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THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 

DAVID W. BISHOP, Carnegie Institution of JOHN H. LOCHHEAD, University of Vermont 

Washington y L LooSANOFF) n- s> Fish and wi i d iif e 

FRANK A. BROWN, JR., Northwestern University Service 

JAMES CASE, State University of Iowa C. L. PROSSER, University of Illinois 

JOHN W. GOWEN, Iowa State College BERTA SCHARRER, Albert Einstein College of 
LIBBIE H. HYMAN, American Museum of Medicine 

Natural History FRANZ SCHRADER, Duke University 

J. LOGAN IRVIN, University of North Carolina WM. RANDOLPH TAYLOR, University of Michigan 

DONALD P. COSTELLO, University of North Carolina 
Managing Editor 



VOLUME 121 

JULY TO DECEMBER, 1961 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE & LEMON STS. 

LANCASTER, PA. 




11 

THE BIOLOGICAL BULLETIN is issued six times a year at the 
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- 
sylvania. 

Subscriptions and similar matter should be addressed to The 
Biological Bulletin, Marine Biological Laboratory, Woods Hole, 
Massachusetts. Agent for Great Britain: Wheldon and Wesley, 
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, 
W. C. 2. Single numbers $2.50. Subscription per volume (three 
issues), $6.00. 

Communications relative to manuscripts should be sent to the 
Managing Editor, Marine Biological Laboratory, Woods Hole, 
Massachusetts, between June 1 and September 1, and to Dr. 
Donald P. Costello, P.O. Box 429, Chapel Hill, North Carolina, 
during the remainder of the year. 



Second-class postage paid at Lancaster, Pa. 



LANCASTER PRESS, INC., LANCASTER, PA. 




CONTENTS 

^ MASS. /$/ 

^K ^X- 

No. 1. AUGUST, 1961 

PAGE 

Annual Report of the Marine Biological Laboratory 1 

BANG, FREDERIK B. 

Reaction to injury in the oyster (Crassostrea virginica) 57 

BEERS, C. DALE 

The obligate commensal ciliates of Strongylocentrotus drobachiensis : 

Occurrence and division in urchins of diverse ages ; survival in sea water 

in relation to infectivity 69 

DALES, R. PHILLIPS 

Observations on the respiration of the sabellid polychaete Schizobranchia 

insignis 82 

DAVID, CHARLES N., AND ROBERT J. CONOVER 

Preliminary investigation on the physiology and ecology of luminescence 

in the copepod, Metridia lucens 92 

DETHIER, V. G., AND D. R. EVANS 

The physiological control of water ingestion in the blowfly 108 

DINGLE, HUGH 

Flight and swimming reflexes in giant water bugs 117 

DUNHAM, PHILIP B., AND F. M. CHILD 

Ion regulation in Tetrahymena 129 

GIESE, ARTHUR C. 

Further studies on Allocentrotus fragilis, a deep-sea echinoid 141 

GOLDSMITH, MARY HELEN M., AND HOWARD A. SCHNEIDERMAN 

A dual effect of carbon dioxide on insects poisoned by oxygen 151 

Goss, RICHARD J. 

Metabolic antagonists and prolonged survival of scale homografts in 

Fundulus heteroclitus 162 

IWASAKI, HIDEO 

The life-cycle of Porphyra tenera in vitro \73 

JENKINS, MARIE M. 

Respiration rates in planarians. III. The effect of thyroid compounds on 

oxygen consumption 188 

SCHARRER, BERTA, AND MARIANNE VON HARNACK 

Histophysiological studies on the corpus allatum of Leucophaea maderae. 

III. The effect of castration 193 

No. 2. OCTOBER, 1961 

BARLOW, GEORGE W. 

Intra- and interspecific differences in rate of oxygen consumption in gobiid 
fishes of the genus Gillichthys 209 



79674 



iv CONTENTS 

BLINKS, L. R., AND CURTIS V. GIVAN 

The absence of daily photosynthetic rhythm in some littoral marine algae. . 230 
BUCK, JOHN, AND JAMES F. CASE 

Control of flashing in fireflies. I. The lantern as a neuroeffector organ. . . 234 

BURBANCK, W. D., AND MADELINE P. BURBANCK 

Variations in the dorsal pattern of Cyathura polita ( Stimpson ) from estu- 
aries along the coasts of eastern United States and the Gulf of Mexico .... 257 

CARLSON, ALBERT D. 

/ Effects of neural activity on the firefly pseudoflash 265 

COOK, JAMES R. 

Euglena gracilis in synchronous division. II. Biosynthetic rates over the 
life cycle 277 

GROSS, WARREN J. 

Osmotic tolerance and regulation in crabs from a hypersaline lagoon 290 

GUNTER, GORDON, L. L. SULYA AND B. E. Box 

Some evolutionary patterns in fishes' blood 302 

HAYES, DORA K., AND W. D. ARMSTRONG 

The distribution of mineral material in the calcified carapace and claw 
shell of the American lobster, Homarus americanus, evaluated by means 
of microroentgenograms 307 

MAYNARD, DONALD M. 

Thoracic neurosecretory structures in Brachyura. I. Gross anatomy 316 

RITTER, HOPE, JR. 

Glutathione-controlled anaerobiosis in Cryptocercus, and its detection by 
polarography 330 

RULON, OLIN 

Cobalt and glutathione in the preservation of fertility and life of sand 
dollar eggs 347 

SCHONE, HERMANN 

Learning in the spiny lobster Panulirus argus 354 

Abstracts of papers presented at the Marine Biological Laboratory 366 

No. 3. DECEMBER, 1961 

ALLEN, KENNETH 

The effect of salinity on the amino acid concentration in Rangia cuneata 
(Pelecypoda) 419 

BAUER, G. ERIC, AND ARNOLD LAZAROW 

Studies on the isolated islet tissue of fish. IV. In vitro incorporation of 
C 14 - and HMabeled amino acids into goosefish islet tissue proteins 425 

BERNARD, FRANCIS J., AND CHARLES E. LANE 

Absorption and excretion of copper ion during settlement and metamor- 
phosis of the barnacle, Balanus amphitrite niveus 438 

CASE, JAMES, AND G. F. GWILLIAM 

Amino acid sensitivity of the dactyl chemoreceptors of Carcinides maenas 449 

DETHIER, V. G. 

Behavioral aspects of protein ingestion by the blowfly Phormia regina 
Meigen 456 



CONTENTS v 

GEORGE, J. C, AND A. K. SUSHEELA 

A histophysiological study of the rat diaphragm 471 

GROSSO, LEONARD L. 

The effect of thiourea, administered by immersion of the maternal organ- 
ism, on the embryos of Lebistes reticulatus, with notes on the adult gonadal 
changes 481 

DE LUQUE, ORLANDO, ALICE S. HUNTER AND F. R. HUNTER 

Osmotic studies of amphibian eggs. III. Ovulated eggs 497 

MARTIGNONI, MAURO E., AND ROBERT J. SCALLION 

Preparation and uses of insect hemocyte monolayers in vitro 507 

PIPA, RUDOLPH L. 

Studies on the hexapod nervous system. IV. A cytological and cyto- 
chemical study of neurons and their inclusions in the brain of a cockroach, 
Periplaneta americana (L. ) 521 

SIMMONS, JOHN E., JR. 

Urease activity in trypanorhynch cestodes 535 

SPIEGEL, MELVIN 

Tryptophan pyrrolase activity in the liver of adult Rana pipiens 547 

STEVENSON, J. Ross 

Polyphenol oxidase in the tegumental glands in relation to the molting 
cycle of the isopod crustacean Armadillidiurn vulgare 554 

WEBB, H. MARGUERITE, AND FRANK A. BROWN, JR. 

Seasonal variations in Oo-consumption of Uca pugnax 561 

WILLIAMS, CARROLL M. 

The juvenile hormone. II. Its role in the endocrine control of molting, 
pupation, and adult development in the Cecropia silkworm 572 



Vol. 121, No. 1 August, 1961 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



THE MARINE BIOLOGICAL LABORATORY 
SIXTY-THIRD REPORT, FOR THE YEAR 1960 SEVENTY-THIRD YEAR 

I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 15, 1960) ... 1 

STANDING COMMITTEES 

II. ACT OF INCORPORATION 4 

III. BY-LAWS OF THE CORPORATION 4 

IV. REPORT OF THE DIRECTOR 6 

Memorials 8 

Addenda : 

1. The Staff 11 

2. Investigators, Lalor and Lillie Fellows, and Students 14 

3. Fellowships and Scholarships 25 

4. Tabular View of Attendance, 1956-1960 25 

5. Institutions Represented 26 

6. Evening Lectures 27 

7. Shorter Scientific Papers (Seminars) 28 

8. Members of the Corporation 29 

V. REPORT OF THE LIBRARIAN 49 

VI. REPORT OF THE TREASURER 50 



I. TRUSTEES 

GERARD SWOPE, JR., President of the Corporation, 570 Lexington Ave., New York City 

A. K. PARPART, Vice President of the Corporation, Princeton University 

PHILIP B. ARMSTRONG, Director, State University of New York, Medical Center at 

Syracuse 

C. LLOYD CLAFF, Clerk of the Corporation, Randolph, Mass. 
JAMES H. WICKERSHAM, Treasurer, 530 Fifth Ave., New York City 



EMERITI 



W. C. CURTIS, University of Missouri 
PAUL S. GALTSOFF, Woods Hole, Mass. 
E. B. HARVEY, Woods Hole, Mass. 



2 MARINE BIOLOGICAL LABORATORY 

M. H. JACOBS, University of Pennsylvania School of Medicine 

F. P. KNOWLTON, Syracuse University 
CHARLES W. METZ, Woods Hole, Massachusetts 
W. J. V. OSTERHOUT, Rockefeller Institute 
CHARLES PACKARD, Woods Hole, Mass. 

A. C. REDFIELD, Woods Hole Oceanographic Institution 
LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y. 

TO SERVE UNTIL 1964 

C. LALOR BURDICK, The Lalor Foundation 

E. G. BUTLER, Princeton University 

K. S. COLE, National Institutes of Health 

S. KUFFLER, Harvard Medical School 

C. B. METZ, Oceanographic Institute, Florida State University 

ROBERTS RUGH, College of Physicians and Surgeons, Columbia University 

G. T. SCOTT, Oberlin College 

E. Z WILLING, Brandeis University 

TO SERVE UNTIL 1963 

L. G. BARTH, Columbia University 

JOHN B. BUCK, National Institutes of Health 

AURIN M. CHASE, Princeton University 

SEYMOUR S. COHEN, University of Pennsylvania School of Medicine 

DONALD P. COSTELLO, University of North Carolina 

TERU HAYASHI, Columbia University 

DOUGLAS A. MARSLAND, New York University, Washington Square College 

H. BURR STEINBACH, University of Chicago 

TO SERVE UNTIL 1962 

FRANK A. BROWN, JR., Northwestern University 

SEARS CROWELL, Indiana University 

ALBERT I. LANSING, University of Pittsburgh Medical School 

WILLIAM D. MCLROY, Johns Hopkins University 

C. LADD PROSSER, University of Illinois 
S. MERYL ROSE, University of Illinois 

MARY SEARS, Woods Hole Oceanographic Institution 
ALBERT TYLER, California Institute of Technology 



TO SERVE UNTIL 1961 

ERIC BALL, Harvard University Medical School 

D. W. BRONK, Rockefeller Institute 

G. FAILLA, Columbia University, College of Physicians & Surgeons 

R. T. KEMPTON, Vassar College 

L. H. KLEINHOLZ, Reed College 

IRVING M. KLOTZ, Northwestern University 

ALBERT SZENT-GYORGYI, Marine Biological Laboratory 

WM. RANDOLPH TAYLOR, University of Michigan 



TRUSTEES 

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES 

GERARD SWOPE, JR., ex officio, Chairman W. D. MCELROY, 1961 

JAMES H. WICKERSHAM, ex officio F. A. BROWN, JR., 1961 

ARTHUR K. PARPART, ex officio JOHN BUCK, 1962 

P. B. ARMSTRONG, ex officio ALBERT I. LANSING, 1962 

KENNETH S. COLE, 1963 STEPHEN KUFFLER, 1963 

THE LIBRARY COMMITTEE 

MARY SEARS, Chairman ANTHONY C. CLEMENT 

SEYMOUR S. COHEN C. LADD PROSSER 

THE APPARATUS COMMITTEE 

ALBERT I. LANSING, Chairman RALPH H. CHENEY 

HARRY GRUNDFEST FREDERIK BANG 

HOWARD K. SCHACHMAN 

THE SUPPLY DEPARTMENT COMMITTEE 

RUDOLF T. KEMPTON, Chairman GROVER C. STEPHENS 

SEARS CROWELL DAVID BISHOP 

THE EVENING LECTURE COMMITTEE 

PHILIP B. ARMSTRONG, Chairman DONALD P. COSTELLO 

H. BURR STEINBACH S. MERYL ROSE 

FRANK A. BROWN, JR. 

THE INSTRUCTION COMMITTEE 

JOHN B. BUCK, Chairman BOSTWICK KETCHUM 

ARNOLD LAZAROW JAMES \V. GREEN 

TERU HAYASHI 

THE BUILDINGS AND GROUNDS COMMITTEE 

EDGAR ZWILLING, Chairman JAMES CASE 

MORRIS ROCKSTEIN DANIEL GROSCH 

THE RADIATION COMMITTEE 

G. FAILLA, Chairman WALTER L. WILSON 

ROGER L. GREIF WALTER S. VINCENT 

CARL C. SPEIDEL 

THE RESEARCH SPACE COMMITTEE 

PHILIP B. ARMSTRONG, Chairman MAC V. EDDS, JR. 

ARTHUR K. PARPART W T ILLIAM D. MCLROY 



4 MARINE BIOLOGICAL LABORATORY 

II. ACT OF INCORPORATION 

No. 3170 

COMMONWEALTH OF MASSACHUSETTS 

Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. 
Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, 
William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves 
with the intention of forming a Corporation under the name of the Marine Biological 
Laboratory, for the purpose of establishing and maintaining a laboratory or station for 
scientific study and investigation, and a school for instruction in biology and natural his- 
tory, and have complied with the provisions of the statutes of this Commonwealth in such 
case made and provided, as appears from the certificate of the President, Treasurer, and 
Trustees of said Corporation, duly approved by the Commissioner of Corporations, and 
recorded in this office; 

Nozv, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- 
setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi- 
ner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, 
their associates and successors, are legally organized and established as, and are hereby 
made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB- 
ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties, 
and restrictions, which by law appertain thereto. 

Witness my official signature hereunto subscribed, and the seal of the Commonwealth 
of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord 
One Thousand Eight Hundred and Eighty-Eight. 

[SEAL] HENRY B. PIERCE, 

Secretary of the Commonwealth. 



III. BY-LAWS OF THE CORPORATION OF THE MARINE 
BIOLOGICAL LABORATORY 

I. The members of the Corporation shall consist of persons elected by the Board of 
Trustees. 

II. The officers of the Corporation shall consist of a President, Vice President, 
Director, Treasurer, and Clerk. 

III. The Annual Meeting of the members shall be held on the Friday following the 
second Tuesday in August in each year at the Laboratory in Woods Hole, Massachusetts, 
at 9:30 A.M., and at such meeting the members shall choose by ballot a Treasurer and a 
Clerk to serve one year, and eight Trustees to serve four years, and shall transact such 
other business as may properly come before the meeting. Special meetings of the mem- 
bers may be called by the Trustees to be held at such time and place as may be designated. 

IV. Twenty-five members shall constitute a quorum at any meeting. 

V. Any member in good standing may vote at any meeting, either in person or by 
proxy duly executed. 

VI. Inasmuch as the time and place of the Annual Meeting of members are fixed by 
these By-laws, no notice of the Annual Meeting need be given. Notice of any special 



BY-LAWS OF THE CORPORATION 

meeting of members, however, shall be given by the Clerk by mailing notice of the time 
and place and purpose of such meeting, at least fifteen (15) days before such meeting, 
to each member at his or her address as shown on the records of the Corporation. 

VII. The Annual Meeting of the Trustees shall be held promptly after the Annual 
Meeting of the Corporation at the Laboratory in Woods Hole, Mass. Special meetings 
of the Trustees shall be called by the President, or by any seven Trustees, to be held at 
such time and place as may be designated, and the Secretary shall give notice thereof by 
written or printed notice, mailed to each Trustee at his address as shown on the records 
of the Corporation, at least one ( 1 ) week before the meeting. At such special meeting 
only matters stated in the notice shall be considered. Seven Trustees of those eligible to 
vote shall constitute a quorum for the transaction of business at any meeting. 

VIII. There shall be three groups of Trustees: 

(A) Thirty-two Trustees chosen by the Corporation, divided into four classes, each 
to serve four years. After having served two consecutive terms of four years each. 
Trustees are ineligible for re-election until a year has elapsed. In addition, there shall 
be two groups of Trustees as follows : 

(B) Trustees ex officio, who shall be the President and Vice President of the Cor- 
poration, the Director of the Laboratory, the Associate Director, the Treasurer, and the 
Clerk : 

(C) Trustees Emeriti, who shall be elected from present or former Trustees by the 
Corporation. Any regular Trustee who has attained the age of seventy years shall con- 
tinue to serve as Trustee until the next Annual Meeting of the Corporation, whereupon 
his office as regular Trustee shall become vacant and be filled by election by the Corpora- 
tion and he shall become eligible for election as Trustee Emeritus for life. The Trustees 
ex officio and Emeriti shall have all the rights of the Trustees except that Trustees 
Emeritus shall not have the right to vote. 

The Trustees and officers shall hold their respective offices until their successors are 
chosen and have qualified in their stead. 

IX. The Trustees shall have the control and management of the affairs of the Cor- 
poration ; they shall elect a President of the Corporation who shall also be Chairman of 
the Board of Trustees and who shall be elected for a term of five years and shall serve 
until his successor is selected and qualified ; and shall also elect a Vice President of the 
Corporation who shall also be the Vice Chairman of the Board of Trustees and who shall 
be elected for a term of five years and shall serve until his successor is selected and 
qualified ; they shall appoint a Director of the Laboratory ; and they may choose such 
other officers and agents as they may think best ; they may fix the compensation and define 
the duties of all the officers and agents ; and may remove them, or any of them, except 
those chosen by the members, at any time ; they may fill vacancies occurring in any 
manner in their own number or in any of the offices. The Board of Trustees shall have 
the power to choose an Executive Committee from their own number, and to delegate to 
such Committee such of their own powers as they may deem expedient. They shall from 
time to time elect members to the Corporation upon such terms and conditions as they 
may think best. 

X. The Associates of the Marine Biological Laboratory shall be an unincorporated 
group of persons (including associations and corporations) interested in the Laboratory 
and shall be organized and operated under the general supervision and authority of the 
Trustees. 



MARINE BIOLOGICAL LABORATORY 

XI. The consent of every Trustee shall be necessary to dissolution of the Marine 
Biological Laboratory. In case of dissolution, the property shall be disposed of in such 
manner and upon such terms as shall be determined by the affirmative vote of two-thirds 
of the Board of Trustees. 

XII. The account of the Treasurer shall be audited annually by a certified public 
accountant. 

XIII. These By-laws may be altered at any meeting of the Trustees, provided that the 
notice of such meeting shall state that an alteration of the By-laws will be acted upon. 



IV. REPORT OF THE DIRECTOR 

To : THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY 
Gentlemen : 

I submit herewith the Report of the seventy-third session of the Marine Bio- 
logical Laboratory. 

1. Policy 

During the past year there were several extended discussions on the advisability 
of developing year-round programs in Marine Biology at the Laboratory. Several 
alternatives were suggested with reservations expressed on the advisability of 
establishing a year-round program staffed with permanent personnel, if these staff 
members were to be employed by the Marine Biological Laboratory. It was voted 
that the Laboratory should do everything possible to assist in establishing in the 
Woods Hole area an independent institute for basic research in the broad field 
of Marine Biology. 

2. Land Acquisitions 

Four parcels of land were acquired during the year, including the Veeder prop- 
erty, the Broderick property and the Tinkham property on Albatross Street. In- 
cluded were four residences. The small cottage on the Tinkham Property is in 
very bad condition and unfit for housing purposes. The Breakwater Hotel property 
on Bar Neck Road was also purchased and the hotel razed, leaving a free area of 
1.4 acres designed to be used as a site for a combination dormitory-dining hall for 
which tentative plans have been developed. The total land area acquired by these 
purchases increases the land holdings of the Laboratory in the immediate vicinity 
of its campus by 2.2 acres. 

3. New Laboratory Building 

One of the feature events of the year was the completion of the new laboratory 
building in time for summer occupancy. Included in this building are 61 labora- 
tories, 10 being used as general radiobiological service laboratories, 5 constant tem- 
perature rooms, 16 dark rooms, 1 aquarium room, 1 conference room, 1 lecture 



REPORT OF THE DIRECTOR / 

room accommodating 140 people, 1 photo laboratory and 4 dry rooms. The sum- 
mer occupancy demonstrated the adaptability of the new building to a wide variety 
of research activities. Also, the building stood up very effectively through Hurri- 
cane Donna and other severe storms, and proved to be a watertight building, con- 
trary to the Laboratory's experience with buildings of brick construction. 

4. Personnel Changes 

It is the policy of the Laboratory that the heads of the various training programs 
will serve for a period of five years. Dr. Nelson T. Spratt, Jr., will serve as head 
of the training program in Experimental Embryology during the summer of 1961 
and will be succeeded by Dr. James D. Ebert. Dr. Clark P. Read succeeds Dr. 
Grover C. Stephens as head of the training program in Invertebrate Zoology. By 
action of the Executive Committee, three additional training programs at the post- 
doctoral level will be established starting in the summer of 1962. These will be 
in Marine Microbiology, Problems of Fertility, and Comparative Physiology, to be 
headed respectively by Drs. W. D. McElroy, C. B. Metz and C. Ladd Prosser. 

5. Laboratory Fees 

During the past several years, the fees paid by investigators for laboratory space 
and the included services have covered only one-sixth of the cost of operation of 
such a laboratory. It was voted by the Executive Committee to gradually increase 
these fees over a period of two years so as to finally increase the fees to cover one- 
third of these costs. The Executive Committee also voted to have a weekly inclu- 
sive dormitory charge to cover both board and room, patterned after the usual 
operation as seen in the colleges and universities. 

6. Plant Changes 

During the summer of 1960 all of the training programs operated throughout 
the summer instead of two groups, each group operating for half the summer. In 
order to accommodate all of the training programs concurrently, the Old Lecture 
Hall was completely remodeled to accommodate the training program in Experi- 
mental Embryology. The ground floor provides a general student laboratory, the 
second floor a series of laboratories for special research procedures. 

7. Grants, Contracts and Contributions, in Support of Laboratory Activities, 
Including Training Grants 

The total income from these sources of support amounts to $373,000 in 1960. 
This represents 44% of the total income and is made up of the following accounts : 

Training grants for the courses from NIH and NSF, support for regular re- 
search activities from NIH, NSF, AEC and ONR and gifts from the MBL Asso- 
ciates, Josephine C. Crane Foundation, The Rockefeller Foundation, The George 
F. Jewett Foundation and the following pharmaceutical companies : The Merck Co. 
Foundation, Carter Products, Inc., C. I. B. A. Pharm. Products Inc., Abbott 
Laboratories, Schering Foundation, Inc., Eli Lilly & Company, the Upjohn Com- 
pany and E. R. Squibb & Sons. 



8 MARINE BIOLOGICAL LABORATORY 

8. Future Plans 

With the acquisition of the Breakwater Property the Laboratory now has a 
proper site for the location of the projected dormitory-dining hall. The Officers 
of the Corporation are exploring various sources of funds for this construction 
which is so necessary for the solution of problems of congestion and parking difficul- 
ties in our campus. In addition funds are being sought for the construction of 
additional cottages in the Devil's Lane Tract. 

Respectfully submitted, 
PHILIP B. ARMSTRONG, 

Director 

MEMORIALS 

Ross GRANVILLE HARRISON 

:' ' ' ., by ; 

Chester L. Yntema 

Ross Granville Harrison died September 30, 1959, after a full life of 89 years. During 
his lifetime biology became a modern science. His contributions based on critical experi- 
mentation were a great factor in this maturation and his example has been an inspiration 
to biologists. 

Dr. Harrison was born January 13, 1870, in Germantown, Pennsylvania. His under- 
graduate and graduate work was done at Johns Hopkins ; he received his Doctorate of 
Philosophy in 1894. The thesis on the embryological origin of the rays of the fins in 
teleosts was done with Dr. Brooks as his teacher. Five years later, after intermittent 
study in Germany, he was awarded the degree of Doctor Medicine by the University 
of Bonn. 

After receiving his Ph.D., Dr. Harrison taught at Bryn Mawr for a year and then 
studied for a year in Germany. In 1896 he returned to Johns Hopkins to join the depart- 
ment of anatomy headed by Dr. Mall. In 1907 he accepted the Bronson professorship of 
comparative anatomy at Yale and the remainder of his career continued with Yale as 
its base. 

Early in his stay at Yale, the Osborn Laboratories were built for the Departments 
of Botany and Zoology and these buildings continue to serve the departments. In addi- 
tion, the science departments at Yale became University departments as he demanded. 
This recognition is so generally given today that it is difficult to realize that the issue 
once had to be made and pressed. 

Dr. Woodruff and Dr. Petrunkevitch joined Dr. Coe and Dr. Harrison; these four 
became central and lasting figures in a zoology department which was outstanding in 
both its undergraduate and graduate programs. An increasing number of graduate 
students and foreign fellows came to the Osborn Laboratories and for many years there 
was a group of students pursuing their thesis work under Dr. Harrison. 

In his scientific research Dr. Harrison furthered the concept of an experimental 
approach to embryology initiated by Roux and Driesch and he devised means of analyzing 
development. In part, his genius consisted of picking a critical experiment bearing on 
a basic problem and performing the experiment in an uncomplicated way. This approach 
is illustrated by his cultivation of neuroblasts from the neural tube of the frog embryo 
in hanging drops of frog lymph, and following the growth of the processes from these 
cells by repeated microscopic observations. By this one procedure he settled the con- 
troversy over the origin of nerve cell processes, and in addition devised the technique 



REPORT OF THE DIRECTOR 9 

of tissue culture for animal cells and tissues which has come to be a standard biological 
procedure. After other pioneer studies with explanation, he developed and refined 
means of transplantation for amphibian embryos which he and many others have used. 
The analyses of development he undertook included studies of the lateral line organs, 
the neural crest, the polarization of the limb and the internal ear, and growth rates in 
heteroplastic transplants. 

In each of his many reports, the same standard of perfection is maintained. In a 
clear and obvious way, a basic problem is resolved by results from simple experiments 
ingeniously devised and applied. 

Dr. Harrison was granted an honorary master's degree from Yale University in 1907 
and honorary doctor's degrees from Yale and Cincinnati in 1920. These honors were 
followed by similar recognition from several other institutions in this country and in 
Europe. He was a member of many learned societies and the recipient of awards given 
in recognition of his achievements. 

Dr. Harrison's memberships in societies and academies of other countries indicate 
the regard held for him. He was a member of the Royal Society of Lund, the Royal 
Society of Uppsala, and the German Anatomical Society, of which he was a president. 
He was a corresponding member of the Gottingen Academy of Science, the German 
Academy of Sciences, the Bavarian Academy of Science, the Academy of Sciences of 
the Institute of France, and the Society of Biology of Paris. He was an honorary 
member of the Royal Academy of Turin and the Royal Academy of Belgium. He was 
a foreign correspondent of the Academy of Science of the Institute of Bologna. He was 
a foreign associate of the Academy of Medicine of Paris. He was a foreign member of 
the Royal Netherlands Academy of Science, the Norwegian Academy of Science, the 
National Academy of Rome, the Royal Swedish Academy of Stockholm, the Zoological 
Society of London and the Royal Society of London. 

The professional activities of Dr. Harrison included many administrative responsi- 
bilities in addition to those that he met at Yale. His interest in marine laboratories is 
evident from his connections with such institutions. He served as a Trustee of this 
Laboratory from 1908 to 1940 and then became a Trustee Emeritus. In addition he was 
a Trustee of the Woods Hole Oceanographic Institution and the Bermuda Biological 
Station. He served as president of several scientific societies. He was a member of 
the boards of the Rockefeller Institute of Medical Research and the Jane Coffin Childs 
Fund for Medical Research. 

From 1903 to 1946, Dr. Harrison was Managing Editor of the Journal of Experi- 
mental Zoology; he imprinted upon this publication a standard of excellence which is a 
challenge to contributors. 

For many years, Dr. Harrison was a councilor and member of the executive com- 
mittee of the National Academy of Sciences. After his retirement from Yale he was 
chairman of the National Research Council from 1938 to 1946. During this period, 
which included the Second World War, he handled responsibilities for the national gov- 
ernment with which the National Academy and the National Research Council were 
faced. During the same period he was a member of the science committee of the 
National Resources Planning Board. Later he served on the United States National 
Committee for the United Nations Educational Scientific and Cultural Organization. 

With all his accomplishments, Dr. Harrison was modest, self-contained and retiring. 
He had a deep regard for the individualities of others. This was particularly evident to 
those who were graduate students under him. He himself set an example of application 
and devotion to his work ; others could determine their own pace and ways without com- 
ment or persuasion. He had no understanding of incompetence but he overlooked human 
foolishness and foolhardiness. His practice of sharing his luncheon hour with students 
was of great value which came to be appreciated more fully with passing years. During 



10 MARINE BIOLOGICAL LABORATORY 

this hour of sandwiches and tea no mention of scientific interests was recognized. Con- 
sequently a variety of topics was covered under the wise and sympathetic aegis of Dr. 
Harrison. We came to appreciate and be influenced by his wide range of knowledge and 
interests, his great understanding, and his whimsical humor. 

Dr. Harrison is survived by his wife, Mrs. Ida Lange Harrison, whom he met in 
Germany and married in 1896. Also surviving him are their three daughters and two 
sons. In honoring the memory of Dr. Harrison we wish to convey to his family a sense 
of our indebtedness and appreciation for his many years with us. 

LEWIS VICTOR HEILBRUNN 

by 
H. B. Steinbach 

This is a note in memory of Lewis Victor Heilbrunn who died in an automobile acci- 
dent early last fall. If a memorial could echo the nature of the man, it would be vig- 
orous, terse, highly intelligent and very human. 

Lewis Victor Heilbrunn was one of the most influential figures of modern cellular 
biology, not only through his books and scientific papers but through his impact on his 
students and associates. He had the special knack of bringing out and fostering the 
intellectual best of those who worked with him. In large part this must have been due 
to the fact that he spent his life as an eager searcher after truth, not as a repository of 
the truth. Thus, those who talked to him of their problems, scientific and otherwise, 
found themselves discussing the problems and arriving at conclusions rather than being 
told answers. Surely this is at the heart of all good teaching, and Heilbrunn was its 
best exponent. 

His life was intimately associated with the Marine Biological Laboratory, and he 
loved the institution with a fierce devotion. 

The records show him first appearing here as a student investigator in 1912 at the 
age of twenty. He was elected a Member of the Corporation in 1915. The Director's 
report for this year records that Heilbrunn, with a few others, was responsible for raising 
the sum of twelve dollars to enable the library to subscribe to the British Journal of 
Physiology. 

He was elected a Trustee in 1931 and to the Executive Committee in 1934, and over 
the years continued to serve the MBL in a variety of capacities. While his services to 
the Laboratory may lose their sharpness with the death of the man, they do not cease. 
At least eighteen active members of the Corporation received their doctorate degrees 
under his direction, as did four who have served or are serving on the Executive Com- 
mittee. An equal number of workers active in the interests of the Laboratory gladly 
would acknowledge their direct debt to his training. 

In 1917 and 1918 his name does not appear on the attendance records of the Labora- 
tory. During these years he served as a pilot in the then new air force of his country. 
In a parenthetical way it could be noted that it is quite consistent with the essential 
daring of the man that he should be an accomplished pilot of an aircraft fifteen years 
before he learned to drive a car. 

In 1919 the record shows him in attendance as an Independent Investigator from 
Brooklyn, New York, his home town. The record does not spell out the circumstances 
but one can be sure that he paused but briefly at home upon demobilization and then 
took off at once for his beloved Woods Hole. 

Heilbrunn's professional career is recorded in other places and need not be repeated 
here. He was a great man, not to be illustrated by a recital of data. Time may dim 
the memory but his influence will be great for years to come. In the absence of ade- 



REPORT OF THE DIRECTOR 11 

quate words the true nature of the man will be found residing in the memories of those 
who had the pleasures and the jolts of working with him. He was a catalyst, an arouser. 
Some awoke to anger, to difference but this was productive; some he awoke to curiosity, 
to the equable search; some he awoke to fire, to the necessity of looking and thinking 
and doing day and night, in dreams as in waking, for the truth that man must seek in 
the laboratory, in the university, in life. 

To work with Heilbrunn was to be a part of his family. The interests of the world 
were the subjects of his cosmic classroom, housed alike in the laboratory, ice cream 
socials and the soft-ball field. We extend to his widow, Ellen Donovan Heilbrunn and 
to his daughter Constance our understanding sympathy and our gratitude for sharing 
him with us. 

ZOOLOGY 

I. CONSULTANTS 

F. A. BROWN, JR., Professor of Zoology, Northwestern University 
LIBBIE H. HYMAN, American Museum of Natural History 

A. C. REDFIELD, Woods Hole Oceanographic Institution 

II. INSTRUCTORS 

GROVER C. STEPHENS, Associate Professor of Zoology, University of Minnesota, in charge 
of the course 

MILTON FINGERMAN, Assistant Professor of Zoology, Newcomb College, Tulane Uni- 
versity 

BERNARD L. STREHLER, Chief, Cellular and Comparative Physiology, Division of Geron- 
tology, National Institutes of Health 

PAUL P. WEINSTEIN, Laboratory of Tropical Disease, National Institutes of Health 

RICHARD C. SANBORN, Professor of Zoology, Department of Biological Sciences, Purdue 
University 

JAMES CASE, Associate Professor of Zoology, State University of Iowa 

A. FARMANFARMAIAN, Research Associate, University of California 

G. F. GWILLIAM, Assistant Professor of Biology, Reed College 

III. ASSISTANTS 

ROBERT ASHMAN, Wabash College 
JONATHAN P. GREEN, University of Minnesota 

EMBRYOLOGY 
I. INSTRUCTORS 

MAC V. EDDS, JR., Professor of Biology, Brown University, in charge of the course 
PHILIP GRANT, Assistant Professor of Pathobiology, The Johns Hopkins University 
LIONEL I. REBHUN, Assistant Professor of Biology, Princeton University 
JOHN W. SAUNDERS, JR., Professor of Zoology, Marquette University 
NELSON T. SPRATT, JR., Professor of Zoology, University of Minnesota 
MAURICE SUSSMAN, Associate Professor of Biology, Brandeis University 

II. LABORATORY ASSISTANTS 

DAVID SONNEBORN, Brandeis University 
RICHARD WHITTAKER, Yale University 



12 MARINE BIOLOGICAL LABORATORY 

PHYSIOLOGY 

I. CONSULTANTS 

MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania 
OTTO LOEWI, Professor of Pharmacology, New York University School of Medicine 
ARTHUR K. PARPART, Professor of Biology, Princeton University 

ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Marine Biological 
Laboratory 

II. INSTRUCTORS 

W. D. MCELROY, Director, McCollum-Pratt Institute, The Johns Hopkins University; 

in charge of the course 

FRANCIS D. CARLSON, Associate Professor of Biophysics, The Johns Hopkins University 
BERNARD D. DAVIS, Professor of Pharmacology, Harvard Medical School 
DONALD R. GRIFFIN, Professor of Zoology, Harvard University 
TIMOTHY H. GOLDSMITH, Society of Fellows, Harvard University 
ROBERT B. LOFTFIELD, Massachusetts General Hospital 

BOTANY 
I. CONSULTANT 
WILLIAM RANDOLPH TAYLOR, Professor of Botany, University of Michigan 

II. INSTRUCTORS 

RICHARD C. STARR, Associate Professor of Botany, Indiana University, in charge <>! Ihr 

course 

WALTER R. HERNDON, Associate Professor of Botany, University of Alabama 
JOHN M. KINGSBURY, Assistant Professor of Botany, Cornell University 

III. COLLECTOR 
JOYCE FLETCHER, New York Botanical Garden 

IV. LABORATORY ASSISTANTS 

MELVIN GOLDSTEIN, Indiana University 

PHILIP COOK, Botany Department, Indiana University 

ECOLOGY 

I. CONSULTANTS 

PAUL GALTSOFF, U. S. Fish and Wildlife Service, W'oods Hole 

ALFRED S. REDFIELD, Woods Hole Oceanographic Institution 

BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution 

EDWIN T. MOUL, Rutgers University 

CHARLES E. JENNER, University of North Carolina 

HOWARD T. ODUM, University of Texas 



REPORT OF THE DIRECTOR 13 

II. INSTRUCTORS 

EUGENE P. ODUM, Alumni Foundation Professor of Zoology, University of Georgia, in 

charge of the course 

JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution 
HOWARD L. SANDERS, Woods Hole Oceanographic Institution 
WALTER R. TAYLOR, Chesapeake Bay Institute, The Johns Hopkins University 

III. LABORATORY ASSISTANTS 

RICHARD B. WILLIAMS, Harvard University and Marine Institute, University of Georgia 
ELIJAH V. SWIFT, Swarthmore College 

1. THE LABORATORY STAFF, 1960 
HOMER P. SMITH, General Manager 

MRS. DEBORAH LAWRENCE HARLOW, Librarian ROBERT KAHLER, Superintendent, 

CARL O. SCHWEIDENBACK, Manager of the Buildings and Grounds 

Supply Department ROBERT B. MILLS, Manager, DC- 
IRVINE L. BROADBENT, Office Manager velopment of Research Service 

GENERAL OFFICE 

MRS. LILA S. MYERS MRS. MARION C. CHASE 

MRS. VIVIEN R. BROWN MRS. VIVIAN I. MANSON 

MRS. VIRGINIA M. MOREHOUSE MRS. RUTH MAYO 

LIBRARY 

MRS. GWENDOLYN S. BLOMBERG JANICE PARENT 

ALBERT K. NEAL 

MAINTENANCE OF BUILDINGS AND GROUNDS 

ROBERT ADAMS RALPH H. LEWIS 

ELDON P. ALLEN RUSSELL F. LEWIS 

GARDNER GAYTON ALAN G. LUNX 

ROBERT GUNNING ALTON J. PIERCE 

ROBERT W. HAMPTON ROBERT H. WALKER, JR. 

WALTER J. JASKUN JAMES S. THAYER 
DONALD B. LEHY 

DEPARTMENT OF RESEARCH SERVICE 

GAIL M. CAVANAUGH CAROLINE MCDAXIEL 

SEAVER R. HARLOW 

SUPPLY DEPARTMENT 

DONALD P. BURNHAM BRUNO F. TRAPASSO 

MILTON B. GRAY MRS. PATRICIA TRAVARES 

GEOFFREY J. LEHY JOHN J. VALOIS 

ROBERT O. LEHY JARED L. VINCENT 

ROBERT M. PERRY HALLETT S. \\~AGSTAFF 



14 MARINE BIOLOGICAL LABORATORY 

2. INVESTIGATORS; LALOR AND LILLIE FELLOWS; AND STUDENTS 

Independent Investigators, 1960 

ADAMS, RALPH G., National Institutes of Health 

ADELMAN, WILLIAM J., JR., Physiologist, National Institutes of Health 

ALLEN, M. JEAN, Professor of Biology and Chairman, Wilson College 

AMATNIEK, ERNEST, Biophysicist, College of Physicians and Surgeons 

AMBERSON, WILLIAM R., Marine Biological Laboratory 

ANDERSON, JOHN MAXWELL, Professor of Zoology, Cornell University 

ARMSTRONG, PHILIP B., Professor and Chairman, Department of Anatomy, Upstate Medical 

Center 

ATWOOD, KIMBALL C, Associate Professor of Medical Genetics, University of Chicago 
BALTUS, ELYANE, Research Associate, Free University of Brussels, Belgium 
BANG, FREDERIK B., Chairman, Department of Pathobiology, Johns Hopkins School of Hygiene 
BARTH, L. G., Professor of Zoology, Columbia University 
BAYLOR, MARTHA B., Marine Biological Laboratory 
BEEVERS, HARRY, Professor of Biology, Purdue University 
BELTON, PETER, College of Physicians and Surgeons, Columbia University 
BENNETT, MICHAEL V. L., Assistant Professor of Neurology, College of Physicians and Surgeons 
BERENDSEN, HERMAN J. C., Staff Member, Massachusetts Institute of Technology 
BERGER, CHARLES A., Chairman, Biology Department, Fordham University 
BERNSTEIN, MAURICE H., Assistant Professor, Wayne State University 
BORGESE, THOMAS A., Research Fellow in Medicine, Harvard Medical School 
BRADLEY, DAN F., Scientist, Commissioned Corps of U. S. Public Health Service 
BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University 
BUCK, JOHN B., Physiologist, National Institutes of Health 
BUDINGER, THOMAS F., Woods Hole Oceanographic Institution 
BURBANCK, W. D., Professor of Biology, Emory University 
BURKE, JOSEPH A., Assistant Professor of Biology, Loyola College 
CABRERA, GUILLERMO, Assistant Professor of Biochemistry, New York University College of 

Dentistry 

CARLSON, FRANCIS D., Associate Professor, The Johns Hopkins University 
CASE, JAMES, Assistant Professor of Zoology, State University of Iowa 
CHAET, ALFRED B., Associate Professor of Biology, The American University 
CHALAZONITIS, N., Charge de Recherches, Faculte des Sciences, Lyon, France 
CHANDLER, WILLIAM K., Medical Officer, Public Health Service 
CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College 
CHILD, FRANK M., Instructor in Zoology, University of Chicago 
CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School 
CLARK, ARNOLD M., Professor of Biology, University of Delaware 
CLEMENT, ANTHONY C., Professor of Biology, Emory University 
COHEN, BERNARD, Visiting Scholar, Columbia University 

COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health 
COLLIER, JACK R., Marine Biological Laboratory 

COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve School of Medi- 
cine 

COPELAND, EUGENE, Chairman, Zoology Department, Tulane University 
COSTELLO, DONALD P., Kenan Professor of Zoology, University of North Carolina 
CRANE, ROBERT K., Associate Professor of Biological Chemistry, Washington University Medi- 
cal School 

CROWELL, SEARS, Associate Professor in Zoology, Indiana University 
DAVIS, BERNARD D., Head, Department of Bacteriology, Harvard Medical School 
DAVISON, JOHN A., Assistant Professor of Zoology, Florida State University 
DAVSON, HUGH, Medical Research Council and University College, London 
DETTBARN, WOLF D., Research Associate, College of Physicians & Surgeons 
DOWBEN, ROBERT M., Assistant Professor of Medicine, Northwestern University 



REPORT OF THE DIRECTOR 15 

DuBois, ARTHUR B., Associate Professor of Physiology, University of Pennsylvania, Graduate 

School of Medicine 

EDDS, MAC V., JR., Professor of Biology, Brown University 
EDWARDS, CHARLES, Associate Professor of Physiology, University of Minnesota 
EHRENPREIS, SEYMOUR, Assistant Professor, College of Physicians & Surgeons 
EICHEL, HERBERT J., Research Associate, Hahnemann Medical College 
FAILLA, G., Professor, Columbia University 
FAILLA, PATRICIA, Columbia University 

FARMANFARMAIAN, ALLAHVERDI, Research Associate, University of California 
FELDHERR, CARL, Physiologist, College of Physicians & Surgeons 
FIELD, JAMES B., Senior Investigator, National Institutes of Health 

FINGERMAN, MILTON, Assistant Professor of Zoology, Newcomb College of Tulane University 
FISCHER, SIEGMUND, Research Associate, Albert Einstein College of Medicine 
FLAKS, JOEL G., Associate, Department of Biochemistry, University of Pennsylvania School of 

Medicine 

FULTON, CHANDLER M., Assistant Professor of Biology, The Rockefeller Institute 
FUORTES, M. G. F., Chief, Neurophysiology Section, National Institutes of Health 
FURSHPAN, EDWIN J., Instructor in Neurophysiology, Harvard Medical School 
GLADE, RICHARD W., Assistant Professor of Zoology, University of Vermont 
GOLDSMITH, TIMOTHY H., Junior Fellow, Harvard University 
GRANT, PHILIP, Assistant Professor of Pathobiology, The Johns Hopkins University School 

of Hygiene 

GREEN, JAMES W., Associate Professor of Physiology, Rutgers University 
GREGG, JAMES H., Associate Professor of Biology, University of Florida 
GRIFFIN, DONALD R., Professor of Zoology, Harvard University 
GROSCH, DANIEL S., Professor of Genetics, North Carolina State College 
GROSS, PAUL R., Associate Professor of Biology, New York University 
GRUNDFEST, HARRY, Associate Professor of Neurology, College of Physicians & Surgeons 
GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College 
GWILLIAM, GILBERT F., Assistant Professor of Biology, Reed College 
HAGERMAN, DWAIN D., Associate in Biological Chemistry, Harvard Medical School 
HAGINS, WILLIAM A., Physiologist, National Institutes of Health 

HARDING, CLIFFORD V., Assistant Professor of Physiology, College of Physicians & Surgeons 
HARVEY, ETHEL BROWNE, Marine Biological Laboratory 
HAYASHI, TERU, Professor of Zoology, Columbia University 

HEGYELI, ANDREW F., Biochemist, Institute for Muscle Research, Marine Biological Laboratory 
HEINMETS, FERDINAND, Head, Physiology Laboratory, Research and Engineering Center, Natick 
HENLEY, CATHERINE, Research Associate, University of North Carolina 
HERNDON, WALTER R., Associate Professor of Botany, University of Alabama 
HERVEY, JOHN P., Senior Electronic Engineer, Marine Biological Laboratory 
HIRSHFIELD, HENRY I., Associate Professor of Biology, New York University 
HOLTZER, HOWARD, Associate Professor of Anatomy, University of Pennsylvania, School of 

Medicine 

HOLZ, GEORGE G., JR., Associate Professor of Zoology, Syracuse University 
HOSKIN, FRANCIS C. G., Assistant Professor, College of Physicians & Surgeons 
HURWITZ, JERARD, Assistant Professor of Microbiology, New York University 
ISENBERG, IRVIN, Institute for Muscle Research, Marine Biological Laboratory 
JOHNSON, SISTER MARIA BENIGNA, Professor of Biology, Saint Joseph College 
KAAN, HELEN W., Marine Biological Laboratory 

KAMINER, BENJAMIN, Senior Lecturer in Physiology, University of Witwatersrand, Johannes- 
burg, S. A. 

