estimation of phytoplankton production in the ocean from chlorophyll and light data. Limnol. and Occanogr., 2 : 281-286. YENTSCH, C. S., AND J. H. RYTHER, 1957. Short-term variations in phytoplankton chlorophyll and their significance. Limnol. and Oceanogr., 2 : 140-142. THE FORMATION OF SUBNUCLEAR AGGREGATES IN NORMAL AND SYNCHRONIZED PROTOZOAN CELLS1 OTTO H. SCHERBAUM, ALLAN L. LOUDERBACK AND THEODORE L. JAHN Department of Zoology, University of California, Los Angeles 24, Cal. Since Biitschli (1876) established the nuclear dualism in ciliates, there has been much speculation about the biological role of the micro- and macronucleus. The mitotic behavior of the micronucleus, with its delicate apparatus for chromo- somal segregation, led to the generally accepted view of the importance of the micronucleus in inheritance and reproduction. The macronucleus, on the other hand, which was found to divide "simply" by pinching in two, was considered to be concerned "only" with the regulation of metabolic functions in the cell. This concept of the duality of nuclear function as formulated by Hertwig in 1889, was substantiated by Goldschmidt (1904) and Popoff (1908). These authors distinguished between the genetically-active idiochromatin and the tropho- chromatin, which was concerned exclusively with the cellular metabolism. In the uninuclear protists both types of chromatin \vere considered to be present in one nucleus, while in ciliates the idiochromatin was confined to the micronucleus and the trophochromatin was found in the macronucleus only. The view of the dualistic function of the nuclei in ciliates was abandoned after experimental data accumulated showing the controlling role the macronucleus plays in the processes of cell division and regeneration (Grell, 1950). The genetic importance of the macronucleus in ciliates stimulated cytological studies of its structure. A considerable body of evidence has been accumulated during the past 30 years showing Feulgen-positive bodies in the cytoplasm, which could not be accounted for by "macronuclear fragmentation," the process of disintegration of the macronucleus upon conjugation of two cells. These bodies often have a spheri- cal shape, and after Feulgen staining show the homogeneous appearance of micro- nuclei. Very often these bodies have been erroneously described as micronuclei, a fact which was pointed out by Kidder (1933). Diller (1936) observed simple fragmentations of the macronucleus in Paraniecium aurelia, and he used the term "hemixis" to denote such autonomous changes of the macronucleus which are not related to sexual phenomena or binary fission. It is believed now that the macronucleus of the ciliates consists of many diploid subnuclei (Sonneborn, 1947). We therefore propose the term "subnuclear aggre- gates" (SNA's) for the Feulgen-positive material lost or expelled from the macro- nucleus into the cytoplasm. The formation of SNA's may occur 1) by simple extrusion of Feulgen-positive material from the macronucleus, or 2) by loss during the process of binary fission of the macronucleus. An example of extrusion of chromatin masses from the macronuclear anlagen in the exconjugates of Ancis- truma isseli was described by Kidder (1933), and a spontaneous "budding" of 1 This work was supported by grant G-2490 of the National Science Foundation. 269 270 SCHERBAUM, LOUDERBACK AND JAHN macronuclei, independent of cell division, in Ichthyophthirius multifiliis was found by Haas (1933). In the course of binary fission of the macronucleus of Colpidium colpoda Kidder and Diller (1934) described how some of the nuclear material is left behind in the fission plane. This material becomes condensed and finally disappears. A similar phenomenon was described by Furgason (1940) in the amicronucleate strain T of Tctrahyincna pyriformis and by McDonald (1958) in Tetrahymena pyriformis H. These frequently observed chromatin extrusions from the macronucleus led Kidder and Diller (1934) to the suggestion of a presumptive role for this phe- nomenon. It wras thought extrusion might be a manifestation of a universal principle of nuclear reorganization which, in turn, could account for a high division rate. However, no quantitative studies have so far been carried out on the forma- tion of the SNA's. their frequency of formation, and their absolute size in various phases of population growth. The system of synchronous cell division in Tetrahymena pyriformis, strain GL, as worked out by Scherbaum and Zeuthen (1953, 1955), was used for the study of this phenomenon. During the induced synchrony about 85 per cent of the cells are in the visible stage of fission and all stages of SNA formation