KANE, ROBERT E., Assistant Professor of Biochemistry, Brandeis University 
KEMPTON, RUDOLF T., Professor of Zoology, Vassar College 
KEOSIAN, JOHN, Professor of Biology, Rutgers University 

KESSEL, RICHARD G., Instructor in Anatomy, Bowman Gray School of Medicine of Wake Forest 
KINGSBURY, JOHN M., Assistant Professor of Botany, Cornell University 
KISHIMOTO, UICHIRO, Rockefeller Fellow, National Institutes of Health 
KITAI, STEPHEN T., Graduate Student, Wayne State University and Lafayette Clinic 



16 MARINE BIOLOGICAL LABORATORY 

KRANE, STEPHEN M., Assistant in Medicine, Massachusetts General Hospital 

KUFFLER, STEPHEN W., Professor of Neurophysiology, Harvard Medical School 

KURY, LIVIA REV., Institute for Muscle Research, Marine Biological Laboratory 

LAMBERT, FRANCIS L., Jacques Loeb Associate, Rockefeller Institute 

LANSING, ALBERT I., Professor of Anatomy, University of Pittsburgh School of Medicine 

LAURIE, JOHN S., Assistant Professor of Experimental Biology, University of Utah 

LAZAROW, ARNOLD, Professor and Head, Department of Anatomy, University of Minnesota 

LEVY, MILTON, Professor and Chairman, Department of Biochemistry, New York University 

College of Dentistry 

LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont 
LOEWENSTEIN, WERNER R., Associate Professor of Physiology, Columbia University 
LOFTFIELD, ROBERT B., Associate Biochemist, Massachusetts General Hospital 
LONDON, IRVING M., Professor of Medicine, Albert Einstein College of Medicine 
LOOMIS, WILLIAM F., Professor of Biochemistry, Brandeis University 

DELoRENZO, A. J., Director Anatomical Research Laboratories, Johns Hopkins Medical School 
LOVE, WARNER E., Assistant Professor of Biophysics, The Johns Hopkins University 
Luco, J. V., Professor of Neurophysiology, Catholic University Medical School, Santiago, Chile 
LYNCH, REV. WILLIAM F., St. Ambrose College 
MAHLER, HENRY R., Professor of Chemistry, Indiana University 
MANN, DAVID E., Professor of Pharmacology, Temple University 

MANN, THADDEUS R. R., Director, Unit of Reproductive Physiology and Biochemistry, Uni- 
versity of Cambridge, England 

MARGALEF, RAMON, Institute Investigaciones Pesqueras, Paseo Nacional, Barcelona, Spain 
MARSLAND, DOUGLAS, Professor of Biology, New York University, Washington Square College 
MATEYKO, G. M., Assistant Professor of Biology, New York University, Washington Square 

College 

MATHEWS, MARTIN B., Associate Professor, University of Chicago 

MCELROY, W. D., Chairman, Department of Biology and Director, McCollum-Pratt Institute 
METZ, CHARLES B., Professor of Zoology, Florida State University 
METZ, CHARLES W., Marine Biological Laboratory 

MIDDLEBROOK, W. ROBERT, Institute for Muscle Research, Marine Biological Laboratory 
MILLER, JAMES A., Professor of Anatomy, Emory University 
MITCHISON, J. M., Reader, University of Edinburgh, Scotland 

MOORE, JOHN W., Associate Chief, Laboratory of Biophysics, National Institutes of Health 
MORI, SYUITI, Professor, Zoological Institute, Kyoto University, Kyoto, Japan 
MORRILL, JOHN B., Assistant Professor of Biology, Wesleyan University 
MOSCONA, A. A., Associate Professor, University of Chicago 
MOYED, HARRIS S., Associate in Bacteriology, Harvard Medical School 
MULLINS, L. J., Associate Professor of Biophysics, Purdue University 
MUSACCHIA, X. J., Associate Professor in Biology, Saint Louis University 
NACE, PAUL FOLEY, Associate Professor of Zoology, McMaster University 
NELSON, LEONARD, Associate Professor of Physiology, Emory University 
ODUM, EUGENE P., Alumni Foundation Professor of Zoology, University of Georgia 
OIKAWA, TOSHIHIKO, Assistant Professor, Department of Physiology, Tohoku University School 

of Medicine, Sendai, Japan 
OSTERHOUT, W. J. V., Rockefeller Institute 

PALINCSAR, EDWARD E., Instructor in Biology, Loyola University 
PAPENFUSS, GEORGE F., Professor of Botany, University of California 
PARKER, JOHNSON, Assistant Professor in Plant Physiology, Yale University 
PARPART, ARTHUR K., Chairman and Professor of Biology, Princeton University 
PATERSON, MABEL C., Assistant Professor of Zoology, Vassar College 
PERSON, PHILIP, Veterans Administration Hospital, Brooklyn 

PO-CHEDLEY, DONALD S., Professor and Chairman of Biology Department, D'Youville College 
POLGAR, GEORGE, Research Fellow, University of Pennsylvania, Graduate School of Medicine 
POTTER, DAVID D., Instructor in Neurophysiology, Harvard Medical School 
RAPPORT, MAURICE M., Professor of Biochemistry, Albert Einstein College of Medicine 
REBHUN, LIONEL I., Assistant Professor of Biology, Princeton University 
REUBEN, JOHN P., Research Associate, Columbia University 



REPORT OF THE DIRECTOR 17 

REYNOLDS, LESLIE B., Assistant in Physiology, Medical College of South Carolina 
ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of Medi- 
cine 

ROSE, S. MERYL, Professor of Zoology, University of Illinois 

ROSENBERG, EVELYN K., Associate Professor, New York University-Bellevue Medical Center 
ROSENBERG, PHILIP, Special Postdoctoral Trainee Fellow, College of Physicians & Surgeons 
ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College 
RUGH, ROBERTS, Associate Professor of Radiology, Columbia University 
RUSHTON, W. A. H., Chief, Neurophysiology Section, National Institutes of Health 
RUSTAD, RONALD C, Assistant Professor of Physiology, Florida State University 
RYTHER, JOHN H., Marine Biologist, Woods Hole Oceanographic Institution 
SANBORN, RICHARD C., Professor of Zoology, Purdue University 
SANDERS, HOWARD L., Research Associate, Woods Hole Oceanographic Institution 
SATO, HIDEMI, Dartmouth Medical School 

SAUNDERS, JOHN W., Professor and Chairman, Department of Biology, Marquette University 
SCHACHMAN, HOWARD K., Professor of Biochemistry, University of California 
SCHUH, JOSEPH E., Associate Professor of Biology, Saint Peter's College 
SCOTT, ALLAN C., Professor of Biology, Colby College 
SCOTT, SISTER FLORENCE MARIE, Professor and Chairman, Department of Biology, Seton Hill 

College 

SCOTT, GEORGE T., Professor and Chairman, Department of Zoology, Oberlin College 
SEGAL, JOHN R., Physiologist, Veterans Administration Hospital, Boston 
SELIGER, HOWARD H., Research Associate, The Johns Hopkins University 
SMELSER, GEORGE K., Professor of Anatomy, Columbia University 
SMITH, LESLIE FRANK, Department of Biochemistry, Cambridge, England 
SONNENBLICK, B. P., Professor of Biology, Rutgers University 
SPECTOR, ABRAHAM, Instructor, Howe Laboratory 

SPEIDEL, CARL C., Professor of Anatomy, University of Virginia, School of Medicine 
SPIEGEL, MELVIN, Assistant Professor of Zoology, Dartmouth College 
SPINDELL, WILLIAM, Associate Professor of Chemistry, Rutgers University 
SPRATT, NELSON T., JR., Chairman and Professor of Zoology, University of Minnesota 
SPYROPOULOS, CONSTANTINE S., Neurophysiologist, National Institutes of Health 
STARR, RICHARD C., Associate Professor of Botany, Indiana University 
STEIN, MYRON, Instructor in Medicine, Harvard Medical School 

STEINBACH, H. BURR, Professor and Chairman, Department of Zoology, University of Chicago 
STEPHENS, GROVER C., Associate Professor of Zoology, University of Minnesota 
STONE, WILLIAM, JR., Director, Ophthalmic Plastics Laboratory, Massachusetts Eye and Ear 

Infirmary 

STREHLER, BERNARD L., Chief, Cellular and Comparative Physiology Section, National Insti- 
tutes of Health 
STRITTMATTER, CORNELIUS F., Assistant Professor of Biological Chemistry, Harvard Medical 

School 
STRITTMATTER, PHILIPP, Assistant Professor of Biochemistry, Washington University Medical 

School 

STRUMWASSER, FELIX, Neurophysiologist, National Institutes of Health 
STUNKARD, HORACE W., Research Associate, The American Museum of Natural History 
STURTEVANT, A. H., Professor of Genetics, California Institute of Technology 
SUDAK, FREDERICK N., Assistant Professor of Physiology, Albert Einstein College of Medicine 
SUSSMAN, MAURICE, Associate Professor, Brandeis University 

SZENT-GYORGYI, ALBERT, Director, Institute for Muscle Research, Marine Biological Laboratory 
SZENT-GYORGYI, ANDREW, Institute for Muscle Research, Marine Biological Laboratory 
TAKEUCHI, AKIRA, Fellow of Rockefeller Foundation, University of Utah 
TASAKI, ICHIJI, Chief, Special Senses Section, National Institutes of Health 
TAYLOR, ROBERT E., Physiologist, National Institutes of Health 
TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan 

TAYLOR, W. ROWLAND, Assistant Professor of Oceanography, The Johns Hopkins University 
TEWINKEL, Lois E., Professor of Zoology, Smith College 
DETERRA, NOEL, Post-doctoral Fellow, Rockefeller Institute 



18 MARINE BIOLOGICAL LABORATORY 

TORCH, REUBEN, Assistant Professor of Zoology, University of Vermont 

TRACER, WILLIAM, Associate Professor, Rockefeller Institute 

TRAVIS, DAVID M., Assistant Professor of Pharmacology, University of Florida 

TROLL, WALTER, Associate Professor, New York University 

TWEEDELL, KENYON S., Assistant Professor of Biology, University of Notre Dame 

VAN NORMAN, EARL, SR., Princeton University 

DEVILLAFRANCA, GEORGE W., Associate Professor of Zoology, Smith College 

VILLEE, CLAUDE A., Associate Professor of Biological Chemistry, Harvard University 

VINCENT, WALTER S., Assistant Professor of Anatomy, Upstate Medical Center 

WARREN, LEONARD, Visiting Scientist, National Institutes of Health 

WATANABE, AKIRA, Research Associate, College of Physicians & Surgeons 

WEBB, H. MARGUERITE, Associate Professor, Goucher College 

WEINSTEIN, PAUL P., Senior Scientist, National Institutes of Health 

WEISS, LEON P., Associate Professor of Anatomy, Johns Hopkins Medical School 

WERMAN, ROBERT, Visiting Scholar, College of Physicians & Surgeons 

WHITING, ANNA R., Guest Investigator, University of Pennsylvania 

WICHTERMAN, RALPH, Professor of Biology, Temple University 

WIERCINSKI, FLOYD J., Associate Professor of Biological Science, Drexel Institute of Technology 

WILBER, CHARLES G., Professorial Lecturer in Biology, Loyola College 

WILLEY, CHARLES H., Professor and Chairman, Department of Biology, New York University 

WILSON, THOMAS HASTINGS, Assistant Professor of Physiology, Harvard Medical School 

WILSON, WALTER L., Assistant Professor of Physiology, University of Vermont College of 

Medicine 
WITTENBERG, JONATHAN B., Associate Professor of Physiology, Albert Einstein College of 

Medicine 

WRIGHT, PAUL A., Associate Professor of Zoology, University of New Hampshire 
YEANDLE, STEPHEN, Assistant Professor, George Washington University 
ZIGMAN, SEYMOUR, Research Associate, Massachusetts Eye and Ear Infirmary 
ZIMMERMAN, ARTHUR M., Instructor in Pharmacology, Downstate Medical Center 
ZWILLING, EDGAR, Professor of Biology, Brandeis University 

Lalor Fellows, 1960 

MANN, THADDEUS R. R., Molteno Institute, Cambridge, England 
DOWBEN, ROBERT M., Northwestern University 
FLAKS, JOEL G., University of Pennsylvania School of Medicine 
HURWITZ, JERARD, New York University College of Medicine 
STRITTMATTER, CORNELIUS F., Harvard Medical School 
WARREN, LEONARD, National Institutes of Health 

Lillie Fellow, 1960 

MOSCONA, A. A., University of Chicago 

Grass Fellows, 1960 

KITAI, STEPHEN T., Massachusetts General Hospital 

Luco, J. V., Catholic University Medical School, Santiago, Chile 

REYNOLDS, LESLIE B., Medical College of South Carolina 

Beginning Investigators, 1960 

ALSUP, PEGGY ANN, Harvard Medical School 
BEEBE, CURT, University of Vermont, College of Medicine 
BINSTOCK, LEONARD, National Institutes of Health 
BOLEYN, BRENDA J., University of Rhode Island 
BROWN, GEORGE W., North Carolina State College 



REPORT OF THE DIRECTOR 19 

CAMPBELL, JAMES W., Rice Institute 

CIUCHTA, HENRY, Temple University, School of Pharmacy 

COOKE, IAN M., Harvard University 

CORRIDEN, FRANKLIN E., University of Delaware 

COSTELLO, ROBERT CHARLES, University of North Carolina 

CURTIS, DAVID R., Australian National University 

DOOLITTLE, RUSSELL F., Harvard University 

DUBNAU, DAVID, Columbia University 

DUDEL, JOSEF, Harvard Medical School 

DUNHAM, PHILIP B., University of Chicago 

EISENBERG, ROBERT S., Harvard College 

FAUST, ROBERT G., Princeton University 

FILOSA, MICHAEL F., Johns Hopkins University 

FRUMENTO, A. S., University of Buenos Aires 

GASSELING, MARY T., Marquette University 

HANLORE, MARY S., University of California 

HEX SHAW, EDGAR C, Harvard Medical School 

HUVER, CHARLES W., Yale University 

HWANG, JOSEPH CHI-CHIU, University of Oregon 

ISSELBACHER, KURT J., Massachusetts General Hospital 

JACKSON, JAMES A., Western Reserve University 

KATZ, GEORGE M., College of Physicians & Surgeons 

KROPF, ALLEN, Amherst College 

MORAN, JOSEPH F., JR., Russell Sage College 

ORKAND, RICHARD K., University of Utah 

PETERSON, R. PRICE, University of Pennsylvania, School of Medicine 

RUDOMIN, PEDRO N., Rockefeller Institute 

SCHUEL, HERBERT, New York University 

SHEPARD, DAVID, University of Chicago 

SIMMONS, JOHN E., Rice Institute 

SJODIN, R. A., Purdue University 

SUDDUTH, SOLON S., Johns Hopkins School of Medicine 

WHITELEY, GEORGE C., JR., The Hill School 

WHITFIELD, SYLVIA G., Tulane University 

WILF, RUTH T., University of Illinois 

WOOD, ROBERT W., Sloan-Kettering Division, Cornell University 

WORMSER, EVA H., Johns Hopkins University 

Research Assistants, 1960 

ABBOTT, JOAN, University of Pennsylvania Medical School 

ANTLEY, RAY MILLS, Emory University 

AREND, WILLIAM P., Columbia Medical School 

ASHMAN, ROBERT F., Wabash College 

BAIRD, SPENCER, Institute for Muscle Research, Marine Biological Laboratory 

BARNWELL, FRANKLIN H., Northwestern University 

BAUER, ADELIA C., Marine Biological Laboratory 

BAUER, G. ERIC, University of Minnesota 

BERMAN, LAWRENCE JOSEPH, Harvard Medical School 

BERMAN, PAUL ELIOT, Upstate Medical Center 

BIANCHI, CARLA, Northwestern University 

BITO, LASZLO Z., Columbia University 

BLEYMAN, LEA K., Columbia University 

BLUMSTEIN, JOYCE R., Albert Einstein College of Medicine 

BOSLER, ROBERT, Harvard Medical School 

BRANHAM, JOSEPH M., Florida State University 

BURDICK, CAROLYN, Harvard Medical School 

BYRNE, PAUL M., National Institutes of Health 



20 MARINE BIOLOGICAL LABORATORY 

CANBY, DIANE MARIE, Smith College 

CECCARINI, COSTANTE, St. Peter's College 

CICAK, ANNA, Albert Einstein College of Medicine 

CLARK, ELOISE E., Columbia University 

COOK, PHILIP WILLIAM, Indiana University 

CORDES, EUGENE, Brandeis University 

COUSINEAU, GILLES H., New York University 

CROWE, PRISCILLA, Seton Hill College 

DEWEL, WILLIAM C, Wesleyan University 

DIETRICH, THOMAS S., Wayne State University College of Medicine 

DINGLE, AL. D., University of Illinois 

DOWNS, PATRICIA, Colby College 

DUBIN, DONALD, Harvard Medical School 

DUBNAU, EUGENIE J., Columbia University 

EDWARDS, JOAN F., Wilson College 

EIGNER, ELIZABETH ANN, Massachusetts General Hospital 

ELEFANT, HELENE, Bellevue Medical Center 

ELEK, MARIA E., Johns Hopkins School of Hygiene 

ERSKINE, LOUISE, Institute for Muscle Research, Marine Biological Laboratory 

EVAN, GERALD L., University of Vermont 

EWING, RICHARD D., Reed College 

FEHRENBAKER, LAWRENCE G., Wayne State University, College of Medicine 

FELDSHUH, DANA, Massachusetts Eye and Ear Infirmary 

FINKEL, ARNOLD, New York University College of Medicine 

FISHER, SYLVIA S., Saint Louis University 

FLATHERS, ANN R., University of New Hampshire 

FLETCHER, JOYCE, New York City 

FONG, BETTY ANN, New York University 

FORAN, ELIZABETH H., Smith College 

GIBBON, CHARLOTTE A., Indiana University 

GOLDSTEIN, MELVIN E., Indiana University 

GRABSKE, ROBERT, Kansas University 

GRANT, DAVID C., Yale University 

GREEN, JONATHAN, University of Minnesota 

GREEN, SAMUEL A., JR., Claymont, Delaware 

HALEY, BARBARA, Brandeis University 

HALL, ZACH W., Emory University 

HALPERN, EVELYN, Western Reserve University Medical School 

HAMMOND, CONSTANCE, Radcliffe College 

HANSON, FRANK E., JR., State University of Iowa 

HATHAWAY, RALPH R., Florida State University 

HAYWARD, GEORGE, National Institutes of Health 

HENRY, ELEANOR, Hahnemann Medical College 

HESSLER, ANITA Y., Woods Hole, Massachusetts 

HIRSCH, CARL A., Harvard Medical School 

HOLSTEIN, IRMA, University of Pennsylvania, Graduate School of Medicine 

HOLSTEN, GEORGE H., Ill, Yale University 

HUFNAGEL, LINDA, University of Vermont 

HUMPHREYS, TOM D., University of Chicago 

HUTTRER, ANNICK, Mount Holyoke College 

JACKSON, THOMAS J., Lehigh University 

JAFFREY, IRA S., New York State University 

KELLOCK, MARGERY, College of Physicians & Surgeons 

KENNEN, DANE E. M., American University 

KIMBALL, SALLY P., Columbia University 

KREWSON, CARRIE R., Vassar College 

LAUFENBERG, HENRY J., Saint Peter's College 

LEHV, JANE WENDY, Vassar College 



REPORT OF THE DIRECTOR 21 

LEINING, JUDITH M., Massachusetts Eye and Ear Infirmary 

LEMMA, AKLILU, Johns Hopkins University School of Hygiene 

LENOX, MARILYN, Philippi, West Virginia 

LIBBIN, DICK, University of Cincinnati 

LOOMIS, WILLIAM F., JR., Loomis Laboratory 

LORING, JANET M., Harvard Medical School 

MCKENZIE, SHARON G., American University 

MAcNicHOL, EDWARD F., JR., Johns Hopkins University 

MAKINEN, PAULA M., University of Minnesota 

MILLER, HEDWIG B., Wellesley College 

MILLS, NANCY L., College of Physicians & Surgeons 

MINGIOLI, ELIZABETH S., Harvard University 

MUSICK, ROY, American University 

NAGABHUSHANAM, R., Tulane University 

NAUMANN, DOROTHY C., Smith College 

NORRIS, ELAINE, Wesleyan University 

GETTING, BONNALIE J., Northwestern University 

OTERO-VILARDEBO, Luis R., University of Puerto Rico 

OWENS, DEAN PAUL, Johns Hopkins University 

PALMER, JOHN D., Northwestern University 

PHILPOTT, CHARLES W., Tulane University 

PHILPOTT, LORALEE, Tulane University 

POLLACK, MATTHEW, National Institutes of Health 

RANLETT, MARY, Dartmouth College 

RAY, FRANCES L., Bellevue Medical Center 

ROBERTS, MARY Lou, Washington University Medical School 

RODGERS, PATRICIA E., New York University 

ROSENBLUTH, RAJA, Columbia University 

ROSSMAN, RONALD E., Princeton University 

SCOTT, NANCY F., University of Vermont 

SCRICCO, ELAINE ANN, Howe Laboratory 

SEIDMAN, AARON, Brandeis University 

SIMON, BARBARA, Rutgers University 

SMALLER, BERNARD, Argonne National Laboratory 

SMITH, ISSAR, Columbia University 

SONNEBORN, DAVID R., Brandeis University 

SPENCER, JOYCE, Harvard Medical School 

SPRITZER, RUTH C., New York University School of Medicine 

SRINIVASAN, DOBLI, College of Physicians & Surgeons 

STEINBERG, SONIA N., Brandeis University 

STERN, EDWARD L., University of Chicago 

SUTHERLAND, KERSTIN E., Institute for Muscle Research, Marine Biological Laboratory 

SWIFT, ELIJAH, Swarthmore College 

SZENT-GYORGYI, EVE, Institute for Muscle Research, Marine Biological Laboratory 

SZENT-GYORGYI, MARTA, Institute for Muscle Research, Marine Biological Laboratory 

THOMAS, CYNTHIA, Massachusetts Eye and Ear Infirmary 

TULCHIN, NATALIE, New York University 

WATKINS, DUDLEY T., Oberlin College 

WATTERS, CHRISTOPHER, Notre Dame University 

WEINTRAUB, ARTHUR H., New York University 

WEIS, PEDDRICK, New York University College of Dentistry 

WELLINGTON, FREDERICA M., Harvard Medical School 

WHITTAKER, J. RICHARD, Yale University 

WILKENS, JERREL L., Tulane University 

WILLIAMS, RICHARD B., University of Georgia 

WILSON, JOAN, Rice Institute 

ZAMBERNARDI, JOSEPH, Tulane University 

ZIMINSKY, ALVIN C., National Institutes of Health 



22 MARINE BIOLOGICAL LABORATORY 

Library Readers, 1960 

ARVANITAKI, ANGELIQUE, Director, Faculte de Sciences, Lyon, France 

BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School 

BECK, LYLE V., University of Pittsburgh School of Medicine 

BLUM, HAROLD F., Physiologist, National Cancer Institute and Princeton University 

BODANSKY, OSCAR, Chief, Division of Enzymology and Metabolism, Sloan-Kettering Institute 

BOVEE, EUGENE C, Associate Professor, University of Florida 

BRIDGMAN, ANNA JOSEPHINE, Professor of Biology, Agnes Scott College 

BUTLER, ELMER G., Professor of Zoology, Princeton University 

CHANUTIN, ALFRED, Professor of Biochemistry, University of Virginia School of Medicine 

CHASE, AURIN M., Associate Professor of Biology, Princeton University 

CLARK, ELIOT R., University of Pennsylvania 

COHEN, SEYMOUR S., Professor of Biochemistry, University of Pennsylvania 

EDER, HOWARD, Professor of Medicine, Albert Einstein College of Medicine 

EISEN, HERMAN N., Professor of Medicine, Washington University 

FLAVIN, MARTIN, National Heart Institute, National Institutes of Health 

FLESCH, PETER, Associate Professor of Research Dermatology, University of Pennsylvania 

FRIES, E. F. B., Associate Professor, The City College of New York 

GINSBERG, HAROLD S., Associate Professor, Western Reserve University 

GOLDTHWAIT, DAVID A., Assistant Professor of Biochemistry, Western Reserve University 

GREEN, MAURICE, Assistant Professor, Saint Louis University School of Medicine 

HERRMANN, ROBERT L., Assistant Professor of Biochemistry, Boston University School of 

Medicine 

HOBERMAN, HENRY D., Professor of Biochemistry, Albert Einstein College of Medicine 
HURWITZ, CHARLES, Chief, General Medical Research Laboratory, Veterans Administration 

Hospital 

JACOBS, M. H., Professor Emeritus, University of Pennsylvania 

JENNISON, MARSHALL W., Chairman, Department of Bacteriology and Botany, Syracuse Uni- 
versity 

KARUSH, FRED, Professor of Immunochemistry, University of Pennsylvania School of Medicine 
KLEIN, MORTON, Professor of Microbiology, Temple University School of Medicine 
KLOTZ, IRVING M., Professor of Chemistry and Biology, Northwestern University 
LENHOFF, HOWARD M., Howard Hughes Memorial Institute 
LEVINE, RACHMIEL, Chairman, Department of Medicine, Michael Reese Hospital 
LIONETTI, FABIAN J., Associate Professor of Biochemistry, Boston University School of Medicine 
LUBIN, MARTIN, Assistant Professor of Pharmacology, Harvard Medical School 
MCDONALD, SISTER ELIZABETH SETON, Chairman, Department of Biology, College of Mt. St. 

Joseph 

MOUL, EDWIN T., Associate Professor of Botany, Rutgers University 
NELSON, THOMAS C., Senior Microbiologist, Eli Lilly and Company 
NOVIKOFF, ALEX B., Research Professor, Albert Einstein College of Medicine 
PEABODY, RICHARD A., Assistant Professor of Biochemistry, Albany Medical College 
PICK, JOSEPH, Professor of Anatomy, New York University Medical Center 
PULLMAN, BERNARD, Professor, University of Paris 

ROOT, WALTER S., Professor of Physiology, College of Physicians & Surgeons 
ROTH, REV. OWEN H., Associate Professor of Zoology, St. Vincent College 
SCHLAMOWITZ, MAX, Associate Cancer Research Scientist, Roswell Park Memorial Institute 
SCHWARZ, KLAUS, Chief, Section on Experimental Liver Diseases, National Institutes of Health 
SPIRTES, M. A., Associate Professor of Pharmacology, Hahnemann Medical College 
STETTEN, DEWiTT, Associate Director in Charge of Research, National Institutes of Health 
STETTEN, MARJORIE R., Biochemist, National Institutes of Health 
SULKIN, S. EDWARD, Professor and Chairman, Department of Microbiology, University of Texas 

Southwestern Medical School 

TOLKSDORF, SIBYLLE, Senior Biochemist, Schering Corporation 
TRURNIT, HANS J., Principal Scientist, Research Institute for Advanced Study 
VILLANI, FRANK J., Senior Research Chemist, Schering Corporation 
WAINIO, WALTER W., Professor of Biochemistry, Rutgers University 



REPORT OF THE DIRECTOR 23 

WARNER, ROBERT C., Associate Professor of Biochemistry, New York University College of 

Medicine 
WEIGLE, WILLIAM O., Assistant Professor of Immunochemistry in Pathology, University of 

Pittsburgh School of Medicine 

WEXLER, HARRY, Director of Research, U. S. Weather Bureau 
WHEELER, GEORGE E., Assistant Professor of Biology, Brooklyn College 
YNTEMA, CHESTER L., Professor of Anatomy, Upstate Medical Center at Syracuse 

Students, 1960 

All students listed completed formal course program, June 21-July 30. Asterisk indicates 
students completed Post Course Research Program, August 1-September 3. 

BOTANY 

AUYANG, SHIH-CHEN, Clark University 
*BONAMO, PATRICIA M., Cornell University 

BROOKS, AUSTIN E., Wabash College 
*CYRUS, RODNEY V., University of Michigan 

DEUTSCH, ELIZABETH J., Radcliffe College 

ERICKSON, PAUL A., Clark University 

FALCON, GISELA, Ave. Galipan No. 16, San Bernardino, Caracas, Venezuela 

FRANKLIN, SANDRA E., Acadia University 
*HALL, NANCY V., Vassar College 

KOETZNER, KENNETH L., Lycoming College 

KREMER, PETER R., Cornell University 

LANG, NORMA J., Indiana University 

MITCHELL, ROBERT A., Cornell University 

MULLIN, MICHAEL M., Harvard University 
*NICHOLS, HERBERT W., University of Alabama 

NOODEN, LARRY D., Harvard University 
*WATERS, ANNETTE, Indiana University 
*WESTERDALE, THOMAS H., University of Michigan 

EMBRYOLOGY 

*ALLISON, WILLIAM S., Brandeis University 

ARNOLD, JOHN M., University of Minnesota 
*CLARKE, RICHARD B., University of Illinois 
*COHEN, NICHOLAS, University of Rochester 

EISENSTADT, JEROME, Brandeis University 
*ESPER, HILDEGARD, Columbia University 
*GREEN, SANDRA J., University of Minnesota 

HOVINGH, PETER, Johns Hopkins University 
*JAFFEE, ROBERT L., University of Rochester 
*KIMMEL, DONALD L., JR., Temple University 

LICHTENBERG, INGEBORG, University of Chicago 

MARSHALL, LEE ANN, University of Michigan 

ORLOFF, SERVE, Brussels, Belgium 
*PLATT, JOHN R., University of Chicago 

RACE, JAMES, JR., State University of Iowa 
*RITTENHOUSE, ELIZABETH W., University of Michigan 

SLATER, DONALD W., Indiana University 

SWEENY, PHILLIP R., Brown University 

WILLE, JOHN J., JR., Indiana University 
*WINESDORFER, JOHN E., Johns Hopkins University 



24 MARINE BIOLOGICAL LABORATORY 

ECOLOGY 

*ALEXANDER, DOUGLAS G., University of North Carolina 

BEARDOW, JANE M., Drew University 
*BROUGHTON, WILLIAM S., University of Georgia 
*DE LA CRUZ, ARMANDO, University of the Philippines, Pasay City, Philippines 

GOLD, KENNETH, New York University 
*GUSTAFSON, ALTON H., Bowdoin College 
*GUTKNECHT, JOHN W., University of North Carolina 
*KRAMER, DANA D., City College of New York 
*PLATZMAN, SARA J., Yale University 
*STERNS, CAROL W., Peekskill, New York 

VANDENACK, SISTER JULIA MARIE, Catholic University of America 
*WILKENS, JERREL L., Tulane University 
*ZiEG, ROGER G., University of Nebraska 

PHYSIOLOGY 

ALBERTS, BRUCE M., Harvard College 
*BOASS, AGNA, Radcliffe College 
*BRODY, STUART, Stanford University 
*COLLIER, ROBERT J., Harvard University 
*DOLAN, MICHAEL F., Johns Hopkins University 
*FORREST, HELEN F., Rutgers University 
*FREEMAN, ALAN R., Hahnemann Medical College 

FRIDOVICH, IRWIN, Duke University 
*HALL, ZACH W., Emory University 
*HEMPFLING, WALTER P., Yale University 

HOLTZMAN, ERIC, Columbia University 
*MADDUX, WILLIAM S., Princeton University 

McEwEN, BRUCE S., Rockefeller Institute 

NADING, Louis K., Oberlin College 
*NATHENSON, STANLEY G., Washington University 
*NORRIS, JOHN L., Vanderbilt University 

ORR, CHARLES W. M., Johns Hopkins University 
*PATRICK, NOEL V., Columbia University 

RICHARDSON, G. S., Harvard Medical School 

ROSENFIELD, CAROL, New York University College of Medicine 
*RozE, ULDIS, Washington University Medical School 

SCHINDLER, FREDERICK J., University of Pennsylvania 
*SCHWARTZ, NORMAN M., Syracuse University 

SNIPES, CHARLES A., Duke University 
*STONE, HENRY O., Duke University 

TANG, JIEN-NAN JORDAN, Oklahoma Medical Research Foundation 
*TURNEY, TULLY, JR., University of North Carolina 

WEBB, GEORGE D., University of Colorado Medical School 

INVERTEBRATE ZOOLOGY 

ALEXANDER, KATHLEEN, University of North Carolina 
*BALLARD, JULIET L., Drew University 

BOTTOMLY, GAIL, University of Massachusetts 

BRIGGS, RICHARD G., Cornell University 
*BROCH, EDMUND S., Cornell University 
*BRUNO, MERLE S., Syracuse University 

CHAICHARN, AIMORN, University of New Hampshire 

CLARRIDGE, JILL E., University of Michigan 



REPORT OF THE DIRECTOR 25 

*CLELAND, CHARLES F., Wabash College 

COSTELLO, ROBERT C., University of North Carolina 
*D'AcosTiNO, ANTHONY S., New York University 

DRUMMOND, SISTER THERESE, Catholic University of America 

EAGLESON, LOUISE J., Spellman College 

EDLIN, GORDON J., University of Oregon 

EMLEN, JOHN M., University of Wisconsin 

FARRELL, CAROLYN ROSE, Marquette University 
*FENNER, BARBARA, Vassar College 

FOURCADE, MIGUEL, S. J., Fordham University 

GAUTHIER, GERALDINE F., Harvard Medical School 
*HADDAD, LAMIA, Brown University 

HARMAN, MARY, Radcliffe College 

HOLLAND, NICHOLAS D., Carleton College 

HOLT, PORTIA, Colorado College 
*HOPPER, FRED A., JR., University of Oklahoma 

JARVIS, SISTER JULIE, Catholic University of America 

KANESHIRO, EDNA S., Syracuse University 

KECK, CARL W., Lafayette College 

KEE, JAMES W., JR., Massachusetts Institute of Technology 
*KIRCHENBERG, RALPH J., DePaul University 

KNOWLTON, ROBERT E., Bowdoin College 

KREWSON, CARRIE R., Vassar College 

LUCKENBILL, LOUISE M., Washington University 

MAHOWALD, ANTHONY P., S. J., Johns Hopkins University 

MORRISON, ROBERTA A., Smith College 

PORCARO, CAROL A., Marymount College 
*SCHOPF, THOMAS J. M., Oberlin College 

SMITH, STEPHEN D., Wesleyan University 

SQUADRONI, JOSE, S. J., Fordham University 

THEROUS, ROGER B., Bureau of Commercial Fisheries 

VOGEL, STEVEN, Tufts University 

WAUGH, MARY, Wilson College 

WESTHOFF, DAVID D., St. Louis University 

ZWEIG, CHARLES H., Brandeis University 

3. FELLOWSHIPS AND SCHOLARSHIPS, 1960 

Calkins Scholarship : 

RICHARD BRIGGS, Invertebrate Zoology Course 

Bio Club Scholarship : 

DANA KRAMER, Ecology Course 

Lucretia Crocker Fellowships : 

KENNETH GOLD, Ecology Course 
GISELA FALCON, Botany Course 

4. TABULAR VIEW OF ATTENDANCE, 1956-1960 

1956 1957 1958 1959 1960 

INVESTIGATORS TOTAL 304 326 410 427 458 

Independent 184 186 203 215 231 

Under Instruction 20 23 39 45 42 

Library Readers 50 42 54 51 50 

Research Assistants 50 75 114 116 135 



26 



MARINE BIOLOGICAL LABORATORY 



STUDENTS TOTAL 140 139 138 134 122 

Invertebrate Zoology 55 55 55 49 43 

Embryology 28 27 22 23 20 

Physiology 30 30 27 27 28 

Botany 18 18 18 20 18 

Ecology 9 9 16 15 13 

TOTAL ATTENDANCE 444 465 548 561 580 

Less persons represented as both investigators and 

students 2 3 5 4 2 

442 462 543 557 578 

INSTITUTIONS REPRESENTED TOTAL 130 129 142 143 144 

By Investigators 97 94 110 98 83 

By Students 33 35 74 73 61 

SCHOOLS AND ACADEMIES REPRESENTED 

By Investigators 1 1 2 8 5 

By Students 3 5 12 2 

FOREIGN INSTITUTIONS REPRESENTED 

By Investigators 9 11 20 29 11 

By Students 6 5 6 9 3 



5. INSTITUTIONS REPRESENTED, 1960 



Acadia University 

American Museum of Natural History 

American University 

Amherst College 

Bowdoin College 

Brandeis University 

Brown University 

Carleton College 

Catholic University of America 

City College of New York 

Clark University 

Colby College 

Colorado College 

Columbia University 

Columbia University College of Physicians 

and Surgeons 
Cornell University 
Dartmouth College 
DePaul University 
Drew University 
Duke University 
D'Youville College 
Drexel Institute of Technology 
Emory University 
Florida State University 
Fordham University 
George Washington University 
Goucher College 
Hahnemann Medical School 
Harvard University 
Harvard University Medical School 
Indiana University 
Institute for Muscle Research 
Johns Hopkins University 



Lafayette College 

Loyola College 

Marquette University 

Marymount College 

Massachusetts Institute of Technology 

McMaster University 

Medical College of South Carolina 

National Institutes of Health 

New York University Heights 

New York University, College of Dentistry 

New York University, College of Medicine 

New York University, Washington Square 

College 

North Carolina State College 
Notre Dame University 
Oberlin College 

Oklahoma Medical Research Foundation 
Princeton University 
Purdue University 
Queens College 
Radcliffe College 
Reed College 
Rice Institute 
Rockefeller Institute 
Rockefeller Foundation 
Russell Sage College 
Rutgers University 
Saint Joseph College 
Saint Louis University 
Seton Hill College 
Single Cell Research Foundation 
Smith College 
Spellman College 
State University of Iowa 



REPORT OF THE DIRECTOR 



27 



State University of New York, Upstate Medi- 
cal College 

State University of New York, Downstate 
Medical College 

Syracuse University 

Temple University 

Tufts College 

Tulane University 

University of Alabama 

University of California 

University of Chicago 

University of Colorado Medical School 

University of Delaware 

University of Florida 

University of Georgia 

University of Illinois 

University of Massachusetts 

University of Michigan 

University of Minnesota 

University of Nebraska 

University of New Hampshire 



University of North Carolina 
University of Oregon 
University of Pennsylvania 
University of Pennsylvania School of Medicine 
University of Pittsburgh School of Medicine 
University of Utah 
University of Vermont 
U. S. Bureau of Commercial Fisheries 
Vassar College 

V. A. Administration Hospital at Brooklyn 
Wabash College 
Washington University 
Washington University Medical School 
Wayne State University 
Wesleyan University 

Western Reserve University School of Medi- 
cine 

Wilson College 

Woods Hole Oceanographic Institution 
Yale University 



FOREIGN INSTITUTIONS REPRESENTED, 1960 



Free University of Brussels, Belgium 
Faculte Des Sciences, Lyon, France 
University College, London 
University of Witwatersrand, Johannesburg, 

South Africa 
Catholic University Medical School, Santiago, 

Chile 

University of Cambridge, England 
University of Edinburgh, Scotland 



Kyoto University, Kyoto, Japan 

McMaster University, Canada 

Tohoku University School of Medicine, Sen- 

dai, Japan 

University of Buenos Aires 
University of the Philippines, Pasay City, 

Philippines 
Institute Investigaciones, Barcelona 3, Spain 



SUPPORTING INSTITUTIONS AND AGENCIES, 1960 



Associates of the Marine Biological Labora- 
tory 

Atomic Energy Commission 
Josephine B. Crane Foundation 
The Grass Foundation 
The Lalor Foundation 



The Merck Company Foundation 
National Institutes of Health 
National Science Foundation 
Office of Naval Research 
The Rockefeller Foundation 
Schering Foundation, Inc. 



CORPORATE ASSOCIATES 



Abbott Laboratories 

CIBA Pharmaceutical Products, Inc. 

Carter Products, Inc. 



Eli Lilly and Company 
E. R. Squibb & Sons 
The Upjohn Company 



6. EVENING LECTURES, 1960 



June 24 G. ADRIAN HORRIDGE 

St. Andrews University, Scotland 

July 1 A. A. MOSCONA 

University of Chicago, Frank R. 
Lille Fellow at MBL 



"Electrophysiological and anatomical anal- 
ysis of primitive ganglia" 

"Experimental studies on tissue synthe- 
sis : problems and prospects" 



28 



MARINE BIOLOGICAL LABORATORY 



July 8 THADDEUS R. R. MANN 

Molteno Institute, University of 
Cambridge, Senior Lalor Fellow 
at MBL 
July 15 CLIFFORD V. HARDING 

Columbia University, College of 
Physicians & Surgeons 

July 22 J. V. Luco 

Catholic University of Chile, Alex- 
ander Forbes Lecturer at MBL 

July 25 J. V. Luco 

July 29 DEWITT STETTEN, JR. 

National Institutes of Health 

August 5 LASZLO LORAND 

Northwestern University 

August 12 SEVERO OCHOA 

New York University School of 
Medicine 

August 19 ERNST A. SCHARRER 

Albert Einstein College of Medicine 
August 26 GEORGE L. CLARKE 

Harvard University 



"Comparative aspects of sperm physiol- 
ogy" 



"The control of cell division" 



"Physiological studies during Wallerian 
degeneration" 

"The trophic effect of neuron activity" 
"The metabolism of gout" 

"The chemical basis of the clotting of 
blood" 

"Metabolism of propionic acid in animal 
tissues" 

"Neurosecretion" 

"Ecological aspects of daylight and bio- 
luminescence in the sea" 



7. TUESDAY EVENING SEMINARS, 1960 



July 5 MARTIN B. MATHEWS 

HAROLD F. BLUM 
July 12 RONALD C. RUSTAD 

DONALD P. COSTELLO 

C. C. SPEIDEL 
R. H. CHENEY 

July 19 ALBERT SZENT-GYORGYI 

IRVIN ISENBERG 

BENJAMIN KAMINER 

ANDREW HEGYELI 
July 26 W. ROBERT MIDDLEBROOK 

HERMAN J. C. BERENDSEN 
ALEX B. NOVIKOFF 



August 2 PHILIP PERSON 
ALBERT FINE 

KLAUS SCHWARZ 
S. EHRENPREIS 



"Some comparative biochemistry of con- 
nective tissue ground substance" 

"Complexity and organization" 

"X-ray induced dissociation of the mi- 
totic and micromere 'clocks' " 

"The giant cleavage spindle of the egg 
of Polychocnis cannelcnsis" 

"Motion pictures of radiation-induced 
modifications of fertilization and early 
development of the sea urchin Arbacia" 

"Energy and charge transfer" 

"Spin resonance studies of riboflavin 
semiquinones and riboflavin complexes" 

"Contractile responses in the presence of 
charge transfer complexes" 

"Detection of electron donors" 

"The action of trypsin on acetylated myo- 
sin" 

"The structure of water in tissue, as stud- 
ied by nuclear magnetic resonance" 

"Phagocytosis, pinocytosis and lysosomes : 
Cytochemical and electron microscopic 
studies" 

"The role of free radical formation during 
indophenol blue synthesis by respira- 
tory enzymes" 

"A role of trivalent chromium in glucose 
utilization" 

"A receptor protein : Isolation and drug 
binding properties" 



REPORT OF THE DIRECTOR 29 

August 9 JAMES CASE "Excitation of firefly light organ" 

JOHN BUCK 

R. A. SJODIN "Cation permeability in muscle" 

P. BELTON "Effects of ions on potential in lepidop- 

teran muscle fibers" 
August 16 F. D. CARLSON "A scheme for the mechanochemistry of 

muscle" 
A. G. SZENT-GYORGYI "Studies on actin. I. Reversibility of 

actin depolymerization in presence of 

KI" 

T. HAYASHI "Studies on actin. II. Polymerization and 

RAJA ROSENBLUTH the bound nucleotide" 

8. MEMBERS OF THE CORPORATION, 1960 
1. LIFE MEMBERS 

BRODIE, MR. DONALD M., 522 Fifth Avenue, New York 18, New York 

CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania 

CARVER, DR. GAIL L., Mercer University, Macon, Georgia 

COLE, DR. ELBERT C., 2 Chipman Park, Middlebury, Vermont 

COWDRY, DR. E. V., Washington University, St. Louis, Missouri 

CRANE, MRS. W. MURRAY, Woods Hole, Massachusetts 

GOLDFARB, DR. A. J., College of the City of New York, New York City, New York 

KNOWLTON, DR. F. P., 1356 Westmoreland Avenue, Syracuse, New York 

LEWIS, DR. W. H., Johns Hopkins University, Baltimore, Maryland 

LOWTHER, DR. FLORENCE DEL., Barnard College, New York City, New York 

MALONE, DR. E. F., 6610 North llth Street, Philadelphia 26, Pennsylvania 

MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts 

MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pennsylvania 

PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana 

PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania 

RIGGS, MR. LAWRASON, 74 Trinity Place, New York 6, New York 

SCOTT, DR. ERNEST L., Columbia University, New York City, New York 

SCHRADER, DR. FRANZ, Duke University, Durham, N. C. 

SCHRADER, DR. SALLY, Duke University, Durham, N. C. 