can readily be found. METHOD The amicronucleate strain GL of Tetrahymena pyriformis was grown princi- pally as described earlier (Scherbaum and Zeuthen, 1955). The growth medium was two per cent proteose peptone (Difco) with 0.5 per cent glucose and 0.1 per cent liver fraction L (Wilson Laboratories) in glass-distilled water. Salts were added as in the basal medium A of Kidder and Dewey, except that phosphates were omitted. The medium was filtered and autoclaved at 15 pounds for 15 minutes. One ml. of a three-day-old stock culture (approximately 2 X 105 cells per ml.) was used for the inoculation of 150 ml. of culture medium in a 500-ml. culture flask. The flask was submerged in a temperature-controlled water bath, which was mounted on a shaker. Samples of 5 ml. were removed from the experimental flask at regular intervals for counting (Scherbaum, 1957) and for nuclear preparations. For the latter, the samples were concentrated by centrifugation in a hand centrifuge and the supernatant removed by suction. The concentrated cell suspension wras fixed in one per cent aqueous osmic acid for two minutes. The cells were removed from the fixative by centrifugation, washed in water, and passed through alcohol (30 per cent to 100 per cent). The cells were then pipetted onto albuminized cover- slips, slightly dried to affix the cells to the glass surface, and transferred to absolute alcohol for ten minutes. The cover slips wrere stored in 70 per cent alcohol. For the Feulgen reaction the samples were hydrolyzed in 1 N HC1 at 60° C. for 12 minutes and exposed to the Schiff reagent for one hour. RESULTS At an approximate population density of 5 X 103 cells per ml. the first sample was removed. This served as the control for normal exponential multiplication. The second sample was removed during the synchronous division. For the indue- SUBNUCLEAR AGGREGATES IN PROTOZOA 271 A FIGURE 1. Photomicrographs of Feulgen-stained cells from various growth phases: Normal exponential multiplication (A), after temperature treatment (B), during and after the first synchronous division (C to K), and in maximum stationary phase (L). The distance between the two lines on the scale in (A) is 10 /*; "n" denotes a subnuclear aggregate (SNA). Further explanation in the text. 272 SCHERBAUM, LOUDERBACK AND JAHN tion of synchrony the culture was exposed to seven temperature cycles. The temperature was changed every half hour between 28° and 33.9° C. One hour and 15 minutes after the end of the seventh cycle 80 to 85 per cent of the cells were in the visible stage of fission. The third sample was removed after 48 hours of sub- sequent growth when the cells were in the early stationary phase. Figure 1 shows photomicrographs of cells in various stages of population growth and of the formation of the SNA's during division. In A the deeply stained SNA (n) is close to the macronucleus and resembles the micronucleus as shown by Holz, Scherbaum and Williams (1957) in mating type 1, variety 1, of Tetrahymcna pyri- formis. Figure 1, B shows a typical enlarged cell and nucleus after the end of the temperature treatment. No SNA from a previous division can be seen, al- though some were found in other preparations. In C the macronucleus elongates during the onset of synchronous division. A distinct portion of the nucleus seems to be "suspended" between the macronuclear halves pulling apart amitotically. In TABLE I Size and number of subnuclear aggregates (SNA's) in various growth phases Sample No. Number of cells with SNA (%) Mean macronuclear volume in p? Mean volume of SNA in fi3 SNA/macronucl. volume ratio in % 1 Control exponent, multiplication 16 265.0 1.9 0.72 2 Prior to synchr. division 22 1063.0 X X 3 After synchr. division 55 430.0 12.2 2.84 4 Max. station, phase 6 122.0 1.8 1.48 In order to determine the average percentage cells with SNA's, 100 cells of each type were examined. The macronuclear volume for each growth stage is the average for the 100 cells measured. The mean volume given for the SNA is the average of 50 measurements; "x" denotes that no measurements were made. D to G this macronuclear remnant can be seen at various stages of cell division. In these phases of division the fragment still shows the typical granular compo- sition of the macronucleus. However, somewhat later, when the fibrous connection between the macronucleus and the fragment disappears, the fragment tends to be- come spherical, the granular structure disappears, and the fragment becomes a dense homogeneous mass, resembling the micronucleus in this respect (I-L). Figure 1, J and K shows cells immediately after division. In cells of the early stationary phase of growth, SNA's were also found (L). For a quantitative estimation of the size and number of the SNA's the experi- mental culture was sampled in various growth phases. The result is shown in Table I. The number of cells with SNA's is relatively constant in exponentially growing cultures (16 per cent) and increases slightly in the course of the heat treatment. SUBNUCLEAR AGGREGATES IN PROTOZOA 273 However, after synchronous division SNA's were found in 55 per cent of the cells. On the assumption that the SNA's observed in the cells prior to division are carried through the synchronous division step, one can calculate that in approximately 45 per cent of the cells undergoing division new formation of SNA's took place. The mean volume of the SNA's is relatively constant in the logarithmic phase and stationary phase of growth. It is approximately 2.0 /A This value is 0.7 per cent and 1.5 per cent of the macronuclear volume at these two growth phases, respectively. After the synchronous division the average SNA volume is 12 //,3, showing a six-fold increase as compared to normal values. EVALUATION OF THE RESULTS AND DISCUSSION In almost all cells examined only one SNA was found, but in some cases two or three SNA's could be observed in one cell. From the frequency with which FIGURE 2. Photomicrograph of a Feulgen-stained cell during the first division after the heat treatment. The distance between the two lines on the scale is 10 /JL. Further explanation in the text. the SNA's occur at various growth phases it seems as if that they are broken down to Feulgen-negative material or are extruded from the cell. However, there is no evidence which might serve to evaluate either of these possibilities. The ab- normally large nuclei of synchronized cells, together with the larger size of the SNA's of synchronized cells, might suggest that the size of the SNA's depends to some degree on the volume of the parent macronucleus. However, the size of the newly formed SNA's may vary, as can be seen in Figure 1, E-H. Further- more, the size appears to be a function of the age of the SNA, since when first formed it is granular, similar to the macronucleus, and it then becomes homo- geneous and smaller, apparently by condensation, before it disappears. In the present analysis we followed the formation of the SNA's with the Feulgen method for DNA only. However, nothing is known about the concentration of the basic proteins in these bodies. Basic proteins are normally found to be associated with 274 SCHERBAUM, LOUDERBACK AND JAHN the DNA in the nucleus. A difference in stainability of the basic proteins in the micro- and macronucleus was observed by Alfert and Goldstein (1955) in mating types I and II of Tetrahymcna. One could imagine that the original DNA basic protein ratio, as characteristic for the macronucleus and for the young SNA's in Tetrahymcna GL, could change by preferential degradation of DNA in the course of the presumed condensation process occurring after formation of the SNA's. Although the extrusion of macronuclear material in non-dividing cells and the formation of SNA's have been observed in various protozoan cells (see introduc- tion), there is no conclusive evidence concerning the role which these phenomena play in the metabolism of the cells. Following the concept of strict equal distri- bution of parental DNA to the daughter cells one might be somewhat puzzled by this phenomenon. However, the high degree of polyploidy in the macronucleus suggests that such an equal distribution may not be a "conditio sine qua non." A slight imbalance in timing of nuclear and cell division could cause this loss of DNA in the fission plane, and the failure for it to be incorporated into the daughter nuclei. In rare instances an "imbalance" of nuclear and cell division was observed in synchonized cells. For instance, Figure 2 shows a cell during synchronous division, dividing into three instead of two daughters. In the right part of the cell, nuclear division is completed, while cellular division lags slightly behind. In the left part of the cell the macronucleus is in division, while cyto- plasmic division is far more advanced than in a normal cell with a comparable nuclear figure. That such irregularities hardly affect the viability of the cells is not surprising in view of the fact that the protozoan macronucleus is a highly polyploid system. Sonneborn (1947) concludes, from genetic evidence, that the macronucleus of Paramecium aurclia must contain about 40 diploid "subnuclei." These observations suggest the interesting problem of to what extent this high polyploidy of the macronucleus could be reduced experimentally. For instance, in starving