TURNER, DR. C. L., Northwestern University, Evanston, Illinois 

WAITE, DR. F. G., 144 Locust Street, Dover, New Hampshire 

WALLACE, DR. LOUISE B., 359 Lytton Avenue, Palo Alto, California 

WARREN, DR. HERBERT S., 610 Montgomery Avenue, Bryn Mawr, Pennsylvania 

2. REGULAR MEMBERS 

ABELL, DR. RICHARD G., 7 Cooper Road, New York City, New York 
ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts 
ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York 
ADOLPH, DR. EDWARD F., University of Rochester School of Medicine and Dentis- 
try, Rochester, New York 

ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota 

ALLEN, DR. M. JEAN, Department of Biology, Wilson College, Chambersburg, 
Pennsylvania 



30 MARINE BIOLOGICAL LABORATORY 

ALLEN, DR. ROBERT D., Department of Biology, Princeton University, Princeton, 

New Jersey 
ALSCHER, DR. RUTH, Department of Physiology, Manhattanville College, Purchase, 

New York 
AMATNIEK, DR. ERNEST, Department of Neurology, College of Physicians and 

Surgeons, New York City, New York 
AMBERSON, DR. WILLIAM R., Woods Hole, Massachusetts 
ANDERSON, DR. J. M., Department of Zoology, Cornell University, Ithaca, New 

York 

ANDERSON, DR. RUBERT S., Medical Laboratories, Army Chemical Center, Mary- 
land (Box 632, Edgewood, Maryland) 
ANDERSON, DR. T. F., Institute for Cancer Research, Fox Chase, Philadelphia 11, 

Pennsylvania 
ARMSTRONG, DR. PHILIP B., Department of Anatomy, State University of New 

York College of Medicine, Syracuse 10, New York 
ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory, 

Oak Ridge, Tennessee 
ATWOOD, DR. KIMBALL C., Department of Microbiology, University of Illinois, 

Urbana, Illinois 

AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts 
AYERS, DR. JOHN C., Department of Zoology, University of Michigan, Ann Arbor, 

Michigan 
BAITSELL, DR. GEORGE A., Osborn Zoological Laboratories, Yale University, New 

Haven. Connecticut 

BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medi- 
cal School, Boston 15, Massachusetts 
BALLARD, DR. WILLIAM W., Department of Zoology, Dartmouth College, Hanover, 

New Hampshire 

BALTUS, DR. ELYANE, Laboratory of Animal Morphology, Brussels, Belgium 
BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School of 

Hygiene. Baltimore 5, Maryland 

BARD, DR. PHILLIP, Johns Hopkins Medical School, Baltimore, Maryland 
EARTH, DR. L. G., Department of Zoology, Columbia University, New York 27, 

New York 
EARTH, DR. LUCENA, Department of Zoology, Barnard College, New York 27, 

New York 
BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana, 

Illinois 
BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa 

City, Iowa 
BECK, DR. L. V., Department of Physiology and Pharmacology, University of 

Pittsburgh School of Medicine, Pittsburgh 13, Pennsylvania 
BEHRE, DR. ELINOR M., Black Mountain, North Carolina 
BENESCH, DR. REINHOLD, College of Physicians and Surgeons, New York 32, 

New York 
BENESCH, DR. RUTH, College of Physicians and Surgeons, New York 32, New York 



REPORT OF THE DIRECTOR 31 

BENNETT, DR. MICHAEL V., Department of Neurology, College of Physicians and 
Surgeons, New York 32, New York 

BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet 
Briar, Virginia 

BERG, DR. WILLIAM E., Department of Zoology, University of California, Berkeley 
4, California 

BERMAN, DR. MONKS, Institute for Arthritis and Metabolic Diseases, National 
Institutes of Health, Bethesda 14, Maryland 

BERNHEIMER, DR. ALAN W., New York University College of Medicine, New 
York 16, New York 

BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne State University Col- 
lege of Medicine, Detroit 7, Michigan 

BERTHOLF, DR. LLOYD M., Illinois Wesleyan University, Bloomington, Illinois 

BEVELANDER, DR. GERRIT, New York University School of Dentistry, 477 First 
Avenue, New York 16, New York 

BIGELOW, DR. HENRY B., Museum of Comparative Zoology, Harvard University, 
Cambridge 38, Massachusetts 

BISHOP, DR. DAVID W., Department of Embryology, Carnegie Institution of Wash- 
ington, Baltimore 5, Maryland 

BLANCHARD, DR. K. C, Johns Hopkins Medical School, Baltimore, Maryland 

BLOCK, DR. ROBERT, 518 South 42nd Street, Apartment C7, Philadelphia 4, Penn- 
sylvania 

BLUM, DR. HAROLD F., Department of Biology, Princeton University, Princeton, 
New Jersey 

BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center, 
444 East 68th Street, New York 21, New York 

BODIAN, DR. DAVID, Department of Anatomy, Johns Hopkins University, 709 
North Wolfe Street, Baltimore 5, Maryland 

BOELL, DR. EDGAR J., Osborn Zoological Laboratories, Yale University, New 
Haven, Connecticut 

BOETTIGER, DR. EDWARD G., Department of Zoology, University of Connecticut, 
Storrs, Connecticut 

BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin 12, 
Texas 

BOREI, DR. HANS, Department of Zoology, University of Pennsylvania, Philadel- 
phia 4, Pennsylvania 

BOWEN, DR. VAUGHAN T., Woods Hole Oceanographic Institution, Woods Hole, 
Massachusetts 

BRADLEY, DR. HAROLD C., 2639 Durant Avenue, Berkeley 4, California 

BRIDGMAN, DR. ANNA J., Department of Biology, Agnes Scott College, Decatur, 
Georgia 

BRONK, DR. DETLEV W., Rockefeller Institute, 66th Street and York Avenue, 
New York 21, New York 

BROOKS, DR. MATILDA M., Department of Physiology, University of California, 
Berkeley 4, California 

BROWN, DR. DUGALD E. S., Department of Zoology, University of Michigan, Ann 
Arbor, Michigan 



32 MARINE BIOLOGICAL LABORATORY 

BROWN, DR. FRANK A., JR., Department of Biological Sciences, Northwestern 

University, Evanston, Illinois 
BROWNELL, DR. KATHERINE A., Department of Physiology, Ohio State University, 

Columbus, Ohio 
BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of Health, 

Bethesda 14, Maryland 

BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia 
BULLOCK, DR. T. H., Department of Zoology, University of California, Los An- 
geles 24, California 

BURBANCK, DR. WILLIAM D., Box 834, Emory University, Atlanta 22, Georgia 
BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilmington, 

Delaware 
BURKENROAD, DR. M. D., c/o Lab. Nal. de Pesca, Apartado 3318, Estofeta #1, 

Olindania, Republic of Panama 
BUTLER, DR. E. G., Department of Biology, P. O. Box 704, Princeton University, 

Princeton, New Jersey 

CAMERON, DR. J. A., Baylor College of Dentistry, Dallas, Texas 
CANTONI, DR. GIULLIO, National Institutes of Health, Mental Health, Bethesda 14, 

Maryland 
CARLSON, DR. FRANCIS D., Department of Biophysics, Johns Hopkins University, 

Baltimore 18, Maryland 

CARPENTER, DR. RUSSELL L., Tufts University, Medford 55, Massachusetts 
CARSON, Miss RACHEL, 11701 Berwick Road, Silver Spring, Maryland 
CASE, DR. JAMES, Department of Zoology, State University of Iowa, Iowa City, 

Iowa 
CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York Avenue, 

New York City, New York 

CATTELL, MR. WARE, Cosmos Club, Washington 5, District of Columbia 
CHAET, DR. ALFRED B., Department of Biology, American University, Washington 

16, District of Columbia 
CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical 

School, Coral Gables, Florida 
CHANG, DR. JOSEPH J., Akademiestrasse 3, Physiologisches Institut, Postfach 201, 

Heidelberg, Germany 
CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton, 

New Jersey 
CHENEY, DR. RALPH H., Biological Laboratory, Brooklyn College, Brooklyn 10. 

New York 

CLAFF, DR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts 
CLARK, DR. A. M., Department of Biological Sciences, University of Delaware, 

Newark, Delaware 

CLARK, DR. E. R., 315 South 41st Street, Philadelphia 4, Pennsylvania 
CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, 

New York 
CLARKE, DR. GEORGE L., Biological Laboratories, Harvard University, Cambridge 

38, Massachusetts 



REPORT OF THE DIRECTOR 33 

CLELAND, DR. RALPH E., Department of Botany, Indiana University, Bloomington, 

Indiana 
CLEMENT, DR. A. C, Department of Biology, Emory University, Atlanta 22, 

Georgia 

COE, DR. W. R., 183 Third Avenue, Chula Vista, California 
COHEN, DR. SEYMOUR S., Department of Biochemistry, University of Pennsylvania 

School of Medicine, Philadelphia, Pennsylvania 
COLE, DR. KENNETH S., National Institutes of Health (NINDB), Bethesda 14, 

Maryland 

COLLETTE, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts 
COLLIER, DR. JACK R., Marine Biological Laboratory, Woods Hole, Massachusetts 
COLTON, DR. H. S., Box 601, Flagstaff, Arizona 
COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, New 

York 
COLWIN, DR. LAURA H., Department of Biology, Queens College, Flushing, New 

York 
COOPER, DR. KENNETH W., Department of Cytology, Dartmouth Medical School, 

Hanover, New Hampshire 

COOPERSTEIN, DR. S HER WIN J., Department of Anatomy, Western Reserve Uni- 
versity Medical School, Cleveland, Ohio 

COPELAND, DR. D. E., 5820 Hurst Street, Apartment 8, New Orleans 18, Louisiana 
COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine 
COPLEY, DR. A. L., Medical Research Laboratories, Charing Cross Hospital, 8 Ex- 
change Court, Strand, London W. C. 2, England 

CORNMAN, DR. IVOR, Hazleton Laboratories, Box 333, Falls Church, Virginia 
COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 

COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro- 
lina, Chapel Hill, North Carolina 
CRANE, MR. JOHN O., Woods Hole, Massachusetts 

CRANE, DR. ROBERT K., Department of Biological Chemistry, Washington Univer- 
sity Medical School, St. Louis, Missouri 

CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire 
CROUSE, DR. HELEN V., Department of Botany, Columbia University, New York 

27, New York 
CROWELL, DR. P. S., JR., Department of Zoology, Indiana University, Bloomington, 

Indiana 
CSAPO, DR. ARPAD I., Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 

CURTIS, DR. MAYNIE R., University of Miami, Box 1015, South Miami, Florida 
CURTIS, DR. W. C., University of Missouri, Columbia, Missouri 
DAN, DR. JEAN CLARK, Misaki Biological Station, Misaki, Japan 
DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan 
DANIELLI, DR. JAMES F., Department of Zoology, King's College, London, England 
DAVIS, DR. BERNARD D., Harvard Medical School, 25 Shattuck Street, Boston 15, 
Massachusetts 



34 MARINE BIOLOGICAL LABORATORY 

DAWSON, DR. A. B., Biological Laboratories, Harvard University, Cambridge 38, 

Massachusetts 

DAWSON, DR. J. A., 129 Violet Avenue, Floral Park, Long Island, New York 
DEANE, DR. HELEN W., Albert Einstein College of Medicine, New York 61, New 

York 
DILLER, DR. IRENE C., Institute for Cancer Research, Fox Chase, Philadelphia 11, 

Pennsylvania 

DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania 
DIXON, DR. FRANK J., Department of Pathology, University of Pittsburgh School 

of Medicine, Pittsburgh 13, Pennsylvania 
DODDS, DR. G. S., West Virginia University School of Medicine, Morgantown, 

West Virginia 

DOLLEY, DR. WILLIAM L., Department of Biology, Randolph-Macon College, Ash- 
land, Virginia 
DOTY, DR. MAXWELL S., Department of Biology, University of Hawaii, Honolulu, 

Hawaii 
DURYEE, DR. WILLIAM R., George Washington University School of Medicine, 

Department of Physiology, Washington 5, District of Columbia 
EDDS, DR. MAC V., JR., Department of Biology, Brown University, Providence 12, 

Rhode Island 
EDWARDS, DR. CHARLES, Department of Physiology, University of Minnesota, 

Minneapolis 14, Minnesota 

EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania 
EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis, 

Missouri 
ELLIOTT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann 

Arbor, Michigan 
ESSNER, DR. EDWARD S., Department of Pathology, Albert Einstein College of 

Medicine, New York 61, New York 
EVANS, DR. TITUS C., State University of Iowa College of Medicine, Iowa City, 

Iowa 

FAILLA, DR. G., Building 203, Argonne National Laboratory, Argonne, Illinois 
FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France 
FERGUSON, DR. F. P., Division of General Medical Sciences, National Institutes of 

Health, Bethesda 14, Maryland 
FERGUSON, DR. JAMES K. W., Connought Laboratories, University of Toronto, 

Ontario, Canada 
FIGGE, DR. F. H. J., University of Maryland Medical School, Lombard and Green 

Streets, Baltimore 1, Maryland 

FINGERMAN, DR. MILTON, Department of Zoology, Newcomb College, Tulane Uni- 
versity, New Orleans 18, Louisiana 

FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Rich- 
mond, Virginia 
FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, 

Toronto, Canada 
FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto, 

Canada 



REPORT OF THE DIRECTOR 35 

FORBES, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge 
38, Massachusetts 

FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois, 
Urbana, Illinois 

FREYGANG, DR. WALTER H., JR., Box 516, Essex Fells, New Jersey 

FRIES, DR. ERIK F. B., Box 605, Woods Hole, Massachusetts 

FRISCH, DR. JOHN A., Canisius College, Buffalo, New York 

FURSHPAN, DR. EDWIN J., Department of Neurophysiology, Harvard Medical 
School, Boston 15, Massachusetts 

FURTH, DR. JACOB, 183 Cleveland Avenue, Buffalo, New York 

FYE, DR. PAUL M., Woods Hole Oceanographic Institution, Woods Hole, Massa- 
chusetts 

GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn 10, 
New York 

GAFFRON, DR. HANS, Department of Biology, Florida State University, Conradi 
Building, Tallahassee, Florida 

GALL, DR. JOSEPH C, Department of Zoology, University of Minnesota, Minneapo- 
lis 14, Minnesota 

GALTSOFF, DR. PAUL S., Woods Hole, Massachusetts 

GASSER, DR. HERBERT S., Rockefeller Institute, 66th Street and York Avenue, New 
York 21, New York 

GILMAN, DR. LAUREN C., Department of Zoology, University of Miami, Coral 
Gables, Florida 

GINSBERG, DR. HAROLD S., Department of Microbiology, University of Pennsyl- 
vania School of Medicine, Philadelphia 4, Pennsylvania 

GOLDSMITH, DR. TIMOTHY H., Department of Zoology, Yale University, New 
Haven, Connecticut 

GOLDSTEIN, DR. LESTER, Department of Zoology, University of Pennsylvania, 
Philadelphia 4, Pennsylvania 

GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, Atlanta 
22, Georgia 

GOODRICH, DR. H. B., Department of Biology, Wesleyan University, Middletown, 
Connecticut 

GOTSCHALL, DR. GERTRUDE Y., Rockefeller Institute, 66th Street and York Avenue, 
New York 21, New York 

GRAHAM, DR. HERBERT, U. S. Fish and Wildlife Service, Woods Hole, Massachu- 
setts 

GRAND, MR. C. G., Cancer Institute of Miami, 1155 N. W. 15th Street, Miami, 
Florida 

GRANT, DR. M. P., Sarah Lawrence College, Bronxville, New York 

GRANT, DR. PHILIP, Department of Pathobiology, Johns Hopkins University School 
of Hygiene, Baltimore 5, Maryland 

GRAY, DR. IRVING E., Department of Zoology, Duke University, Durham, North 
Carolina 

GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New Bruns- 
wick, New Jersey 



36 MARINE BIOLOGICAL LABORATORY 

GREEN, DR. MAURICE, Department of Microbiology, St. Louis University Medical 

School, St. Louis, Missouri 
GREGG, DR. JAMES H., Department of Biological Sciences, University of Florida, 

Gainesville, Florida 
GREGG, DR. JOHN R., Department of Zoology, Duke University, Durham, North 

Carolina 

GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical Col- 
lege, New York 21, New York 
GRIFFIN, DR. DONALD R., Biological Laboratories, Harvard University, Cambridge 

38, Massachusetts 
GROSCH, DR. DANIEL S., Department of Genetics, Gardner Hall, North Carolina 

State College, Raleigh, North Carolina 
GROSS, DR. PAUL, Department of Biology, New York University, University 

Heights, New York 53, New York 
GRUNDFEST, DR. HARRY, Columbia University College of Physicians and Surgeons, 

New York 32, New York 

GUDERNATSCH, DR. FREDERICK, 41 Fifth Avenue, New York 3, New York 
GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn 10, 

New York 

HAJDU, DR. STEPHEN, National Institutes of Health, Bethesda 14, Maryland 
HALL, DR. FRANK G., Department of Physiology, Duke University Medical School, 

Durham, North Carolina 
HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St. 

Louis, Missouri 
HAMILTON, DR. HOWARD L., Department of Zoology, Iowa State College, Ames, 

Iowa 

HANCE, DR. ROBERT T., Box 108, R. R. #3, Loveland, Ohio 
HARDING, DR. CLIFFORD V., JR., 300 Knickerbocker Road, Tenafly, New Jersey 
HARNLY, DR. MORRIS H., Washington Square College, New York University, New 

York 3, New York 
HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research, 66th Street 

and York Avenue, New York 21, New York 

HARTMAN, DR. FRANK A., Ohio State University, Hamilton Hall, Columbus, Ohio 
HARVEY, DR. ETHEL BROWNE, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 
HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo 

3, New York 

HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institution of Ocean- 
ography, University of California, La Jolla, California 
HAYASHI, DR. TERU, Department of Zoology, Columbia University, New York 27, 

New York 

HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesley 81, Massachusetts 
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts 
HENDLEY, DR. CHARLES D., 615 South Second Avenue, Highland Park, New Jersey 
HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 



V REPORT OF THE DIRECTOR 37 

HERNDON, DR. WALTER R., Biology Department, University of Alabama, Univer- 
sity, Alabama 

HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts 
HESS, DR. WALTER N., 309 Aiken Street, Rock Hill, South Carolina 
HIATT, DR. HOWARD H., Department of Medicine, Harvard Medical School, Boston 

15, Massachusetts 

HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio 
HILL, DR. SAMUEL E., 135 Brunswick Road, Troy, New York 
HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge 38, 

Massachusetts 
HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge 38, 

Massachusetts 
HODGE, DR. CHARLES, IV, Department of Biology, Temple University, Philadelphia, 

Pennsylvania 
HOFFMAN, DR. JOSEPH, National Heart Institute, National Institutes of Health, 

Bethesda 14, Maryland 
HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia 4, 

Pennsylvania 
HOLLAENDER, DR. ALEXANDER, Biology Division, Oak Ridge National Laboratory, 

Oak Ridge, Tennessee 
HOLZ, DR. GEORGE G., JR., Department of Zoology, Syracuse University, Syracuse, 

New York 

HOPKINS, DR. HOYT S., 59 Heatherdell Road, Ardsley, New York 
HUNTER, DR. FRANCIS R., University of the Andes, Calle 18-a Carrera 1-E, Bogota, 

Colombia, South America 

HUTCHENS, DR. JOHN O., Department of Physiology, University of Chicago, Chi- 
cago 37, Illinois 

HYDE, DR. BEAL B., Department of Plant Sciences, University of Oklahoma, Nor- 
man, Oklahoma 
HYMAN, DR. LIBBIE H., American Museum of Natural History, Central Park 

West at 79th Street, New York 24, New York 

IRVING, DR. LAURENCE, U. S. Public Health Service, Anchorage, Alaska 
ISENBERG, DR. IRVIN, Institute for Muscle Research, Marine Biological Laboratory, 

Woods Hole, Massachusetts 

ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts 
JACOBS, DR. M. H., University of Pennsylvania School of Medicine, Philadelphia 

4, Pennsylvania 
JACOBS, DR. WILLIAM P., Department of Biology, Princeton University, Princeton, 

New Jersey 
JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 
JOHNSON, DR. FRANK H., Department of Biology, Princeton University, Princeton, 

New Jersey 
JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of 

Florida, Gainesville, Florida 

JONES, DR. RAYMOND F., Department of Biology, Princeton University, Princeton, 
New Jersey 



38 MARINE BIOLOGICAL LABORATORY 

KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachusetts 
KABAT, DR. E. A., Neurological Institute, College of Physicians and Surgeons, 

New York 32, New York 

KARUSH, DR. FRED, Department of Pediatrics, University of Pennsylvania, Phila- 
delphia 4, Pennsylvania 
KAUFMANN, DR. B. P., Carnegie Institution, Cold Spring Harbor, Long Island, 

New York 
KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann 

Arbor, Michigan 
KEMPTON, DR. RUDOLF T., Department of Zoology, Vassar College, Poughkeepsie, 

New York 
KEOSIAN, DR. JOHN, Department of Biology, Rutgers University, Newark 2, New 

Jersey 
KETCHUM, DR. BOSTWICK, Woods Hole Oceanographic Institution, Woods Hole, 

Massachusetts 

KILLE, DR. FRANK R., State Department of Education, Albany 1, New York 
KIND, DR. C. ALBERT, Department of Zoology, University of Connecticut, Storrs, 

Connecticut 

KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia 
KING, DR. JOHN W., Morgan State College, Baltimore 12, Maryland 
KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa 
KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca, New 

York 

KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York 
KLEIN, DR. MORTON, Department of Microbiology, Temple University, Philadel- 
phia, Pennsylvania 
KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland 2, 

Oregon 
KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston, 

Illinois 
KOLIN, DR. ALEXANDER, Department of Biophysics, California Medical School, 

Los Angeles 24, California 
KORR, DR. I. M., Department of Physiology, Kirksville College of Osteopathy, 

Kirksville, Missouri 
KRAHL, DR. M. E., Department of Physiology, University of Chicago, Chicago 37, 

Illinois 
KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Baltimore, 

Maryland 

KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois 
KUFFLER, DR. STEPHEN, Department of Pharmacology, Harvard Medical School, 

Neurophysical Laboratory, Boston 15, Massachusetts 
KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New York 

21, New York 

LACKEY, DR. JAMES B., Box 497, Melrose, Florida 
LAMY, DR. FRANCOIS, Department of Anatomy, University of Pittsburgh School of 

Medicine, Pittsburgh 13, Pennsylvania 
LANCEFIELD, DR. D. E., Queens College, Flushing, New York 



REPORT OF THE DIRECTOR 39 

LANCEFIELD, DR. REBECCA C, Rockefeller Institute, 66th Street and York Avenue, 

New York 21, New York 

LANDIS, DR. E. M., Harvard Medical School, Boston 15, Massachusetts 
LANSING, DR. ALBERT I., Department of Anatomy, University of Pittsburgh Medi- 
cal School, Pittsburgh 13, Pennsylvania 

LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts- 
burgh, Pennsylvania 

LAVIN, DR. GEORGE I., 6200 Norvo Road, Baltimore 7, Maryland 
LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota Medical 

School, Minneapolis 14, Minnesota 
LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford University Medical 

School, Stanford, California 
LEE, DR. RICHARD E., Cornell University College of Medicine, New York City, 

New York 
LEFEVRE, DR. PAUL G., University of Louisville School of Medicine, Louisville, 

Kentucky 

LEHMANN, DR. FRITZ, Zoologische Institut, University of Berne, Berne, Switzer- 
land 

LEVINE, DR. RACHMIEL, Michael Rees Hospital, Chicago 16, Illinois 
LEVY, DR. MILTON, Department of Biochemistry, New York University School of 

Dentistry, New York 10, New York 

LEWIN, DR. RALPH A., Scripps Institution of Oceanography, La Jolla, California 
LEWIS, DR. IVEY F., 1110 Rugby Road, Charlottesville, Virginia 
LING, DR. GILBERT, 307 Berkeley Road, Merion, Pennsylvania 
LITTLE, DR. E. P., 216 High Street, West Newton, Massachusetts 
LLOYD, DR. DAVID P. C., Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 

LOCHHEAD, DR. JOHN H., Department of Zoology, University of Vermont, Burling- 
ton, Vermont 

LOEB, DR. R. F., 950 Park Avenue, New York 28, New York 
LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York 
LOFTFIELD, DR. ROBERT B., Associate Biochemist, Massachusetts General Hospital, 

Boston, Massachusetts 

LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans- 
ton, Illinois 
DELORENZO, DR. ANTHONY, Anatomical and Pathological Research Laboratories, 

Johns Hopkins Hospital, Baltimore 5, Maryland 
LOVE, DR. Lois H., 1043 Marlau Drive, Baltimore 12, Maryland 
LOVE, DR. WARNER E., 1043 Marlau Drive, Baltimore 12, Maryland 
LUBIN, DR. MARTIN, Department of Pharmacology, Harvard Medical School, 

Boston 15, Massachusetts 
LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 
LYNCH, DR. WILLIAM, Department of Biology, St. Ambrose College, Davenport, 

Iowa 

LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America, 
Washington 17, District of Columbia 



40 MARINE BIOLOGICAL LABORATORY 

MACDOUGALL, DR. MARY STUART, Mt. Vernon Apartments, 423 Clairmont Avenue, 

Decatur, Georgia 
McCANN, DR. FRANCES, Department of Physiology, Dartmouth Medical School, 

Hanover, New Hampshire 
McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School, 

Philadelphia 4, Pennsylvania 
MCDONALD, SISTER ELIZABETH SETON, Department of Biology, College of Mt. St. 

Joseph, Mt. St. Joseph, Ohio 
MCDONALD, DR. MARGARET H., Carnegie Institution of Washington, Cold Spring 

Harbor, Long Island, New York 
McELROY, DR. WILLIAM D., Department of Biology, Johns Hopkins University, 

Baltimore 18, Maryland 
MAAS, DR. WERNER K., New York University College of Medicine, New York 

City, New York 
MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 135 

Harrison Avenue, Boston, Massachusetts 

MANWELL, DR. REGINALD D.. Department of Zoology, Syracuse University, Syra- 
cuse, New York 
MARSHAK, DR. ALFRED, Department of Radiology, Jefferson Medical College, 

Philadelphia 7, Pennsylvania 
MARSLAND, DR. DOUGLAS A., New York University, Washington Square College, 

New York 3, New York 
MARTIN, DR. EARL A., Department of Biology, Brooklyn College, Brooklyn 10, 

New York 
MATHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College, 

Williamstown, Massachusetts 

MAYOR, DR. JAMES W., 8 Gracewood Park, Cambridge 38, Massachusetts 
MAZIA, DR. DANIEL. Department of Zoology, University of California. Berkeley 4, 

California 

MEDES, DR. GRACE, 303 Abington Avenue, Philadelphia 11, Pennsylvania 
MEINKOTH, DR. NORMAN A., Department of Biology, Swarthmore College, 

Swarthmore, Pennsylvania 
MENKIN, DR. VALY, University of Kansas City School of Dentistry, 1108 East 

10th Street, Kansas City, Missouri 
METZ, DR. C. B., Oceanographic Institute, Florida State University, Tallahassee, 

Florida 

METZ, DR. CHARLES W., Box 714, Woods Hole, Massachusetts 
MIDDLEBROOK, DR. ROBERT, Institute for Muscle Research, Marine Biological Lab- 
oratory, Woods Hole, Massachusetts 
MILLER, DR. J. A., JR., Department of Anatomy, Tulane University Medical School, 

New Orleans 18, Louisiana 
MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire, 

Durham, New Hampshire 
MOE, MR. HENRY A., Guggenheim Memorial Foundation, 551 Fifth Avenue, New 

York 17, New York 
MONROY, DR. ALBERTO, Institute of Comparative Anatomy, University of Palermo, 

Italy 



REPORT OF THE DIRECTOR 



41 



MOORE, DR. GEORGE M., Department of Zoology, University of New Hampshire, 

Durham, New Hampshire 
MOORE, DR. JOHN A., Department of Zoology, Columbia University, New York 

27, New York 
MOORE, DR. JOHN W., Laboratory of Biophysics, NINDB, National Institutes of 

Health, Bethesda 14, Maryland 

MORRILL, DR. JOHN B., JR., Department of Biology, Wesleyan University, Middle- 
town, Connecticut 
MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick, 

New Jersey 

MOUNTAIN, MRS. J. D., Charles Road, Mt. Kisco, New York 
MULLINS, DR. LORIN J., Biophysical Laboratory, Purdue University, Lafayette, 

Indiana 
MUSACCHIA, DR. XAVIER, JR., Department of Biology, St. Louis University, St. 

Louis 4, Missouri 
NABRIT, DR. S. M., President, Texas Southern University, 3201 Wheeler Avenue, 

Houston 4, Texas 
NACE, DR. PAUL FOLEY, Department of Biology, Hamilton College, McMaster 

University, Hamilton, Ontario 

XACHMANSOHN, DR. DAVID, Columbia University, College of Physicians and Sur- 
geons, New York 32, New York 

NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts 
NELSON, DR. LEONARD, Department of Physiology, Emory University, Atlanta 22, 

Georgia 
NEURATH, DR. H., Department of Biochemistry, University of Washington, Seattle 

5, Washington 

NICOLL, DR. PAUL A., BMSI/USOM/P-K, APO 271, New York City, New York 
Niu, DR. MAN-CHIANG, Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 
XOVIKOFF, DR. ALEX B., Department of Pathology, Albert Einstein College of 

Medicine, New York 61, New York 
OCHOA, DR. SEVERO, New York University College of Medicine, New York 61, 

New York 
ODUM, DR. EUGENE, Department of Zoology, University of Georgia, Athens, 

Georgia 
OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn 

Mawr, Pennsylvania 
OSTERHOUT, DR. W. J. V., Rockefeller Institute, 66th Street and York Avenue, 

New York 21, New York 
OSTERHOUT, DR. MARION IRWIN, Rockefeller Institute, 66th Street and York 

Avenue, New York 21, New York 
PACKARD, DR. CHARLES, Woods Hole, Massachusetts 
PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio 
PARPART, DR. ARTHUR K., Department of Biology, Princeton University, Princeton, 

New Jersey 

PASSANO, DR. LEONARD M., Osborn Zoological Laboratories, Yale University, New 
Haven, Connecticut 



42 MARINE BIOLOGICAL LABORATORY 

PATTEN, DR. BRADLEY M., University of Michigan School of Medicine, Ann Arbor, 
Michigan 

PERKINS, DR. JOHN F., JR., Department of Physiology, University of Chicago, 
Chicago 37, Illinois 

PERSON, DR. PHILIP, Chief, Special Dental Research Program, VA Hospital, 
Brooklyn 9, New York 

PETTIBONE, DR. MARIAN H., Department of Zoology, University of New Hamp- 
shire, Durham, New Hampshire 

PHILPOTT, MR. DELBERT E., 496 Palmer Avenue, Falmouth, Massachusetts 

PICK, DR. JOSEPH, Department of Anatomy, New York University-Bellevue Medi- 
cal Center, New York 16, New York 

PIERCE, DR. MADELENE E., Department of Zoology, Vassar College, Poughkeepsie, 
New York 

PLOUGH, DR. HAROLD H., Department of Biology, Amherst College, Amherst, 
Massachusetts 

POLLISTER, DR. A. W., Department of Zoology, Columbia University, New York 
27, New York 

POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut 

POTTER, DR. DAVID, Department of Neurophysiology, Harvard Medical School, 
Boston 15, Massachusetts 

PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti- 
more 12, Maryland 

PROSSER, DR. C. LADD, 401 Natural History Building, University of Illinois, Ur- 
bana, Illinois 

PROVASOLI, DR. LUIGI, Haskins Laboratories, 305 East 43rd Street, New York 17, 
New York 

RAMSEY, DR. ROBERT W., Medical College of Virginia, Richmond, Virginia 

RAND, DR. HERBERT W., 7 Siders Pond Road, Falmouth, Massachusetts 

RANKIN, DR. JOHN S., Department of Zoology, University of Connecticut, Storrs, 
Connecticut 

RANZI, DR. SILVIO, Department of Zoology, University of Milan, Milan, Italy 

RATNER, DR. SARAH, Public Health Research Institute of the City of New York, 
Foot of East 15th Street, New York 9, New York 

RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta 22, 
Georgia 

READ, DR. CLARK P., Department of Biology, Rice University, Houston, Texas 

REBHUN, DR. LIONEL I., Department of Biology, Box 704, Princeton University, 
Princeton, New Jersey 

RECHNAGEL, DR. R. O., Department of Physiology, Western Reserve University, 
Cleveland, Ohio 

REDFIELD, DR. ALFRED C., Woods Hole, Massachusetts 

RENN, DR. CHARLES E., 509 Ames Hall, Johns Hopkins University, Baltimore 18, 
Maryland 

REUBEN, DR. JOHN P., Department of Neurology, College of Physicians and Sur- 
geons, New York 32, New York 

REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue, 
New York 16, New York 



REPORT OF THE DIRECTOR 43 

RICHARDS, DR. A., 2950 E. Mabel Street, Tucson, Arizona 

RICHARDS, DR. A. GLENN, Department of Entomology, University of Minnesota, 
St. Paul 1, Minnesota 

RICHARDS, DR. OSCAR W., American Optical Company, Research Center, South- 
bridge, Massachusetts 

ROCKSTEIN, DR. MORRIS, Department of Physiology, New York University College 
of Medicine, New York 16, New York 

ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York 

ROMER, DR. ALFRED S., Museum of Comparative Zoology, Harvard University, 
Cambridge 38, Massachusetts 

RONKIN, DR. RAPHAEL R., Department of Physiology, University of Delaware, 
Newark, Delaware 

ROOT, DR. R. W., Department of Biology, College of the City of New York, New 
York City, New York 

ROOT, DR. W. S., Columbia University College of Physicians and Surgeons, De- 
partment of Physiology, New York 32, New York 

ROSE, DR. S. MERYL, Department of Biology, Wesleyan University, Middletown, 
Connecticut 

ROSENBERG, DR. EVELYN K., Department of Pathology, New York University- 
Bellevue Medical Center, New York 16, New York 

ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pittsburgh 
Medical School, Pittsburgh 13, Pennsylvania 

Rossi, DR. HAROLD H., Department of Radiology, Columbia University, 630 West 
168th Street, New York 32, New York 

ROTH, DR. JAY S., Department of Zoology and Entomology, University of Connec- 
ticut, Storrs, Connecticut 

ROTHENBERG, DR. M. A., Scientific Director, Dugway Proving Ground, Dugway, 
Utah 

RUGH, DR. ROBERTS, Radiological Research Laboratory, College of Physicians and 
Surgeons, 630 West 168th Street, New York 32, New York 

RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden 

RUTMAN, DR. ROBERT J., General Laboratory Building, 215 S. 34th Street, Phila- 
delphia 4, Pennsylvania 

RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole, 
Massachusetts 

SANBORN, DR. RICHARD C., Department of Biological Sciences, Purdue University, 
Lafayette, Indiana 

SANDEEN, DR. MURIEL I., Department of Zoology, Duke University, Durham, 
North Carolina 

SAUNDERS, MR. LAWRENCE, West Washington Square, Philadelphia 5, Pennsylvania 

SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of Cali- 
fornia, Berkeley 4, California 

SCHARRER, DR. ERNST A., Department of Anatomy, Albert Einstein College of 
Medicine, 1710 Newport Avenue, New York 61, New York 

SCHLESINGER, DR. R. WALTER, Department of Microbiology, St. Louis University 
School of Medicine, 1402 South Grand Boulevard, St. Louis 4, Missouri 



44 MARINE BIOLOGICAL LABORATORY 

SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio 

SCHMITT, DR. FRANCIS O., Department of Biology, Massachusetts Institute of 
Technology, Cambridge, Massachusetts 

SCHMITT, DR. O. H., Department of Physics, University of Minnesota, Minneapo- 
lis 14, Minnesota 

SCHNEIDERMAN, DR. HOWARD A., Department of Zoology, Cornell University, 
Ithaca, New York 

SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, California 

SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, 
Massachusetts 

SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington, 
Indiana 

SCOTT, DR. ALLAN C., Colby College, Waterville, Maine 

SCOTT, DR. D. B. McNAiR, Botany Annex, Cancer Chemotherapy Laboratory, 
University of Pennsylvania, Philadelphia 4, Pennsylvania 

SCOTT, SISTER FLORENCE MARIE, Seton Hill College, Greensburg, Pennsylvania 

SCOTT, DR. GEORGE T., Department of Zoology, Oberlin College, Oberlin, Ohio 

SEARS, DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massa- 
chusetts 

SENFT, DR. ALFRED W., Woods Hole, Massachusetts 

SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and 
Surgeons, New York 32, New York 

SHANES, DR. ABRAHAM, Experimental Biological and Medicine Institute, National 
Institutes of Health, Bethesda 14, Maryland 

SHAPIRO, DR. HERBERT, 5800 North Camac Street, Philadelphia 41, Pennsylvania 

SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East 
Lansing, Michigan 

SHEDLOVSKY, DR. THEODORE, Rockefeller Institute, 66th Street and York Avenue, 
New York 21, New York 

SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont 

SICHEL, MRS. F. J. M., 38 Henderson Terrace, Burlington, Vermont 

SILVA, DR. PAUL, Department of Botany, University of California, Berkeley 4, 
California 

SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa 
City, Iowa 

SMITH, DR. DIETRICH C., Department of Physiology, University of Maryland 
School of Medicine, Baltimore, Maryland 

SMITH, MR. HOMER P., Marine Biological Laboratory, Woods Hole, Massachusetts 

SMITH, MR. PAUL FERRIS, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 
SMITH, DR. RALPH I., Department of Zoology, University of California, Berkeley 

4, California 
SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington, 

Indiana 

SONNENBLICK, DR. B. P., Rutgers University, 40 Rector Street, Newark 2, New 
Jersey 



REPORT OF THE DIRECTOR 45 

SPEIDEL, DR. CARL C., Department of Anatomy, University of Virginia, University, 
Virginia 

SPIEGEL, DR. MELVIN, Department of Zoology, Dartmouth College, Hanover, New 
Hampshire 

SPRATT, DR. NELSON T., JR., Department of Zoology, University of Minnesota, 
Minneapolis 14, Minnesota 

SPYROPOULOS, DR. C. S., Building 9, Room 140, National Institutes of Health, 
Bethesda 14, Maryland 

STARR, DR. RICHARD C., Department of Botany, Indiana University, Bloomington, 
Indiana 

STEINBACH, DR. H. BURR, Department of Zoology, University of Chicago, Chicago 
37, Illinois 

STEINBERG, DR. MALCOLM S., Department of Biology, Johns Hopkins University, 
Baltimore 18, Maryland 

STEINHARDT, DR. JACINTO, Operations Evaluation Group, Massachusetts Institute 
of Technology, Cambridge, Massachusetts 

STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota, Min- 
neapolis 14, Minnesota 

STETTEN, DR. DE\ITT, Director in Charge of Research, NIAMD, National Insti- 
tutes of Health, Bethesda 14, Maryland 

STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois 

STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College, South 
Hadley, Massachusetts 

STONE, DR. WILLIAM, Ophthalmic Plastics Laboratory, Massachusetts Eye and 
Ear Infirmary, Boston, Massachusetts 

STRAUS, DR. W. L., JR., Department of Anatomy, Johns Hopkins University Medi- 
cal School, Baltimore 5, Maryland 

STREHLER, DR. BERNARD L., Cellular and Comparative Physiology Section, National 
Institutes of Health, Bethesda 14, Maryland 

STUNKARD, DR. HORACE W., American Museum of Natural History, Central Park 
West at 79th Street, New York 24, New York 

STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena 4, 
California 

SUDAK, DR. FREDERICK N., Department of Physiology, Albert Einstein College of 
Medicine, New York 61, New York 

SULKIN, DR. S. EDWARD, Department of Bacteriology, University of Texas, South- 
western Medical School, Dallas, Texas 

SWOPE, MR. GERARD, JR., 570 Lexington Avenue, New York 22, Xew York 

SZENT-GYORGYI, DR. ALBERT, Institute for Muscle Research, Marine Biological 
Laboratory, Woods Hole, Massachusetts 

SZENT-GYORGYI, DR. ANDREW G., Institute for Muscle Research, Marine Biological 
Laboratory, Woods Hole, Massachusetts 

TASAKI, DR. ICHIJI, Laboratory of Neurophysiology, NINDB, Bethesda 14, Mary- 
land 

TASHIRO, DR. SHIRO, University of Cincinnati Medical College, Cincinnati, Ohio 

TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology, NINDB, Bethesda 14, 
Maryland 



46 MARINE BIOLOGICAL LABORATORY 

TAYLOR, DR. WM. RANDOLPH, Department of Botany, University of Michigan, Ann 
Arbor, Michigan 

TEWINKEL, DR. Lois E., Department of Zoology, Smith College, Northampton, 
Massachusetts 

TOBIAS, DR. JULIAN, Department of Physiology, University of Chicago, Chicago, 
Illinois 

TRACY, DR. HENRY C, General Delivery, Oxford, Mississippi 

TRACER, DR. WILLIAM, Rockefeller Institute, 66th Street and York Avenue, New 
York 21, New York 

TRINKAUS, PR. J. PHILIP, Department of Zoology, Osborn Zoological Labora- 
tories, Yale University, New Haven, Connecticut 

TROLL, DR. WALTER, Department of Industrial Medicine, New York University, 
College of Medicine, New York 16, New York 

TWEEDELL, DR. KEN YON S., Department of Biology, University of Notre Dame, 
Notre Dame, Indiana 

TYLER, DR. ALBERT, Division of Biology, California Institute of Technology, Pasa- 
dena 4, California 

UHLENHUTH, DR. EDWARD, University of Maryland School of Medicine, Balti- 
more, Maryland 

URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago, Chicago, 
Illinois 

DE VILLAFRANCA, DR. GEORGE W., Department of Zoology, Smith College, Nor- 
thampton, Massachusetts 

VILLEE, DR. CLAUDE A., Department of Biological Chemistry, Harvard Medical 
School, Boston 15, Massachusetts 

VINCENT, DR. WALTER S., Department of Anatomy, State University of New York 
School of Medicine, Syracuse 10, New York 

WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New 
Brunswick, New Jersey 

WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge 38, 
Massachusetts 

WARNER, DR. ROBERT C., Department of Chemistry, New York University College 
of Medicine, New York 16, New York 

WATERMAN, DR. T. H., Department of Zoology, 272 Gibbs Research Laboratory, 
Yale University, New Haven, Connecticut 

WEBB, DR. MARGUERITE, Department of Physiology and Bacteriology, Goucher 
College, Towson, Baltimore 4, Maryland 

WEISS, DR. PAUL A., Laboratory of Developmental Biology, Rockefeller Institute, 
66th Street and York Avenue, New York 21, New York 

WENRICH, DR. D. H., Department of Zoology, University of Pennsylvania, Phila- 
delphia 4, Pennsylvania 

WERMAN, DR. ROBERT, Department of Neurology, College of Physicians and Sur- 
geons, New York 32, New York 

WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut 

WHITAKER, DR. DOUGLAS M., Rockefeller Institute for Medical Research, 66th 
Street and York Avenue, New York 21, New York 



REPORT OF THE DIRECTOR 47 

WHITE, DR. E. GRACE, 1312 Edgar Avenue, Chambersburg, Pennsylvania 

WHITING, DR. ANNA R., Department of Zoology, University of Pennsylvania, 
Philadelphia 4, Pennsylvania 

WHITING, DR. PHINEAS, Department of Zoology, University of Pennsylvania, 
Philadelphia 4, Pennsylvania 

WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York 

WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, 
Pennsylvania 

WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts 

WIERCINSKI, DR. FLOYD J., Department of Biological Sciences, Dre:xU Institute of 
Technology, 32nd and Chestnut Streets, Philadelphia 4, Pennsylvania 

WIGLEY, DR. ROLAND L., U. S. Fish and Wildlife Service, Woods Hole, Massa- 
chusetts 

WILBER, DR. C. G., Medical Laboratories, Applied Physiology Branch, Army 
Chemical Center, Maryland 

WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore 
18, Maryland 

WILSON, DR. J. WALTER, Department of Biology, Brown University, Providence 12, 
Rhode Island 

WILSON, DR. WALTER L., Department of Physiology, University of Vermont Col- 
lege of Medicine, Burlington, Vermont 

WITSCHI, DR. EMIL, Department of Zoology, State University of Iowa, Iowa 
City, Iowa 

WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry, 
Albert Einstein College of Medicine, New York 61, New York 

WOLF, DR. ERNST, Pendelton Hall, Wellesley College, Wellesley, Massachusetts 

WOODWARD, DR. ARTHUR A., Army Chemical Center, Maryland (Applied Physiol- 
ogy Branch, Army Chemical Corps, Medical Laboratory) 

WRIGHT, DR. PAUL A., Department of Zoology, University of New Hampshire, 
Durham, New Hampshire 

WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton, 
Massachusetts 

YNTEMA, DR. C. L., Department of Anatomy, State University of New York Col- 
lege of Medicine, Syracuse 10, New York 

YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts 

ZIMMERMAN, DR. A. M., Department of Pharmacology, State University of New 
York, Downstate Medical Center, Brooklyn 3, New York 

ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings- 
ton, Rhode Island 

ZIRKLE, DR. RAYMOND E., Department of Radiobiology, University of Chicago, 
Chicago 37, Illinois 

ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie, 
New York 

ZWEIFACH, DR. BENJAMIN, New York University Bellevue Medical Center, New 
York 16, New York 

ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham 54, 
Massachusetts 



48 



MARINE BIOLOGICAL LABORATORY 



3. ASSOCIATE MEMBERS 



ALTON, DR. AND MRS. BENJAMIN H. 

ARMSTRONG, DR. AND MRS. P. B. 

BACON, MRS. ROBERT 

BAITSELL, MRS. GEORGE 

BALL, MRS. ERIC 

BARBOUR, MR. Lucius H. 

BARTOW, MR. AND MRS. CLARENCE 

BARTOW, MRS. FRANCIS D. 

BARTOW, MR. AND MRS. PHILIP K. 

BELL, MRS. ARTHUR W. 

BRADLEY, MR. AND MRS. ALBERT L. 

BRADLEY, MR. AND MRS. CHARLES 

BROWN, MRS. THORNTON 

BURDICK, DR. C. LALOR 

BURLINGAME, MRS. F. A. 

CAHOON, MRS. SAMUEL, SR. 

CALKINS, MRS. GARY N. 

CALKINS, MRS. G. NATHAN, JR. 

CALKINS, MR. AND MRS. SAMUEL W. 

CARLTON, MR. AND MRS. WINSLOW 

CLAFF, DR. AND MRS. C. LLOYD 

CLARK, DR. AND MRS. ALFRED HULL 

CLARK, MRS. LEROY 

CLARK, MR. AND MRS. W. VAN ALAN 

CLOWES, MR. ALLEN W. 

CLOWES, MRS. G. H. A. 

CLOWES, DR. AND MRS. G. H. A., JR. 

COLTON, MR. AND MRS. H. SEYMOUR 

COWDRY, DR. AND MRS. E. V. 

CRANE, MR. AND MRS. BRUCE 

CRANE, MR. JOHN 

CRANE, Miss LOUISE 

CRANE, MRS. MURRAY 

CRANE, MR. STEPHEN 

CRANE, MRS. W. CAREY 

CROSSLEY, MR. AND MRS. ARCHIBALD M. 

CROWELL, MR. AND MRS. PRINCE S. 

CURTIS, DR. AND MRS. W. D. 

DANIELS, MR. AND MRS. F. HAROLD 

DAY, MR. AND MRS. POMEROY 

DRAPER, MRS. MARY C. 

DREYER, MR. AND MRS. FRANK A. 

ELSMITH, MRS. DOROTHY 

ENDERS, MRS. FREDERICK 

EWING, MR. AND MRS. FREDERIC 



EWING, MR. WILLIAM 
FAY, MR. AND MRS. HENRY H. 
FISHER, MR. AND MRS. B. C. 
FRANCIS, MRS. LEWIS H., JR. 
FROST, MRS. FRANK J. 
GALTSOFF, MRS. PAUL S. 

GlFFORD, MR. AND MRS. JOHN A. 
GlFFORD, MR. AND MRS. PROSSER 
GlLCHRIST, MR. AND MRS. JOHN M. 
GlLDEA, DR. AND MRS. E. F. 

GREEN, Miss GLADYS M. 
GULESIAN, MRS. PAUL J. 
HAIG, MRS. R. H. 