cultures of Tetrahymcna pyrifonnis strain S, Weis (1954) found a re- duction in cell size to less than 10 per cent of the normal volume. These cells "regulated" back to their normal size upon addition of nutrients to the culture medium. If one assumes an almost constant nucleo/cytoplasmic ratio and 40 diploid "subnuclei" (as found for Paramecium'), one might expect the starved cells to carry only 4 diploid "subnuclei." Opposed to the view that the loss of subnuclei during binary fission is an arbitrary phenomenon, based on mere chance, is the idea which attributes a strict regulatory function to these processes. Findings by Kidder and Claff (1938) seem to substantiate this point of view. These authors investigated the life cycle of Colpoda cue nil us and described chromatin extrusion following each division in regular and predictable fashion. This "budding" of the macronuclei occurs almost synchronously in the two daughter cells. In contrast to the loss of DNA during the fission process, as described for synchronized cells of Tetrahymena, we have in Colpoda cuciillus an example of active regulation or reorganization of some sort after the daughter cells are formed. The authors wish to thank Prof. W. H. Furgason, Department of Zoology, U.C.L.A., for his criticism and advice in preparation of the manuscript. Prof. W. Balamuth, Department of Zoology, University of California, Berkeley, Prof. Fur- gason and Dr. J. Loefer, O.N.R., Pasadena, kindly supplied samples of Tetra- hymena pyrifonnis, strain GL. SUBNUCLEAR AGGREGATES IN PROTOZOA 275 SUMMARY 1. The formation of "subnuclear aggregates" (SNA's) is studied quantita- tively in synchronously-dividing cells of Tetrahymena pyriformis strain GL. 2. In normal cultures approximately 16 per cent of the cells were found to contain SNA's. This value rises to 55 per cent after synchronous division. The SNA/macronuclear volume ratio is 0.72 per cent in normal cells and 2.8 per cent in cells after synchronous division. 3. The possible significance of the formation of SNA is discussed. LITERATURE CITED ALFERT, M., AND N. O. GOLDSTEIN, 1955. Cytochemical properties of nucleoproteins in Tetra- hymena pyriformis ; a difference in protein composition between macro and micronuclei. /. E.rp. ZooL, 130: 403-419. BUTSCHLI, O., 1876. Studien iiber die ersten Entwicklungsvorgange der Eizelle, der Zellteilung und die Conjugation der Infusoricn. Abh. Scnckenbcrg. natitrforsch. Gcs. Frank jurt, 10: 1-250. DILLER, W. F., 1936. Nuclear reorganization processes in Paramccium aurelia, with descrip- tions of autogamy and "hemixis." /. Morphol., 59: 11-67. FURGASON, W. H., 1940. The significant cytostomal pattern of the "Glaucoma-Colpidium group" and a proposed new genus and species, Tetrahymena gelcii. Arch. f. Protist., 94: 224-266. GOLDSCHMIDT, R., 1904. Die Chromidien der Protozoen. Arch. f. Protist., 5 : 126-144. GRELL, K., 1950. Der Kerndualismus der Ciliaten und Suctorien. Natuntnss., 37 : 347-356. HAAS, G., 1933. Beitrage zur Kenntnis der Cytologie von Ichthyophthirius multifiliis. Arch, f. Protist., 81 : 88-137. HERTWIG, R., 1889. Uber die Konjugation der Infusorien. Abhandl. Bayr. Akad. Wiss., 17: 150-233. HOLZ, G. G., O. H. SCHERBAUM AND N. WILLIAMS, 1957. The arrest of mitosis and stomato- genesis during temperature induction of synchronous division in Tetrahymena pyri- jormis, mating type 1, variety 1. E.rp. Cell Res., 13: 618-621. KIDDER, G. W., 1933. On the genus Ancistruma Strand II. The conjugation and nuclear re- organization of A. isscli. Arch. f. Protist., 81 : 1-18. KIDDER, G. W., AND C. L. CLAFF, 1938. Cytological investigations of Colpoda cncitllus. Biol. Bull., 74: 178-197. KIDDER, G. W., AND W. F. DILLER, 1934. Observations on the binary fission of four species of common free-living ciliates, with special reference to the macronuclear chromatin. Biol. Bull., 67: 201-219. MCDONALD, BARBARA B., 1958. Quantitative aspects of deoxyribose nucleic acid (DNA) metabolism in an amicronucleate strain of Tetrahymena. Biol. Bull., 114: 71-94. POPOFF, M., 1908. Die Gametenbildung und die Konjugation von Carchesium polypinum L. Zcitschr. iviss. ZooL, 89: 478-524. SCHERBAUM, O., 1957. The application of a standard counting method in estimation of growth in normal and heat-treated cultures of Tetrahymena pyriformis. Acta path, microbiol. scand., 40: 7-12. SCHERBAUM, O., AND E. ZEUTHEN, 1953. Induction of synchronous cell division in mass cul- tures of Tetrahymena pyriformis. E.rp. Cell Res., 6 : 221-227. SCHERBAUM, O., AND E. ZEUTHEN, 1955. Temperature induced synchronous divisions in the ciliate protozoon Tetrahymena pyriformis growing in synthetic and proteose-peptone media. Exp. Cell Res., Suppl. 