HAMLEN, MR. AND MRS. J. MONROE 
HARRELL, MR. AND MRS. JOEL E. 
HARRINGTON, MR. AND MRS. ROBERT 
HARVEY, DR. ETHEL B. 
HERRINGTON, MRS. A. W. S. 
HERVEY, DR. AND MRS. JOHN P. 
HlRSCHFELD, MRS. NATHAN B. 
HOUSTON, MR. AND MRS. HOWARD 
JEWETT, MRS. G. F. 
JOHLIN, MRS. JACOB M. 
KEITH, MR. AND MRS. HAROLD C. 
KING, MR. AND MRS. FRANKLIN 
KOLLER, MR. AND MRS. LEWIS 
LAURENCE, MR. AND MRS. THOMAS E. 
LEMANN, MRS. BENJAMIN 

LlNEAWEAVER, MR. THOMAS, III 

LOBB, MRS. JOHN 

LOEB, DR. AND MRS. ROBERT F. 

McCuSKER, MR. AND MRS. PAUL J. 

MCKELVY, MR. JOHN E. 
MARSLAND, MRS. DOUGLAS A. 
MARVIN, MRS. WALTER T. 
MAST, MRS. S. O. 
MEIGS, DR. AND MRS. J. WISTER 
MITCHELL, MRS. JAMES McC. 
MIXTER, MRS. W. JASON 
MOSSER, MRS. BENJAMIN D. 
MOTLEY, MRS. THOMAS 
NEWTON, Miss HELEN 
NICHOLS, MRS. GEORGE 
NIMS, MRS. E. D. 
PACKARD, MRS. CHARLES 



REPORT OF THE LIBRARIAN 



49 



PARK, MR. AND MRS. M. S. 
PENNINGTON, Miss ANNE H. 
REDFIELD, DR. AND MRS. ALFRED C. 
REZNIKOFF, DR. AND MRS. PAUL 
RIGGS, MR. AND MRS. LA \YRASON 
RIVINUS, MRS. F. M., JR. 
ROBINSON, DR. MILES 
RUDD, MR. AND MRS. H. W. DWIGHT 
SANDS, Miss ADELAIDE G. 
SAUNDERS, MR. AND MRS. LAWRENCE 
SHIVERICK, MRS. ARTHUR 
SINCLAIR, MR. AND MRS. W. RICHARD- 
SON 

SPEIDEL, DR. AND MRS. CARL 
STONE, MR. AND MRS. LEO 



STONE, MR. AND MRS. S. M. 
STRAUSS, DR. AND MRS. DONALD B. 
STUNKARD, MRS. HORACE W. 
SWIFT, MR. E. KENT 
SWOPE, MR. AND MRS. GERARD, JR. 
SWOPE, Miss HENRIETTA 
TOMPKINS, MR. AND MRS. B. A. 
WEBSTER, MRS. EDWIN S. 
WHITELEY, Miss MABEL W. 

WlCKERSHAM, MR. AND MRS. JAMES H. 
WlLHELM, DR. AND MRS. HlLMER J. 

WILLISTON, MR. SAMUEL 
WILLISTON, Miss EMILY 
WILSON, MRS. EDMUND B. 

WOLFINSOHN, MRS. WOLFE 



V. REPORT OF THE LIBRARIAN 

At the close of the year, the Library received currently 1717 journals, 56 new 
titles having been added in 1960. Of the total, the Marine Biological Laboratory 
subscribed to 502, received 653 in exchange and 190 as gifts. The Woods Hole 
Oceanographic Institution subscribed to 109, received 200 in exchange and 63 
as gifts. 

The Laboratory purchased 121 books, received 92 complimentary copies (9 
from authors and 83 from publishers), and accepted 74 miscellaneous gifts. The 
Institution purchased 38 books and received 9 as gifts. The total number of books 
accessioned totalled 334. 

Through purchase, exchange and gift, the Laboratory completed 12 journal sets 
and partially completed 13. The Institution completed two sets and partially com- 
pleted one. There were 3825 reprints added to the collection, of which 1749 were 
of current issue. 

At the close of the year there were 77,525 bound volumes and 216,452 reprints. 
The number of requests for inter-library loans increased over 1959. There were 
469 volumes sent out and 57 were borrowed. About 1000 volumes were bound. 

Many valuable books and reprints were received from Drs. P. W. Whiting, 
L. H. Hyman, Walter S. Root, Roberts Rugh, Ethel B. Harvey, Irvine H. Page, 
Robt. F. Loeb, H. J. Humm, and the Department of Microbiology, University of 
Pennsylvania Medical School. The Library extends grateful acknowledgment to 
these generous contributors. 

Many duplicate books and reprints were sent to the Department of Cytology, 
Warsaw L'niversity, thus furthering our exchange relationship abroad. 

The year was an exceptionally busy one due to the increase in acquisitions, to 
the larger number of scientists using the library, and to several changes in the staff. 

Respectfully submitted, 

DEBORAH L. HARLOW, 

Librarian 



50 MARINE BIOLOGICAL LABORATORY 

VI. REPORT OF THE TREASURER 

The market value of the General Endowment Fund and the Library Fund at 
December 31, 1960, amounted to $1,796,571 as against a book value of $1,146,393. 
This compares with values of $1,786,262 and $1,023,297 at the end of the preceding 
year. The average yield on the securities was 3.60% of the market value and 5.65% 
of book value. The total uninvested principal cash in the above accounts as of 
December 31, 1960, was $255.16. Classification of the Securities held in the 
Endowment Funds appears in the Auditor's report. 

The market value of the pooled securities as of December 31, 1960, was $333,218 
with uninvested principal cash of $120.35 ; the market value at December 31, 1959, 
being $309,251. The book value of the securities in this account was $274,294 on 
December 31, 1960, compared with $257,576 a year earlier. The average yield on 
market value was 3.75% and 4.56% of book value. 

The proportionate interest in the Pool Fund Account of the various Funds as 
of December 31, 1960, is as follows: 

Pension Funds 23.979% 

General Laboratory Investment 53.681 

Other : 

Bio Club Scholarship Fund 1.536 

Rev. Arsenius Boyer Scholarship Fund 1.881 

Gary N. Calkins Fund 1.760 

Allen R. Memhard Fund 342 

F. R. Lillie Memorial Fund 5.933 

Lucretia Crocker Fund 6.423 

E. G. Conklin Fund 1.088 

M. H. Jacobs Scholarship Fund 774 

Jewett Memorial Fund 572 

Anonymous Gift 2.031 

The special custodian account yielded an income last year of $9,619 and this 
amount is being reserved for capital improvements. 

Donations from the M. B. L. Associates for 1960 were $4,320 as compared with 
$4,170 for 1959. Unrestricted gifts from foundations, societies and companies 
amounted to $18,035. 

We are administering 15 grants for investigators in addition to those grants 
made directly to the Marine Biological Laboratory. The amounts of grants vary 
in accordance with the investigator's project of research. An amount of 15% based 
on amount expended is allowed the Laboratory as overhead. 

The Lillie Fellowship Fund with a market value of $88,415 and a book value 
of $92,789, as well as the investment in the General Biological Supply House with 
a book value of $12,700, is carried in the Balance Sheet, item "Other Investments." 
The General Biological Supply House for the fiscal year ended June 30, 1960, had 
a profit after taxes of $314,034 as compared to $303,300 in 1959 and $218,210 in 
1958, and $123,430 in 1957. In the fiscal year 1960, the Marine Biological Labora- 
tory received dividends from the General Biological Supply House of $30,480 as 
against $30,480 in 1959 and $25,400 in 1958. 



REPORT OF THE TREASURER 51 

Following is a statement of the auditors : 
To tJic Trustees of the Marine Biological Laboratory, Woods Hole, Massachusetts: 

\Ye have examined the balance sheets of the Marine Biological Laboratory as 
at December 31, 1960 and 1959, the related statements of operation expenditures, 
income and current fund for the years then ended, and statement of funds for the 
year ended December 31, 1960. Our examination was made in accordance with 
generally accepted auditing standards, and accordingly included such tests of the 
account records and such other auditing procedures as we considered necessary in 
the circumstances. 

In our opinion, the accompanying financial statements present fairly the assets, 
liabilities and funds of the Marine Biological Laboratory at December 31, 1960, 
and the results of its operations for the year then ended. 

Boston, Massachusetts 

May 31, 1961 LYBRAND, Ross BROS. & MONTGOMERY 

JAMES H. WICKERSHAM, 

Treasurer 



52 MARINE BIOLOGICAL LABORATORY 

MARINE BIOLOGICAL LABORATORY 

BALANCE SHEETS 
December 31, 1960 and 1959 

Investments 

1960 19 5 

Investments held by Trustee: 

Securities, at cost (approximate market quotation 1960 $1,796,000) $1,146,393 $1,023,297 
Cash . 255 2,990 



$1,146,648 $1,026,287 
Investments of other endowment and unrestricted funds : 

Pooled investments, at cost (approximate market quotation 1960, 

$333,218; less $5,728 temporary investment of current fund cash) $ 268,566 $ 251,848 

Other investments 137,742 132,882 

Cash 10,839 13,973 

Accounts recrivable 21 1,510 



$1,563,816 $1,426,500 



Plant Assets 

Land, buildings, library and equipment ( note) $3,280,059 $3,204,017 

Less allowance for depreciation ( note ) 1,142,879 1,109,716 



$2,137,180 $2,094,301 

Construction in progress 1,455,811 776,628 

Cash 82,042 3,699 

Accounts receivable : 1,196 

U. S. Government obligations, at cost : 

$275,000 Treasury bills, due 1/15/60 273,258 



$3,675,033 $3,149,082 



Current Assets 

Cash $ 77,546 $ 113,588 

U. S. Government obligations, at cost : 

$75,000 Treasury bills, due 1/15/60 74,525 

Temporary investment in pooled securities 5,728 5,728 

Accounts receivable (U. S. Government, 1960, $43,443; 1959, $35,554) .. 59,889 61,629 

Inventories of specimens and Bulletins 47,641 4,->,400 

Prepaid insurance and other 16,778 12,953 



$ 207,582 $ 313,823 



$5,446,431 $4,889,405 



REPORT OF THE TREASURER 53 

MARINE BIOLOGICAL LABORATORY 

BALANCE SHEETS 
December 31, 1960 and 1959 

Endoii-'incnt Funds 

1960 1959 

Endowment funds given in trust for benefit of the Marine Biological Lab- 
oratory $1,146,648 $1,026,287 



Endowment funds for awards and scholarships : 

Principal $ 126,302 $ 126.193 

Unexpended income 7,285 5,399 



$ 133,587 $ 131,592 

Unrestricted funds functioning as endowment 206,378 206,378 

Retirement fund 71,449 61,640 

Pooled investments accumulated gain 5,754 603 



$1,563,816 $1,426,500 



Plant Liability and Funds 

Funds expended for plant, less retirements $4,668,475 $3,832,639 

Less allowance for depreciation charged thereto 1,142,879 1,109,716 



$3,525,596 $2,722,923 
Unexpended plant funds 82,042 276,957 



$3,607,638 $2,999,880 
Accounts payable 67,395 149,202 



$3,675,033 $3,149,082 



Current Liabilities and Funds 



Accounts payable . $ 41,106 $ 46,154 

Unexpended research grants 51,726 131,146 

Unexpended balances of gifts for designated purposes 9,663 10,799 

Current fund . 105,087 125,724 



$ 207,582 $ 313,823 



$5,446,431 $4,889,405 



Note The Laboratory has since January 1, 1916, provided for reduction of book amounts of 
plant assets and funds invested in plant at annual rates ranging from \% to 5% of the original 
cost of the assets. 



54 MARINE BIOLOGICAL LABORATORY 

MARINE BIOLOGICAL LABORATORY 

STATEMENTS OF OPERATING EXPENDITURES, INCOME AND CURRENT FUND 
Years Ended December 31, 1960 and 1959 

Operating Expenditures 

1960 1959 * 

Research and accessory services $ 250,578 $ 222,624 

Instruction 219,234 79,396 

Library and publications 61,462 58,190 

Direct costs on research grants 182,899 221,436 



$ 714,173 $ 581,646 

Administration and general 70,037 63,947 

Plant operation and maintenance 117,980 113,454 

Dormitories and dining 162,713 154,405 

Additions to plant from current income 78,654 23,448 



$1,143,557 $ 936,900 
Less depreciation included in plant operation and dormitories and 

dining above but charged to plant funds 48,086 46,604 



$1,095,471 $ 890,296 

Income 

Research fees $ 56,408 $ 50,242 

Accessory services (including sales of biological specimens, 1960, $48,817, 

1959, $66,742) 151,109 141,022 

Instruction fees 23,905 21,395 

Grants for instruction and research training 185,571 40,478 

Library fees, Bulletin subscriptions and other 35,174 25,225 

Reimbursements and allowances for direct and indirect costs on research 

grants 221,197 246,326 

Dormitories and dining income 105,086 106,424 



$778,450 $ 631,112 

Gifts used for current expenses 48,300 36,590 

Grants used for current expenses 143,018 107,500 

Investment income used for current expenses 105,066 100,432 



Total current income $1,074,834 $ 875,634 



Excess of operating expenditures over current income $ 20,637 $ 14,662 

Current fund balance January 1 125,724 140,386 



Current fund balance December 31 $ 105,087 $ 125,724 

* 1959 amounts have been reclassified for purposes of comparison. 



REPORT OF THE TREASURER 55 



MARINE BIOLOGICAL LABORATORY 

STATEMENT OF FUNDS 
Year Ended December 31, 1960 

Balance Gifts and Invest- Used for Other Balance 

Jan.l, Other ment Current Expendi- Dec. 31, 

1960 Receipts Income Expenses titres 1960 

Invested funds . . $1,426,500 $ 142,749 $110,875 $103,074 $ 13,234 $1,563,816 



Unexpended plant funds . $ 276,957 556,456 9,619 760,990 82,042 



Unexpended research 

grants $ 131,146 470,366 549,786 $ 51,726 



Unexpended gifts for 

designated purposes . $ 10,799 48,355 48,300 1.191 $ 9,663 



Current fund $ 125,724 20,637 $ 105,087 



$1,217,926 $120,494 $721,797 $775,415 



Gifts $ 603,616 

Grants for research, train- 
ing and support .... 470,366 

Net gain on sales of 

securities 125,620 

Appropriated from current 

income and other . . . 18,324 



$1,217,926 

Expended for construction 

of new building .... $760,990 

Scholarship awards 3,185 

Payments to pensioners .... 10,049 

Other 1,191 

$775,415 



56 



MARINE BIOLOGICAL LABORATORY 



MARINE BIOLOGICAL LABORATORY 
SUMMARY OF INVESTMENTS OF ENDOWMENT FUNDS 



December 31, 1960 



Securities held by Trustee : 
General endowment fund : 



% of Market 
Cost Total Quotations 



Investment 

% of Income 
Total 1060 



U. S. Government Securities .... $ 
Corporate bonds 


35,164 
572 187 


3 


.6 

Q 


$ 


36,619 
^47653 


2.5 
373 


$ 


1,272 

18984 


Preferred stocks 


84,778 


8 


Q 




70 138 


48 




3370 


Common stocks 


263,894 


27 


6 




813 572 


554 




29474 




















$ 


956,023 


100 


.0 


$1 


,467,982 


100.0 


.$ 


53,100 


General educational board endowment 
fund : 
U. S. Government securities $ 


31,060 


16 




$ 


32434 


99 


$ 


1 205 


Other bonds 


94888 


40 


8 




88450 


269 




3480 


Preferred stocks 


26745 


14 







24058 


73 




1063 


Common stocks 


37,677 




Q 




183,647 


55.9 




5,972 




















$ 


190,370 


100 


.0 


$ 


328.589 


100.0 


$ 


11,720 



Total securities held by 
Trustee 



$1,146,393 



$1,796,571 



Investments of other endowment and un- 
restricted funds : 
Pooled investments : 



$ 137,742 

Total investments of other en- 
dowment and restricted 
funds $ 412,036 



Total investment income 

Custodian's fees charged thereto 

Income of current funds temporarily invested 
in pooled securities 

Investment income distributed to funds . . . 



$ 64,820 



U. S. Government securities . . . 


.. . $ 1,018 


.4 


$ 1,048 


.3 


$ 180 


Corporate bonds 


149866 


54.6 


147,782 


44.3 


6 575 


Preferred stocks 


3,214 


1.2 


3,075 


1.0 


112 


Common stocks 


120,196 


43.8 


181,314 


54.4 


5,645 
















$ 274,294 


100.0 


$ 333,219 


100.0 


$ 12,512 


Other investments : 
U S Government securities 


.. $ 7,000 








$ 981 


Other bonds 


47,971 








1,675 


Preferred stocks 


3,728 








131 


Common stocks 


46,530 








31,552 


Real estate 


32,513 























$ 34,339 



$ 46,851 

$111,671 

(546) 

(250) 
$110,875 



REACTION TO INJURY IN THE OYSTER 
(CRASSOSTREA VIRGINICA) 

FREDERIK B. BANG 

Mitriuc Biological Laboratory, Woods Hole, Mass., and flic Department of Pathobioloc/y, 
The Johns Hopkins School of Hygiene and Public Health, Baltimore 5, Md. 

The comparative approach to pathology, which uses both cold-blooded verte- 
brates and invertebrates to advantage, was pioneered brilliantly by Metchnikov 
(1891) and has since been continued, somewhat sporadically, in France (Canta- 
cuzene, 1923) and elsewhere (Cameron, 1932; Schlumberger, 1952). There still 
remains a tremendous dearth of information concerning the reaction of various 
invertebrates to injury and infection. A partial exception to this is the study of 
insect pathology (Steinhaus, 1949). 

In a continuing study of pathological processes in the oyster we have found 
that the initial phase of phagocytosis of bacteria by the oyster amebocyte is often 
preceded by adhesion of the bacteria to the amebocyte so that the cell surface is 
literally covered with bacteria, and the sticking may be limited to the contact 
of the flagellum of the organism with the amebocyte so that the still motile bac- 
terium becomes anchored. Secondly, the classical cellular clot formed by the 
agglutination of these amebocytes may be accompanied by an extracellular clot 
which immobilizes bacteria. And finally, the cellular clot may lie directly observed 
within the vascular system of the living oyster and may be produced by the 
injection of an extract of oyster tissue. 

MATERIAL AND METHODS 

Most of the experiments were done on a so-called half shell preparation in which, 
after an edge of shell was knocked off, the shell was pried open with a knife until 
the adductor muscle was seen ; then, with as little trauma as possible, the adductor 
muscle was cut and the upper shell removed. In good preparations this meant 
that a portion of the mantle and the muscle was cut, and the pericardium was left 
intact. Such preparations (Fig. 1 ) were kept in running sea water and used during 
the next several days. Some of these lived as long as a week or ten days, but had by 
that time gradually deteriorated, showing a loss of leucocytes from the blood and 
progressive infection and disintegration of the muscle. Heart blood was readily 
obtained from them at any time, and direct examination of the various vessels of the 
mantle, palps and gills was satisfactory under a Zeiss dissecting microscope (40 X ). 
Intracardiac injections were usually done directly into the ventricle, and blood was 
withdrawn from the auricle. 

During these operations, the animals must obviously be damaged to a greater 
degree than were Stauber's preparations (1950) in which a window was made 
directly over the heart. However, they allowed direct examination of the entire 
gill and vascular system, and were used only as acute preparations. A limited 

57 



58 



FREDERIK B. BANG 



number of observations were made on oysters in which a hole was carefully drilled 
near the pericardium and the shell was then picked away until the sheath was 
exposed. 

Observations on phagocytosis were made with a Zeiss phase microscope both 
at 500 X and 1250 X. A drop of freshly obtained blood was placed on a slide, 
then either a drop of bacterial suspensions from a freshly grown culture of marine 
bacteria was added to it, or a small portion of the colony itself was added with a 
loop directly to the drop of blood. The preparation was covered with a #1 cover- 




anterior aorta- 
aortic bulb 

FIGURE 1. Diagram of half-shell preparation of oyster. Circulatory system indicated in 
heavy black line. Injections were made directly into the heart (H). Observations were made 
principally on mantle arteries. 

slip, and if observations were to be continued, the entire preparation was ringed 
with Vaseline to prevent evaporation. Amebocytes remained viable for 12 hours 
or more under these conditions. 



RESULTS 
Phagocytosis 

The amebocyte, which has been extensively studied in this and other molluscs 
(Faure-Fremiet, 1927), is a granular round cell floating freely in the blood stream. 
On contact with glass it flattens out and moves continually over the surface of the 
slide. This motion under phase may clearly be seen to begin by the extrusion of a 



REACTION TO INJURY IN OYSTER 59 

series of filamentous pseudopodia which may be resolved with high power phase 
microscopy (Fig. 2) and which is shown in the accompanying electron micro- 
scope pictures. The spread of ectoplasm, illustrated in the accompanying figures 
(Figs. 3, 4, 5), then flows afterward, filling up the spaces between. Granules and 
other portions of the cell then flow into this region. A variety of cell forms may be 
observed on these slides ; some of them lack granules, others contain large wavy 
frills of pseudopodia, other large amorphous but refractile inclusions. Since the 
amebocyte may both lose its granules and may ingest large amounts of material, 
we are unable to say whether these represent different types of cell or physiological 
variants of one type. Most of the cells observed during the process of phagocytosis 
were granular cells. 

One of the most remarkable facts which was observed early in the study was 
the absence of phagocytosis. Frequently an amebocyte was seen to approach a 
bacterium with its fibrous processes, then either to reverse its flow or turn aside. 
During the course of several hours this behavior was repeated continuously and no 
phagocytosis was observed. Since it is so obviously contrary to established ideas 
of the importance of phagocytosis, and specific studies on phagocystosis of food 
particles by the oyster, the observations were repeated with a number of bacteria, 
and it was found that excellent phagocytosis might be obtained with a certain 
bacterium, yet little if any phagocytosis was observed in amebocytes from the same 
oyster if another preparation of bacteria was introduced. Repeated attempts were 
made to determine whether such failure of phagocytosis was due to the strain of 
bacteria, or to a combination of certain bacteria with amebocytes from certain 
oysters. It was not possible from day to day to find a combination of amebocytes 
and bacteria which did not phagocytize, but no observations were repeated within 
a few hours of each other and it remains likely that there is an undiscovered factor 
important in phagocytosis which is responsible for this variation. 

When a successful combination was obtained, and particularly when the bac- 
terium used was motile, phagocytosis was usually preceded by a massive sticking 
of the bacteria to the amebocyte, so that the amebocyte resembled a porcupine. 
Ingestion followed this phase (Fig. 4). Some incidental observations on phago- 
cytosis by amebocytes of the marine worm, Urcchis, showed the adhesion of bac- 
teria to be limited to the portion of the cell which had spread out on the glass. 1 
In the oyster, however, since extrusions of the cell appeared from all sides it was 
not possible to determine whether all portions of the amebocyte were equally sticky. 

The curious anchoring of motile bacteria to amebocytes which renders them 
unable to leave the amebocyte while they seem at the same time to have no contact 
with it, was explained by a fortunate electron microscope picture (Fig. ^,5). The 
presence of the unipolar flagellum wrapped around the filamentous pseudopodia 
fully explains the continual tugging and jerking at an invisible anchor. 

Extracellular clot formation 

During the summer of 1956, and in the two succeeding summers, the formation 
of a definite extracellular clot was observed (Fig. 6). It could be seen only under 
phase microscopy and seemed similar in texture and formation to a clot described 

1 These observations were made originally by Mr. Stuart Krassner, to whom we are 
indebted for permission to include this material. 



60 



FREDERIK B. BANG 




FIGURES 2-3. 



REACTION TO INJURY IN OYSTER 61 

by Gregoire (1952) in a variety of insect bloods. The clot was found in only 
about one-third of the oysters which were examined, and was not present in the 
same oyster at all times. Furthermore, we have been unable to determine the con- 
ditions under which it may be consistently formed in an individual oyster. How- 
ever, we were able to reproduce this extracellular clot throughout the summer 
months of the last three years, and have been clearly able to rule out artifacts 
of preparation. 

The clot was first noticed in slides which had been kept for continued observa- 
tion of phagocytosis. It tended to occur near the occasional small air bubble 
which was entrapped beneath the coverslip in the sealed slide. It was not present 
immediately but usually appeared in 15 minutes to half an hour, so that it was 
suspected of being a local drying phenomenon until it was seen in slides which 
had no appreciable air bubbles, and in sealed hanging drops and direct preparations. 
Attempts to relate it to the time of day, time since opening of the oyster, amount 
of trauma, and sex of oyster, failed. A mixture of pericardial fluid and blood did 
not affect the process. A higher percentage of extracellular clots seemed to lie 
obtained from oysters which had been forced-starved by keeping them out of the 
sea water, and then replacing them ; but this produced positive results in only 
about half of the cases. Since the clot had been originally observed in a preparation 
to which bacteria had been added, repeated comparisons were made in the presence 
and absence of the bacteria. In most cases, when the clot was obtained in the 
presence of the bacteria, it was also found in the control slide to which bacteria 
had not been added, though it was usually less extensive. 

It seems to have a real role in the repair of traumatized tissue, for : 1) it was 
found already developing in small cellular clots taken directly from the heart ; 
2) it occurred predominantly around clumps of cells; 3) bacteria were immobilized 
by its development (Fig. 7) ; and, 4) it was obtained both immediately after 
opening an oyster and from some preparations which had been on the half shell 
as long as 24 hours. Its possible relation to cycles of feeding by the amebocytes 
is unknown. 

I ntrai'ascnlar clots 

There is a rapid clumping of cells when oyster blood is withdrawn in glass 
vessels, which is also the case with many other invertebrate bloods. Direct obser- 
vation of traumatized blood vessels shows the formation of the same type of cellular 
clot at the open end of the vessel, so that within a few minutes of cutting, the clotted 
cells have effectively sealed the end. Similar clumps of amebocytes are observed 
directly covering the cut end of the adductor muscle in our preparations, and 
oysters which had been repeatedly bled develop a shaggy pericarditis which consists 
of masses of these clumped cells. However, in oysters which have had minimal 
trauma and have remained in clean running sea water for several hours after being 
opened, the circulation may be fully effective and direct examination of the distended 
vascular system was possible. In such a view, the cells are seen moving to and fro, 

FIGURE 2. Phase micrograph of oyster amebocyte on glass, approximately 600 times. 

FIGURE 3. Electron micrograph of whole cell preparation of amebocyte. Filamentous 
pseudopods extend away from the cell edge. This and succeeding electron micrographs were 
made by allowing the amebocytes to spread out on a collodion film. The cells were fixed with 
osmium vapor, washed and the film placed on grids ; 9,000 times. 



62 



FREDERIK B. BANG 




I 1 






5 



FIGURES 4-5. 



REACTION TO INJURY IN OYSTER 63 

and relatively few of them are clumped. When the circulation is sluggish they 
may be found lining the lower side of a vessel, but they readily move from one 
portion to another as the oyster is tilted. The obvious question whether a par- 
ticular portion of the tissue was responsible for the formation of the cellular clot 
was tested by making a sea- water extract of gill tissue, centrifuging the extract and 
injecting about 0.1 cc. of the relatively clear supernatant directly into the heart. 
The material immediately spread throughout the animal and an interesting series 
of events set in. If the oyster had relatively large numbers of cells so that the 
blood was milky in appearance, the first reaction was the formation of large curd- 
like clumps of loosely aggregated cells. These became more dense, soon ceased 
to flow back and forth, and within 10 to 15 minutes were stuck in tight clumps to 
the edge of the vessel, and the fluid itself appeared perfectly clear. The vessel 
frequently decreased in size, particularly if the heart happened to cease beating. 
In many cases, some flow back and forth in the mantle vessels continued even 
though there was no visible heart beat, presumably from the action of the accessory 
heart (Fig. 1). Within about two hours after the injection, most of the effects 
had worn off: the heart was beating, the blood was again flowing freely, and rela- 
tively few clumps were seen. Individual cells were observed flowing freely in 
the large vessels or moving in and out of the fine branches of the palps or the gills. 
When these oysters were reinjected with the original extract, an apparently full- 
fledged repetition of the reaction was observed. 

The reaction was not obtained by the injection of sea water, of suspensions of 
carmine, or of bacteria of several sorts, though a moderate "curdling" of the blood 
was seen after the injection of heavy suspensions of bacteria. 

India ink of two sorts was then injected in suspensions of sea water. The 
usual preparation of colloidal ink when injected caused prompt clumping of cells, 
a cessation of heart beat, and the probable development of intravascular clumps 
like those seen following the injection of tissue extracts. However, the black masses 
of material which were partially phagocytized, as described by Stauber (1950), 
obscured the observation. A preparation of "Pelican" India ink, which lacks 
the gum coating present in most commercial India inks, produced a much milder 
reaction (Muller, 1927a, 1927b). The particles were soon phagocytized as small 
particles or clumps without major changes in the circulation itself, just as carmine 
particles had been. 

DISCUSSION 

The capacity to react to injury, an essential function of living cells, is basic to 
studies in pathology. Following Metchnikov (1891), who began with a marine 
echinoderm embryo, the greatest attention of pathologists when studying inverte- 
brates has concentrated on the wandering cells or amebocytes. From a comparative 
pathological point of view, at least three phenomena are contained in the oyster 
in this one cell. These are phagocytosis of invading bacteria, inflammation, and 
thrombosis. Since the amebocyte is the only circulating cell of the blood in the 
oyster, and since cellular clots are the common mechanism of closing gaps in the 

FIGURE 4. Beginning phagocytosis of uniflagellate bacterium. The flagellum is coiled 
around several pseudopods of the amebocyte ; 14,000 times. 

FIGURE 5. An electron micrograph showing later stage in phagocytosis of bacterium ; 
9000 times. 



64 



FREDERIK B. BANG 




FIGURE 6. Phase micrograph showing extracellular clotting of amebocytes ; 1000 times. 
FIGURE 7. Similar extracellular clot with bacteria involved in clot ; 1000 times. 



REACTION TO INJURY IN OYSTER 65 

vascular system among invertebrates (Geddes, 1880; Cuenot, 1891), it is of 
course impossible to separate the function of inflammation whereby a white cell 
in vertebrates becomes adherent to a vessel wall and migrates through it, and that 
of the adherence of many amebocytes together to form a tight clump which blocks 
the free flow of blood. 

An ideal invertebrate in which to follow the above processes would allow direct 
observation of the vascular channels without trauma, and would from the bacterio- 
logical point of view allow for external sterilization and thus obtaining of blood 
without contamination by the surrounding fluid or air. In this regard the oyster 
and other molluscs have no external surface which may be sterilized and then 
punctured, and have no extension of the vascular system that may be observed 
without the introduction of trauma. Thus, though a variety of studies have been 
done on diseased oysters (Herdman and Boyce, 1899; Roughley, 1926; Stauber, 
1945; Mackin, 1951; Mackin, et al., 1952), there is little direct information on 
the pathogenesis of any of the disease states. 

Phagocytosis of food material for transport through the oyster, and of particulate 
matter has been studied rather extensively (Yonge, 1926; Takatsuki, 1934), 
primarily by following the events in sequence by histological sections. 

Phagocytosis itself was first observed about a hundred years ago, by Haeckel 
(1862), \vho injected particulate dyes into molluscs so that the distribution of the 
vascular system might be determined. He pointed to the potential importance of 
the phenomenon in nutrition. Then came the disclosure by Metchnikov of the role 
of phagocytosis as a defense mechanism (1884, 1891). In the oyster and other 
molluscs the importance of the amebocyte in digestion, transfer of food, and repair, 
has been firmly established (Yonge, 1928; Wagge, 1955). Recently Tripp (I960), 
has followed the fate of several species of bacteria in oyster tissue following intra- 
cardiac injection of large numbers. He has shown that phagocytosis may be 
apparent in the cells circulating within the vessels and subsequently in the tissues. 
Intracellular digestion appeared to be a major mechanism of disposal of the bac- 
teria. In several infectious diseases of the oyster the presumptive agent is thought 
to be disseminated by the amebocyte (Orton, 1923). It was therefore a surprise 
to us to find that there was a marked variation in the phagocytosis of different 
preparations of bacteria by leucocytes of the same oyster. Attempts to show in- 
creased phagocytosis in the presence of mucus from the gill, of disintegrating 
crystalline style material, or of extracts of the hepato-pancreas failed. 

Although "surface phagocytosis" (Wood, 1951-1952) took place in the process 
of the flow of amebocytic protoplasm around bacterium, it was not always the 
explanation, for masses of bacteria were found stuck to the surface of amebocytes 
in most of the cases where phagocytosis was apparent. The direct adherence of 
the amebocyte to the bacterium itself was highlighted by the observation of the 
flagellar adherence of the bacteria to the amebocyte so that it was unable to escape 
from the amebocyte. 

The evolutionary need for extracellular clot formation becomes greater when 
the amebocytes or leucocytes have much less direct contact with each other because 
of the presence of large numbers of red cells. However, extracellular clot or gel 
formation is well developed in several invertebrates (Gregoire and Florkin, 1950; 
Loeb, 1910; Gregoire, 1952; Bang, 1956) in which the predominant circulating 



66 



FREDERIK B. BANG 



cells are directly involved in clot formation (Yonge, 1926). The presence of this 
extracellular gel, which seemed fully able to limit bacterial motion in many of the 
oysters which we examined, may indicate that additional advantage is to be gained 
from such mechanisms of thrombosis which extend beyond the cell. The possible 
role of this extracellular material in rendering bacteria more susceptible to phago- 
cytosis needs further study. The origin of this extracellular gel from the extru- 
sion of the many cellular granules is an obvious possibility which has not been 
investigated. 

Direct observations of the formation of the cellular clot at a point of traumatic 
rupture of a vessel, the accumulation of great numbers of these cells on the heart 
when it is exposed to sea water by opening the pericardium, and the accumulation 
of amebocytes at the cut edge of the adductor muscle, led to the question as to the 
effect of tissue extracts. It was soon found that a fresh crude sea-water extract 
of ground gill tissue, when injected directly into the heart, caused a rapid clumping 
of cells and the tight adherence of these cells to the vessel wall, so that the circula- 
tion was greatly slowed or stopped. Injection of sea water, of bacterial suspensions, 
and of carmine, failed to cause similar marked effects. Thrombosis accompanied 
by phagocytosis was rapidly produced by the injection of certain preparations of 






1mm. 

FIGURE 8. Diagram of observations of mantle artery: (1) shows the clear appearance of 
the vessel under normal conditions. Individual amebocytes may be seen poorly and are not 
indicated here. (2) Beginning clumping amebocytes within the vessel. They are loosely 
clumped and move rather freely in the vessel. (3) Amebocyte clumps which have contracted 
into tight balls of thrombus and are adherent to the vessel wall. 



REACTION TO INJURY IN OYSTER 67 

India ink, but not by a preparation which is stated to lack the shellac coating which 
in itself causes extensive thrombosis. 

Our experiments have been limited to acute short term experiments. Several 
other molluscs have been used in the study of chronic processes (Drew and de 
Morgan, 1910; Zawarzin, 1927), and the importance of epithelial tissues, mucous 
sheets and chronic fibrous tissue "repair" needs extensive exploration (Kedrowsky, 
1925; Labbe, 1929). 

SUMMARY 

1. In vitro phagocytosis of marine bacteria by fresh oyster leucocytes, though 
readily demonstrable in most cases, was by no means an invariable phenomenon. 
When it occurred, it was frequently accompanied by a massive sticking of bacteria 
to the leucocytes. The flagellar portion of the bacterium might be so caught by 
the amebocyte that the bacterium was unable to escape, even though the body was 
not in contact with the amebocyte. 

2. An irregular but repeated formation of an extracellular clot is described as 
seen in vitro by phase microscopy. Reasons for believing that it is a true phe- 
nomenon in the oyster are given. 

3. Intravascular clotting or thrombosis was produced by the intracardiac 
injection of tissue extracts. The clotting disappeared spontaneously within two 
hours after the injection. 

LITERATURE CITED 

BANG, F. B., 1956. A bacterial disease of Linutlus polyphemus. Bull. Johns Hopkins Hosp., 

98: 325-351. 

CAMERON, G. R., 1932. Inflammation in earthworms. /. Path, and Bact., 35: 933-972. 
CANTACUZENE, J., 1923. Le probleme de I'immunite chez les invertebres. C. R. Soc. Biol. 

(75th ami.) : 48-119. 
CUENOT, L., 1891. fitude sur le sang et les glandes lympathiques dans le serie animate. 

Arch, de Zool. E.vp. Gen.. 19: 13-90. 
DREW, G. H., AND W. DE MORGAN, 1910. The origin and formation of fibrous tissue produced 

as a reaction to injury in Pcctcn maximus, as a type of Lamellibranchiata. Quart. J. 

Micr. Sci., 55: 595-610. 
FAURE-FREMIET, E., 1927. Les amibocytes des invertebres a 1'etat quiescent et a 1'etat actif. 

Arch. d'Anat. Micr., 23: 99-173. 

GEDDES, P., 1880. On the coalescence of ameboid cells into plasmodia and the so-called coagu- 
lation of invertebrate fluids. Proc. Roy. Soc. London, Ser. B, 30 : 252-254. 
GREGOIRE, CH., 1952. Sur le coagulation du sang de Limulus polyphemus (Arachnida). Arch. 

Intern, de Physiol, 60: 97-99. 
GREGOIRE, CH., AND M. FLORKIN, 1950. Blood coagulation in arthropods. Plivsiol. Comp. et. 

Oecol, 2: 126-139. 

HAECKEL, E., 1862. Die Radiolarien. Berlin : Geo. Reimer. Pp. 104-106. 
HERDMAN, W. A., AND R. BOYCE, 1899. Oysters and disease. Lancashire Sea Fisheries 

Mem. #1. 
KEDROWSKY, B., 1925. Reactive Veranderung in den Geweben der Teichmuschel (Anodonta 

sp.) bei Einfuhrung von sterilem Zelloidin. I'irchow's Arch'w., 257: 815-845. 
LABBE, A., 1929. Reactions experimentales des Mollusques a 1'introduction de stylets de celloi- 

dine. C. R. Soc. Biol.. 100: 166-168. 

LOEB, L., 1910. t v ber die Blutgerinnung bei Wirbellosen. Biochem. Zeitschr., 24: 478-495. 
MACKIN, J. G., 1951. Histopathology of infection of Crassostrea virginica by Dermocystidiwn 

marinum. Bull. Alar. Sci. Gulf and Caribb., 1 : 72-87. 
MACKIN, J. G., P. KORRINGA AND S. H. HOPKINS, 1952. Hexamitiasis of Ostrca cdulis and 

Crassostrea virginica. Bull. Mar. Sci. Gulf and Caribb., 1 : 266-277. 



68 FREDERIK B. BANG 

METCHNIKOV, E., 1884. t)ber eine Sprosspelzkrankheit der Daphnien. Vir chow's Archiv., 

96: 177-195. 
METCHNIKOV, E., 1891. Lectures on the comparative pathology of inflammation. Trans, by 

F. A. Starling and E. H. Starling. London : Keagan Paul, Trench, Trubner and Co. 
MULLER, G. L., 1927a. Normoblastosis produced by India ink. /. Exp. Med., 45 : 399-410. 
MULLER, G. L., 1927b. Polycythemia, normoblastosis and erythrocytic hyperplasia of the bone 

marrow produced by gum shellac. /. Exp. Med., 45 : 753-770. 
ORTON, J. H., 1923. Summary of an account of investigations into the cause or causes of the 

unusual mortality among oysters in English oyster beds during 1920 and 1921. /. Mar. 

Biol. Assoc., 13: 1-23. 
ROUGHLEY, T. C, 1926. An investigation of the cause of oyster mortality on George's River, 

New South Wales, 1924-25. Proc. Linn. Soc. N. S. Wales, 51 : 446-491. 
SCHLUMBERGER, H. G., 1952. A comparative study of the reaction to injury: the cellular 

response to methycholanthrene and to talc in the body cavity of the cockroach (Peri- 

planeta amcricana). Arch. Path., 54: 98-113. 
STAUBER, L. A., 1945. Pinnotheres ostreum, parasites on the American oyster, Ostrca 

(Gryphaea) virginica. Biol. Bull, 88: 269-291. 
STAUBER, L. A., 1950. The fate of India ink injected intracardially into the oyster, Ostrea 

virginica (Gmelin). Biol. Bull, 98: 227-241. 

STEINHAUS, E., 1949. Insect Pathology. McGraw-Hill Book Co., New York. 
TAKATSUKI, S., 1934. On the nature and function of the amebocytes of Ostrea ednlis. Quart. 

J. Micr. Sci., 76 : 377-436. 
TRIPP, M. R., 1960. Mechanisms of removal of injected micro-organisms from the "American 

oyster Crassostrca virginica (Gmelin). Biol. Bull., 119: 273-282. 
WAGGE, L. E., 1955. Amebocytes. Int. Rev. Cyt., 4: 31-75. 
WOOD, W. B., 1951-1952. Studies on the cellular immunology of acute bacterial infections. 

Harvey Lectures., pp. 72-98. 
YONGE, C. M., 1926. Structure and physiology of the organs of feeding and digestion in Ostrea 

edulis. J. Mar. Biol. Assoc., 14: 295-386. 
YONGE, C. M., 1928. The absorption of glucose by Ostrea edulis. J. Mar. Biol. Assoc., 15: 

643-658. 
ZAWARZIN, A., 1927. tiber die reactiven Veranderungen des Epithels bei der Einfuhrung eines 

Fremdkorpers in den Alantel von Anodonta. Zeitschr. f. Mikr. Anat. Forsch., 11: 

215-282. 



THE OBLIGATE COMMENSAL CILIATES OF STRONGYLOCEN- 

TROTUS DROBACHIENSIS : OCCURRENCE AND DIVISION IN 

URCHINS OF DIVERSE AGES; SURVIVAL IN SEA WATER 

IN RELATION TO INFECTIVITY 

C. DALE BEERS 

Department of Zoology, University of North Carolina, Chapel Hill, North Carolina, 
and the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 

Seven species of ciliated protozoa have been reported from the alimentary tract 
of the sea urchin Strongylocentrotus drobachicnsis (O. F. Miiller) in the coastal 
waters of Mt. Desert Island, Maine (Powers, 1933a). Three of them are holo- 
trichs which have no known free-living congeners and are restricted to echinoid 
hosts. They are Entodiscus borealis (Hentschel, 1924) Madsen, 1931 ; Madsenia 
indomita (Madsen, 1931) Kahl, 1934; and Biggaria gracilis (Powers, 1933) Kahl, 
1934. In the words of Kirby (1941, p. 921), such ciliates "may be supposed to 
have evolved in the shelter of these hosts" and they are thus regarded as obligate 
commensals. The relation of the remaining four to their host is not entirely clear, 
owing to inadequate study. Powers (1933a, p. 119) regards them "as chance or 
vagrant ciliates, which, after being engulfed with food, are able to survive" and 
multiply as entozoic commensals. Two of the four are holotrichs, namely, Plagio- 
pyla minuta Powers, 1933, and Cyclidium stercoris Powers, 1935 ; one is a hypo- 
trich which Beers (1954) identified as Euplotes balteatus (Dujardin, 1841) Kahl, 
1932, and the final and least common is an undetermined species of the peritrich 
Trichodina. Reference may be made to Beers ( 1948) for further details concerning 
the taxonomy of the ciliates. In order to avoid the constant repetition of the 
unwieldy binomial Strongylocentrotus drobachiensis, the terms "urchin" and "urch- 
ins" are substituted in the following pages and refer without exception to this 
echinoid. 

To return to the three obligate commensals, which are the subject of the present 
study, Power (1933a) notes that adult urchins at Mt. Desert Island are almost 
invariably infected with them and indeed may harbor them in almost incredible 
abundance. In the summer of 1947, Beers (1948) extended Powers' investigations 
by making a quantitative study of the occurrence and morphogenetic condition of 
the ciliates in 182 urchins, the tests of which varied in diameter from 30 to 60 mm. 
All the urchins were infected with E. borealis and M. indomita, and 181 of them 
with B. gracilis. Counts of the ciliates in fresh samples of enteric fluid showed 
that the vast majority of the urchins harbored infections of each species that varied 
in intensity from "moderate" (M) to "heavy" (H), M meaning 50-500 individuals 
of the species per 0.1 ml. of fluid and H meaning 500-1000 or more per 0.1 ml. 
The remaining infections were designated as "light" (L), meaning fewer than 50 
individuals of a species per sample. A regional distribution of the ciliates was also 

69 



70 C. DALE BEERS 

noted, in that E. borealis occurred primarily in the stomach (inferior spiral or 
intestine of some authors), M. indomita in the intestine (superior spiral or large 
intestine ) , and B. gracilis in the rectum. However, the foregoing distribution of 
E. borealis and M. indomita prevailed as a rule only in well-fed urchins ; in inade- 
quately fed urchins they tended to shift aborally and in extreme cases of hunger 
to commingle with B. gracilis. The factors that were responsible for the regional 
distribution of the ciliates were unexplained. 

In any flourishing population of ciliates, whether free-living or associated in 
any way with a host, one might reasonably expect to find at almost any time a 
significant percentage of individuals that are dividing. It is therefore remarkable 
that dividing specimens of E. borealis and M. indoinita are extremely rare, even in 
ciliate populations of great density. With reference to the division of E. borealis, 
Powers (1933b, p. 130) comments as follows: "A study of about 600 specimens 
fixed during the day gave but three individuals showing any signs of fission." In 
1947 the writer made a special effort to find dividing specimens of E. borealis and 
M. indomita in the 182 urchins that have been mentioned. The urchins were 
collected and examined without delay at practically all hours of the day and night, 
but only six of them revealed dividing individuals of E. borealis. Concerning 
M. indomita, neither Madsen (1931) nor Powers (1933a) mentioned its division, 
and the task of finding dividing specimens was especially difficult. In the 88 urchins 
that were examined in July, only one dividing individual was found ; in 94 studied 
in August, dividing specimens were found in only three. It was concluded that 
division in E. borealis and M. indoinita was a periodic phenomenon : that long 
intervals of non-divisional life alternated with brief periods of intense divisional 
activity. In retrospect it became apparent that both Powers and the writer, in 
their efforts to find dividing ciliates, inadvertently restricted their studies to rela- 
tively large mature urchins, in which the ciliate populations were already well- 
established and probably somewhat stabilized. It will be seen in the following 
pages that when some of the younger urchins are examined, divisional stages can 
be found in abundance. Turning finally to B. gracilis, it was evident that this 
ciliate differed markedly in its reproductive activities from the preceding two. Of 
the 181 infected urchins, all contained dividing specimens, and there was thus no 
evidence of long periods of non-divisional life. 

The relation of the size of the urchins to the condition of their respective ciliate 
infections was not considered in the earlier study (Beers, 1948). Actually, there 
is often great diversity of size in an aggregation of urchins on a rocky ledge or 
in a tide pool. For example, urchins taken by the writer from a single tide pool 
at Long Ledge, Mt. Desert Island, on July 10, 1960, varied in diameter from 8 to 
65 mm. To some extent these differences merely reflected different rates of growth, 
but Grieg (1928) concluded that size (diameter of test) is a fairly reliable measure 
of the age of the urchins. Basing his studies on urchins taken from the Folden 
Fjord and the Bals Fjord of Norway, and on other materials, he concluded that 
the following relations of size to age prevail, at least in a general way : diameter 
0.5 mm., metamorphosis just completed; 1-2.5 mm., "the same year-group" as 
the foregoing, meaning urchins in their first summer of life ; 56 mm., about 1 year 
old; 15 mm., about 2 years old; 24 mm., 3 years old; 40 mm., 4 years; 50 mm., 
5 years; 60 mm., 6 years; 78 mm. (the largest specimen), "probably about 8 years 



CILIATES OF STRONGYLOCENTROTUS 71 

old." In the region of Mt. Desert Island, the spawning of urchins begins in 
February and ends in April. Since the urchins of northern Europe have a similar 
spawning period (Mortensen, 1943, p. 211), there is little doubt that Grieg's esti- 
mates of age are equally applicable to Mt. Desert Island urchins. 

The present paper is a record of further observations on E. borcalis, M. indomita 
and B. gracilis as found in 152 urchins taken at Mt. Desert Island in the summer 
of 1960. It is based on seven collections or small populations of urchins, each of 
which consisted of specimens of as many different sizes (age-groups) as were 
available at the respective sites of collection. The study concerns in particular the 
following aspects of the biology of the ciliates. 

(1) Their occurrence and morphogenetic condition (whether dividing or not) 
in "small" urchins, meaning urchins 8-14 mm. in diameter and presumably about 
1.5 years old. (A minimal size of 8 mm. was fixed solely by the unavailability of 
any urchins of smaller size.) This aspect attempts to answer these questions: At 
what age do urchins become infected with the respective ciliates ? Once established 
in the urchin, do the ciliate populations build up immediately or does a delay ensue 
following their ingestion by the host ? 

(2) Their occurrence and morphogenetic condition in "larger" urchins, mean- 
ing urchins 15-65 mm. in diameter and representing five age-groups, namely, 2.5 
to 6.5 years, in increments of one year. This aspect attempts to answer these 
questions : Do the infections become progressively more intense (ciliates more 
plentiful) as the urchins increase in age? Is the division of the ciliates, in par- 
ticular that of E. borealis and M. indomita, correlated in any way with the age 
of the urchins? 

(3) Their morphogenetic condition throughout a population of urchins. That 
is to say, does division of the ciliates occur simultaneously in all the urchins of 
a population or does it affect only certain age-groups or random individuals? 

(4) Their capability to survive in sea water outside the body of the urchin, 
bearing in mind that cysts are unknown in all echinoid ciliates and that young 
urchins undoubtedly become infected by the ingestion of the usual trophic forms ; 
thus, such survival affects directly their transmission from urchin to urchin. 

MATERIAL AND METHODS 

Of the seven collections of urchins, three were taken at low tide from the 
rocks of Emery Cove Ledge on July 2, 13 and 24. The remaining four were 
taken from four different tide pools at Long Ledge on July 10, August 8 and 26, 
and September 1. Each collection consisted of about 40 individuals. Of the 
urchins of each collection, 10 to 15, representing as many age-groups (sizes) as 
were available, were opened and examined without delay on the day of collection, 
and a like number was examined on the following day. The remaining ones were 
excluded from consideration, since it seemed advisable to use only urchins that 
were relatively recently collected. 

The total number of individuals of each species of ciliate was actually counted 
in the small urchins, but this procedure was usually impracticable with reference 
to larger urchins, in view of the enormous numbers of ciliates in them. Thus, 
0.05-ml. or 0.1 -ml. samples of enteric fluid were taken from these urchins, and 



72 



C. DALE BEERS 



the number of ciliates of each species was estimated in the samples. If the size 
of the urchin permitted, five 0.1 -ml. samples were taken from the stomach, five 
from the intestine, and two from the rectum. In 0.1-ml. samples, the three degrees 
of infection that have been defined were again distinguished with reference to each 
species. In 0.05-ml. samples, half the aforementioned numbers of individuals was 
employed to distinguish the respective degrees of infection. 

With reference to the survival of the ciliates in pure sea water, details of the 
procedure will follow. 

RESULTS 

1. Occurrence and morphogenetic condition of the ciliates in small urchins (diame- 
ter of test, 8-14 mm.} 

Unfortunately, only nine urchins of this size were available for study. Never- 
theless, it is believed that they furnish information that is significant (Table I). 

TABLE I 

Total numbers of ciliates of three species in each of nine small urchins (age about 1.5 years) 
taken at Long Ledge, Mt. Desert Island, in 1960 



Diameter of test 

in mm. 


Date collected 


Entodiscus 
borealis 


Madsenia 
indomita 


Biggaria 
gracilis 


8 


July 10 











8 


August 26 











8 


September 1 











9 


July 10 


3 





1 


9 


August 26 


9 


12 


2 


9 


September 1 


1 


8 





12 


August 8 


10 


8 


1 


13 


August 26 


26 


15 


4 


14 


July 10 


9 


28 


2 



Since urchins attain a diameter of 5-6 mm. at the end of one year and of 15 mm. 
at the end of two years, it is assumed that these nine urchins emerged as plutei in 
February or March of 1959 and were thus about 1.5 years old in the summer of 
1960. 

A very careful examination of the contents of the digestive tracts of three 
S-mm. urchins revealed no ciliates whatsoever, although the digestive tract of each 
was well filled with algal food. A similar examination of three 9-mm. urchins 
that were collected on the same dates as the foregoing revealed only small numbers 
of ciliates, though M. indomita was absent in one of them and B. gracilis in another. 
None of the ciliates was dividing. Evidently these three urchins, at the time in 
their second summer of life, were in the process of acquiring their respective ciliate 
infections. Finally, an examination of three urchins that had diameters of 12, 13, 
and 14 mm., respectively, showed ciliates of all three species in each urchin. On 
the average, these urchins contained two to three times as many individuals of 
each species as the 9-mm. urchins, even though the infections with B. gracilis were 
extremely light. Again, no dividing specimens were observed. 



CILIATES OF STRONGYLOCENTROTUS 



73 



2. Occurrence and morphogenetic condition of the ciliates in larger urchins (di- 
ameter of test, 15-65 mm.} 

Urchins 15-23 nun. in diaui. Twelve urchins of this size, assumed to be 
about 2.5 years old and thus in their third summer of life, were available for study. 
Whereas the urchins of the preceding age-group (1.5 years) were either uninfected 
or at best only lightly infected, all the urchins of the present group were infected 
with the three ciliates, and about half of the infections qualified either as moderate 
or heavy. The status of the respective infections in the 12 urchins was as follows : 
E. borealis, 1 H, 5 M, 6 L; M. indomita, 1 H, 4 M, 7 L; B. gracilis, 6 M, 6 L. 
Thus, a marked increase in the intensity of infection was clearly demonstrable in 
the 2.5-year-old urchins. It is evident that such an increase could have come 
about either by the ingestion of additional individuals or by the division of those 
already ingested. Manifestly, no comment can be made concerning the ingestion 

TABLE II 

Incidence of division of three species of ciliates in urchins of five different age-groups taken at 
Ml. Desert Island, summer 1960. All the urchins were infected with the three species. 









Number (and percentage) of urchins that contained 


Number of 


Diameter of test 


Approximate age 


dividing ciliates of species indicated 


examined 


in mm. 


years 


Rntodiscus 


Madsenia 


Biggaria 








borealis 


indomita 


gracilis 


12 


15-23 


2.5 


9 (75.0) 


7 (58.3) 


12 (100) 


32 


24-39 


3.5 


2 (6.3) 


1 (3.1) 


32 (100) 


35 


40-49 


4.5 


2 (5.7) 


2 (5.7) 


35 (100) 


37 


50-59 


5.5 


3 (8.1) 


3 (8.1) 


37 (100) 


27 


60-65 


6.5 


2 (7.4) 


1 (3.7) 


27 (100) 



of ciliates during the one-year interim, but it is significant that a remarkably high 
percentage of the 2.5-year-old urchins contained dividing individuals, showing 
conclusively that the respective ciliate populations were undergoing rapid augmen- 
tation by binary fission. The data concerning division in these urchins are sum- 
marized in Line 1 of Table II, reference to which shows that E. borealis was 
dividing in 9 of the 12 urchins, M. indomita in 7 of them, and B. gracilis in all 
of them. Furthermore, dividing specimens were relatively abundant, the incidence 
amounting to about one in every 25-50 individuals of each species. With reference 
to the division of E. borealis and J\l. indomita, it may be said now for purposes 
of emphasis that in none of the remaining age-groups was there such a high per- 
centage of urchins that contained the two ciliates in division. Concerning B. 
gracilis, it has been pointed out that this ciliate differs in its reproductive activities 
from the aforementioned two, in that long periods of non-divisional life are absent. 
Thus, B. gracilis was dividing in all 12 urchins. 

Urchins 24-39 mm. in diam. Urchins of this size, assumed to be about 3.5 
years old, were available in almost unlimited numbers at both collecting sites, as 
were indeed those of all succeeding age-groups. Of 32 urchins of this size that 
were examined, all were infected with the three ciliates, as were all the urchins 
of the age-groups subsequently to be discussed. The respective degrees of infec- 



74 C. DALE BEERS 

tion among the 32 hosts were as follows : E. borealis, 20 H, 10 M, 2 L; M. indomita, 
18 H, 11 M, 3 L; B. gracilis, 7 H, 23 M, 2 L. In terms of percentages, 91 to 94 f> 
of the urchins harbored infections of each species that qualified as moderate to 
heavy. Thus, these urchins were distinctly more heavily infected than those of 
the preceding two groups, and it will be seen, when older age-groups are considered, 
that the infections had now attained their maximal intensities. 

The findings relative to division are summarized in Table II, Line 2. Of the 
32 urchins, only two contained dividing specimens of E. borcalis, and even in them 
division was somewhat sparse and affected no more than one specimen in every 
100. In spite of an exceptionally thorough examination of the samples, dividing 
individuals of M. indomita could be found in only one of the urchins (a different 
one from the foregoing two). In accordance with expectations, B. gracilis was 
dividing in all the urchins of the group. 

Urchins 40-49 mm. in diam. Thirty-five urchins of this size, assumed to be 
about 4.5 years old, were examined. The respective degrees of infection follow : 
E. borealis, 15 H, 17 M, 3 L; M. indomita, 17 H, 16 M, 2 L; B. gracilis, 5 H,27 M. 
3 L. Again, 91 to 94% of the urchins harbored infections that varied from moderate 
to heavy. Data relative to the occurrence of division are summarized in Table II, 
Line 3, where it is seen that only two urchins contained dividing specimens of 
E. borealis and a like number (actually another two) those of M. indomita. All 
contained dividing specimens of B. gracilis. 

Urchins 50-59 mm. in diam. An examination of 37 urchins of this size, 
assumed to be about 5.5 years old, yielded the following degrees of infection: 
E. borealis, 12 H, 22 M, 3 L; M. indomita, 19 H, 16 M, 2 L; B. gracilis, 12 H. 
21 M, 4 L. With reference to each of the species, 90 to 95% of the urchins had 
infections that varied in intensity from moderate to heavy. The data concerning 
the incidence of division, summarized in Table II, Line 4, show that three of the 
urchins had dividing specimens of E. borealis and three (one of the foregoing 
plus two others) had M. indomita in division. As usual, all the urchins contained 
dividing forms of B. gracilis. 

Urchins 60-65 mm. in diam. Twenty-seven urchins of this size (age about 
6.5 years) were examined. Their respective degrees of infection were the follow- 
ing: E. borealis, 13 H, 12 M, 2 L; M. indomita, 15 H, 11 M, 1 L; B. gracilis, 9 H. 
16 M, 2 L. Again, with reference to each species, moderate to high infections 
comprised more than 90% of the total. Of the 27 urchins, two harbored divisional 
stages of E. borealis and a third one contained At. indomita in division (Table II. 
Line 5), whereas B. gracilis was dividing, as expected, in all of them. 

3. MorpJiogenetic condition of the ciliates in the respective collections of urchins 

It has been mentioned that the urchins of the present study comprised seven 
collections, each of which may be regarded as a small population ; each at least is 
believed to be a fairly representative sample of a natural population. And it has 
been shown, within the limits of the available material, (1) that urchins 8-14 mm. 
in diameter (age 1.5 years) may or may not be infected, but that if infected, they 
contain no dividing ciliates (Table I) ; and (2) that all urchins 15-23 mm. in 
diameter or larger (2.5 years of age or older) are infected with the ciliates, that 
B. gracilis is constantly dividing in all of them, but that E. borealis and M. indomita 



CILIATES OF STRONGYLOCENTROTUS 



75 



can be found in division in only a limited, though variable, number of them 
(Table II). However, the data concerning the division of E. borealis and M. 
indoniita in certain urchins of ages 2.5-6.5 years, as presented in Table II, tell 
nothing about the distribution of these particular urchins in the respective collections 
or population samples. Thus, one may ask : If E. borealis and J\I. indoniita are 
dividing in most of the urchins of one age-group of a collection for example, the 
2.5-year group are they also dividing in a like percentage of urchins of the remain- 
ing age-groups of the same collection? 

This aspect can be adequately presented by considering in detail the compo- 
sition of two typical collections of urchins of ages 2.5-6.5 years and the condition 
of the two ciliates therein. The collections are those taken at Long Ledge on 
July 10 and August 8. The results are presented in Table III, in which the left 
column under the headings beginning "No. of urchins" refers to the collection of 



TABLE III 

Incidence of division of two species of ciliates in two collections of urchins taken at Long Ledge, 
Mt. Desert Island, on July 10 and August 8, 1960. All the urchins were infected 

with both ciliates. 



No. of urchins 
examined 


Range in size of 
urchins in mm. 


Approximate age of 
urchins in years 


No. of urchins that contained dividing 
ciliates of species indicated 


Entodiscus borealis 


Madsenia indomita 


5 4 


15-23 


2.5 


4 3 


4 3 


5 5 


24-39 


3.5 


1 1 





5 5 


40-49 


4.5 





2 


5 5 


50-59 


5.5 


2 


1 1 


5 4 


60-65 


6.5 


1 


1 



July 10, the right to that of August 8. Line 1 shows that five urchins of the 
size and age indicated were taken on July 10 and four on August 8. Of the five, 
four contained E. borealis in division and four had M. indoniita in division. (Three 
of the five contained dividing individuals of both species.) Of the four urchins 
taken August 8, three had E. borealis and three had M. indoniita in division. (Two 
had both species.) If the nine urchins are considered as a group, seven of them 
or 77.7% contained dividing individuals of E. borealis and seven contained M. 
indomita in division. 

What was the condition of the two ciliates in the remaining age-groups of the 
two collections ? Was division as widespread in the urchins of these groups ? The 
answer is conclusively in the negative, as shown in the remaining four lines of 
Table III. For example, Line 2 shows that five 3.5-year-old urchins of each 
collection were examined. Only one urchin of each collection contained E. borealis 
in division; in none of the ten was M. indomita dividing. The urchins of the 
remaining age-groups revealed essentially similar findings (Lines 3-5). Thus, 
division in E. borealis and M. indomita, when it occurs in a population of urchins, 
does not necessarily affect uniformly all the urchins of the different age-groups of 
the population. 



76 C. DALE BEERS 

4. Survival of the ciliates in sea zvater and its relation to infectivity 

It has been pointed out that cysts are unknown in ciliates of echinoids and that 
young hosts undoubtedly acquire their faunules by the ingestion of trophic forms 
that escape among the fecal pellets. This conclusion implies that echinoid ciliates 
can live in sea water outside the body of the host, although information on their 
survival is meager. Powers (1933b, p. 123) states that specimens of E. borealis 
when transferred to sea water "appear normal" and "live for various lengths of 
time," and he was able to keep specimens in hanging-drop preparations at 7 C 
for periods that varied from 15 to 23 days. It is doubtful that the survival of a 
large entozoic ciliate in the restricted confines of a small hanging drop reveals 
anything of special significance about its survival under natural conditions, and 
Powers himself states that the animals seemed "merely to exist." Since the 
capability of echinoid ciliates to survive in sea water is inseparably related to the 
infection of new hosts, a study of the survival of the three entocommensals of 

5. drobachiensis was undertaken, but the procedure differed radically from that 
of Powers. 

The sea water was taken from Frenchman Bay (mean annual salinity, 31.8) 
well beyond the intertidal zone and was passed through Whatman No. 43 filter 
paper to remove the predatory or unwanted plankters. Each of the three species 
was dealt with separately in the following manner, as illustrated by E. borealis. 
A clean pre-cooled Syracuse watch glass was placed on the stage of a dissecting 
binocular and filled with 10 ml. of sea water (temperature 15 C., approximately 
that of Salisbury Cove sea water in the summer of 1960). Then, about 75 specimens 
of E. borealis from a recently collected urchin were placed in the watch glass near 
its right margin. The ciliates usually dispersed rapidly, so that many of them 
soon arrived in relatively pure sea water at the left margin of the watch glass, 
whereupon 25 of them were transferred by means of a small pipette to 1 ml. of 
fresh sea water in a Columbia culture dish (square plate-glass depression slide, 
measuring 42 mm. on a side). The culture dish was placed in a covered Stender 
dish which was outfitted as a small moist chamber and kept in a tray of running 
sea water to maintain the temperature at 15 C. The condition of the ciliates was 
observed and recorded at the end of 6, 9, 24, 48, 72, and 96 hours, reckoning from 
the beginning. The experiment as just described was repeated some 20 times, 
using ciliates from more than a dozen different urchins. The method was decidedly 
superior to the use of hanging-drop preparations, in that the ciliates were first 
allowed to wash themselves relatively free of intestinal materials and were then 
transferred to 1 ml. of fresh sea water, which is a relatively large volume for only 
25 ciliates. In most of the experiments the final culture dishes were exposed 
to the natural light of the laboratory, but in some they were kept in darkness 
(Stender dishes painted black on the outside) except during the brief intervals of 
observation. Since the histories of the cultures were identical, there was no 
evidence that moderate illumination was detrimental to the ciliates or that darkness 
was beneficial. 

The procedure that has been described for E. borealis was likewise employed 
with M. indomita and B. gracilis. To facilitate comparisons, the results obtained 
with 300 individuals of each species, representing 12 culture-dish experiments, will 
be considered (Table IV). 



CILIATES OF STRONGYLOCENTROTUS 



77 



E. borealis. Upon transfer to sea water, the ciliates, in agreement with Powers' 
findings, showed little or no heightened irritability and suffered no observable 
distortion in shape. At the end of 6 hours, 297 of the original 300 were present 
in the cultures, and at end of 9 hours, 296. It is likely that the death and dis- 
appearance of a few resulted from injuries that accompanied the process of washing 
and transfer. At the end of 24 hours, 281 were present (survival, 93.7%). Some 
were swimming normally and others were creeping on the bottom of the dish or 
against the surface film. However, the many food vacuoles which they originally 
contained had disappeared, and thus the cytosome was relatively transparent. At 
the end of 48 hours, 256 (85.3% of the original number) were still present, but 
they were distinctly smaller, quite transparent, very slow of movement, and evi- 
dently much weakened from lack of food. During the succeeding 24-hour period 

TABLE IV 

Survival of three species of urchin ciliates in sea water. Total number of individuals of 
each species at beginning of experiment was 300. Hours cited are reckoned 

from the beginning. 





Ciliate 




Kntodiscus borealis 


Madsenia indomita 


Biggaria gracilis 


No. of survivors after 








6 hours 


297 


298 


217 


9 hours 


296 


298 


103 


24 hours 


281 


295 





48 hours 


256 


272 




72 hours 


17 


36 




96 hours 











the animals suffered drastic mortality, since only 17 (5.7%) remained at the end 
of 72 hours. These few survivors were much smaller than formerly and were 
barely able to swim. At the end of 96 hours, there were no survivors. 

M. indomita. Unlike E. borealis, this ciliate when transferred to sea water 
displayed greatly heightened irritability, for the animals swam rapidly and errati- 
cally. However, their intense activity subsided within 5 to 10 minutes, and with 
no ill effects, to judge by their survival. In general, the results paralleled those 
obtained with E. borealis, although there were slightly more survivors throughout 
the first three days. At the end of 24 hours all the food vacuoles had disappeared 
from the cytoplasm, but the animals were still swimming normally. At the end 
of 48 hours they were considerably diminished in size and were very transparent, 
and their locomotion was extremely sluggish. Again, a high mortality occurred 
during the third 24-hour period, such that only 36 were present after 72 hours. 
No survivors remained at the end of 96 hours. 

B. gracilis. The outcome of the experiments with this ciliate was entirely 
unexpected. Upon transfer to sea water, B. gracilis swam rapidly and quite 
erratically, as if the medium were distinctly unfavorable. Of the original 300 speci- 
mens, only 217 (72.3%) were present after 6 hours, and at the end of 9 hours 
this number was reduced to 103. Many of these were vacuolated and clearly 



78 C. DALE BEERS 

abnormal in structure, and the remains of others were visible in the culture dishes. 
Since nearly all the survivors contained food vacuoles, the many deaths among 
the animals could not be attributed to starvation, but must have resulted from the 
properties of the medium. At the end of 24 hours there were no survivors. 

DISCUSSION 

A comprehensive investigation of the relation of the three ciliates to their host 
in the Mt. Desert Island region would require a study of urchins of practically all 
sizes taken during all the months of the year. Unfortunately, such a study has 
not been feasible, and the present one is admittedly incomplete. Nevertheless, the 
results are of special interest and are fully adequate, it is believed, to support the 
conclusions that are advanced in the following sections. 

1. Acquisition of infections by young urchins and the delayed onset of division 

The absence of ciliates in 8-mm. urchins (age about 1.5 years) indicates that 
young urchins do not acquire their infections during their first summer of life, or 
even during the first year. The presence of relatively small numbers of ciliates in 
urchins 9-14 mm. in diameter (age likewise about 1.5 years, but no doubt some- 
what older than the foregoing) indicates that the urchins first acquire their ciliates 
during their second summer when they are at least 9 mm. in diameter and about 
1.5 years old. 

It might reasonably be assumed that all urchins would become infected not 
long after metamorphosis and that all would contain fairly dense populations of 
ciliates by the middle of their second summer. Actually, at least four factors mili- 
tate against the early acquisition of infections by young urchins at Mt. Desert 
Island. The first three are of general occurrence ; the fourth is to some extent 
peculiar to the region of the Island. They are the following. (1) The ciliate 
losses that accompany the extrusion of fecal pellets are relatively small, to judge 
by earlier experience (Beers, 1948), as if each ciliate resists dislodgement from its 
preferred segment of the gut. Thus, urchin ciliates are extremely scarce and very 
difficult to find in the waters of the urchin's natural habitat, though they can be 
found with no difficulty in the bottom sediments of an aquarium that is well-stocked 
with urchins. (2) The period of survival of the ciliates in a healthy condition in 
sea water outside the body of the host is relatively short, varying from 6 to 48 
hours. Although the ciliates tend to adhere loosely to any substratum and to 
creep upon it. thereby facilitating to some extent their ingestion by a new host, 
the length of time available for their chance discovery and ingestion by an urchin 
is distinctly limited. (3) Some of the ciliates are no doubt destroyed by predators. 
(4) Tidal extremes are great, the mean tidal range being 10.6 ft. (3.23 m.) at 
Salisbury Cove. Thus, enormous quantities of water ebb and flow twice daily 
over the urchins and undoubtedly carry away many of the extruded ciliates. In 
view of the existence of these inimical factors, it is perhaps not so surprising to 
find that the infection of the young urchins is appreciably delayed. 

The absence of dividing ciliates in urchins 9-14 mm. in size suggests that the 
respective ciliate populations do not undergo augmentation by cell division imme- 
diately after the infection of the host, but are increased during the second summer 



CILIATES OF STRONGYLOCENTROTUS 79 

only by the ingestion of additional specimens. Significant augmentation by division 
appears to be delayed until the third summer, when the urchins are about 2.5 
years old. 

2. Establishment of t!ic ciliate populations; division of the ciliates in urchins of 

diverse ages and in populations of urchins 

Whereas none of the infected 1. 5-year-old urchins contained dividing ciliates. 

* o 

it has been seen that of 12 infected urchins of age 2.5 years, 9 had E. borealis in 
division, 7 had M. indomita, and all had B. gracilis. These findings indicate, as 
has been said, that it is during the third summer of the urchin's life that the respec- 
tive ciliate populations first experience augmentation by division, resulting in the 
establishment of populations of maximum density. 

Once the populations of E. borealis and M. indomita are established, infrequent 
eruptions of divisional activity seem adequate to maintain them in the host ; thus, 
relatively few (3.1 to S.\ c / f ) of the older urchins (age 3.5 to 6.5 years) harbor 
them in division. The factors that are responsible for the seemingly long intervals 
of non-divisional life and the occasional, intense outbreaks of division are unex- 
plained, as has been said. It has been seen that division in the two cannot be 
correlated with the age of the host ; it seems to occur randomly in older urchins, 
irrespective of their age. Neither does their division affect en masse the individuals 
of an urchin population, even though the urchins seem to be living under similar 
conditions. Various possibilities present themselves by way of explanation, for 
example : ( 1 ) There is an inherent rhythm of long frequency in the reproductive 
activities of the ciliates. (2) Division is correlated, either qualitatively or quanti- 
tatively, with the food of the urchin and with the nature of the intestinal flora. 
Although sea-weeds are the preferred food of 6". drobachiensis, it is actually omnivo- 
rous, and the nature of the intestinal contents is somewhat unpredictable. Thus, 
urchins of a collection from one and the same tide pool at Long Ledge were found 
to have fed on a variety of materials. Some contained principally filamentous green 
algae in their alimentary tracts; others, bladder wrack (Fucns and the like) and 
sea lettuce (Ulva*) ; still others, calcareous algae; and finally some contained non- 
descript materials that seemed to consist of barnacle remains and bottom sediments. 
To what extent these diverse food materials affect the ciliate fauna has not been 
ascertained. (3) Division is correlated with the physiological state of the urchin, 
though practically nothing can be said at present concerning this point. Of the 
foregoing possibilities, the second would seem to be the most readily amenable 
to experimental analysis, and it is planned that progress in this direction will be 
attempted within the near future. 

The probable significance of the constant and uninterrupted division of 
B. gracilis is mentioned below. 

3. Survival of the ciliates in sea zvater in relation to infectivity 

It has been seen that under the conditions of the experiments both E. borealis 
and M. indomita can tolerate pure sea water for about 48 hours. Evidently this 
interval of time, assuming that it also prevails under natural conditions, is adequate 
to insure the eventual ingestion of a sufficient number of individuals to perpetuate 
the two species in the host. 



80 C. DALE BEERS 

Much in contrast with the two-day survival of the foregoing species is the 
seeming incapability of B. gracilis to tolerate sea water longer than 6-12 hours. 
Although little can be said with certainty in explanation of this pecularity, certain 
aspects of the autecology of B. gracilis seem worthy of mention. Of all the species 
of ciliates that occur in S. drobachiensis at Mt. Desert Island, B. gracilis is the only 
one that is primarily an inhabitant of the rectum. In this disadvantageous site, it 
is expelled in greater numbers than any of its confreres (Beers, 1948). But it is 
also the only one of the ciliates, with the exception of Enplotes balteatus, which is 
probably nothing more than a facultative commensal (Beers, 1954), that is con- 
stantly dividing within the urchin. Thus, its loss in greater numbers is offset by 
frequent division, and its continued survival in the host is reasonably assured. In 
sea water outside the body of the host, B. gracilis experiences a further disadvantage 
from the standpoint of survival, in that it has relatively little tolerance for sea water. 
But it is lost in greater numbers from its host, as has just been said. The escape 
of larger numbers of individuals into the external world would seem to compensate 
adequately for the briefer period of survival of each ; thus, a relatively constant 
number of individuals is presumably maintained in the external environment, where 
they can be ingested by new hosts. Though vulnerable to excessive losses both 
within the urchin and without, B. gracilis nonetheless maintains itself by the agency 
of constant division. 

SUMMARY 

1. The first part of the study concerns certain relationships of the ciliates 
Entodiscus borealis, Madsenia indomita and Biggaria gracilis to their host, the 
sea urchin Strongyloccntrotus drobachiensis. It is based on an examination of 
152 urchins taken at Mt. Desert Island, Maine, in the summer of 1-960. The respec- 
tive ages of the urchins are estimates based on size (diameter of test). The second 
part concerns the survival of the ciliates in sea water, since their survival is insepa- 
rably related to the infection of new hosts. 

2. Nine urchins measuring 8-14 mm. in diameter (age 1.5 years) were either 
uninfected or very lightly infected, and none of the ciliates was dividing. Urchins 
evidently acquire their ciliates at this age (second summer). 

3. All the urchins of the remaining age-groups were infected with all 3 ciliates. 
Of 12 urchins that measured 15-23 mm. in diameter, all contained dividing speci- 
mens of B. gracilis, 9 contained dividing individuals of E. borealis, and 7 contained 
M. indomita in division. The results indicate that the respective ciliate popula- 
tons build up rapidly to maximal densities in the third summer of the urchin's life 
(age about 2.5 years). 

4. The remaining urchins were assigned by size to 4 age-groups. The number 
of urchins in each group, their range in size, and their estimated ages follow : 32 
urchins, 24-39 mm., 3.5 years; 35, 40-49 mm., 4.5; 37, 50-59 mm., 5.5; 27, 
60-65 mm., 6.5. All the urchins harbored dividing specimens of B. gracilis; thus 
this ciliate remains in constant division once infection is well established. But in 
each group only a small percentage of the urchins (3 to 8%) contained dividing 
specimens of E. borealis and M. indomita. Thus, their division, though evidently 
cyclical, could not be correlated with the age of the urchins. 

5. In a natural population of urchins, the division of E. borealis and M. indomita 
does not affect simultaneously any large percentage of the urchins. Except in 2.5- 



CILIATES OF STRONGYLOCENTROTUS 81 

year-old urchins, it appears to occur randomly. Since the urchins of a population 
practice dissimilar food habits, it is possible that division is correlated with the 
nature of the food and the subsequent intestinal flora. 

6. In pure sea water most specimens of E. borealis and M. indoinita can survive 
about 48 hours, and their death is due to starvation. Individuals of B. gracilis can 
survive no longer than 6-12 hours, and death does not result from starvation but 
seemingly from the properties of the medium. It is suggested that the constant 
voiding of B. gracilis among the fecal pellets of the host compensates for its rela- 
tively brief period of survival in sea water. 

LITERATURE CITED 

BEERS, C. D., 1948. The ciliates of Strongylocentrotus drb'bachicnsis: Incidence, distribution 

in the host, and division. Biol. Bull., 94: 99-112. 
BEERS, C. D., 1954. Plagiopyla minuta and Euplotes baltcatits, ciliates of the sea urchin 

Strongylocentrotus drobachiensis. J. Protosool., 1 : 86-92. 
GRIEG, J. A., 1928. The Folden Fjord. Echinodermata. Tromso Mus. Skrijtcr, 1 (Part 7) : 

1-12. 
KAHL, A., 1932. Urtiere oder Protozoa. I: Wimpertiere oder Ciliata (Infusoria). In Dahl : 

Die Ticrwclt Deutschlands. Lief. 25, 399-650. Jena: Verlag von Gustav Fischer. 
KAHL, A., 1934. Ciliata entocommensalia et parasitica. In Grimpe u. Wagler : Die Ticnvelt 

dcr Nord- und Ostsce. Lief. 26, Teil II. C4, 147-183. Leipzig: Akademische Ver- 

lagsgesellschaft. 
KIRBY, H., 1941. Relationships between certain protozoa and other animals. In Calkins and 

Summers: Protozoa in Biological Research, 890-1008. New York: Columbia Uni- 
versity Press. 
MADSEN, H., 1931. Bemerkungen iiber einige entozoische und freilebende marine Infusorien 

der Gattungen Uronema, Cyclidium, Cristigera, Aspidisca und Entodiscus gen. nov. 

Zoo/. Anz., 96: 99-112. 
MORTENSEN, T., 1943. A monograph of the Echinoidea. III. 3. Camarodonta. II. Echinidae, 

Strongylocentrotidae, Parasaleniidae, Echinometridae. Copenhagen : C. A. Reitzel. 
POWERS, P. B. A., 1933a. Studies on the ciliates from sea urchins. I. General taxonomy. 

Biol. Bull., 65: 106-121. 
POWERS, P. B. A., 1933b. Studies on the ciliates from sea urchins. II. Entodiscus borealis 

(Hentschel) (Protozoa, Ciliata), behavior and morphology. Biol. Bull., 65: 122-136. 
POWERS, P. B. A., 1935. Studies on the ciliates of sea-urchins. A general survey of the infesta- 
tions occurring in Tortugas echinoids. Carnegie lust, of Wash., Publ. 452: 293-326. 



OBSERVATIONS ON THE RESPIRATION OF THE SABELLID 
POLYCHAETE SCHIZOBRANCHIA INSIGNIS 

R. PHILLIPS DALES 
Bedford College, University of London, London N.1V. 1 

Schisobranchia insignis Bush lives in tough fibrous tubes, mostly 10-20 cm. 
long and 510 mm. in diameter, attached to the underside of floating wharves, to 
pilings and to rocks on the Pacific northwest coast of America (Fig. 1. A). It may 
also be dredged from muddy bottoms. 

The dense crown of orange, purple or grey branched filaments, which is used 
both for feeding and for respiration, may be expanded beyond the opening of the 
tube for long periods when the worm is undisturbed. For shorter periods the 
worm lies wholly within the tube. Worms also irrigate their tubes by waves of 
muscular contraction of the body wall which may pass in either direction. Irriga- 
tion occurs when the crown is expanded as well as when the worm is retracted 
within the tube. 

That the crown of all sabellids is used for feeding may readily be confirmed by 
simple observation, but its importance in respiration appears to vary from one 
species to another. Zoond (1931) found a 63% fall in oxygen uptake after ampu- 
tation of the crown in Bispira rolutacornis (Montagu) and Fox (1938) found the 
same decrease when Sabclla spallansanii (Viviani) was similarly treated. On the 
other hand, Wells (1952) found that bisected Sabclla f>ai'oiiina Savigny showed 
no significant fall in total rate of oxygen uptake of the two parts, but that My.ricola 
infundibulum Renier did, there being a sharp drop in total uptake when bisected, 
and the posterior part giving relatively lower values than those of Sabclla paroniiia. 
He concluded that in Sabclla, while the current caused by the crown provides for 
the crown's own respiratory needs, it is the irrigation current which is of importance 
to the rest of the body. Myxicola, on the other hand, does not irrigate its tube and 
is wholly dependent on the crown which functions not only in feeding but as a gill. 

These differences suggested that it might be of interest to investigate the activities 
of another sabellid under conditions as natural as possible. The importance of the 
crown in respiration has only hitherto been assessed by the drastic procedure of 
amputation, and the rate of oxygen uptake has never been measured with the rate 
of water transport through the crown. Consequently, I have made measurements 
of oxygen uptake by the worm when expanded and when wholly withdrawn within 
the tube. The volumes of water passed (1) through the tube and (2) through 
the crown have also been measured, and the percentage utilization of oxygen by 
the crown and by the remainder of the body estimated under normal circumstances. 
All measurements have been at 12-13 C. 

All the observations were made on animals from wharves in the vicinity of 
Friday Harbor. Washington. I am glad of this opportunity to thank Dr. Robert L. 
Fernald and the Staff of the Friday Harbor Laboratories of the University of 

82 



RESPIRATION OF SCHIZOBRANCHIA 



83 



Washington for their hospitality and help. I also wish to thank Professor H. 
Munro Fox, F.R.S., for helpful criticism of this paper. 

\Yorms were stripped of their own tubes and accommodated in pieces of trans- 
parent plastic or transparent rubber tubing of suitable length and diameter. Such 
tubes reveal the activities of the worm, readily enable the tube to be linked to 
recording apparatus, and permit measurement of oxygen uptake under nearly 



M 

chamber 



transparen 
plastic 
tubin 





D 



FIGURE 1. A, appearance of colony of Schizobranchia in nature; B, apparatus used to 
record irrigation; C, irrigation prevented; D, expansion prevented. 

normal conditions. The work of Hyman (1932) and Fox (1938) emphasises the 
importance of simulating natural conditions as far as possible. Worms used in the 
experiments had been acclimatised to plastic or rubber tubes for at least one or 
two weeks. 

EXTENSION AND WITHDRAWAL 

To obtain some idea of the amount of time spent by the worm with the crown 
expanded, and the amount of time passed wholly withdrawn within the tube, worms 



84 R. PHILLIPS DALES 

accommodated in plastic tubes were attached to a recording apparatus (Fig. 1, B) 
similar to that used by Wells (1951) for Sabella. The apparatus was immersed 
in a tank through which a circulation of sea water was maintained and in which 
the water level (1) remained constant. By adjusting the size of the capillary leak 
(a) the movement of the worm could be recorded on a slowly revolving kymograph 
by the lever actuated by changes in the level of the water in the float chamber (f). 
By selecting a larger capillary which allowed a more rapid flow 7 than could be 
maintained by the worm irrigating under normal conditions, it was possible to 
adjust the capillary so that the float would be affected only by relatively rapid 
movements of the whole body, as in extension or withdrawal. Two typical traces, 
each of 12 hours duration, made by different worms are shown in Figure 2. It will 




FIGURE 2. Continuous record of expansion and withdrawal by two different worms. 
Duration of each trace, 12 hours. Read from left to right. Upward spikes represent withdrawal 
within the tube, downward spikes represent extension from the tube with expansion of the 
crown. Extension is more gradual than withdrawal, as is shown by the stepped trace. The 
horizontal parts of the trace represent the times when the worm remained with the crown 
expanded beyond the opening of the tube. 

be seen that extension and withdrawal occur at intervals of some regularity. The 
long period when the crown is expanded following extension after midnight was 
common to many worms and records. It may have been due merely to absence of 
stimulation by workers in the laboratory, although many other sabellids have been 
seen to expand their crowns more at night (Mclntosh, 1922, Fox, 1938). It will 
be noticed that each period of expansion exceeded each period spent wholly with- 
drawn. The time spent retracted on any one occasion did not exceed 10-15 min- 
uts, while periods of expansion were 20-60 minutes or longer. The interpretation 
of the records was confirmed by frequent observation. 

IRRIGATION 

Each tube, though firmly attached by mucus at or near the base to wharves or 
pilings, has one or more small openings 1 mm. or so in diameter near the hind end 
(Fig. 1, A). Mucus can be secreted through these to regain attachment, or new 
apertures made as occasion demands, and the orientation of the tube somew ? hat 
changed, as Fox (1938) observed in Sabella spallanzanii. Apart from these possi- 
bilities, which enable a small amount of re-orientation within the clump of animals 
so that each has room to expand the crown, Schizobranchia is completely sessile and 



RESPIRATION OF SCHIZOBRANCHIA 85 

is unable to turn around in the tube except on its own longitudinal axis. Unlike 
Sabclla, however, it rarely does so. 

Irrigation of the tube is effected by muscular swellings passing down the body, 
most commonly from head to tail. Occasionally the direction of irrigation is 
reversed. These activities may occur when the crown is expanded or when the 
worm is withdrawn into the tube. The volume of the tube containing a worm of 
average size (2 grams fresh weight) is about 1.5 ml., the volume of such a tube 
empty being about 3.5 ml. Some idea of the irrigation rate may be obtained by 
injecting a suspension of carbon into the tube by means of a hypodermic syringe, 
and observing the rate of travel of the particles along a horizontally fixed graduated 
tube sealed on to the hind end. Under otherwise normal conditions the fluid in 
the tube may be completely renewed in 30-60 seconds of activity. 



A 



B 



C 



FIGURE 3. A-C, continuous record of irrigatory activity of a single worm over a period of 36 
hours. Read from left to right. Each line represents 12 hours. Further explanation in text. 

A continuous record of irrigation may be obtained with the apparatus already 
described by attaching a finer capillary at (a), such that the flow into or from the 
tank causes a slight rise or fall of 1-3 mm. to occur in the float chamber. A pressure 
difference of this magnitude may easily be recorded, but is unlikely to be great 
enough to modify the behaviour of the worm. A record of such activity is shown 
in Figure 3, C. The details of such traces were interpreted by watching worms 
from time to time while the trace was being made. 

In the records elevation represents irrigation headwards ; depression, tailwards. 
Irrigation will be seen to be somewhat irregular in rate, but to be fairly continuous. 
The volume passed can be calculated, knowing the dimensions of the capillary and 
the rest of the apparatus ("Wells and Dales, 1951), or may be determined empiri- 
cally. The average rate was found to be 0.3-0.5 ml./min. for a 2-gram (fresh 
weight) worm. Wells (1952) found a similar rate for Sabella pavonina of compa- 
rable weight. By inserting a small bung into the opening of a worm's tube attached 
to the recording apparatus, extension of the worm and irrigation could be stopped. 
If the period of closure did not exceed 10-15 minutes (Fig. 3, A : 1 ; Fig. 3, B : 3) 
normal activity was resumed after release. If this period was exceeded (20-45- 



86 



R. PHILLIPS DALES 



minute closure) as in Figure 3, A: 2; Figure 3, B: 1, 2, release was followed by 
very vigorous irrigation, as much as 0.75 ml. min. being passed for an hour or 
more by a 2-gram worm. 

By connecting another piece of tubing to both the open end of a plastic tube 
in which a worm had been accommodated and attached to a recording apparatus 
(Fig. 1, B), and to the jet from the float chamber instead of the capillary leak, 
the circulation could be closed without preventing the worm from irrigating. Under 



0-20 



c 
u 



X 
O 



0.10 



008 



006 



004 



0.0?. 



2.0-4.0 ce.Ot/l. 

O 6.0-8.0 

A 60-8.0 fellow,n S 41. /, 

at less tKan 1.0cc/L. 
A 8.0-140 

(b= I2-I5C) 




10 



5-0 



6.0 



2.0 >0 40 

FresVi Weio^t 

FIGURE 4. Rate of oxygen uptake under different external oxygen concentrations. 



RESPIRATION OF SCHIZOBRANCHIA 87 

normal conditions worms underwent short bursts of "testing" activity, driving first 
in one direction and then in the other, and on release continued vigorous irrigation 
for some hours. Wells (1951) found that Sabclla spallanzanii responded similarly. 
In Figure 3, B : 3, a hurst of testing irrigation in a headward direction was in prog- 
ress on release, and this direction was maintained for the following two hours, the 
worm gradually returning to its normal behaviour pattern. 

OXYGEN UPTAKE 

The rate of oxygen uptake was measured by the modification of Fox and 
Wingfield (1938) of the well-known Winkler technique. The rates of uptake made 
under different oxygen concentrations plotted against total fresh weight are pre- 
sented in Figure 4. 

Uptake was measured in closed bottles after a period long enough to ensure 
accurate estimation, but normally not so long that the quantity of oxygen in the 
bottle was reduced to not less than -15 cc./l. or the quantity of carbon dioxide 
increased to a level at which the rate might be affected. Bottles of approximately 
270 ml. capacity were used and most measurements were made after a 1-4-hour 
period, according to the size of the animal. Worms thoroughly acclimatised to 
plastic tubing were used, as measurements so made might be expected to be closest 
to those under entirely natural conditions; Fox (1938) found that the oxygen 
uptake in Sabclla spallanzanii was 20-30% lower in worms freshly deprived of 
their tubes. Each animal was therefore enclosed in a bottle large enough to ensure 
normal activity ; worms were able to expand the crown, to withdraw and to irrigate. 
The bottles were occasionally inverted to ensure thorough mixing ; most of the worms 
were acclimatised to being disturbed and their behaviour appeared to be affected 
only momentarily. All the determinations were made at 1213 C. 

(a) Response to raised or lowered o.vygen content of the water 

The normal rate of oxygen uptake could be maintained by the worm when the 
oxygen content of the water was lowered to 2 cc./l., either gradually by the animal 
itself over an extended period in a closed bottle, or by bubbling nitrogen through 
the water before the experiment. When the oxygen content \vas slowly reduced 
by the animal itself, the rate of uptake was significantly reduced between 1.3 
2.5 cc. O 2 /l. 

If enclosed in a small chamber so that the rate of pulsation of the branchial 
vessels could be observed, the normal rate of 9-10 pulsations/min. (13C.) fell 
off rapidly below about 2.5 cc. O 2 /l., and ceased altogether around 1.3 cc. O 2 /l. 
Fox (1938) found the same effect in Sabella spallanzanii. 

The rate of oxygen uptake was not affected by raising the oxygen content of 
the water, similar values being obtained up to 14.0 cc. O 2 /l. It is interesting, how- 
ever, in confirmation of the findings of Fox and Taylor (1955), that worms were 
not adversely affected by these high concentrations, and survived indefinitely in 
the laboratory circulation which had a high oxygen content (7-8 cc. O 2 /l.) owing 
partly to the action of the pumps. These sabellids are indeed usually found in 
habitats with a good circulation of water where the concentration of oxygen is 
likely to be high. Fox (1932) and Ewer and Fox (1940) have shown that the 
chlorocruorin of Sabclla spallanzanii blood is adapted for oxygen transport only at 



88 



R. PHILLIPS DALES 



high outside concentrations, and C. Manwell (private communication) has found 
the same in Schisobranchia insignis. 

(b) Uptake of oxygen zvhen extended 

When the crown was removed, and after the animal had recovered from the 
operation for a day or two, the oxygen uptake by the rest of the body was measured ; 
the value obtained was about 25% that of the normal animal. 

With normal animals irrigation, and hence normal respiratory exchange across 
the body wall, could be stopped by plugging the hind end of the tube (Fig. 1, C). 
Observation suggested that such worms remained extended more continuously, and 
measurement of oxygen uptake in closed bottles showed that the values obtained 
were not significantly different from those of normally irrigating worms. In other 

TABLE I 

Rate of oxygen uptake under normal conditions, when irrigation is prevented (as in Fig. 1, C), 
and when confined within the tube (as in Fig. 1, D) 



Worm number 


Total fresh weight 


O2 uptake under 
normal conditions 
(cc./g./hr.) 


O2 uptake without 
irrigation 
(Fig. 1. C) 


O> uptake under 
forced withdrawal 
(Fig. 1, D) 


16 


0.460 


0.1920 


0.1790 


0.0782 


17 


0.520 


0.2330 


0.2266 


0.0819 


18 


0.600 


0.2210 


0.2165 


0.0759 


19 


1.205 


0.1132 


0.1568 


0.0463 


20 


0.565 


0.2212 


0.2298 


0.0696 


21 


0.765 


0.1684 


0.1851 


0.0855 


22 


2.100 


0.0916 


0.1114 


0.0316 



words, normal oxygen uptake can be maintained by the crown alone (i.e., without 
uptake through the body wall), though it may have to remain expanded to do so. 
Wells (1952) found that crownless Sabclla paronina perished if unable to irrigate. 