3: 312-325. SONNEBORN, T. M., 1947. Recent advances in the genetics of Paramccium and Euplotes. Adv. in Genetics, 1 : 263-358. WEIS, D., 1954. Observations on size reversibility in cultures of Tetrahymena pyriformis. J. Protozool., 1. STUDIES ON DIGENETIC TREMATODES OF THE GENERA GYMNOPHALLUS AND PARVATREMA HORACE W. STUNKARDi AND JOSEPH R. UZMANN 2 U. S. Fisli and Wildlife Service HISTORICAL REVIEW The genus Gymnophallus was erected by Odhner (1900) to contain Distomum deliciosum Olsson, 1893 and other small species from the gall bladder, intestine and bursa Fabricii of shore-birds. Subsequent observations have shown that the asexual generations of these worms occur in bivalve mollusks and that the cercariae, which are produced .in sporocysts, belong to the Dichotoma group of furcocercous larvae. Typically, these cercariae have eye-spots and short bifid tails, although either or both may be reduced or absent. Cercaria dichotoma emerges and swims as a furcocercous larva. In certain species the tail undergoes regression and is lost before the larva emerges from the sporocyst whereas, in others, apparently no tail is formed. On emergence from the first intermediate host, the cercariae attach to the mantle or body wall of bivalve or gastropod mollusks where as unencysted metacercariae, they develop to almost definitive size. The metacercariae may pro- duce lesions on the mantles of their hosts and such injuries stimulate proliferation of tissues, especially of the secreting layer of the mantle, and deposition of nacreous material. Despite the observations of many investigators over a period of more than fifty years, no complete life-history has yet been worked out and the specific relations between particular cercariae, metacercariae, and sexually mature worms remain undetermined. The presence of pearly formations in the mantle of Mytilus edulis has been known for at least three hundred years. According to Giard (1907), they were reported by Olaus Worm in 1655 from mussels taken at Roeskild, near Copen- hagen. Robert Garner observed them in M. edulis from the English coast and he (1872) recognized that they were formed as a reaction by the mollusk to a small distome parasite on the mantle. Baron d'Hamonville (1894) found pearls (sans valeur} in M. edulis at Billiers (Morbihan) France, although the infection was limited to the area of the port. Giard (1897) reported small distomes, often asso- ciated with irregularly shaped calcareous deposits, between the mantle and shell of Dona.v trunculus L., Tcllina fabiila Gronov, Tcllina tennis DaCosta and Telllna solidula (= T. balthica L., ex parte} from Boulogne-sur-Mer. The worms were 0.5 mm. long, with rudiments of testes, but no ovary, and Giard suspected that they might be stages in the life-cycle of Brachycoelium luteum (van Beneden), a para- site of the common dogfish, Scyllium canlcula. The larger specimens were often less active, more opaque, and filled with sporozoans (Glugidees). Dubois (1901) studied the parasites of M. edulis at Billiers and found them in reddish brown spots, 1 The American Museum of Natural History, New York 24, N. Y. 2 Fisheries Center, University of Washington, Seattle 5, Wash. 276 DIGENETIC TREMATODES 277 which were the loci of pearl formation. The worms measured 0.4-0.6 mm. in length and for them he proposed the name, Distomum margaritarum. He found the same or a similar parasite in Mytilus gallopr ovine ialis from the coast of Provence. Jameson also studied the parasites at Billiers; he (1902) described and figured the worms which he stated closely resembled Distomum somateriae, whose mature stages had been described by Levinsen (1881) from the intestine of the eider duck, Somateria mollissima, taken near Egedesminde, Greenland. He recalled that Mobius (1857) had reported a trematode associated with pearls in the pearl oyster, Margaritifera margaritifcra, from the west coast of N. America. Follow- ing the action of Stossich (1899), Jameson referred the species from M. edulis to Lecithodendrium Looss, but misspelled the name, Lcucithodendriiim. He reported that he had found larvae, similar to those in M. cdulis, in sporocysts in Tapes decussatus and that he had infected M. edulis with these sporocysts. The larvae in the sporocysts were tailless ; they differed from those in M. edulis only in their smaller size, paler color, more distended excretory organs and empty gut, and in the possession of special sense organs and eyes. The sporocysts were present in all of almost 200 Tapes examined, located chiefly in the margin of the mantle where it is attached at the pallial line. The sporocysts were spherical to oval, about 0.5 mm. in diameter,