(c) Uptake of oxygen when withdrawn 

When withdrawn within the tube the crown is not well displayed for respiratory 
exchange or feeding, as the numerous branched filaments are tightly rolled together. 
By fitting a narrower tube to the opening (Fig. 1, D) extension could be prevented 
but irrigation continued. Under these conditions oxygen uptake was reduced, 
usually to about 40% of the normal value. On release, worms remained extended 
for some time. Values obtained for seven worms in which measurements of oxygen 
uptake were made under normal conditions, with irrigation prevented, and when 
wholly withdrawn, are compared in Table I. 

RATE OF FILTERING BY THE CROWN 

When the crown is extended water flows through the filaments as a result of 
ciliary activity. A measure of the volume of water strained through the crown 
may be obtained by the use of colloidal graphite suspensions, since the particles 
coming into surface contact are removed from suspension by mucus. Removal of 
particles is exponential if the system remains constant in volume and the particles 



RESPIRATION OF SCHIZOBRANCHIA 89 

remaining in suspension are evenly distributed (Dales, 1957). Particles may be 
ingested or rejected, but in either case are removed from suspension. The rate at 
which unit volume is cleared of particles may be calculated by measuring the de- 
crease in density at known intervals against controls (J^rgensen, 1949). The 
filtering rate in ten experiments was calculated over a three-hour period using 
worms of 0.5-2.0 grams fresh weight at 12-13 C. The mean rate of filtering was 
70.7 ml./g./hr. This is of the same order of magnitude as has been found in other 
sabellids (Dales, 1957). 

DISCUSSION 

All these observations suggest that the life of Scliisobranchia insignis is very 
similar to that of Sabclla spallansanii. Both species may be found in tubes open 
at the hind end attached to rocks and wharves. Both irrigate their tubes with equal 
facility in either direction, and pause in this activity for periods rarely exceeding 
10 minutes. 

It is difficult to assess the part played by the crown in supplying the respiratory 
needs of the rest of the body since, as Wells (1951, 1952) has pointed out, the 
needs of the crown itself are high owing to its activity. The vascular supply to 
the crown may be of service both in conveying oxygen away and in conveying 
nutrients to the ciliated epithelium and other tissues of the crown. The ability to 
continue to live, and to regenerate the crown when this is amputated, provided 
that the worm is able to irrigate its tube, suggests that the crown is not essential, 
although under normal circumstances it may well supply part of the body's needs. 

Schizobranclua cannot autotomize the crown as Sabella does, so that decapita- 
tion results in a more serious loss in the total blood volume and perhaps a more 
unusual derangement of metabolism than in Sabclla. While some individuals did 
regenerate their crowns, many died under laboratory conditions ; Sabella seems 
better adapted for this contingency. The crown is also relatively larger in Schiso- 
branchia, and the reduction in oxygen uptake to 25% of the normal value may 
well be partly due to the loss of that part of the uptake accounted for by the crown 
itself. When the worm is retained in the tube (or when, in nature, the worm is 
wholly withdrawn) the irrigation current alone supplies oxygen to the animal and 
removes carbon dioxide and other waste products. Under these conditions the 
oxygen uptake is 40% of that when extended. While the crown is not then 
expanded and the animal's need for oxygen may be somewhat less, the cilia on the 
crown do not cease to move, and the muscular contractions causing irrigation of 
course continue. It could be argued that if crownless worms can continue to live, 
providing that they are able to irrigate, and that the oxygen uptake of crownless 
worms is 25% of what it was before decapitation, then the requirement of oxygen 
for maintaining irrigation may be met by 25% of the normal total oxygen uptake. 
Other activities may well be interrupted after decapitation, but the major part of 
the remaining 75% of the normal oxygen uptake may thus be accounted for by the 
activity of the crown itself. That worms wholly withdrawn (Fig. 1, D) have an 
oxygen uptake of 40% of the value for expanded though not continuously irrigating 
worms (Fig. 1, C) suggests that perhaps a value approaching 60% of the total 
oxygen uptake is due to the activity of the crown alone. While the circulation of 
the blood from the crown can supply the respiratory needs of the rest of the body 
if irrigation is not possible, under normal circumstances it need not do so. Uptake 



90 R. PHILLIPS DALES 

of oxygen will ensure, through the intermediacy of the vascular system, a supply 
of oxygen to all parts at all times, for activity of the crown and irrigation are 
independent activities. 

Pulsation of the branchial vessels was observed by Fox (1938) and Wells 
(1951) in Sabclla to cease after some time when totally enclosed in a small chamber 
or in a tube, and cessation may be seen also in ScJiizobrancliia. As already noted, 
pulsations occur under normal circumstances at a rate of about 10/min. at 13 C., 
but these cease altogether when the oxygen content of the water has fallen to about 
1.3 cc. C>2/1. Fox (1938) suggested that this effect may be due to accumulation of 
carbon dioxide, but as Wells (1951) points out, this is unlikely to occur under 
normal conditions owing to irrigation. On the other hand, if the worm is wholly 
withdrawn and, while ceasing to irrigate, its uptake of oxygen remains at 40% 
of its normal value (0.1 cc. O 2 /hr. for a 2-gram worm), the oxygen contained in 
the 1.5 ml. of water within the tube would be used up in 15 minutes. The possibility 
that the factor which ends a short rest from irrigation might be lack of oxygen or 
accumulation of carbon dioxide should not, therefore, be dismissed. In Scliizo- 
branchia, however, pauses were never observed to be as long as this, and in any 
case when the irrigatory waves cease the oxygen requirement should be less. In 
addition, there should be sufficient oxygen in the blood to provide for such brief 
pauses as occur (Ewer and Fox, 1940) and it seems more likely from the work of 
Wells (1951, 1955) that the resumption of irrigation is spontaneous. 

The measurement of filtration rate showed that 70 ml. of water/g./hr. was 
moved across the filaments by the activity of the crown cilia, while the normal irri- 
gation rate through the tube was about 12 ml. g./hr. While it would be unwise 
to draw too close a comparison, these figures suggest that the crown is in fact 
achieving more effectual work in water transport than the body when irrigating, 
so that it is not surprising to find that the total oxygen uptake is reduced to 40% 
when the crown is not expanded. The utilization of oxygen from the water passed 
through the crown may be obtained from the filtration rate (70 ml./g./hr.) and the 
rate of oxygen uptake (0.05 cc. O 2 /g./hr.). Under laboratory conditions (external 
oxygen content of 7.0 cc. Oo/l. ) this can be estimated at about 10%. When the 
worm is wholly withdrawn the oxygen consumption falls, as we have just noted, 
to 40% of the normal value or 0.02 cc. Oo/g./hr., which is withdrawn from only 
12 ml., giving a utilisation of about 24%. The rather low utilisation by the crown 
rather suggests that the flow is maintained more for feeding than for respiration. 

Wells (1951) suggested that in Sabclla pavonlna feeding was at least a possi- 
bility when the crown is withdrawn. While this may be so in S. f>ai'oniiia, which 
has a singularly delicate and "open" crown, it seems far less likely to occur in Schizo- 
brancJiia in which the crown is more complicated, much branched, and closely 
furled when contracted. 

The results discussed here suggest that ScJrizobrancIiia is able to maintain its 
respiratory needs when withdrawn within the tube, and that it emerges to feed in 
response to some spontaneous mechanism such as Wells (1955) has described in 
other polychaetes. This ability to meet the demand for oxygen by irrigation when 
withdrawn within the tube is a factor with obvious survival value. It is interesting 
that sabellicls such as My.ricola (Wells, 1952) and Chonc, which do not irrigate 
their tubes, have exceptionally well developed giant fibre systems and retraction 
responses. This should increase their chances of survival, for not only are these 



RESPIRATION OF SCHIZOBRANCHIA 91 

worms dependent on their crowns for respiratory exchange but the crowns must, 
therefore, be more constantly displayed. 

SUMMARY 

1. Observations on the life of a sabellid Scliizobraiichia insignis have been made 
under conditions resembling as far as possible those found in nature. 

2. The amount of time spent with the cro\vn expanded and the amount passed 
wholly withdrawn within the tube have been measured, and the utilisation of oxygen 
under these two conditions estimated. 

3. The volume of water passed through the crown for respiratory and feeding 
purposes, as well as the volume pumped through the tube, have also been measured, 
and the part played by each in respiratory exchange discussed. It was found that 
about 70 ml./hr./g. animal (fresh weight) is passed through the crown by the action 
of the filamentary cilia, and the volume pumped through the tube is about 12 
ml./hr./g. 

4. Utilisation of oxygen by the crown is relatively low (10%) ; utilisation by 
the whole worm when withdrawn is about 24%, and the large volume strained by 
the crown is probably related to the food requirements rather than to the respiratory 
needs of the worm. 

5. It is suggested that the oxygen taken up by the crown is largely utilised in its 
own activity although it can, and does, provide for the needs of the rest of the body 
during pauses in irrigation when expanded. 

LITERATURE CITED 

DALES, R. P., 1957. Some quantitative aspects of feeding in sabellid and serpulid fan worms. 

/. Alar. Biol. Assoc., 36 : 309-316. 
EWER, R. F., AND H. M. Fox, 1940. On the function of chlorocruorin. Proc. Roy. Soc. London, 

Scr. B, 129: 137-153. 
Fox, H. M., 1932. The oxygen affinity of chlorocruorin. Proc. Roy. Soc. London, Scr. B, 

111: 356-363. 
Fox, H. M., 1938. On the blood circulation and metabolism of sabellids. Proc. Rov. Soc. 

London, Scr. B, 125: 554-569. 
Fox, H. M., AND A. E. R. TAYLOR, 1955. The tolerance of oxygen by aquatic invertebrates. 

Proc. Roy. Soc. London, Scr. B, 143 : 214-225. 
Fox, H. M., AND C. A. WINGFIELD, 1938. A portable apparatus for the determination of 

oxygen dissolved in a small volume of water. /. E.vp. Biol., 15 : 437445. 
HYMAN, L. H., 1932. Relation of oxygen tension to oxygen consumption in Nereis virens. 

J. E.\-p. Zoo/., 61 : 209-221. 
J0RGENSEN, C. B., 1949. The rate of feeding by Mvtilns in different kinds of suspension. /. 

Mar. Biol. Assoc., 28: 333-344. 

MclNTOSH, W. C., 1922. A Monograph of the British Marine Annelids, 4, London (Ray Soc.). 
WELLS, G. P., 1950. Spontaneous activity cycles in polychaete worms. /. E.rp. Biol. Sym- 
posium IT. Physiological mechanisms in animal behaviour : 127-142. 
WELLS, G. P., 1951. On the behaviour of Sabclla. Proc. Roy. Soc. London, Ser. B, 138: 

278-299. 
WELLS, G. P., 1952. The respiratory significance of the crown in the polychaete worms 

Sabclla and My.ricola. Proc. Roy. Soc. London, Scr. B, 140: 70-82. 
WELLS, G. P., AND R. P. DALES, 1951. Spontaneous activity patterns in animal behaviour: the 

irrigation of the burrow in the polychaetes Chactoptcrus variopedatus Renier and 

Nereis d'rccrsicolor O. F. Muller. /. Mar. Biol. Assoc., 29 : 661-680. 
ZOOND, A., 1931. Studies in the localization of respiratory exchange in invertebrates. II. The 

branchial filaments of the sabellid, Bispira z'oluticornis. J. E.rp. Biol., 8: 258-266. 



PRELIMINARY INVESTIGATION ON THE PHYSIOLOGY AND 

ECOLOGY OF LUMINESCENCE IN THE COPEPOD, 

METRIDIA LUCENS 1 

CHARLES N. DAVID AND ROBERT J. CONOVER 

Harvard College, Cambridge, Mass., and Woods Hole Oceanographic Institution, 

Woods Hole, Mass. 

Since the advent of the photomultiplier tube with greatly increased sensitivity 
to low light intensity, it has been possible to measure marine luminescence quanti- 
tatively. Luminescent flashes have been found to be far more prevalent at all depths 
in the sea than had generally been suspected (Clarke and Backus, 1956; Clarke 
and Breslau, 1959, 1960; Clarke and Hubbard, 1959; Clarke and Wertheim, 1956; 
Boden and Kampa, 1957, 1958; Kampa and Boden, 1957). Attempts to identify 
the source of this flashing, using tl^e luminescence camera built by Breslau and 
Edgerton (1958), suggest that most of the luminescence is produced by planktonic 
organisms less than a centimeter long (Clarke and Breslau, 1959; Clarke, personal 
communication). Certain planktonic species whose luminescence has been in- 
vestigated do not show spontaneous luminescence in the laboratory. Probably some 
of the luminescence which has been'-measured at sea may be artificially stimulated 
by the unavoidable motion of the photometer suspended from a research vessel. 
However, Kampa and Boden (1957) have concluded that some luminescence ap- 
pears to be "natural" or "spontaneous." 

Bioluminescence in a small planktonic animal has been examined particularly 
with a view toward evaluating its potential as a source of luminescence in the 
natural environment and determining the significance of the luminescence for the 
organism. The calanoid copepod, Metridia lucens, was the animal chosen. 

This copepod was recognized a luminescent by Boeck (1865) who described 
the species. Several additional workers have made microscopic or field observa- 
tions on the luminescent Copepoda (Dahl, 1893, 1894; Kiernik, 1908; Vanhoffen, 
1895; Giesbrecht, 1895), but very little experimental work has been done. 

In the present work preliminary investigation of certain physical properties of 
the luminescent emission and of the physiology of the luminescent mechanism has 
been attempted, in addition to the experiments designed to ascertain what ecological 
significance luminescence may have for this copepod. 

The authors are indebted to Dr. George L. Clarke for his advice and criticism 
in planning the work and in the preparation of the manuscript. The authors also 
wish to express their thanks to Dr. W. D. McElroy, Dr. James F. Case, Dr. Edward 
R. Baylor and members of the staff of the Woods Hole Oceanographic Institution 
for their cooperation and assistance. 

1 Contribution No. 1183 from the Woods Hole Oceanographic Institution. Research sup- 
ported by National Science Foundation Grants 3838 and 8913. 

92 



LUMINESCENCE OF A MARINE COPEPOD 



93 



MATERIALS AND METHODS 

The copepods used in the experiment were obtained in Cape Cod Bay about 3-5 
miles northeast of the mouth of the Cape Cod Canal in 20-30 m. of water. Col- 
lections were made on two occasions, July 7 and August 16, 1960. with a %-meter 
#00 plankton net towed near the bottom. The Metridia were isolated from the 
catch and maintained in the laboratory approximately 40 animals to 1000 ml. of food 
culture. Laboratory cultures of the diatom Tlialassiosira fluriatilis were used as 
food diluted 1 : 20 by volume with millipore-filtered sea water from Cape Cod Bay. 
This gave a concentration of 6000 to 10,000 cells/ml, in the final food culture. 

CARBON 
ELECTRODE 



OBSERVATION 
CHAMBER 




SALT 
AGAR PLUG 



FIGURE 1. Electrode chamber used for stimulation of Metridia. For details see text. 

Either streptomycin or penicillin (50 mg./l.) was added to inhibit bacterial growth. 
For specific experiments, smaller groups of Metridia were kept in proportionately 
smaller volumes of food medium. All groups of animals were kept in a darkened 
refrigerator at 5-7 C. 

Measurements of luminescence were made in a "black box" consisting of a tar- 
paper covered wooden frame built on top of a table. A large opening on one side 
of the box covered with a black cloth sleeve and drawstring permitted the investi- 
gator's head to remain inside the box for observations or to monitor recording and 
stimulating instruments outside the box. 

The measurements of luminescence were made with the portable bathyphotom- 
eter designed and built by Breslau (1959), which employs a RCA 5819 photo- 
multiplier tube with 1200 v. battery power and a transistor amplifier circuit. Ex- 
perimental material was placed directly in front of the photomultiplier about 18 
cm. from the sensitive surface. A Texas Instruments, Inc. single-channel, strip- 
chart recorder ("Recti/Riter") was used to record intensity (in yu.w./cm. 2 ) against 
time during each flash. 



94 CHARLES N. DAVID AND ROBERT J. CONOVER 

Since the Mctridia do not generally luminesce spontaneously in the laboratory, 
mechanical or electrical stimulation must be applied to study the characteristics of 
the flashing. In order to standardize the stimulus delivered to the animals, a 
simple electrode chamber was constructed as shown in Figure 1. The device was 
cut out of a piece of Incite and the connecting holes between the two side chambers 
and the central chamber were filled with 3 c /c agar made with millipore-filtered sea 
water. For experiments, carbon electrodes wired to a pulse regulator were placed 
in the side chambers and the whole device was filled with cooled sea water to com- 
plete the circuit. Metridia, either individually or in groups, were then placed in the 
central chamber for stimulation. 

All stimulation was performed with alternating current controlled through an 
electronic switch and a continuously adjustable autotransformer, Variac Type 
W10MT. The switch regulated the duration of pulses to one-tenth of a second 
and the interval between pulses to two-tenths of a second. Although slightly sensi- 
tive to changes in salinity and temperature, the current was regulated accurately 
to one-tenth of an ampere. The chart speed of the recorder was varied for different 
experiments. The slower speed (6 in. hr. ) was used to record the frequency and 
intensity of flashes. The faster speeds (6 or 12 in./min.) were used when a measure 
of total luminescent flux (area under intensity curve) or the duration of a flash was 
required. 

DESCRIPTION AND DISTRIBUTION OF METRIDIA LUCENS 

Mctridia litccns is a medium-sized copepod, virtually colorless in the living 
state. Its size varies between 2.4-3.0 mm. for females and 1.8-2.5 mm. for males. 

Although Metridia luccns is a common copepod of temperate and boreal water, 
very little is known regarding its seasonal abundance or life-history. In the Gulf 
of Maine, Bigelow (1924) noted an increased abundance in the spring and again 
in September and October. Bigelow (1924) and Clarke (1933, 1934) observed 
extensive diurnal vertical migrations in this species. In the waters off the coast of 
Ireland it also seemed to have a period of maximum abundance in May and a smaller 
period of increase in the fall (Farran, 1920). During the spring it has been 
reported to be responsible for brilliant phosphorescence on the Irish coast (Farran, 
1903, in Bigelow, 1924). 

Little is known regarding the internal anatomy of the copepods. Among the 
calanoicls only Calaints finiiiarchicns has been studied in detail (by Lowe, 1935). 
It is presumed that in the general features of its morphology Mctridia does not 
differ greatly from Calami s although there doubtless are certain differences in 
structural detail. 

The only light-sensitive organ in most copepods, including Metridia, is a 
single naupliar eye. It seems very doutbful that this organ can have any role in 
behavior requiring recognition of other organisms because it cannot form images. 
However, it can presumably detect intensity gradients (as in vertical migration) 
and possibly the plane of polarization of incident light. 

Luminescent glands 

The earliest workers recognized that the luminescence produced by Mctridia 
was primarily external. Boeck (1865), who described Mctridia luccns, noted that 



LUMINESCENCE OF A MARINE COPEPOD 



95 



the light seemed to be produced in the head region and also from the abdomen. 
Vanhoffen (1895), working with the larger Jl/. longa, observed luminescence dis- 
tributed over most of the thorax as well as the head. In addition to the external 
secretion, he also felt that some light was produced internally which indicated the 
position of the secretory glands. 

The authors observed that Mctridia liicens seemed to produce luminescence, 
when stimulated electrically, chiefly from the anterior part of the head and from 
the region of the caudal rami. The separation of these two regions was sufficiently 
distinct that the light produced persisted sometimes as two discrete points for 
some seconds. 




\ 



FIGURE 2. Mctridia litccns: left, a dorsal view; right, a lateral view. Arrows indicate the 
general regions of the body where luminescent glands were found. 

Due to the kindness of Dr. Robert Hessler, some histological preparations of 
Mctridia luccns, fixed in Zenker's and stained with hematoxylin and eosin were 
available for study of the glands and their distribution. Figure 2 shows the regions 
of the body seen microscopically to produce luminescence in observations on living 
animals. Glands were located in the histological preparations in most of these 
places with definite concentrations on the anterior surface of the head and on the 
posterior portion of the abdomen. 

The glands varied in shape somewhat depending on their location in the body. 
Those in the urosome had a long connecting duct between the glands and the 
external pore while those in the thorax opened directly to the outside through a 



96 



CHARLES N. DAVID AND ROBERT J. CONOVER 



short duct. In several cases masses of dark material which might be the lumi- 
nescent substance were observed in these ducts. 

Sewell (1932, 1947) describes the presence of external pores on the cuticle, 
presumably associated with glandular structures, in several groups of copepods 
including Metridia. It is not certain, however, that these are the openings to 



CM 
2 

o 



ICT 2 

icr 3 



10 



-4 



10 



-5 



10 



10 



-6 



-7 



10 



10 



-3 



-4 



10 



-5 



10 



-6 



10 



-7 



A 




B 





SEC 10 

t 



20 



30 

t 



40 



50 



60 



10 



20 



30 



FIGURE 3. Luminescence of single Metridia when stimulated in the electrode chamber. 
Arrows along the time scale indicate instant of stimulation (0.7 amp.). Chart speed is 6 in./min. 
A shows curve for an animal tested six hours after capture ; B shows curve for an animal tested 
after being kept in the laboratory for one month. 

luminescent glands, particularly since such structures are found in several genera 
not presently known to be luminescent, notably Eucalanns and Tcmora. The dis- 
tribution of these pores has not been worked out in detail for Metridia lucens. 

Physical characteristics of the luminescence 

The luminescent emission of Metridia lucens is generally a bright flash of vary- 
ing duration. According to Harvey (1952), luminescence in copepods results 
from the simultaneous discharge of substrate and enzyme into the surrounding 



LUMINESCENCE OF A MARINE COPEPOD 



Q7 



medium ; presumably the immediate peak emission occurs at the instant of initial 
contact between the reacting substances in the presence of oxygen. Generally a 
gradual decay follows as enzyme and substrate diffuse away into the medium, or 
perhaps as the substrate is used up. 

The absolute intensity of the highest peak of the luminescent emission is in 
doubt because of the relatively slow response time of the equipment used. Further- 
more, the maximum emission intensity varied to some extent for individual animals. 
However, the maximum intensity measured for Metridia was 1.2 X 10~ 3 /AW./cm. 2 







550 



600 



425 482" 580 

FIGURE 4. Spectrum of luminescent emission at C. from crushed animals. 

at the working distance of 18 cm. (Fig. 3). With groups of 5 Metridia, there was 
an additive effect giving maximum intensities of up to 4.5 X 10" 3 . The intensity 
of the response decreased after successive stimuli. 

The duration of individual responses varied even more widely than the maxi- 
mum intensities, and ranged from 3 seconds to 50 seconds for all luminescent 
responses with intensities between 10~ 3 and 10' 4 /AW ./cm 2 . There was no apparent 
relationship between the intensity of a luminescent emission and its duration. For 
example, two Metridia, exactly similar in laboratory history, both gave responses 
of 3 >: 10~ 4 /AW./cm. 2 , one emission having a duration of 10 seconds, the other a 
duration of 50 seconds. If responses of a lower maximum intensity than 
10~ 4 /AW./cm. 2 are considered, durations as short as 1 second have been measured, 
particularly at the end of fatigue experiments when the Metridia had already re- 
sponded to 10 or 15 electrical stimuli. 



98 CHARLES N. DAVID AND ROBERT J. CONOVER 

Through the kindness of Dr. W. D. McElroy at the Marine Biological Labora- 
tory, Woods Hole, Massachusetts, it was possible to measure the spectrum of 
Metridias luminescent emission (Fig. 4). The apparatus used was an Aminco 
spectrophotofluorometer in circuit with a drum recorder ("x-y" recorder) and an 
oscilloscope. Because of the rapid decay in intensity of Metridia's luminescence, 
it was necessary to cool a number of the animals in crushed ice in order to slow 
down the enzyme reaction producing the luminescence. Then, by immediately 
crushing the animals in a small test tube directly in the spectrophotofluorometer, 
the luminescence remained at one intensity long enough to record the entire 
spectrum. 

The peak of the spectrum for Aletridia is around 482 m/x and is therefore similar 
to that of Cypridina and certain other luminescent Crustacea (Nicol, 1960). The 
curve is slightly skewed toward longer wave-lengths with about half the spectral 
energy falling in the range between 440 nip. and 525 nip.. The entire spectrum 
lies between 425 nip. and 580 in/*. This spectrum with its peak at 482 nip. coincides 
closely with the wave-lengths having maximum transmission through clear, oceanic 
sea water (Clarke, Chap. 6, 1954). 

Experiments on physiology 

In order to determine whether laboratory culture had any effect on the lumi- 
nescence of Mctridia, freshly captured specimens and some which had been main- 
tained in the laboratory for a month were repeatedly stimulated until failure to 
respond to two successive stimuli indicated the onset of fatigue. A representative 
experiment shown in Figure 3 indicates that the maximum intensity and the rate 
of fatigue were not markedly different for the two specimens. The difference in 
flash duration is not significant considering the wide range of variation shown by 
this characteristic. 

To study the effect of strength of the stimulating pulse on the luminescence, 
the current was increased from .3 amp. to .7 amp. which caused a significant increase 
in the intensity of the luminescence and in the number of responses to stimuli. 
However, pulses stronger than .7 amp. did not cause further increase in lumines- 
cence intensity but seemed to reduce the number of successive responses. Variations 
in the duration of the pulse over the range tested (.10-1.0 second) had little effect 
on the intensity or number of successive responses. However, short intervals, 
i.e., 3 seconds, between pulses induced two or three times as many successive re- 
sponses as were observed using longer intervals between pulses, i.e., 1045 seconds. 

The effect of previous light- or dark-adaptation was tested with separate groups 
of animals kept at about 5 C. in the dark, in the light, and in a room exposed 
to diurnal light changes. The experiment was begun at 1700 on August 3 and 
the luminescence produced by each group was tested on August 5 and again on 
August 8 between 1000-1300. No statistically detectable difference was found 
between the three sets of animals. In another experiment twenty animals kept in 
a water bath at 5 C., where they were exposed to daily light variation, were tested 
at night (2330-0030) and during the day (1300-1400). In the case of a few 
animals the day-time response was somewhat lower than at night but there is no 
evidence in any of the data for a marked inhibition of luminescence by light or for 
a daily rhythm. 



LUMINESCENCE OF A MARINE COPEPOD 

Having established the fact that the experimental techniques used had no 
appreciable effect on the luminescent response of Metridia, two more physiological 
experiments \\ere performed. The first was designed to investigate an observation 
by the authors that animals which fed poorly still luminesced as vigorously as 




10 MILLISECONDS 




10 MILLISECONDS 




20 MILLISECONDS 




FIGURE 5. Lag time between stimulus and luminescent response. In all four cases the 
stimulus was 150 v./5 msc. represented by the break in the smooth horizontal trace. Downward 
deflection of the jagged upper trace (lower trace in D) represents luminescence measured by 
the photomultiplier. The lag times were : A, 8 msc. ; B, 7 msc. ; C, 8 msc. D shows the same 
type of measurement but includes more of the intensity curve and the stepwise rise to maximum 
intensity. 

animals that fed well. Two groups of animals were set up, one fed on the regular 
culture medium and the other starved in millipore-filtered sea water. After one 
week, single stimulus tests were performed. On the basis of the total area under 
the intensity vs. time curve, the results showed no difference between the two 



100 CHARLES N. DAVID AND ROBERT J. CONOVER 

groups. However, when maximum intensity was considered, the results indicated 
statistically (Wilcoxen Ranked Sum) better luminescence for the starved group. 

After the second week, repeated stimulus experiments were conducted on the 
two groups. Single stimulus data showed no statistical difference in the intensity 
of the response between the fed and starved groups, nor did the number of suc- 
cessive responses to repeated stimuli show a significant difference. At the end 
of the third week, however, experiments did demonstrate that the fed Metridia 
had a stronger luminescent response and the same group was able to respond to 
the electric stimulus a greater number of times than the starved animals. 

In another series of experiments the length of time from the beginning of a 
stimulus to the beginning of a response (the lag time) was measured. For the 
necessary guidance and equipment to make these measurements, the authors are 
indebted to Dr. James F. Case at the Marine Biological Laboratory, Woods Hole, 
Mass. Single animals \vere tested in a small cell consisting of a 3-cm. piece of 
glass tubing with agar plugs and silver electrodes at either end. The electrodes 
were connected to a Grass S4 stimulator and the luminescence was measured with 
a RCA 931 A photomultiplier in circuit with an oscilloscope. An automatic camera 
photographed the oscilloscope screen to record the results. 

Using a stimulating pulse of 150 v. for 5 or 10 msc., the Metridia demonstrated 
a lag time (at room temperature) of 8-10 msc. to the beginning of the luminescent 
response and a lag time of 15-24 msc. to the maximum intensity of the response. 
The time to maximum intensity varied widely, depending on whether or not the 
rise to maximum intensity was direct or in a step-wise fashion, the latter giving 
lag times as long as 60 msc. (see Fig. 5). By observing the Metridia through a 
microscope during these experiments it was noticed that for a stimulus of 10 
msc./150 v. usually both head and tail luminesced while for a stimulus of 5 
msc./150 v. only the organs in the head region responded. 

Experiments on behavior 

Because it has often been suggested that luminescence functions as an escape 
mechanism for marine animals that luminesce by means of an extracellular dis- 
charge, the authors decided to investigate the behavior of Metridia in the presence 
of a predator. A series of experiments was conducted in the dark, in which possible 
planktonic predators on Metridia were placed individually with 10 Metridia in 
600-ml. beakers. The species tested were: Paraenchaeta norvegica (Copepoda), 
ParatJietnisto (Eiitlicmisto} gaudichandii (Amphipoda), and euphausiids, Eu- 
phausia krohnii, Thysanoessa incrmis, Nematoscelis mcgalops, and Meganyctiphanes 
norvegica. 

Each experiment \vas continued for at least two days and counts of the number 
of Metridia present were made at intervals of 12 to 16 hours. Only in the case of 
the two euphausiids, Thysanoessa and Meganyctiphanes, was there any predation 
on the Metridia. Although not every individual tested fed on Metridia with the 
same rapacity, MeganyctipJiancs was by far the most successful predator. The best 
predators among the animals tested were then chosen for further examination. 

The predator was placed in a 600-ml. beaker with 10 Metridia and this beaker 
was placed in the black box in front of the photometer. A cool water bath was 
used to keep the temperature in the experimental vessel between 10-12 C. The 



LUMINESCENCE OF A MARINE COPEPOD 



101 



photometer response was recorded at slow speed (6 in./hr.) so that each flash gave 
a spike indicating maximum intensity and time of occurrence. Analyzing the 
results of these experiments was complicated by the fact that both prey (Me- 
tridia) and predator (Meganyctiphanes') were luminescent. However, compari- 
son of the characteristics of Mctridia and Meganyctiphanes luminescence records 
when the animals were stimulated electrically showed that high intensity responses 



ICT 

icr 3 

I0' 4 
ICr 5 



10 



-6 



-7 



10 




* io- 2 




10 



-3 



10 



-4 



10 



-5 



10 



10 



-6 



-7 



(CONTINUED) 



SEC 10 



20 



30 



40 



f. 



50 



60 



10 



20 



30 



FIGURE 6. Luminescence of a single Meganyctiphanes norvegica when stimulated in the 
electrode chamber. Arrows along time scale indicate instant of stimulation. Chart speed is 
6 in./min. The stimulus (0.7 amp.) was as strong as any ever used for Mctridia. Low inten- 
sity of luminescence is notable in comparison to Mctridia's bright flash. 

were almost surely due to Mctridia. Even using a maximum stimulus (1.1 amp.), 
Mcganyctiplianes never produced a response higher than 1 : : 10~ 4 /xw./'cm. 2 and 
it was generally much lower. Furthermore, the luminescent emission of Mega- 
nycti phones was usually a prolonged irregular glow (Fig. 6). The Mctridia, by 
contrast, always gave a single flash that appeared as a perpendicular spike on 
the slow-speed record (see Fig. 7). Using these two criteria, a reasonable inter- 
pretation of the records could be made. 



102 



CHARLES N. DAVID AND ROBERT J. CONOVER 



io-* 

I0' s 



* 



ID' 6 
ID' 7 



1600 



HOURS 



1700 



1800 



2 
o 

*v 

S 
a. 



IO" 



10- 



10- 



ICT 8 
ID' 5 



5 
u 

510- 



ID' 





(CONTINUED) 




























































ll 












1 1 



1900 



2000A A 



(CONTINUED) 










2100 



A 2200 



2300 



io- s 
IQ-" 

io- s 

ID' 7 






(CONTINUED) 
















1 






III 




* i ML , . i 




V 0200 A 0300 \l 0600 A 



FIGURE 7. Record of behavior experiment 3 (see Table I). At the chart speed of 6 in./hr., 
luminescent flashes appear as spikes indicating maximum intensity and time of occurrence. Solid 
triangles indicate successful predation under interpretation outlined in text. The decreasing 
background intensity between 1600 and 1900 hours is due to the setting sun which reduced the 
ambient light in the laboratory. The increased background at 2045-2100 and 2150-2300 hours 
was caused by lights in the laboratory used to monitor the recorder. 



LUMINESCENCE OF A MARINE COPEPOD 



103 



A sample record from an experiment with both prey and predator present is 
show in Figure 7. All the experiments are summarized in Table I. Experiments 
6 and 7 in the table show quite clearly that the two species when separated from 
each other ordinarily do not produce any spontaneous luminescence. Only a single 
weak flash (4 X 10~ 7 ju,w./cm. 2 ), which may have been caused by some accidental 
mechanical stimulus, was observed for the group of Metridia alone. The Mega- 
nyctiphanes alone produced no luminescence at all. This corroborates Mauchline's 
(1959) observation that Meganyctiphanes does not luminesce spontaneously in 
the laboratory except during the breeding season (Dec.-Feb.). On the other 



TABLE I 
Summary of behavior experiments. 

The table shows the interrelationship between luminescence and predation in Metridia lucens. 

For detailed explanation see text and Figure 8. 









Predator 


No. of Metridia 


Number of luminescent 












responses 


Expt. 


Date 


Total 
time 


















No. 




(hrs.) 


No. 


Species 


In expt. 


Eaten 


Above 10~ 7 

jiw./cm. 2 


Above 10-< 
/iw./cm. 2 


1 


8/12-13 


15 


1 


Meganyctiphanes norvegica 


9 


8 


30 


15 


2 


8/17-18 


8 


1 


Meganyctiphanes norvegica 


10 


3 


17 


3 


3 


9/14-15 


16 


1 


Meganyctiphanes norvegica 


10 


10 


33 


14 


4 


9/16-17 


13.5 


1 


Meganyctiphanes norvegica 


11 


5 


41 


21 


5 


9/18 


10.5 


1 


Meganyctiphanes norvegica 


10 


4 


23 


1 


6 


8/13-14 


15.75 








10 





1 





7 


8/14-15 


9.5 


1 


Meganyctiphanes norvegica 














8 


8/11-12 


7.25 


2 


Parathemisto gaudichaudii 


10 





1 





9 


8/19-20 


15.5 


1 


Parathemisto gaudichaudii 


9 











10 


8/18-19 


15.5 


2 


Thysanoessa inermis 


10 


1 


3 





11 


8/24-25 


15 


1 


Nematoscelis megalops 


10 





1 






hand, when the two species were placed in the same container, considerable lumi- 
nescence was observed and some Metridia were eaten (experiments 1-5). Since 
most of the flashes showed up on the record as single spikes, some with an intensity 
greater than 10~ 4 /xw./cni. 2 (see Fig. 7), it was concluded that the copepod was 
primarily responsible for the display. 

On the original records (copied in Fig. 7), it was possible to distinguish two 
kinds of single spikes, ones representing only a single luminescent flash and ones 
where several tracings were actually superimposed. This latter kind represented 
several flashes of different intensities which occurred within an interval short 
enough (30-40 seconds) to prevent their resolution at the slow chart speed. 
Sometimes this multiple-flash sequence was spread out over a longer period of 
time and the smaller flashes were resolved on the record (e.g. 0230 hours in Fig. 7). 
The number of multiple-flash sequences was, in almost every case, exactly equal to 
the number of Metridia eaten. These sequences presumably represent a Metridia s 
capture (large flash) and subsequent struggle to escape (small flashes). The 



104 CHARLES N. DAVID AND ROBERT J. CONOVER 

remaining spikes on the record (caused by single flashes) are presumed to represent 
successful escapes by Metridia. 

In order to determine if the mere mechanical disturbance of another organism 
in the container could cause luminescence, groups of Metridia were tested with 
several other species in the container placed in front of the photometer. A large 
ParatJieinisto (Eutheinisto) gaudichandii, a vigorously swimming hyperiid amphi- 
pod, did not induce any luminescence w r hen placed with Metridia nor did it eat 
any (Table I, experiments 8 and 9). Similar results were obtained with the 
euphausiid Nematoscelis megalops (experiment 11). When TJiysanoessa inennis 
was used (experiment 10) a few flashes were produced and a single Metridia 
was eaten during the experiment. A further test of the effect of mechanical stimula- 
tion was made by vigorously stirring the water in a beaker containing Metridia. 
Considerable disturbance was necessary before any flashing occurred and even 
the most energetic agitation elicited a maximum response of only 6 X 10~ 5 /xw./cm. 2 , 
less than one tenth of the highest responses shown in Table I and Figure 7. 

Direct observation of predation was also attempted in order to determine the 
nature of the luminescence stimulus. An infra-red-sensitive "sniper-scope" (Ed- 
mund Scientific Co.) was used with the infra-red source and a focusing lens placed 
behind the experimental beaker so that the animals appeared in opaque profile 
against a light background. The small Metridia were not always visible with this 
optical arrangement but some individuals were seen to be carried toward the 
euphausiid by the currents set up by the larger animal's pleopods. Sometimes the 
Metridia would dart away before reaching the Meganyctiphanes, but at other times 
the copepod would seem to come in contact with the euphausiid before darting 
away. On a few occasions the euphausiid started off as though in pursuit, but the 
actual act of capture was never observed. 

These observations are in general agreement with those of Mauchline (1959) 
who found Meganyctipliancs capable of filter-feeding on organic detritus and even 
sucking into the "food basket" individual copepods (Paraeuchaeta norvegica) 
and Sagitta by lateral-ventral movements of the thoracic limbs. The animal can 
also seize larger objects by raptorial movements of the appendages but in the 
laboratory "no hunting or stalking of prey takes place" (Mauchline, 1959). 

DISCUSSION 

Over the years there has been considerable speculation regarding the role of 
bioluminescence in the life of various marine organisms. In higher marine forms, 
luminescence has been found associated with either mating behavior, feeding mecha- 
nisms, or defense. Among planktonic species, however, there is less agreement as 
to its functional significance. Besides the three interpretations given above, it 
has been suggested that this phenomenon may often be coupled with other life 
processes in lower animals and therefore might have no function of its own (Russell 
and Yonge, 1928; Harvey, 1929). It has also been suggested that luminescence 
in planktonic and sessile creatures may serve as a "burglar alarm," thereby revealing 
a predator to its own enemies along the food chain (Burkenroad, 1943). 

From the results of the behavior experiments with Metridia, it is apparent that 
there is some relationship between luminescence and the act of predation. Since 
the exact nature of the stimulus is still unknown, it is impossible to determine 



LUMINESCENCE OF A MARINE COPEPOD 105 

positively which, if any, of the above hypotheses is applicable. Nevertheless, some 
of the possibilities may be eliminated. 

Any functional use of luminescence involving species recognition, such as mating 
display or warning systems to other individuals of danger, is doubtful because 
Metridia probably does not have an adequate image-forming eye. Of the remaining 
speculations presented above, the authors currently feel that the defense mechanism 
is the one most consistent with the experimental results. However, Burkenroad's 
hypothesis is not specifically ruled out. 

The reasons for favoring the idea of an escape mechanism arise from : ( 1 ) 
certain of the physical and physiological characteristics of Metridia's luminescent 
emission, and (2) a unique pattern of behavior associated with luminescence in 
this copepod. 

The maximum intensity of Metridia's luminescence is surprisingly brilliant. 
At the working distance (18 cm.) used in this study the flash was of the same 
order of magnitude as that of certain coelenterates and of the crustacean Euphausia 
pacified, and greater than that of the teleost Myctoplutm punctatum, all measured 
at 1 cm. (Nicol, 1960). The duration of the flash is long and its spectral composition 
is similar to the spectrum of the transmission of light through sea w r ater with 
the maximum of the two curves at nearly the same \vave-length. It has also been 
shown that Metridia has an extremely short lag time between stimulus and response. 
The animal recovers quickly after stimulation and fatigues rather slowly on re- 
peated stimulation, even after several weeks without food, suggesting that the 
ability to luminesce is important enough to the organism to be maintained under 
adverse conditions. All these characteristics of Metridia's luminescence, both 
physical and physiological, would certainly be selectively advantageous to the 
animal if its luminescence functioned as an escape mechanism. 

The most significant evidence for the defense mechanism hypothesis, however, 
comes from observations of the behavior of single Metridia stimulated in the 
electrode chamber. On stimulation a point of luminescence was immediately pro- 
duced and then in the majority of cases the animal appeared to dart off into the 
dark, leaving a bright luminescent spot at its original position and sometimes a 
trail of tiny luminescent specks that soon disappeared. Although the animal itself 
could not be seen during this reaction, the agitation of the water gave a clue to 
its behavior and its new position could be verified by passing a second electrical 
stimulus through the water and observing the new location of the resulting lumi- 
nescent flash. The original luminescent emission remained a more or less discrete 
point of light for some seconds after stimulation. 

Such a behavior pattern appears to the authors to indicate the manner in which 
Metridia escapes from Meganyctiplwnes. Although the precise role that lumines- 
cence plays in this escape mechanism is still unknown, two speculations are possible. 
The luminescent emission may startle the attacker, interrupting its feeding pro- 
cedure, or it may merely function as an attractive decoy. In either case, the 
Metridia's rapid departure from the spot where it had luminesced would complete 
the escape. 

The possibility that luminescence only occurs when the Metridia is actually 
captured is not entirely eliminated. More definitive proof must await the elucidation 
of the specific stimulus that induces luminescence. Nevertheless the evidence pre- 



106 CHARLES N. DAVID AND ROBERT J. CONOVER 

sented here indicates that luminescence functions on the behavioral level as an 
escape mechanism for Metridia. It would then seem probable that luminescence, 
which is of such widespread occurrence in the oceans, may well have survival 
value in defense against predation in some similar manner for many other animals 
of the plankton. 

SUMMARY 

1. Skin glands believed to be the source of luminescence were found on the 
anterior portion of the head, on the last thoracic segment, and on the posterior 
margins of each segment of the abdomen. 

2. The maximum intensity of the luminescent flash was 1.2 X 10~ 3 /AW. /cm. 2 
(at 18 cm.). The flash rose rapidly to peak intensity and then decayed slowly. 
The total duration of the flashes with peaks greater than 10~ 4 //.w./cm. 2 ranged 
from 3 to 50 seconds. 

3. The peak of the luminescence spectrum occcurred at 482 mp. and the curve 
fell off to one-half the maximum value at 440 m/x, and 525 m/x. 

4. The ability of Metridia to luminesce on stimulation was found to be 
largely unaffected by prolonged laboratory culture. Starvation had little effect 
on the luminescence for the first three weeks and there was never any inhibition 
by previous light- or dark-adaptation. 

5. With an increase in the strength of the electric stimulus from 0.3 amp. to 
0.7 amp., the intensity of the luminescent flash was found to increase. With pulses 
stronger than 0.7 amp. no change in intensity was recorded but the number of 
successive responses to repeated stimuli was reduced. Duration of the pulse had 
little effect on the intensity or the number of successive responses. 

6. Metridia showed a lag time of 8-10 msc. to the beginning of the luminescent 
response. The lag time to the peak of the luminescent response varied from 20 
to 60 msc. 

7. There was no spontaneous luminescence produced by groups of Metridia 
under conditions of constant darkness. However, the presence of certain plank- 
tonic predators, most notably Meganycti phones norvegica, caused a brilliant display 
of luminescence. The number of flashes attributable to Metridia was always 
greater than the number of Metridia eaten by the predator. There was little evi- 
dence that the luminescent euphausiid, MeganyctipJianes, flashed spontaneously 
either in the presence or absence of its prey. 

8. Observations on the behavior of Metridia during and just after luminescence 
suggest that the flashing may be involved in an escape mechanism, but the precise 
effect of the light on the predator has not been determined. 

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LUMINESCENCE OF A MARINE COPEPOD 107 

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MAUCHLINE, J., 1959. The biology of the euphausiid crustacean, Meganyctiphanes norvegica 

(M. Sars). Proc. Roy. Soc. Edinb., 67: 141-179. 

NICOL, J. A. C., 1960. The Biology of Marine Animals. Sir Isaac Pitman, London. 
RUSSELL, F. S., AND C. M. YONGE, 1928. The Seas. Frederick Warne, London. 
SEWELL, R. B. S., 1932. The Copepoda of Indian seas. Calanoida. Mem. Indian Mns., 

10: 407 pp. 
SEWELL, R. B. S., 1947. The free-swimming planktonic Copepoda. Systematic account. Set. 

Repts., John Murray Expd., 8 : 303 pp. 
VANHOFFEN, E., 1895. Das Leuchten von Metridia longa Lubb. Zoo/. Atiz., 18 : 304-305. 



THE PHYSIOLOGICAL CONTROL OF WATER INGESTION 

IN THE BLOWFLY 1 

V. G. DETHIER AND D. R. EVANS 

Zoological Laboratories, University of Pennsylvania, Philadelphia 4, Pa., and 
Department of Biology, The Johns Hopkins University, Baltimore 18, J\Id. 

Because of their small size and terrestrial habitat, insects constantly face a 
pressing problem in water conservation. Recognition of this fact has stimulated 
many investigators to study routes and mechanisms of water loss and adaptations 
for its prevention (rf., Edney, 1957). Some of these studies have dealt with 
behavioral adaptations, such as humidity preferences, that decrease water loss. 
With regard to the uptake of water, however, little is known apart from observa- 
tions of direct water uptake through the integument at very high humidities by 
some insects. One isolated study of a fly (sp. ?) by Bolwig (1953) showed a 
negative correlation between response to water by drinking and the vapor pressure 
of the blood. Otherwise there appears to be no experimental work on the control 
of drinking by insects (Leclerq, 1946; Edney, 1957). Accordingly, experiments 
were undertaken to reveal the factors underlying thirst and water ingestion in 
the blowfly, Phortnia regina Meigen. 

METHODS 

The blowfly was chosen for study because a considerable body of knowledge 
relating to its sensory physiology and feeding exists. The flies employed were 
taken from a culture maintained in these laboratories since 1947. Flies were 
desiccated or humidified by storage in a sealed vessel containing calcium chloride 
or water, respectively. Measurement of the water intake of individual flies was 
carried out by surgically removing the crop after drinking was complete and 
weighing it. Preferences and volumes consumed of test solutions over periods of 
24 hours and longer were measured by the method of Dethier and Rhoades (1954). 
Injections of fluid into the haemocoel were carried out as before (Evans and 
Dethier, 1957). Flies were designated water-positive or -negative on the basis 
of a uniform response to three tests being obtained ; a positive response consisted 
of proboscis extension upon tarsal contact with water. Bleeding was accomplished 
by cutting off the prothoracic legs close to the thorax and expressing the blood 
by gentle compression of the thorax. 

RESULTS 
Sensory control of drinking 

A fly that has been deprived of water will respond to it in a variety of ways : 
it will orient from a distance to a locus of high humidity ; it will extend its proboscis 

1 This work was aided by National Science Foundation Grants G-6015 and G-5927 and by 
National Institutes of Health Grant E-2358. 

108 



WATER INGESTION IN THE BLOWFLY 



109 



in response to stimulation by water vapor ; it will extend its proboscis in response 
to water applied to the tarsi or labellum ; it will open the labellar lobes and com- 
mence sucking in response to water applied to the labellum. After a period of 
time which is related to the extent of previous water deprivation, drinking will 
cease and the fly will become refractory to further stimulation by water. 

The sense organs on the mouthparts whose stimulation initiates drinking are 
the same chemoreceptive hairs that respond to sugars (Dethier, 1955). Of the 
three sense cells that innervate the hair, one is a mechanoreceptor, one responds 
preferentially to salts, and the other to sugars. Wolbarsht (1957) reported that 
distilled water applied to certain of the hairs caused a firing of nerve impulses in 
both chemosensory cells. Each discharged at an initially high rate but adapted 
rapidly (one had often ceased firing at the end of thirty seconds). 

TABLE I 

The effect of desiccation on water and sugar consumption by the blowfly. Each value is 
based upon tests with thirty individual flies. The figures in parentheses 

represent ranges. 



Experiment 
number 


Treatment of flies 


Av. \vt. 
(mg.) of fly 
minus wings 


Av. duration (sec.) of sucking of each 
solution presented successively 


HjO 


0.1 M 

sucrose 


1.0 M 
sucrose 


1 


Three-day-old flies fed once on 
0.1 M sucrose, starved 24 hrs., 
then desiccated 24 hrs. 


12.1 
(10.2-16.9) 


24 (6-52) 


46 (23-73) 


35 (17-62) 


2 


Three-day-old flies fed once on 
0.1 M sucrose, starved 24 hrs., 
then desiccated 24 hrs. 


11.0 
(9.7-17.0) 








54 (40-90) 


3 


Three-day-old flies fed once on 


23.4 










0.1 M sucrose, starved 24 hrs., 
then humidified 24 hrs. 


(19.6-27.7) 








38 (20-60) 



As has been reported to be the case for the ingestion of sugar solutions (Dethier 
et al., 1956), not only the initiation but also the maintenance and termination of 
water ingestion are dominated by the input of the taste receptors. Water ingestion 
is driven by the input, and adaptation finally terminates it. This control can be 
demonstrated by stimulating one side of the labellum until the receptors adapt, 
whereupon drinking ceases, and then stimulating the remaining receptors, where- 
upon drinking resumes. 

In addition to this primary control of drinking by chemosensory input, other 
factors are involved in the ingestion of water and nutrients. 

Conditions modifying drinking 

Three conditions were considered as possibly modifying drinking behavior : 
namely, starvation and feeding, unacceptable contaminants, and desiccation. Each 
of these was investigated in turn. 



110 



V. G. DETHIER AND D. R. EVANS 



Starvation and feeding. The average life span of Phonnia in the absence of 
food is three days ; accordingly, experiments on starvation were perforce limited to 
this period. The daily intake of water of fourteen individual flies was measured. 
No consistent change in intake was noted over this period. In another experiment 
designed to control the effects of desiccation, thirty flies were starved 24 hours 
and then placed in a humidifier for 24 hours. A control sample was placed in a 
desiccator. The results are summarized in experiments two and three of Table I. 
The humidified flies did not drink even though they had starved 48 hours. In a 
more drastic experiment, flies were kept in the humidifier until the last had died 
of starvation five days later. All remained negative to water till the end. It can 
be concluded that starvation does not induce drinking as long as water loss is 
prevented. 

TABLE II 

The effect of Nad an water intake by the blowfly. Each value is based on tests with ten 
individual flies. The figures in parentheses represent ranges. 



Materials available to the fly 


Mean volume of 
water consumed 
in 3 days 0*1.) 


Mean volume of 
NaCl consumed 
in 3 days (id.) 


Mean total fluid 
intake in 3 days 

0l.) 


Water (no food) 


29 (12-39) 





29 


Water and dry sucrose 


16 (12-61) 





16 


0.1 M XaCl and dry sucrose 





23 (9-36) 


23 


0.5 M NaCl and dry sucrose 





19 (12-38) 


19 


0.5 M NaCl, dry sucrose, and water 


12 (5-18) 


6 (4-7) 


18 


1 M NaCl and dry sucrose 





12 (3-24) 


12 


1 M NaCl, dry sucrose, and water 


14 (6-18) 


4 (0-5) 


18 


2 M NaCl and dry sucrose 





3 (0-12) 


3 


2 M NaCl, dry sucrose, and water 


15 (3-24) 


4 (0-7) 


19 



In order to test whether or not dry food as the only source of nutrition causes 
an increase in drinking, the daily water intake of individuals of two groups was 
measured over a three-day period. One group of flies had free access to water 
but had no food. The other group had free access to water and to a lump of sugar. 
The results are summarized in the first two lines of Table II. Contrary to 
expectations, the ingestion of food did not bring about increased drinking even 
though part of the process of eating solid food involves dissolving it in saliva. 
Whether the reduction of water intake in the presence of sugar is real is not known. 

As a variant of the preceding, the experiments described in Table III can 
equally well be done on flies that have been made water-positive by storage in a 
sealed vessel in contact with anhydrous glucose. They have the opportunity to 
feed continuously, and yet become positive to water after a time. The mechanism 
is very likely the same as that of storage with calcium chloride, but the latter is 
a better desiccant. 

Contaminants. To test the effect of unacceptable taste stimuli on drinking, 
several series of experiments involving the addition of sodium chloride to water 
were undertaken. In one experiment, flies were kept in individual cages equipped 
with two pipettes, one of which contained water, the other a salt solution. The 
salt solutions paired with water ranged in concentration from 10~ 5 M to 5 M. 



WATER INGESTION IN THE BLOWFLY 



111 



The volume of each solution imbibed was measured each day. Results are sum- 
marized in Figure 1. from which it can be seen that the volume of salt solution 
drunk decreases as the concentration increases. Concurrently, the quantity of water 
drunk increases so that the total fluid intake is approximately constant. 

Under more rigorous conditions where the fly was provided with a salt solu- 
tion as its only source of fluid, higher concentrations (as judged by the volume 
imbibed) were tolerated than when water also was present (Table II). As the 



Q 
O 

or - 08 
LU 

CL 



cr 



CJ 

o: 

LJ 

a. 
Q 

LU 



CO 

o 
o 



LJ 

^ 



.06 



.04 



02 



V 



\r 

o 



/ 
,0 




-5 



-4 



-3 -2 

LOG MOLAR CONG. 



-i 



FIGURE 1. Volume of different concentrations of sodium chloride ingested per fly per 24 hours 
in a two-choice situation. Solid line, sodium chloride. Broken line, water. 



concentration of salt was increased, however, the amount of fluid imbibed decreased. 
In still another series of experiments, 60 flies were tested for their responses 
to salt solutions applied to the tarsi, placed in a desiccator with a supply of dry 
sugar for food, then re tested periodically until death. The results obtained with 
1.0 M NaCl illustrate the trend of events. Before being placed in the desiccator 
none of the flies gave any response to this solution. As water loss increased, they 
first would extend the proboscis when the tarsi were stimulated but would not 
open the lobes of the labelluin ; later they would open the labellar lobes but not 
drink ; still later they would drink for a few seconds ; finally, the drinking time 



112 



V. G. DETHIER AND D. R. EVANS 



would increase. In short, as desiccation increased, the rejection threshold of the 
tarsi to salt rose followed by a rise in the rejection threshold of the mouthparts. 

Desiccation. Implicit in all of the foregoing experiments is the idea that water 
loss powerfully affects drinking. The experiments summarized in Table I demon- 
strate the effect of desiccation for 24 hours on response to water. They show 
further that the state of water balance also affects the amount of liquid food ingested. 
Since desiccated flies take more liquid food than do humidified flies, the response is 
clearly directed toward the acquisition of water. 

TABLE III 

The effect of injections on responses of the blowfly to water 



Experiment 
number 


Number 
of flies 


Response before 
treatment 


Injectedf 


Per cent negativ 

after treatment 


1 


35 


+ 


2.5 /il. water 


6 


2 


52 


+ 


8 jul. water 


85 


3 


27 


+ 


2 jul. water 


7 








2 jul. water 


22 








2 jul. water 


70* 


4 


26 


+ 


3 jul. water immediately 


58 








at 10 minutes 


58 








at 60 minutes 


54 


5 


120 





3 n\. 2 M glucose 


82* 


6 


116 





7 jul. 2 M glucose 


96* 


7 


29 





2.4 jul. 4X saline 


100 


8 


53 


+ 


3 jul. 4X saline 


55 


9 


69 


+ 


3 ,ul- 2 M glucose 


55 


10 


48 


+ 


3 jul. 2 M glucose in saline 


56 


11 


40 


+ 


6 jul. 2 M glucose in saline 


85 


12 


92 


+ 


4 jul. mineral oil, moribund 


66 








at 15 min. 




13 


14 


+ 


Fed 2 M glucose 


100 








0-60 minutes 





* Responded subsequently to 0.1 M sucrose. 

f Water indicates distilled water ; the saline was Bodenstein's 10 ; 4 X indicates saline four times 
more concentrated; exps. 1-9 from Evans (1961). 

The control of water responsiveness 

A series of injection experiments was undertaken to assess the effect of blood 
osmotic concentration on the responsiveness of flies to water (Table III). Injec- 
tions of water rendered positive flies negative to water (exps. 1-4). The percentage 
made negative was a function of the volume injected (exps. 1-4). The effects of 
repeated injections were additive (exp. 3), and the effect was immediate (exp. 4). 
Next it was found that injections of even huge volumes of highly concentrated 
solutions did not produce responsiveness to water (exps. 5-7). But these same 
hypertonic injections could abolish water responsiveness (exps. 8-11), indicating 
that volume and not osmotic or dilution factors was the significant feature. Even 
mineral oil, before its toxic effects were apparent, blocked responsiveness to water 



WATER INGESTION IN THE BLOWFLY 113 

(exp. 12). Ingestion of nearly saturated sugar solutions (exp. 13) abolished the 
water responses. 

These results suggested blood volume or pressure as the agent regulating water 
responsiveness ; but since responsiveness had not been induced in any case, the 
effects could have been unspecific even though responsiveness to sugar was not 
affected in the few cases tested (exps. 3, 5, 6). Consequently, it was crucial to 
reduce blood volume and thereby induce water responses. The crude procedure 
of cutting off the abdomen with crop did not make flies positive ; however, bleeding 
did. If a population of flies was desiccated until some responded to water, bleeding 
made most of the remainder (45 of 53) responsive to water. Vigorous responses 
were obtained as quickly as a fly could be tested after the bleeding (a few seconds). 
Bleeding did not alter the response of already responsive flies (12 of 12). And in 
the case of flies given water to satiation, bleeding induced water responses in only 
a few (4 of 13). Apparently there is a threshold such that blood volume must be 
reduced below some particular level before the treatment is effective. 

In another series of experiments, the recurrent nerve was cut in 80 flies which 
were responsive to water before the operation and an additional 50 flies that were 
satiated. Sixty per cent of the former became bloated; 50% of the latter. 

Removal of the corpora allata in 30 flies produced no abnormal water intake. 
Removal of the median neurosecretory cells of the brain from 60 flies caused bloat- 
ing in four. 

Where bloating occurred, the behavior pattern was characteristic. Flies imme- 
diately began drinking and continued to drink intermittently over a period of one 
to two hours. Thereupon no further drinking occurred although the proboscis 
was repeatedly extended. 

DISCUSSION 

As in the case of the ingestion of sugars (Dethier et al., 1956), the act of 
drinking appears to be controlled primarily by sensory input. Stimulation of tarsal 
or labellar taste receptors by water elicits reflexive extension of the proboscis and 
sucking. When adaptation (central and peripheral) has proceeded to some level, 
ingestion is terminated ; the stimulus becomes ineffective. 

The behavioral threshold of the fly to water, however, varies as a function of 
water balance. Specifically, water responsiveness can be abolished by injections 
into the haemocoel and can be induced by bleeding. The chemical nature and the 
concentration of injected material do not matter; the effect is related only to the 
volume and can be altered by injection or bleeding. Since these treatments are 
fully effective as quickly as can be tested, neural mediation of the effect is indicated, 
probably via a mechanoreceptor that signals distension or pressure. Accordingly, 
the input of the mechanoreceptor must act somewhere to set the level of taste 
threshold to water. 

The behavioral threshold to water is also affected by cutting the recurrent nerve 
immediately anterior to the hypocerebral ganglion. Flies undergoing this operation 
became bloated on water. Dethier and Bodenstein (1958) reported that flies in 
which the recurrent nerve had been cut became bloated on sugar solutions. They 
interpreted this effect as interference with the elevation of sugar threshold that 
normally follows sugar ingestion. Evans and Barton Browne (1960) confirmed 



114 V. G. DETHIER AND D. R. EVANS 

the fact of hyperphagia following recurrent nerve section but found that the sugar 
threshold still rose in the normal fashion. Incidental observations suggested to 
them that the effect might be due to a hypersensitivity to water. Day (1943) and 
Thomsen (1952) had observed polydypsia in some flies after removal of the corpus 
allatum. However, allatectomy frequently involves a variable degree of injury 
to the recurrent nerve which could account for the low incidence (ca. 10%) of 
bloating observed by these workers. We have not been able to produce polydypsia 
as a result of allatectomy. Removal of the medial neurosecretory cells of the brain 
also sometimes results in bloating, but this does not necessarily imply a hormonal 
mechanism any more than a neural one since these cells are in neural connection 
with the recurrent nerve. 

Sensory control '(i.e., sensory input to drive and adaptation to stop) of drinking 
still operates in polydypsic flies so that an operated fly becomes bloated through 
repeated rather than continuous drinking. 

It was observed that operated flies kept in contact with water for long periods 
of time no longer imbibed any even though they continued to respond feebly for 
more than 24 hours. If such flies were presented with sugar, they resumed vigorous 
sucking until the crop and abdomen burst. There is obviously in the fly bloated 
on water a strong pressure opposing further intake. The water stimulus is not 
intense enough to produce effective sucking, but sugar, a stronger acceptable 
stimulus, can still produce effective sucking. This suggests that back-pressure was 
responsible for blocking continued imbibition of water. 

Since the behavioral threshold to water is affected by bleeding, by injection, 
and by cutting the recurrent nerve, the simplest explanation is that the recurrent 
nerve carries the neurons that signal blood volume or pressure and set water 
sensitivity. Elucidation of this point will have to await further knowledge of the 
finer details of structures innervated by the recurrent nerve and of connections 
with other parts of the nervous system. 

At this point evidence obtained earlier regarding the control of ingestion of 
sugars bears on the possible mechanisms and greatly complicates the interpretation. 
On the basis of much evidence, Dethier (1955) postulated that each of the two 
chemosensory neurons in the receptor subserved one of the taste qualities, acceptance 
and rejection. According to this hypothesis, water and sugar would be ingested 
because they stimulate the one neuron, and salts would be rejected because they 
stimulate the other (termed, respectively, 5 and L by Hodgson and Roeder, 1956). 
Subsequent electrophysiological studies have supported this view in general. 
Sugars do activate primarily the ^ fiber and salts the L fiber (Hodgson 
and Roeder, 1956). Furthermore, Wolbarsht (1958) and Tateda and Morita 
(1959) have shown that neither fiber exhibits appreciable spontaneous discharge, 
and Wolbarsht (1958) showed that there is no reason to believe that electrical 
responses of the two receptor cells influence one another. Consequently, the hypoth- 
esis explaining the qualities of taste is compatible with this evidence : discharge of 
the 5" fiber initiates and drives feeding, and discharge of the L fiber inhibits the 
feeding reflex somewhere beyond the sense cell level. 

Taste thresholds to sugars vary with feeding and starvation, and the mechanism 
has been studied in some detail (Dethier ct ol., 1956; Evans and Dethier, 1957; 
Dethier and Bodenstein, 1958; Evans and Barton Browne, 1960). All of the evi- 



WATER INGESTION IN THE BLOWFLY 115 

clence suggests that it is the presence of sugar solution in the foregut (exclusive of 
the crop) that sets the level of the sugar threshold. The detector in the foregut 
and the intervening processes have not yet been elucidated, but it was suggested 
(Evans and Dethier,, 1957) that the final effect was to inhibit centrally the effect 
of 5" fiber discharge. The problem now arises as to how taste thresholds to water 
and sugar can be independently regulated as the present results show that they are. 
Now it should be pointed out that the neural explanation of the two taste 
qualities is not really so simple as the discussion above and some of the literature 
suggests. Some data will be cited that show the unexpected complexities that have 
emerged from electrophysiological studies. Wolbarsht (1957) reported that both 
5" and L fibers respond to distilled water. In addition to the two chemosensory 
cells, there is a third sense cell associated with the socket of a chemoreceptor hair 
(Dethier, 1955) that Wolbarsht (1958) has shown to be a mechanoreceptor acti- 
vated by motion of the hair. The distal process of the cell does not enter the hair 
(Dethier and Larsen, personal communication) and therefore chemicals applied 
to the hair tip would not be expected to stimulate it. It is known that bending of 
a hair can evoke proboscis extension in a very starved fly (Dethier, 1955). Hodg- 
son and Barton Browne (1960) reported that bending of a hair influences, albeit 
unpredictably. the electrical response of the L and S fibers to chemicals. The ex- 
periments dealing with ingestion of salt solutions place limitations on hypothetical 
interpretations of the water threshold mechanism. Since the sensory input clue to 
water can drive the ingestion of more and more concentrated salt solutions as the 
flv is made more dehvdrated, the sensorv effects of water are balanced against those 

* / j o 

of salts, just as are the stimuli, sugar and salt. 

In view of the data presented it seems to us that the hypothesis that water and 
sugar act on the same neuron is no longer tenable. Evidence of the existence of a 
specific water receptor is now being sought. 

LITERATURE CITED 

BOLWIG, N., 1953. On the variation of the osmotic pressure of the haemolymph in flies. 6". 

Afr. Ind. Chcm., June. 
DAY, M. F., 1943. The function of the corpus allatum in muscoid Diptera. Biol. Bull., 84: 

127-140. 
DETHIER, V. G., 1955. The physiology and histology of the contact chemoreceptors of the 

blowfly. Quart. Rev. Biol, 30: 348-371. 
DETHIER, V. G., AND D. BODENSTEIN, 1958. Hunger in the blowfly. Zcitschr. Tierpsvchol., 15: 

129-140. 

DETHIER, V. G., D. R. EVANS AND M. V. RHOADES, 1956. Some factors controlling the in- 
gestion of carbohydrates by the blowfly. Biol. Bull.. Ill: 204-222. 
DETHIER, V. G., AND M. V. RHOADES, 1954. Sugar preference-aversion functions for the 

blowfly. /. E.vp. Zool, 126: 177-204. 
EDXEY, E. B., 1957. The Water Relations of Terrestrial Arthropods. Cambridge Monographs 

in Experimental Biology 5, Cambridge University Press. 

EVANS, D. R., 1961. Control of the responsiveness of the blowfly to water. Nature (in press). 
EVANS, D. R., AND L. BARTON BROWNE, 1960. The physiology of hunger in the blowfly. 

Amcr. Mdl. Nat., 64: 282-300. 
EVANS, D. R., AND V. G. DETHIER, 1957. The regulation of taste thresholds for sugars in the 

blowfly. /. Ins. Physiol, 1 : 3-17. 
HODGSON, E. S., AND L. BARTON BROWNE, 1960. Electrophysiology of blowfly taste receptors. 

Anat. Rec., 137 : 365. 



116 V. G. DETHIER AND D. R. EVANS 

HODGSON, E. S., AND K. D. ROEDER, 1956. Electrophysiological studies of arthropod chemo- 
reception. I. General properties of the labellar chemoreceptors of Diptera. J. Cell. 
Comp. Physiol., 48: 51-76. 

LECLERCQ, J., 1946. Des insectes qui boivent de 1'eau. Bull. Ann. Soc. Ent. Beige., 82 : 71-75. 

TATEDA, H., and H. MORITA, 1959. Initiation of spike potentials in contact chemosensory hairs 
of insects. I. The generation site of the recorded spike potentials. /. Cell. Comp. 
Physiol., 54: 171-176. 

THOMSEN, E., 1952. Functional significance of the neurosecretory brain cells and the corpus 
cardiacum in the female blow-fly, Calliphora erythrocephala Meig. /. Exp. Biol., 29 : 
137-172. 

WOLBARSHT, M. L., 1957. Water taste in Phormia. Science, 125: 1248. 

WOLBARSHT, M. L., 1958. Electrical activity in the chemoreceptors of the blowfly. II. Re- 
sponses to electrical stimulation. /. Gen. Physiol., 42: 413-428. 

WOLBARSHT, M. L., AND V. G. DETHIER, 1958. Electrical activity in the chemoreceptors of the 
blowfly. I. Responses to chemical and mechanical stimulation. /. Gen. Physiol., 42 : 
393-412. 



FLIGHT AND SWIMMING REFLEXES IN GIANT WATER BUGS 

HUGH DINGLE 

Department of Zoology, University of Michigan, Ann Arbor, Michigan 

Loss of substrate contact or tactile stimulation initiates a "classic" flight reflex 
in insects (Fraenkel, 1932; Chadwick, 1953). Either one or both factors can 
operate to elicit the reflex ; flight ceases when the legs again make contact with the 
substrate. When giant water bugs were removed from substrate contact, they did 
not fly, but instead swam. If they stopped, they would begin again with direct 
tactile stimulation. In short, they appeared to swim in those situations in which 
terrestrial insects fly. Although a few of the water bugs eventually flew, they did 
so only after a considerable period ; during this time they were swimming. This 
study is an attempt to analyze the swimming and flight reflexes of these giant water 
bugs. 

MATERIALS AND METHODS 

Two species of giant water bug were used, Lcthocerus americanus and Benacus 
griseus. The bugs were captured by light trap (a sheet and a Mercury Vapor 
bulb, General Electric H100 L4) between April and September, 1960, on the Edwin 
S. George Reserve, the wildlife reserve of the University of Michigan, Livingston 
County, Michigan. A total of 60 animals were used ; they were kept in the labora- 
tory on a diet of small fish. 

Giant water bugs are large (about 4.5 to 6.5 cm. long) dorso-ventrally flattened 
predaceous insects. The forelegs are raptorial with enlarged femora and bear only 
a single tarsal claw ; the middle and hind legs are adapted for swimming ; they are 
flattened and bear hairs so arranged as to be raised during the power stroke of the 
leg and depressed during the forward stroke. The swimming legs have the usual 
two tarsal claws. Respiration is accomplished with two retractable tubes which 
protrude from the posterior end of the abdomen (Fig. 4). 

In the analysis of swimming and flight reflexes, the bugs were suspended from 
an applicator stick using a mixture of paraffin, beeswax, and resin to attach the stick 
to the prothorax. They were then placed in the air stream of a wind tunnel and 
given a stick to hold which served as a contact stimulus for the legs. The wind 
tunnel was made from wood and light cardboard and included a cardboard honey- 
comb baffle to cut down turbulence which, as determined by smoke, was slight ; the 
diameter of the tunnel mouth was 10 cm. For wind a fan was used, the speed of 
which could be controlled by a rheostat. Wind speed was calibrated with a Taylor 
Briam's Type Anemometer (No. 3132) ; it ranged up to 7.0 m./sec. In certain 
experiments small jets of water or air, which were directed by attaching a glass 
tube to a rubber hose, were used ; no attempt was made to measure the velocity of 
these. 

117 



118 HUGH DINGLE 

SWIMMING 

Loss of substrate contact almost invariably elicited swimming movements. Tbe 
rate and duration of these movements varied. The initial rate for 19 bugs in quiet 
air ranged from 120 to 320 strokes per minute with an average of 206; the duration 
ranged from 6 seconds to more than 180 seconds with an average of 51 seconds. 
This swimming response was clearly distinguishable from haphazard movement ; 
the forelegs were carried forward of the head, and in intense swimming they were 
stretched forward almost full length. The abdomen was raised, and the middle and 
hind pairs of legs were usually protracted and retracted (see Hughes, 1952, for 
definitions) simultaneously and not alternately as reported by Lauck (1959) for a 
different species. Although alternation was never observed, it was noted that the 
two pairs were sometimes not quite simultaneous. The two legs of each pair 
operated simultaneously as reported by Lauck. 

Swimming could be stopped by giving the bug a stick to hold. Contact with 
any one tarsus was sufficient ; when the bug made contact, the ipselateral leg reached 
for and grasped the stick. Swimming also ceased with contact on other parts of 
the leg, e.g. tibia and femur, especially if tension was applied; Diakonoff (1936) 
reports similar results in a flying cockroach. Sometimes, however, the bug dropped 
the stick or "walked" off it and continued to swim. If the stick was removed care- 
fully, leaving the legs folded under the body, the bug usually remained motionless. 
Swimming in such a situation could be initiated by gently lowering the legs until 
they were outstretched. Bugs also stopped swimming on occasion when they 
presumably saw the stick in front of them, reaching out and seizing it with the 
forelegs. Touching any part of the forelegs resulted in attempts to grasp the stick. 

In experiments testing the effect of increasing wind velocity, the bugs were 
holding a stick which was removed at each higher velocity ; it was returned when 
the bug stopped swimming. After 30 seconds the velocity was increased by about 
1 m./sec. and the process repeated. Rate and duration of swimming increased up 
to a point and then decreased ; this decrease will be discussed in greater detail below. 
The lowest wind velocity measured, 0.5 m./sec., was sufficient to increase rate and 
duration in 50% of the bugs ; for the remainder higher velocities were needed. 
Twenty per cent of the bugs reached their maximum rate at 1.6 m./sec.; maxima 
were attained up to 6.7 m./sec. Maximum durations occurred from 0.5 to 7.0 
m./sec., the total range used in these experiments. Except for one bug which gave 
a brief burst of strokes at around 400 minute, the greatest rate of swimming ob- 
served was 320 strokes/minute which was reached by half the animals ; they could 
not be induced to swim faster. If wind was blown on an animal from the side, 
it often responded with compensatory movements of the legs on the opposite side. 
Figure 1 shows rate and duration with increasing wind speed for three repre- 
sentative bugs. 

If the bug was holding an object, wind alone initiated swimming and consequent 
dropping of the stick in 25% of the cases. Usually, however, swimming occurred 
only when wind was combined with loss of substrate contact. Ordinarily loss of 
contact was the significant stimulus, but often the few bugs that would not swim 
with just loss of contact could be induced to do so if wind was simultaneously 
applied. A bug that had been swimming, but had stopped, would start again when 
wind was applied. 



REFLEXES IN GIANT WATER BUGS 



119 



In addition to loss of contact and wind, direct tactile stimulation, e.g. of the 
abdomen, and vibration or movement of the bug while suspended also caused swim- 
ming. Any movement, whether up and down or to and fro, and any vibration, 
caused either by tapping the stick to which the bug was attached or pounding the 



70 
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a 601 



50 
40 
30 
20 
IO 



c/) 

LL 
O 

z 
o 



a: 
o 



350- 
c" 

6 



o> 

300- 
in 



O 

E 250 



5 

V) 

u_ 
o 

UJ 
5 

cr 



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100 




\ 

OPENED 
WINGS 




FLIGHT 
POSITION 




OPENED 
WINGS 



-0 






/ ---o o 
X 


^FLIGHT 
POSITION 



2345 

WIND VELOCITY ( m /sec) 



FIGURE 1. Graphs of rate and duration of swimming plotted against wind velocity for 
three representative bugs. Circled points indicate beginning of first noticeable flight preparation 
movements ; these were not observed in one animal. 

table with the fist, elicited the swimming. Fraenkel (1932) reports that flight in 
1'cspa, Calliphora, Apis, Schistocerca, etc. resulted from a blow on the abdomen, 
and Diakonoff (1936) found that cockroaches flew if allowed to fall, a phenomenon 
he termed a "fall reflex.' 



120 HUGH DINGLE 

Not too surprisingly, suspended bugs also swim when placed in water, although 
the swimming is very quickly adapting, lasting only a few seconds. Swimming 
can be further induced by directing a current at the bug, moving the animal 
through the water, or by taking the animal out of the water, but again the swim- 
ming is quickly adapting. By far the most rapid swimming comes when the bug 
is allowed to touch some object with its forelegs which it then attempts to grasp. 
This too adapts, but after a longer time. Swimming also follows on occasion when 
the bug presumably sees an object in front of it. A water jet directed at the bug 
from one side causes some compensatory movements of the legs on the opposite 
side. The same results are observed when the bug is rotated through the water in 
a small circle; this phenomenon was also recorded by Hughes (1958) in Dytiscus. 

Sense ore/cms mediating swimming 

Fraenkel (1932) found that his insects would not stop flying when their tarsi 
were removed, which led him to believe that a receptor sensitive to contact was 
located there. Diakonoff (1936), however, was unable to find sensilla on the 
tarsal claws of the cockroach and found that in addition to the tarsi, stimuli on 
the tibiae, femora, and even coxae could stop flight. Since water bugs swam on 
loss of substrate contact, presumably a mechanism similar to that eliciting flight in 
the above cases is involved. It was found that swimming ceased either with tarsal 
contact or with stimuli on the tibiae or femora. 

Touch receptors seem to be implicated in the instance of more rapid swimming 
when the bug touches an object with its forelegs. The leading edges of the femora 
of these bugs are covered with an extensive sensory area, and this area when touched 
is especially apt to elicit increased swimming. The exact nature of these receptors 
and others at the same spot affords a promising line of future investigation. 

Specific receptors, eliciting swimming in response to either air or water currents, 
have not been located. With the forehead, eyes, hair beds behind the eyes, hair 
beds at the bases of the fore femora, and hair beds at the junction of the pro- and 
mesothorax covered individually or all together with paraffin, the swim reflex did 
not appear to be hindered in any way. Although the antennae were not removed 
completely, located as they are in grooves under the eyes, the bugs still began to 
swim in currents after an operation to destroy the brain, indicating that neither 
the antennae nor for that matter any other head receptors innervated by the brain 
are mandatory for the initiation of this swimming. It is suspected that swimming 
in response to current can be initiated by any of several receptors located on the 
body. Certainly the body possesses many groups of hairs located at various joints 
and articulations, and that several of them may be "current receptors" is indicated 
by the fact that a bug will swim in a current coming from virtually any direction. 

When the bugs are in water, however, there do seem to be specific sense organs 
which initiate swimming. The first hint of such receptors came while a bug whose 
nervous connectives had been severed between the pro- and mesothoracic ganglia, 
the cut being made just posterior to the forelegs, was being studied. Such an 
insect loses all muscle tone posterior to the cut, and the legs hang limply. When 
this bug was put in water, the legs began to protract and retract slowly and rhyth- 
mically with enough force to give the bug some forward momentum. Further 
observation revealed that this swimming commenced only when the legs had floated 



REFLEXES IN GIANT WATER BUGS 121 

up so as to be extended almost laterally from the body. Swimming was also 
observed when the bug was held upside down and the legs were in almost the same 
position as when floating, but this was never more than a few strokes. 

In attempts to locate more closely the receptors responsible for this swimming 
response, the leg segments and joints from all four swimming legs were removed 
successively with the following results : 

(1) Removal of first tarsal segment and joint between the two tarsal segments 
bug swam, but kept legs rather sharply bent at tibio-femoral joint. 

(2) Removal of second tarsal segment and tibio-tarsal joint bug swam with 
shorter and more rapid strokes. 

(3) Removal of tibia and tibio-femoral joint bug swam with short, rapid, and 
choppy strokes that were not well co-ordinated. 

These results seemed to indicate that the receptors responsible for the swimming 
response were located somewhere proximal to the tibia. Because of the flotation 
of the legs which seemed to be necessary, the location was suspected to be at either 
the coxo-trochanteral or femoro-trochanteral joint ; the former location appeared 
to be the more likely. Hair beds are located on the trochanters at this joint just 
distal to the trochanteral condyles (Fig. 2). When the legs hung down as they 
did when the bug was suspended, these hair beds were covered by membranous 
cuticular folds present on the coxae ; when the legs floated in water, the hair beds 
were uncovered. 

In bugs with the connectives severed between the pro- and mesothoracic ganglia, 
the trochanteral hair beds on various legs were burned with a hot needle. If these 
were destroyed on all four swimming legs, the bugs showed no response when 
placed in water ; if the hair beds on the middle legs were destroyed, the hind legs 
still swam, with the converse true if the hind leg hair beds were burned. In a bug 
lacking the hair bed on one middle leg, the other three legs swam in the usual 
fashion while the operated leg gave strokes on each alternate stroke of the rest; 
with the hair beds on three legs burned, only the single intact (hind) leg gave 
swimming strokes, and these were slower than previously. In bugs with the central 
nervous system intact, when the hair beds were destroyed on all four swimming 
legs, walking was more or less as usual, but the bugs seemed to have difficulty 
gaining traction on surfaces where normal animals had no difficulty. In both water 
and air, swimming strokes were short and jerky; in air, swimming proved also to 
be more difficult to induce than in normal individuals. These hair beds thus appear 
to be intimately involved with swimming and co-ordination of leg movements. 

The trochanteral hair beds are apparently excited by the cuticular folds which 
cover them when the legs hang do\vn or are folded beneath the body. As the legs 
float up when the bug is in water, these folds roll back progressively until the hair 
beds are uncovered when the legs are extended laterally. Presumably, then, when 
stimulation of the hair bed by the cuticular fold ceases, the leg begins to swim. 
Possibly direct contact with water prompts the swimming movements to some 
extent although this is not the only factor since inverted bugs with severed con- 
nectives also swim. Pringle (1938) described three hair plates on the leg of the 
cockroach, including one at the coxo-trochanteral joint, which he believed were 
also stimulated by a cuticular fold ; the hair plates were incompletely adapting. 



122 



HUGH DINGLE 



Because of the location and action of these sense organs, Pringle considered them 
"position" receptors. The action of the hair beds on the legs of the giant water 
hug seem to have an analogous function, i.e. registering the position of the legs 
until they finally reach swimming position, whereupon the swimming reflex is 
triggered. 

This proposed action of the hair beds helps to explain some aspects of the bugs' 
behavior. As mentioned earlier, a suspended bug tends not to swim when its legs 
are folded under the body as when grasping a stick. This lack of response would, 
on the above explanation, be due to the covering of the hair beds by the cuticular 
folds. In nature the bugs cling to submerged vegetation ; if they were torn free, 
the resultant flotation of the legs would provoke swimming and lead to regaining 
of foothold. 




tr 



1-hb 



FIGURE 2. Ventral view of the coxo-trochanteral joint. The coxa and trochanter have 
been depressed dorsally as they would be if the leg were floating to expose the trochanteral 
condyles and hair beds. When the leg hangs down, the cuticular fold covers these two struc- 
tures ; the fold rolls back as the leg floats up. tr, trochanter ; ex, coxa ; thb, trochanteral hair 
bed ; tc, trochanteral condyles ; cf, cuticular fold. 

Vision also seems to affect swimming. If a suspended bug is rotated through 
the water in a tight circle, the inside legs show compensatory movements that oppose 
the direction of rotation. In a bug with its eyes covered, the compensatory move- 
ments are so reduced as to be almost negligible. Hughes (1958) found reduction 
in the compensatory movements of a rotated Dytiscus when the eyes were covered. 

FLIGHT 

Pre-flight behavior in giant water bugs follows a fairly elaborate and somewhat 
varied pattern. The first sign is usually scraping of the hind legs over the wings 
and depression of the abdomen. There then follows twitching of the legs, which 
in the more advanced stages can be quite violent ; this twitching is often accompanied 
by "shrugging" movements in which the pterothorax and abdomen are moved 
rapidly anterior-posterior at the articulation between the pro- and mesothorax. 
The wings can, at least from the author's observations, be opened at any stage of 
these preparations. 



REFLEXES IN GIANT WATER BUGS 



123 



This rather extensive pre-flight behavior is apparently necessary because of a 
ball and socket mechanism which locks the wings to the pterothorax (Lauck, 1959) ; 
this mechanism is illustrated in Figure 3. The ball protrudes posteriorly from the 
dorsal margin of the mesepimeron and inserts into the socket on the costal margin 
of the hemelytron ; the mesal border of the clavus matches the wing grooves on the 
postnotum. In order to open the wings, the bug must first release the ball and 
socket mechanism, which is probably accomplished, according to Lauck, by a 
combination of contractions of the third axillary muscle and the tergo-sternal 



prsc 



epm 



epm 3 




ro 



sw 



sw 



FIGURE 3. Lcthoccnts: views of pterothcrax and hemelytron to show position of wing ball 
(wb) and wing socket (ws). The posterior margin of the clavus (cl) fits along the wing 
groove (wg). A: Pterothorax with wings on left side removed, prsc, prescutum ; ph, 
phragma ; sc, scutum; sc-scl, scuto-scutellum ; pN, postnotum; epm, epimeron; wb, wing ball; 
wg, wing groove ; T, tergite of abdomen. B : Ventral aspect of left hemelytron. ws, wing 
socket ; co, corium ; cl, clavus ; me, membrane ; we, wing clip. C : Diagram showing wing 
locking mechanism, he, hemelytron ; ra, respiratory apparatus ; ab, abdomen ; sw, swimming 
leg. Arrow points anteriorly. A and B redrawn from Lauck (1959) by permission of the 
publishers. Not drawn to same scale. 

muscles which levate the wings. The various violent leg twitchings, depressions of 
the abdomen, and oscillations of the body characteristic of the pre-flight behavior 
are apparently the result of attempts by the animal to get the wings unlocked. 

There is, however, another possible reason for the pre-flight movements. Krogh 
and Zeuthen (1941) note that lamellicorn beetles "pump" before flight; they 
measured the rise in temperature of the muscles during "pumping" and found that 
not until the temperature was at least 32 C. would the beetles fly. The flight tem- 
perature varied from 32 to 37. Poor fliers like the beetles needed higher body 
temperatures to fly than did sphingid moths which are quite active fliers. Since 
giant water bugs are relatively poor fliers, it is possible that the pre-flight movements 
raise the body temperature enough to fly. 



124 



HUGH DINGLE 



In spite of the extensive pre-flight behavior in most animals used, only a few 
actually flew ; of 44 suspended bugs, four flew while four more opened their wings, 
but did not fly. Several others showed a tendency to assume the flight position, 
but never reached the stage of opening the wings. The flight position is shown in 
Figure 4. The swimming legs are carried folded flat against the underside of the 
body, although not in this illustration ; the abdomen is depressed ; and the respiratory 
apparatus is fully extended and held erect. Those bugs that did fly were, with one 
exception, suspended for five minutes or longer and most of the time in winds of 
greater than 6 m./sec. Weis-Fogh (1956) found that in locusts wind speeds of 
greater than 2 m./sec. were necessary to initiate flight. 




FIGURE 4. (1) Suspended bug holding drinking straw as substrate contact. (2) Swim- 
ming bug; swimming legs are approximately at the end of the backstroke. (3) Bug in flight 
position; note position of swimming legs and respiratory apparatus compared with (2). (4) 
Bug with wings open ; again note position of swimming legs and respiratory apparatus. 

Once a bug had flown, the threshold for further flight or wing opening was 
lowered considerably. Flight could be stopped by bringing the bug into contact 
with the substrate and could usually be initiated again if the animal was suspended. 
If flight was not induced by suspension, it could then be initiated by putting the 
bug into a wind. The contact-loss of contact mechanisms is presumably similar 
to those mentioned above when discussing swimming. 

The stimulation of flight by wind is of some interest. It was found that a jet 
of compressed air delivered through a bit of glass tubing was most effective in 
promoting flight or wing opening (in bugs that had already flown or opened their 
wings ) when it was aimed directly at the bugs' heads from in front. In these bugs 
the wings invariably opened while the air jet was blowing on the head and would 
close when it was removed. If the area of the head above the beak and between 
the eyes was covered with paraffin, the response disappeared ; it reappeared when 
the paraffin was removed. This was true for all 8 of the bugs tested. Partial 



REFLEXES IN GIANT WATER BUGS 125 

covering of the forehead with paraffin did not abolish the response ; so long as part 
of it was exposed, the response was maintained. Examination of a bug's head 
under the dissection microscope revealed that the area being considered was covered 
with fine hairs, virtually invisible to the naked eye, which are presumably responsible 
for the initiation of wing opening or flight when stimulated by air currents. Weis- 
Fogh (1956) found 5 paired groups of wind-sensitive hairs on the head of the 
locust which were sufficient for both the initiation and maintenance of flight, but 
were not necessary for either. Aside from the hair beds, flight in the locust could 
be initiated by loss of tarsal contact, which was also found to be true with giant 
water bugs, and could be maintained by wind on the moving wings, which was not 
observed in this study. In both bugs and locusts the direction of the wind im- 
pinging on the hair beds was not particularly important. 

INTERACTION OF FLIGHT AND SWIMMING 

There seems to be little doubt that the initial response of these insects to loss 
of substrate contact is swimming. As previously mentioned, with increased wind 
speed both rate and duration of swimming increased up to a point, which varied 
from one bug to another, and then decreased. At first it was thought that this was 
due to fatigue or adaptation, but careful observation of the bugs' behavior revealed 
that the most likely possibility was the inhibition of swimming by the pre-flight 
activities even in those bugs, the most usual, in which neither flight nor wing 
opening ever occurred. In the latter cases, however, the bugs often did assume 
flight position with the legs, abdomen, and respiratory apparatus (Fig. 4). Reduc- 
tion of swimming also occurred when the bugs were given successive bursts of 
wind at a constant speed (5.9 m./sec.), although it was not so marked. 

THE CENTRAL NERVOUS SYSTEM 

The anatomy of the giant water bug central nervous system reflects the general 
anatomy and habits of the bug. The sub-oesophageal and prothoracic ganglia 
are fused into one ganglion located between and slightly anterior to the bases of 
the coxae of the raptorial prothoracic legs which are innervated from this ganglion. 
The meso- and metathoracic ganglia are also fused into a common structure 
located between the bases of the mesothoracic legs. This ganglion innervates all 
four swimming legs and the wings. The brain and circumoesophageal connectives 
appear to be grossly similar to those of other insects. 

A bug with its brain destroyed (using a hot needle) moved about apparently 
quite normally. Closer observation, however, revealed certain rather distinctive 
abnormalities. For instance, when walking about, a brainless bug tended to 
lose its balance and fall over on its back when stepping over small objects; once 
on its back it had considerable difficulty righting itself, often being unable to do so. 
An intact animal would, when placed on its back, bridge up with its forelegs and 
give a hard kick with the middle and hind legs on one side pivoting over on the 
tip of the abdomen; a brainless bug, on the other hand, was unable to bridge as 
high with the forelegs or to use the swimming legs effectively to flip over. When 
placed in wind, the brainless bugs differed from the normal in two ways. First, 
they would swim for much longer periods, usually showing no signs of slowing 



126 HUGH DINGLE 

down ; and second, they accepted a stick and thus ceased swimming much more 
readily. Roeder (1937) and Roeder ct al. (1960) note that the praying mantis 
also exhibits hyperactivity with the brain destroyed, walking until exhaustion. 
Bugs with only half the brain destroyed carried out the classic maneuver of circling 
to the intact side. Severing the connectives just behind the forelegs resulted in loss 
of tone in the swimming legs, but the legs continued to swim when the animal 
was placed in water, as noted above ; the forelegs often twitched for a time after 
the cut was made. 

DISCUSSION 

The fact that when the trochanteral hair bed on one mesothoracic leg was 
destroyed, that leg swam on the alternate strokes of its counterpart seems to indi- 
cate transfer of impulses from one side of the mesothoracic ganglion to the other. 
Rowe (1960) has shown electrically that such intraganglionic transfer occurs, 
while several authors (e.g. Diakonoff, 1936; Ten Gate, 1941; and Hughes, 1957) 
have behavioral evidence for it. Destroying the hair beds on both of a pair of 
swimming legs resulted in loss of activity of that pair while the other two con- 
tinued to swim. Thus, as was the case with Pringle (1940), the author was 
unable to demonstrate transfer of a reflex from one thoracic ganglion to another 
even though the meso- and metathoracic ganglia are, in this case, fused. 

Roeder (1937; see also Roeder, 1953) proposed a model for the operation of 
the insect central nervous system ; in this model the brain exercises inhibitory 
control over locomotion, in view of the locomotor hyperactivity of brainless insects. 
Since giant water bugs are also hyperactive when brainless, they appear consistent 
with Roeder's model. Bugs whose connectives had been severed posterior to 
the fused sub-oesophageal and prothoracic ganglia lost all muscle tone in the 
swimming legs, but because of their fusion, it was not possible to separate the two 
ganglia functionally. There is some evidence from studies on cockroaches 
(Diakonoff, 1936; Ten Gate, 1941; Chadwick, 1953) that the prothoracic ganglion 
may be essential for normal co-ordination. 

If Hemiptera are secondarily aquatic, then the swimming reflex of aquatic 
forms like the giant water bugs may be considered a modification of the flight 
reflex of exclusively terrestrial insects. The reflexes, under natural conditions, 
would be triggered by similar sets of circumstances. A floating water bug, for 
instance, is free of substrate contact, and a swimming reflex might result, particu- 
larly since the usual habit of the bug is to cling to floating vegetation. A falling 
terrestrial insect, on the other hand, is also free of substrate contact and generally 
flies. The two situations of floating and being air-borne are essentially the same, 
and the reflexes of a particular insect, be they swimming or flying, are modifications 
to suit the particular medium. 

The escape responses are similarly modified. Strong tactile stimulation, espe- 
cially of the abdomen (Fraenkel, 1932), causes terrestrial insects to leap off the 
substrate and fly. In the aquatic bugs tactile stimuli or vibrations result in violent 
swimming whether the animal is in water or suspended in air. 

But if the swimming reflex is a modification of the flight reflex, why then 
do the water bugs sometimes fly? There appear to be two major possibilities. 
First, the body posterior to the articulation of the pro- and mesothorax of a bug 



REFLEXES IN GIANT WATER BUGS 127 

suspended in air hangs down at a rather sharp angle ; in water this part of the 
body is buoyed up. Diakonoff (1936) reports that movement at the pro-meso- 
thoracic articulation of the cockroach results in a "fall reflex" that elicits flight 
and is apparently due to stimulation of the numerous receptors at the articulation. 
A similar mechanism may stimulate flight in giant water bugs. Second, when 
the bugs are suspended in wind, the hair beds on the head, which have been shown 
to be receptors concerned with flight, are stimulated. This stimulation, if strong 
enough or if summation occurred, would presumably overcome the swimming 
reflex and elicit flight. 

One would predict, on the assumption that swimming with lack of substrate 
contact is a modification of a flight reflex, that it would be a fairly general 
adaptation among aquatic insects. This prediction appears to be largely true. 
Hughes (personal communication) has observed the swimming reflex in Hydro- 
pliilits and Dytiscns, and the author has found it in gyrinids, hydrophilids, dytiscids, 
corixids, and the genus Belostoina, as well as in the giant water bugs discussed 
here. Further investigations of the phenomenon in these groups are now in 
progress. 

The author wishes to thank Drs. L. B. Slobodkin and D. M. Maynard for 
their encouragement and for reading and criticising the manuscript. Dr. F. C. 
Evans kindly permitted the use of the E. S. George Reserve for light trapping, 
Mr. D. F. Owen supplied many of the animals used, and Mr. John Alley took 
the photographs in Figure 4. A special debt is owed to the author's wife, 
Geraldine Dingle, who has read the manuscript several times ; most of her many 
suggestions and criticisms have been included. 

This research was carried out while on a National Science Foundation Co- 
operative Graduate Fellowship. 

SUMMARY 

1. Giant water bugs swim when suspended free of the substrate. This situation 
contrasts with that of terrestrial insects which fly when freely suspended. Swim- 
ming can be stopped by returning contact to the bugs. 

2. Suspended bugs respond to wind with a general increase in rate and duration 
of swimming, followed by a decrease in both. 

3. When bugs are in water, swimming is stimulated by a hair bed located 
on the trochanter at the coxo-trochanteral joint. These hair beds seem to be 
stimulated by cuticular folds which cover them when the legs hang down, but 
roll back and leave them uncovered when the legs float, resulting in swimming. 

4. Flight or wing opening occurred with 8 of 44 suspended bugs. A hair bed 
on the head functions in both the maintenance and initiation of flight in response 
to wind. 

5. The bugs possess an elaborate pre-flight behavior which is apparently 
necessary to unlock a ball and socket mechanism attaching the wings to the ptero- 
thorax. This pre-flight behavior inhibits swimming and causes the decline in rate 
and duration mentioned in (2) above. 

6. In the central nervous system the sub-oesophageal and prothoracic ganglia 
are fused, as are the meso- and methathoracic ganglia. There is behavioral evi- 



128 HUGH DINGLE 

dence for transmission of impulses across a ganglion, but not from one ganglion 
to another, even though the ganglia are fused. 

7. There is evidence that the swimming reflex is a general phenomenon ; appar- 
ently it is an aquatic modification of the flight reflex. 

LITERATURE CITED 

CHADWICK, L. E., 1953. The flight muscles and their control. In : Roeder, K. D., Insect 
Physiology. New York, Wiley. Pp. 648-655. 

DIAKONOFF, A., 1936. Contributions to the knowledge of the fly reflexes and the static sense 
in Periplaneta americana L. Arch. Necrl. Physiol., 21 : 104-129. 

FRAENKEL, G., 1932. Untersuchungen iiber die Koordination von Reflexen und automatisch- 
nervosen Rhythmen bei Insekten. I. Die Flugreflexe der Insekten und ihre Koor- 
dination. Zcitschr. vergleich. Physiol., 16 : 371-393. 

HUGHES, G. M., 1952. The co-ordination of insect movements. I. The walking movements of 
insects. /. Exp. Biol, 29: 267-284. 

HUGHES, G. M., 1957. The co-ordination of insect movements. II. The effect of limb ampu- 
tation and the cutting of commissures in the cockroach (Blatta orientalis). J. Exp. 
Biol., 34 : 306-333. 

HUGHES, G. M., 1958. The co-ordination of insect movements. III. Swimming in Dytiscus, 
Hydrophilus, and a dragonfly nymph. /. Exp. Biol., 35 : 567-583. 

KROGH, AUGUST, AND ERIK ZEUTHEN, 1941. The mechanism of flight preparation in some 
insects. /. Exp. Biol., 18: 1-10. 

LAUCK, DAVID R., 1959. The locomotion of Lcthoccrus (Hemiptera, Belostomatidae). En-t. 
Soc. Amer. Annals, 52 : 93-99. 

PRINGLE, J. W. S., 1938. Proprioception in insects. III. The function of the hair sensilla 
at the joints. /. Exp. Biol., 15: 467-473. 

PRINGLE, J. W. S., 1940. The reflex mechanism of the insect leg. /. Exp. Biol., 17: 8-18. 

ROEDER, K. D., 1937. The control of tonus and locomotor activity in the praying mantis 
(Mantis religiosa L.). J. Exp. Zool., 76: 353-374. 

ROEDER, K. D., 1953. Reflex activity and ganglion function, hi : Roeder, K. D., Insect Physi- 
ology. New York, Wiley. Pp. 463-487. 

ROEDER, K. D., L. TOZIAN AND E. A. WEIANT, 1960. Endogenous nerve activity and behaviour 
in the mantis and cockroach. /. Ins. Physiol., 4 : 45-62. 

ROWE, E. C., 1960. Activity of single nerve cells in an insect thoracic ganglion. Anat. Rec., 
137: 389. 

TEN CATE, J., 1941. Quelques remarques a propos de 1'innervation des mouvements locomo- 
toires de la Blatte (Periplaneta americana L.). Arch. Neerl. Physiol., 25: 401 109. 

WEIS-FOGH, T., 1956. Biology and physics of locust flight. IV. Notes on sensory mechanisms 
in locust flight. Phil. Trans. Roy. Soc., Ser. B, 239: 553-585. 



ION REGULATION IN TETRAHYMENA 1 

PHILIP B. DUNHAM 2 AND F. M. CHILD 
Department of Zoology, University of Chicago, Chicago 37, III. 

Fresh water animals maintain their cells hyperosmotic to their environment 
(Prosser ct al., 1950). In higher animals this is accomplished by specialized 
organs or tissues (e.g. the vertebrate kidney, frog skin, and the anal papillae of 
dipteran larvae) which adjust the osmotic level of body fluids to a level isosmotic 
with the cells. 

Lower invertebrates (protozoa, sponges and coelenterates) have no osmotically 
regulated body fluids. Therefore all cells in fresh-water representatives of these 
groups have the problem of continuous water influx, and must have osmoregulatory 
ability (Kitching, 1954). 

A high potassium content relative to their medium is characteristic of all living 
cells that have been investigated. Most cells are richer in potassium than sodium, 
and there is often less sodium in cells than in the medium. Evidence has been 
offered that in such fresh-water animals as Hydra and Spirostonimn, inorganic ions 
are readily available for exchange with the environment (Lilly, 1955 ; Carter, 1957). 
Table I lists potassium and sodium levels in several fresh-water invertebrates. 
Therefore regulation of body volume in such forms involves regulation of ions as 
well as water. 

This paper reports an investigation of ionic regulation in Tctraliymena pyri- 
jonnis, a fresh-water ciliate. A remarkable ability to maintain a high potassium 
concentration as well as a lower sodium concentration in very dilute medium was 
found. Evidence for a sodium extrusion mechanism was also found. These 
findings will be discussed in terms of a model system for ion regulation in Tctra- 
hyincua, and in terms of relevance to similar problems in other animals. 

METHODS AND MATERIALS 

TctroJiyuiena pyriformis, strain W, was grown axenically in 2% proteose- 
peptone medium (hereafter called normal medium), the ion content of which is 
indicated in Table II. One-liter Roux culture bottles containing 500 ml. of medium 
were innoculated with 5 ml. of a culture in log phase of growth. After four 
days' growth at 22-25 C, the cells were concentrated approximately ten-fold by 
gentle centrifugation. After experimental treatment, which involved either dilu- 
tion of cell suspension in normal medium with distilled water or increasing medium 

1 The methods reported in this paper were worked out initially by the junior author with 
the assistance of Miss Marina Dan. The results and hypotheses are the work of the senior 
author who gratefully acknowledges the help and criticism of Dr. H. B. Steinbach during the 
course of this study. The work has been supported by National Science Foundation grant 
G-4526 and U. S. Public Health Service grant RG-6879. 

2 Trainee in United States Public Health Service Training Grant Program 2G-150. 

129 



130 



PHILIP B. DUNHAM AND F. M. CHILD 



TABLE I 

K and Na (or osmolar) concentrations of some fresh water invertebrates. (See Willmer 
(1958) for a table of osmolar concentrations of some protozoans determined by a 

variety of methods.) 



Organism 


Concentration 


Method of determination 


Literature source 


Acanthamoeba 
(distilled water washed) 


K 26.9 (meq./l. cells) 
Na 14.3 


elemental analysis 


Klein (1959) 


Spirostomum 


K 7 (meq./l. cell water) 
Na 1 


equilibration with isotopic 
tracer 


Carter (1957) 


Tetrahymena 
(in 2% proteose- 
peptone) 


K 31.7 (meq./l. cells) 
Na 12.7 


elemental analysis 


Dunham and Child 
(present report) 


Spongilla 
(summer) 


osmolarity equivalent to 27 
meq. NaCl/1. cell water 


vapor pressure determination 


Zeuthen (1939) 


Pelmatohydra 
(whole animal) 


K 14.4 (meq./l. cell water) 
Na 2.7 


equilibration with isotopic 
tracer 


Lilly (1955) 


Tubifex 
(whole animal) 


K 27.0 (meq. /kg. wet 
Na 23.4 weight) 


elemental analysis 


Dunham 
(unpublished) 


Anodonta 
(muscle) 


K 10.6 (meq. /kg. wet 
Na 5.2 weight) 


elemental analysis 


Hayes and Pelluet 
(1947) 



concentration by adding NaCl or KC1, cell suspensions were reconcentrated when 
necessary so that 10 ml. gave a packed cell volume of about 0.2 ml., as determined 
by centrifugation at a relative centrifugal force of 1600 to constant volume (10 
minutes in 10-ml. Kolmer tubes). 

Dry weight of cells was determined by drying to constant weight at 60 C. 
Cell counts were made in a hemocytometer. 

Cells were extracted for ion analyses by suspending them in dilute acetic acid 
(1 drop glacial acetic acid in 10 ml. water), heating near boiling for 5 minutes 
and allowing them to stand for one hour. K and Na analyses were made with 
a Coleman model 21 flame photometer. Preliminary Cl analyses were made with 
an Aminco-Cotlove chloride titrator. Analysis of nitric acid digests of residues 
after extraction indicated that more than 98% of intracellular K, Na and Cl was 
extracted. Intracellular cation concentrations are expressed in meq./l. cells, after 
appropriate correction for extracellular space as determined by use of radioactive 
iodinated serum albumin (Risa) added immediately prior to centrifugation. Total 
exchangeability and kinetics of intracellular K and Na were determined using trace 

TABLE II 

A' and Na concentrations of Tetrahymena in 2% proteose-peptone (normal) medium. 
K, Na, and Cl concentrations of normal medium. Standard errors and number of 

determinations are given. 



Cells 

K 

Na 

Medium 

K 

Na 

Cl 



meq./l. cells 

31.650.43 (44) 
12.681.3S (44) 

mM 

4.750.13 (60) 
36.50.13 (70) 
28.71.02 (6) 



ION REGULATION IN TETRAHYMENA 



131 



amounts of the appropriate radioisotope, K 42 or Na 24 , obtained from Oak Ridge 
National Laboratories as chlorides in HC1 solution, and neutralized with NaOH 
before use. Counts per minute of wet samples were determined with an end- 
window counter or a Nal-Tl crystal scintillation well counter. Per cent exchanges 
of intracellular K and Na were calculated from the specific activities of the cells 
and of the medium. 



40 



30 





CT 
O> 

E 



oC 

~ 10 




OD 




Ko'.mM 



t 5 



10 



20 



30 



FIGURE 1. K and Na content of Tctrahymena in normal and diluted media. Ordinate: 
cellular concentrations of K and Na (meq./l. cells); abscissas: concentrations of K and Na 
in the medium (MI A/). Open circles: Ki ; solid circles: Nai. Points for Ki and Nai in normal 
medium (see arrows) are averages of 44 determinations; vertical lines delimit the total range 
of the determinations in normal medium. 

RESULTS 

Table II shows the K and Na content of cells in normal medium, and the K, 
Na and Cl content of normal medium. The volume and weight of an average 
cell in normal medium were 1.83 X 1(H ^1. and 1.97 X 1O 2 /*g., respectively, as 
determined from cell counts and packed cell volumes (corrected for Risa space). 
Dry weight of cells in normal medium was determined to be 19.4% of wet weight, 
so cells are 80.6% water. The percentage of Risa space in a volume of packed 
cells in normal medium was 15% (eight determinations ranging from 14% to 
18%, SE = 0.57). This value was not significantly different for cells equilibrated 
with medium diluted eight-fold. 



132 



PHILIP B. DUNHAM AND F. M. CHILD 



40 




t 

initial 



20 



40 60 



80 100 120 
TIME IN MINUTES 



140 160 180 300 



FIGURE 2. Changes in cellular K and Na in Tetrahymena after dilution of normal medium. 
Ordinate: cellular K and Na concentrations (meq./l. cells); abscissa: time (minutes). Open 
symbols: Ki ; solid symbols: Nai. Initial Ki and Na ( (diamonds) are averages of values in 
normal medium. Dilutions were made at zero time. The extents of the dilutions of normal 
medium in the three experiments were as follows : triangles, 6-fold dilution ; circles, 8-fold 
dilution; squares, 13-fold dilution. 

In order to demonstrate how intracellular K and Na are maintained over a 
range of medium concentrations, cells were allowed to equilibrate for at least 
30 minutes in various dilutions of normal medium from two-fold to over 100-fold, 
the extreme dilution involving several distilled water washes. Figure 1 shows 



120 - 




120 140 160 180 200 220 



Normal K Normal Na 



K orNa :mM 



FIGURE 3. K and Na content of Tetrahymena in media concentrated with KC1 or NaCl. 
Ordinate: cellular concentrations of K and Na (meq./l. cells); abscissas: concentrations of K 
and Na in the media. Open circles: Ki ; solid circles: Na,. a and b : ordinate intercepts of 
the linearly increasing portions of the Na and K curves, respectively. Points for Ki and Nai 
in normal and diluted media are taken from Figure 1. 



ION REGULATION IN TETRAHYMENA 



133 



final intracellular concentrations of K and Na (Ki and Na t ) plotted against medium 
concentration (K and Na ). K, and Naj are quite constant over the range of 
medium dilutions investigated : average K 5 = 25.4 meq./l. cells and average Nai = 
5.0 meq./l. cells; Ki/Naj = 5.1. Figure 2 shows the results of three experiments 
in which changes of Kj and Nai were followed after six-fold and greater dilutions 
of normal medium. Changes in Na; take place within the first 30 seconds, after 
which Nai is constant. Kj decreases very slowly after medium dilution, so it is 
difficult to assign an equilibrium level. Therefore it was arbitrarily decided that 
Ki values in cells in dilute medium more than 30 minutes would be reported with 
the reservation that time for equilibration may involve a matter of days. (In 
one experiment, K } in cells in medium diluted 20-fold for two days was about 
half normal KI.) Rates of decrease of IM in cells in dilute medium were never 
greater than 10% per hour, and generally were much slower. 



2OO - 



o> 
o 



cr 
o> 






50 



100 
TIME IN MINUTES 



160 



FIGURE 4. Changes in volume and Na content of Tetrahymena after increasing the NaCl 
concentration of normal medium. Ordinates : cell volume (open circles); Na content per 
unit number of cells (crosses) ; Na content per unit volume of cells (solid circles). Abscissa: 
time (minutes). NaCl concentration of normal medium was increased by 176 inM at zero 
time. Ki per unit number of cells at 160 minutes was not significantly different from initial Ki. 

Cells were equilibrated for 30-120 minutes in media made more concentrated 
than normal in either K or Na (added as chlorides). Figure 3 shows Kj and Na 4 
values from these experiments plotted against K and Na , respectively. Both 
curves are linear above certain medium cation concentrations, with values 
of 0.51 for the slope of Ki./K,, above 11 inM K and 0.48 for the slope of Nai/Na 
above 20 mM Na . Below 20 mM Na , Na s is constant at 5.0 meq./l., whereas 
below 11 mM K , the K curve is roughly sigmoid. 

The kinetics of net influx of cation and the concomitant water movements 
were investigated by subjecting cells to sudden large increases in K or Na . 
Changes in packed cell volume, cation concentration per unit volume of cells, and 
cation concentration per unit number of cells were followed. Figures 4 and 5 
show the results of subjecting cells to increases above normal of 176 mM NaCl 
and 137 mM KC1, respectively. In both cases there is a large initial influx of the 



134 



PHILIP B. DUNHAM AND F. M. CHILD 



elevated cation (shown by increase in //,eq./10 7 cells) and large efflux of water 
(4-5% cell shrinkage in each case) in the first 1.5 minutes. K t per cell increases 
initially about 1.75 times, and subsequently increases slowly to 4.5 times the initial 
level by 200 minutes, accompanied by water re-entry. Na 4 per cell increases 
initially to 6 times initial level, and subsequently slowly increases to 13.7 times 
initial level at 160 minutes, with water re-entry. So equilibration in both cases 
is fast, but much faster in high Na medium. When Na t increased, final Kj per 
cell was not significantly different from the initial value, and likewise for Na } 
per cell after 200 minutes when Kj increased. This lack of reciprocal changes 
means that increases in KI and Nai are accompanied by proportionate increases in 



100 - 




100 
TIME IN MINUTES 



200 



FIGURE 5. Changes in cell volume and K content of Tetrahymcna after increasing KC1 
concentration of normal medium. Ordinates : cell volume (open circles); K content per unit 
number of cells (crosses) ; K content per unit volume of cells, solid circles. Abscissa: time 
(minutes). KC1 concentration of normal medium was increased by 137 mM at zero time. 

per unit number of cells at 200 minutes was not significantly different from initial 



some anion, or decreases in some other cation, if electroneutrality is preserved 
within the cells. (In a few experiments in which both K and Na were increased, 
both Kj and Nai increased in the same way as when studied singly, as described.) 

Preliminary analyses of Cl content of cells show that Cl does not balance 
increases in KI or Naj. Figure 6 shows Clj values in cells in normal, dilute, and 
high NaCl and KC1 media. Clj is quite constant in media ranging from a 2-fold 
dilution (14 mM C1 ) up to 123 mM C1 (99 mM K ) and 73 mM C1 (83.5 
mM Na ) : for 18 determinations, Clj averaged 6.4 meq./l. cells, ranging from 
to 13.2 meq./l. One set of determinations at 150 mM C1 (163 mM Na ) showed 
Qi as high as 37 meq./l., but Na ; was 78.5 meq./l. in this case. For this one 
deviant value, only one-half of Nai could be balanced by Cl, all other determinations 
showing no relationship at all between Clj and K 4 or Na^ 

Preliminary experiments were done on the washout of high K or high Na 
content of cells transferred to normal medium after one hour's equilibration in 



ION REGULATION IN TETRAHYMENA 



135 



tu 





_ 30 










^.20 


- 


E 










5 10 


4 !_J!___! 




A O 




4 i AA i 8 i i i 1 



t 



20 t 40 



60 



80 

CI : mM 



100 



120 



140 



FIGURE 6. Cl content of Tctrahymcna in normal, diluted, and high NaCl and KC1 media. 
Ordinate: cellular concentrations of Cl (meq./l. cells) ; abscissa: Cl concentration of the media. 
Triangles: Ch in normal and diluted media; open circles: Ch in high KC1 media; solid 
circles: Ch in high NaCl media. Arrow indicates Cl concentration of normal medium. Hori- 
zontal line indicates average of all Ch values in medium Cl concentrates ranging from 17 
mM to 123 mM. 

high K or Na medium. Cells were equilibrated in normal medium with the KC1 
concentration increased by 65 mM. Ten minutes after washing with normal 
medium, Ki had decreased from 64 meq./l. to 57 meq./l. , and after 45 minutes to 
48 meq./l. Initial Kj was 33 meq./l. Therefore a portion of elevated cellular K 
washed out much more slowly than it can be increased. This observation is 
consistent with the slow decrease of Kj from cells after dilution of normal medium. 
Cells were also equilibrated for one hour in normal medium plus 170 mM NaCl, 
then washed with normal medium. Ten minutes after washing, Na } was only 
slightly above normal Na ; , indicating that Na s is easily washed out of cells, as a 
portion of Na; is after dilution of normal medium. 



TABLE III 

Kinetics and extent of exchange of cellular K and Na with normal and diluted media 

containing trace amounts of K 42 or Na 24 . Each horizontal row of data represents 

at least two experiments with replicate determinations. 





Medium 
cone. 
(mM) 


Total 
cell cone, 
(meq./l. 
cells) 


Time for 
maximum 
exchange 

(minutes) 


Time for 
i maximum 
exchange 
(minutes) 


Exchangeable 
cell content 
(meq./l. 
cells) 


Unexchange- 
able cell 
content 
(meq./l. 
cells) 


Exchange- 
able 
fraction 


K, normal medium 


4.75 


31.6 


180 


30 


29.2 


2.4 


92.5% 


K, diluted medium 


0.83 


27.4 


180 


30 


25.0 


2.4 


91.0% 


Na, normal medium 


36.5 


12.7 


120 


<1 


11.2 


1.5 


88% 


Na, diluted medium 


3.0 


5.5 


120 


3 


3.25 


2.25 


59% 



136 PHILIP B. DUNHAM AND F. M. CHILD 

Table III summarizes the results obtained when cells were exposed for 5 
or more hours to normal and diluted media containing trace amounts of K 42 or 
Na 24 . Cellular K and Na are largely available for exchange with medium K and 
Na, and exchange is rapid, particularly the exchange of Na. However, small 
amounts of both K and Na do not exchange in a 5-hour period, although the 
exchange reaches a maximum level by three hours for IM and two hours for Na^ 
The amounts of unexchangeable IM and Na ; do not change significantly with 
medium dilution. 

DISCUSSION 

That Tctrahymena is capable of maintaining a high cellular K content relative 
to the K content of normal and diluted media is evident from Figure 1 and Table II. 
Ratios of Kj/Ko are of the order of 100 and higher in very dilute medium. Tetra- 
Jiynnena also retains a small amount of Na in very dilute medium. 

Sizable portions of cellular K and Na are exchangeable with the medium, as 
shown in Table III. Therefore K and Na are not retained in the cells by an 
impermeable membrane. Table III also shows, however, that small and constant 
amounts of both K and Na are unexchangeable. Since net changes are also evi- 
dent, exchange diffusion cannot be responsible for the ready exchange of isotopes. 

A system of internal binding sites with specific affinity for K is suggested to 
explain active K maintenance by Tctrahymcna. Na may be retained by a similar 
mechanism. In addition, a Na extrusion mechanism is proposed. 

Na in Tetrahymena is best explained in terms of a formal model involving 
compartmentalization of Nai. Two components of Na$ are constant, i.e. do not 
vary with Na . One, 1.9 meq./l., is unexchangeable with the medium. The second, 
3,1 meq./l. , is held constant, but is rapidly exchangeable. Forty-eight per cent 
of cell volume is free "Na space," and is available to a mobile Na component which 
is freely diffusible, and proportionate to Na . However, this mobile component 
is maintained 20 meq./l. of water less than Na by Na extrusion. Below 
Na there is no mobile Na component and Na; is constant at 5 meq./l., but at 
20 mAI Na the Na extrusion mechanism is operating maximally and mobile Nai 
begins increasing with Na . Since the other two Na components are constant, total 
Nat increase is linear and represents only the mobile component. There are no 
net exchanges between any of the compartments. These compartments can be 
visualized as physiological entities only, since no morphological significance can be 
attached to them. This model is suggested by the following points of evidence : 

( 1 ) Na extrusion is indicated first, by the constant level of Na 4 up to 20 inM 
Na , and second, by the negative intercept on the ordinate axis of the linearly 
increasing portion of the Naj/Nao curve (indicated in Figure 3). (Permeability 
of the cells to Na precludes passive exclusion of Na.) 

(2) The mobility of Na^ above the constant 5 meq./l. is apparent from the 
rapid equilibration of cells with high Na medium, and the rapid washout of Na 
from cells both when normal medium is diluted and when high Na cells are washed 
in normal medium. 

(3) The linearity of the Nai/Na curve above 20 mM Na and the mobility 
of Nai above 5 meq./l. allow one to conclude that the slope of the Na curve, 0.48, 
represents the fraction of cell volume occupied by the mobile Na component, or 
"Na space." 



ION REGULATION IN TETRAHYMENA 137 

(4) The magnitude of the gradient effected by maximum Na extrusion is pro- 
portional to the magnitude of the negative intercept (about 5 meq./l. cells) cor- 
rected for the constant amount of retained Nat (5 meq./l. cells), or 10 meq./l. cells. 
Since the Na space is 48%, the difference in Na concentration between medium 
and cell water is 10/0.48 = 20.8 meq./l. water. This value should and does corre- 
spond closely to the medium Na concentration at which the Na extrusion mecha- 
nism becomes saturated. This saturation concentration, the "threshold" of Na, 
increase, is analogous to the threshold of glucose excretion in renal tubules at the 
concentration of maximum glucose reabsorption (Shannon and Fisher, 1938), 
which was also interpreted as representing saturation of an accumulating mechanism. 

(5) The evidence for the unexchangeable and exchangeable but constant com- 
ponents of cellular Na has already been presented. 

The nature of the preservation of electroneutrality in the cells upon Na or K 
entry is not at all clear. There is definitely no reciprocal relationship between Na 
and K. There is little intracellular Cl, even upon large increases in the medium 
of either NaCl or KC1. These results also obviate explaining active retention of 
K in terms of a Donnan equilibrium resulting from Na extrusion, unless Cl is 
also specifically excluded from the cells, which seems unlikely at present. If a 
Donnan situation obtained, the relationship Kj/K = Cl /Cli would be expected 
in hold in any medium, and it obviously does not. With increasing medium KG. 
Kj/Ko decreases to less than 1, while Cl /Cli increases to as high as 22. The 
nature of the Cl exclusion is suggested here to be electrostatic rather than a matter 
of specificity or impermeability. Possibly cellular K and Na are associated with 
fixed anionic groups more or less strongly, depending on the ion and the medium 
concentration of the ion. Steinbach (1947) suggested that K is always associated 
with organic components which occupy spaces unavailable to Cl. This still 
does not explain the preservation of electroneutrality. This problem is under 
investigation. 

The KI/KO gradients in normal and diluted media are definite evidence for 
specific K retention. The rapid and nearly complete exchange of K ; and the 
rapid net increase in KI with K make membrane impermeability or any mem- 
brane involvement unlikely explanations for K retention. 

In media with K higher than normal, the slope of the Kj/K curve is steep up 
to 1 1 inM K , above which KI increases less sharply with K , and in a linear fashion. 
The slope of this portion of the curve is 0.51, close to the slope of the Nai/Na 
curve (0.48), suggesting that this increasing KI is occupying a cellular space iden- 
tical with the "Na space." 

However, a portion of this increased cellular K does not readily wash out of 
the cells, and is not nearly as mobile as high cellular Na. Cells equilibrate less 
rapidly with high K medium than with high Na medium. Increased Ki is not 
accompanied by reciprocal Na changes or by Clj increase, as noted above. These 
considerations suggest that the increase in K t involves association with previously 
"empty" K binding sites in the "Na space." (Steinbach (1940) reported K increase 
without Na decrease or Cl increase in Phascolosoina muscle. He suggested (1947) 
that vertebrate skeletal muscle behaves as though there were a limited number 
of groups capable of binding K which are normally saturated, whereas heart and 
invertebrate muscle are normally not saturated with K.) The slower rise of K, 



138 PHILIP B. DUNHAM AND F. M. CHILD 

with K greater than 11 mM and the linearity of this rise suggest that the actively 
maintained, or bound, K is at a maximum above 11 mM K . Then the intercept 
of the linear portion of the Ki/K curve at the ordinate axis, 43 meq./l. (shown in 
Figure 3), should be the level of maximum actively maintained K. In cells equili- 
brated in high K medium, and washed in normal medium, Ki fell to 48 meq./l. 
after 45 minutes, a concentration comparable to the ordinate intercept of the linear 
KI/K O . This constitutes additional evidence for the maximum saturation of bound 
K. Initially it would appear that K maintenance in Tctrahymena, because of the 
sigmoid shape of the Ki/K curve below 11 mM K , does not fit elementary 
Langmuir adsorption theory (applicable to Michaelis enzyme kinetics), which it 
should if a system of binding sites with a saturation level is invoked. However, 
it is likely that this is not the true shape of the curve. The K washout experiment 
described above shows that K i; although it can be rapidly increased, can be only 
slowly washed out. Since the initial K in the experiments involving changing K 
was always that of normal medium, an inflection in the Ki/K curve is expected 
at the K of normal medium, and it is observed (see Figure 2). Therefore the 
explanation of the sigmoid shape of the curve lies not in the nature of the K 
retention mechanism, but in the experimental procedure. Then the real relation- 
ship between actively maintained K and K should fit a Langmuir adsorption iso- 
therm, and a binding sites mechanism for K retention is consistent with the data. 

No comparable obscuring factor exists in the case of Na s since Na apparently 
washes in and out of cells with equal facility in the range of medium concentra- 
tions investigated. The Na retention system is apparently saturated at a very 
low Na . 

The data suggest compartmentalization of K as well as Na : unexchangeable K 
(2.4 meq./l.), exchangeable but bound K (maximum about 43 meq./l.), and freely 
diffusing K, with no threshold K . (See Cowie, Roberts and Roberts, 1949, for 
a discussion of compartmentalization of K in E. coli.) 

The hypothesized ion regulatory machinery of Tctrahymena, shown to be con- 
sistent with the data, consists, first, of a system of internal binding sites which 
specifically accumulate and retain K ; second, a system for retention of a constant, 
low level of Na, and third, a Na extrusion mechanism. Cl plays little role in ion 
balance in Tctrahymena. Cellular K and Na are separable into three components : 
unexchangeable, exchangeable but bound, and mobile components. 

The Na extrusion mechanism may facilitate water removal, and therefore may 
be associated with the contractile vacuole. The water economy of Tctrahymena 
may be analogous to that of other fresh-water animals, in that Tctrahymena may 
not be capable of secreting pure water, but water removal may be facilitated by 
ion secretion (cf. Prosser et al., 1950). 

The relationship of Na retention to Na extrusion and/or K retention, cannot 
be decided from the data. Retained Na may represent lack of complete specificity 
of the K binding sites, but in this case a reciprocal relationship between actively 
retained K and Na would be expected. The retained Na might be a reservoir 
necessary for vacuolar function. This possibility is consistent with the rapid Na 
turnover indicated by rapid exchange, but cannot be easily resolved with the 
constancy of the retained Na relative to Na . A third possibility is a specific 
protoplasmic requirement for a low, constant Na level, for which there is no 



ION REGULATION IN TETRAHYMENA 139 

evidence here and little precedent. (Hydra (Lenhoff and Bovaird, 1960) and 
Chilomonas (Pace, 1941) have possible specific Na requirements.) 

The specificity of K retention in Tetrahymena indicates a protoplasmic K re- 
quirement. (Kidder ct al. (1951) demonstrated a nutritional requirement for K 
in Tetrahymena.} The similarity of levels of K in Tetrahymena and the other 
animals listed in Table I suggests a minimum requisite protoplasmic level of K. 
It is often held that high cellular K is only a reflection of a Donnan equilibrium 
resulting from Na extrusion (see Hodgkin, 1951). Carter (1957) attributes K 
maintenance in Spirostomum to Na exclusion. This has been shown here not to 
be so in Tetrahymena, and Robertson (1957) has shown that in a number of ani- 
mals, including some marine invertebrates, a portion of cellular K cannot be 
accounted for by a Donnan equilibrium, but must be due to specific K retention. 
Steinbach (1947) suggests that cellular K is regulated relative to a constant proto- 
plasmic composition rather than to serve an osmoregulatory function. No doubt 
in marine animals and vertebrates, with their relatively high ionic content, some 
cellular K is held non-specifically to preserve electroneutrality. Cellular K levels 
vary considerably, particularly among fresh-water animals, and probably reflect 
the ability to regulate body fluids, and to an extent the activity of the animal. 
Therefore in vertebrate cells, there may be a K component reflecting a Donnan 
equilibrium plus a component serving a specific protoplasmic role. Since evolu- 
tion of basic cellular mechanisms is generally conservative, a similarity between K 
retention mechanisms in Tetrahymena and other animals is an attractive possibility. 
Tetrahymena affords a system for studying this mechanism without high ion con- 
centrations and large Donnan effects, which would obscure specific K retention in 
other animals. 

Akita (1941) reported data on Na, K and Cl contents of Paramecium which 
were comparable to the data presented above on Tetrahymena. 

SUMMARY 

1. The K and Na content of Tetrahymena pyriformis has been determined, and 
the mechanisms of ionic regulation were investigated. 

2. The main findings were : K and a small amount of Na are maintained in 
very dilute medium. Cellular K and Na are readily exchangeable with K and Na 
of the medium. However, small, constant amounts of each are unexchangeable. 
Cells rapidly equilibrate with media high in K or Na. High K washes out of cells 
slowly, whereas Na enters and washes out of cells with equal facility. There is 
no reciprocal relationship between cellular K and Na. Tetrahymena contains little 
Cl. Increases in cellular K or Na are not accompanied by increases in Cl. 

3. The results are interpretable according to the following proposals : K is 
specifically accumulated and retained by a system of internal binding sites with a 
saturation level. Na is probably retained by a separate mechanism. There is also 
a Na extrusion mechanism which has no relationship with K or Na retention. 
Cellular K and Na are compartmentalized into three components : unexchangeable, 
exchangeable but bound, and freely diffusible components. 

LITERATURE CITED 

AKITA, Y. K., 1941. Electrolytes in Paramecium. Mem. Fac. Sci. Agric., Taihoku hup. U., 
23 : 99-120. 



140 PHILIP B. DUNHAM AND F. M. CHILD 

CARTER, L., 1957. Ionic regulation in the ciliate Spirostomwn ambiguum. J. Exp. Biol., 34: 

71-84. 
COWIE, D. B., R. B. ROBERTS AND I. Z. ROBERTS, 1949. Potassium metabolism in E. coli. 

I. Permeability to sodium and potassium ions. /. Cell. Comp. Physiol., 34 : 243-257. 
HAYES, F. R., AND D. PELLUET, 1947. Electrolytes in mollusc blood and muscle. /. Mar. Biol. 

Assoc., 26 : 580-589. 
HODGKIN, A. L., 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Rev., 

26: 339-409. 
KIDDER, G. W., V. C. DEWEY AND R. E. PARKS, 1951. Studies on the inorganic requirements 

of Tctrahymcna. Physiol. Zool., 24 : 69-75. 
KITCHING, J. A., 1954. Osmoregulation and ionic regulation in animals without kidneys. 

Symp. Soc. Exp. Biol., 8 : 63-75. 
KLEIN, R. L., 1959. Transmembrane flux of K 42 in Acanthamocba. J. Cell. Comp. Physiol., 

53 : 241-258. 
LENHOFF, H. M., AND J. BOVAIRD, 1960. The requirement of trace amounts of environmental 

sodium for the growth and development of Hydra. Exp. Cell Res., 20 : 384-394. 
LILLY, S. J., 1955. Osmoregulation and ionic regulation in Hydra. J. Exp. Biol., 32: 423-439. 
PACE, D. M., 1941. The effects of sodium and potassium on metabolic processes in Chilomonas 

paramccium. J. Cell. Comp. Physiol., 18 : 243-255. 
PROSSER. C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. J. WULFF, 1950. Com- 

partive Animal Physiology. W. B. Saunders Co., Philadelphia. 

ROBERTSON, J. D., 1957. Osmotic and ionic regulation in aquatic invertebrates. In : Inverte- 
brate Physiology. B. Scheer, ed., University of Oregon Publications, Eugene, Oregon, 

pp. 229-2*46. 
SHANNON, J. A., AND S. FISHER, 1938. The renal tubular reabsorption of glucose in the normal 

dog. Amcr. J. Physiol, 122 : 765-774. 
STEINBACH, H. B., 1940. The distribution of electrolytes in Phascolosoma muscle. Biol. Bull. 

78: 444-453. 
STEINBACH, H. B., 1947. Intracellular inorganic ions and muscle action. Ann. N. Y. Acad. 

Sci., 47 : 849-874. 
WILLMER, E. N., 1958. Some further factors affecting the metaplasia of an amoeba (Naeglcria 

(intbcri). J. Embryol. Exp. Morph., 6: 187-213. 
ZEUTHEN, G., 1939. On the hibernation of Spongilla lacustris (L.). Zeitschr. vergl. Physiol. 

26: 537-547. 



FURTHER STUDIES ON ALLOCENTROTUS FRAGILIS, A 

DEEP-SEA ECHINOID 1 

ARTHUR C. GIESE 

Department of Biological Sciences, Stanford University, Stanford, California 

In a previous paper some data on the natural history and breeding of a deep- 
sea echinoid, Alloccntrotus jragilis, were presented (Boolootian ct al., 1959). 
Further studies are reported here, primarily to define more clearly the breeding 
season of the species, as well as to get further information on its nutrition. 

BREEDING SEASON 

The breeding season in the previous study appeared to coincide with winter 
(December to March) but remained uncertain because storms on Monterey Bay 
during the critical period interfered with collecting at the time the boat was avail- 
able. For the present study the urchins were collected from the same beds and by 
the same methods as previously, and the gonad index (the ratio of gonad volume 
to wet weight times 100) was used to estimate the breeding condition of the speci- 
men as before. The breeding activity has now been followed for almost three years, 
and although the data for each year are incomplete, pieced together for the entire 
period in Figure 1, they give support to the notion that a single breeding season 
occurs in this species. Perhaps several periods of spawning and redevelopment 
of eggs occur in a given individual of this species but monthly sampling does not 
give information on this point. However, the appearance of germinal vesicles in 
spawned-out ovaries suggests just this. It proved impossible to keep specimens 
in healthy condition in the laboratory for more than about a month, although it 
was noticed that the red-spot "disease" was much less frequent in animals kept in 
aquaria in the dark (Araki, personal communication). It may prove possible to 
keep the animals in the laboratory for a longer time once the most favorable con- 
ditions are discovered. 

More decisive evidence for an annual cycle is obtained from a study of teased 
pieces of the gonads and from attempts to fertilize the eggs. Such a study indicates 
that although the gonad index may be high and sperms may appear in September, 
October and November, the eggs are almost all in the germinal vesicle stage, each 
with a large nucleus. Such eggs do not mature after shedding and in no case are 
they fertilized on addition of active and presumably mature sperm. In December 
most of the females had ripe eggs and only occasional germinal vesicles were seen 
among them. The eggs fertilized and developed into normal plutei. The same 
was found to be true during February and March. Some of the females examined 

1 1 am indebted to Messrs. George Araki, Peter Glynn and Joseph Balusteri for collecting 
the Allocentrotus; to Messrs. John Lawrence and James Stanley for help with some of the 
chemical determinations ; and to Mr. Albert Towle for help with some of the respirometric 
determinations. 

141 



142 



ARTHUR C. GIESE 



had few eggs but those eggs which remained in the ovaries fertilized and developed 
normally, suggesting that they were only a remnant, the bulk of the gametes having 
been released. During the period immediately following the breeding season, few 
females had eggs and germinal vesicles again became apparent in some. Thereafter, 
all the teased gonads examined microscopically appeared indeterminate as to sex. 
Apparently the tissue had entered a resting stage. The gonads remain indeter- 



X 

LU 

Q 4 



Q 



1957 
1958 
l959 
I960 



GERMINAL 
* VESICLES 



FERTILE EGGS 



INDETERMINATE 




N D 



F M 
MONTH 



M 



FIGURE 1. Reproductive cycle of Allocentrotus fragilis as measured by the gonad index 
(size of gonad relative to body weight) and presence and ripeness of the gametes. The 
gametes were studied closely only during 1959-60. 

minate for several months but at the end of summer sperms can be seen and small 
germinal vesicles again make their appearance, long before the gametes form. 
Active sperms are present over a much wider span of time than mature eggs. 

NUTRITION 

Since the intestines of Allocentrotus brought in from the field are sometimes 
devoid of food, and at other times have very little, it would appear that the urchins 
may go for long periods of time without food. This seems likely since defecation 
may continue for a week or more in the laboratory, indicating slow digestion of an 
ample meal. In the previous study, on only one occasion were we fortunate enough 



STUDIES ON A DEEP-SEA URCHIN 



143 



to obtain specimens richly charged with food when a diatom bloom occurred in the 
area. In the present study many collections yielded animals relatively full of 
diatoms, presumably because of similar blooms. 

On November 7, 1960, a collection was made nearer the edge of the urchin bed 
in an attempt to get smaller specimens for a study of respiration. The intestines 
of these specimens were rilled with bites out of large algae : green, red and brown. 
The fragments of algae were irregular and much larger than the balls of diatoms 
illustrated in the previous paper. In the foregut the algae were undigested and 
had little of the gelatinous material around them. In the hindgut more fully 
digested algae were enclosed in the mass of gelatinous material within which were 

TABLE I 

Chemical composition of gonads and gametes of Allocentrotus fragilis in per cent of dry weight 



Tissue 


Condition 


Lipid 


Non-protein N 


Protein 


Glycogen 


Testis 


gravid 


14.54 
15.34 
13.08 


3.26 
3.62 

4.24 


28.70 
30.25 
31.93 


0.36 


Testis 


starved 
animal 


18.6 

17.7 











Testis 


spent 


12.36 


3.66 


36.13 





Sperm 





3.60 


4.22 


38.7 





Gonad 


indeterminate 


14.50 

17.85 


3.65 
3.66 


23.88 
27.05 


0.83 


Ovary 


gravid 


17.79 
14.61 
15.01 


3.40 
4.40 
2.94 


34.09 
32.47 
27.39 


0.69 


Ovary 


starved 
animal 


20.8 
20.0 











Ovary 


spent 


12.83 


3.17 


20.87 





Eggs 





18.07 


2.44 


28.68 






many bacteria, much as previously described in Strongylocentrotus purpuratus 
(Lasker and Giese, 1954). Fecal pellets collected from animals which had been 
in the laboratory aquaria overnight retained their shape and looked more like the 
rounded pellets previously described from A. fragilis. The algal fragments in many 
of them were almost completely digested, only colorless pieces remaining. In only 
one previous collection of Allocentrotus had individuals with pieces of larger algae 
(Cladophora) in the gut been obtained, all the others having diatoms and fragments 
of various minute materials present at the bottom of the sea. The November 7th 
collection followed rough seas which may have torn algae from the rich offshore 
beds nearby, making them available to the urchins. 

When Allocentrotus, kept in the laboratory without food for a month, were 



144 



ARTHUR C. GIESE 



dissected, they were found to be free of intestinal contents. Since in some col- 
lections urchins were observed free of food, it is likely that in their natural environ- 
ment they are occasionally unable to get food for a comparable period of time. A 
store of nutrients is therefore necessary to maintain the urchins between the sporadic 
droppings of material from the surface waters. The sea urchin has three major 
organs in which storage might occur : gonads, intestine and body wall. The latter 
(hereafter called the test) consists of the test proper, the epidermis, the tissues of the 
coelomic lining and the water vascular system attached to it. Biochemical analyses 2 

TABLE II 

Chemical composition of intestine, intestinal contents, and shell of sea urchins in 

per cent of dry weight 



Tissue 


Condition 


Lipid 


Non-protein N 


Protein 



Allocentrotus fragilis 



Intestine 


well-fed 


28.88 


2.17 


35.38 






26.81 


3.13 


31.22 






23.24 


2.77 


38.84 




starved 


20.6 








Contents 


fresh meal 


8.28 


1.66 


22.83 




feces (rectal) 


1.87 ' 


0.33 


7.15 


Test 


well-fed 


1.79 


0.22 


5.66 




starved 


0.9 












0.8 


- 





Diatoms* 


entire 


8 





28.1 



Strongylocentrotus purpuratus 



Foregut 


well-fed 


12.28 


3.84 


39.33 


Hindgut 


well-fed 


12.52 


3.30 


33.94 


Contents 


fresh meal 


3.88 


0.34 


10.70 



Strongylocentrotus franciscanus 



Gut 


well-fed 


22.2 
18.7 









* From Pease, 1932; 63.2% carbohydrate present in diatoms. 

indicated that the fragile urchin, like the purple sea urchin (Giese et al., 1958), 
stores considerable lipid and a small amount of glycogen in its tissues, as seen in 
the data in Tables I and II. Protein, the main structural constituent of protoplasm, 
is also present in considerable quantity, as expected in any tissue. It would appear 
that the main reserve food is lipid, glycogen being a minor reserve. Since relatively 
little sugar appears in the body fluid, the latter finding is perhaps not surprising. 

2 The methods employed were like those described elsewhere (Giese et al., 1958). Many 
of the measurements were done in triplicate ; later only duplicates were run since the repeats 
were so much alike. 



STUDIES ON A DEEP-SEA URCHIN 



145 



The data in Table I show that considerable lipid is present in the gonads of 
Allocentrotus, a bit more in the gravid than in the spent ones. There is more lipid 
in the eggs than in the ovary but relatively little in the sperms taken alone. With- 
out comment, the data in Table I do not express fully the meaning of the changes 
in the organic content of the gonads of the animals during the breeding season. 
The gonad index varies by a factor of at least 5 (Fig. 1), the maximal variability 
being 9-fold. Therefore, the lipid content per unit dry weight of a gonad is not 
a true measure of the reserves, since the shrunken gonad of a spent or indeterminate 
gonad may be only one-fifth the size of the gravid gonad. The total lipid present 
in the gonad of a gravid animal would be at least 5 times as great as in a spent 
animal. 

TABLE III 
Wet and dry -weight of tissues and tests* 



Tissue 


Wet wt. 


Dry wt. 


% solid 


% water 


GI** 


% body fluid 



Allocentrotus fragilis 



Whole 


35.6 


3.8 


10.6 


89.4 


2.06 


58 


Whole 


66.9 


6.25 


9.4 


90.6 






Whole 


96.6 


11.38 


11.8 


88.2 


2.63 


62 


Gonad (cf) 


2.54 


0.51 


20 


80 






Gut 


2.1 


0.66 


31.3 


68.7 






Lantern 


1.75 


1.06 


60 


40 






Test 


22.7 


7.45 


32.8 


67.2 







Strongylocentrotus purpuratus 



Whole 


98.3 


32.55 


33.2 


66.8 


8.6 


22.3 


Whole 


64.6 


24.5 


38 


62 


10.83 


30.3 


Gonad ( 9 ) 


7 


1.45 


20.6 


79.4 






Gut 


2.64 


0.95 


36 


64 






Lantern 


2.2 


1.6 


73 


27 






Test 


3.2 


20 


61 


39 







* Some data for Strongylocentrotus are given for comparison to Allocentrotus. Note the more 
massive skeleton in Strongylocentrotus, its lesser water content, and the lesser amount of body fluid. 
** GI refers to gonad index (defined in the introduction). 

Lipid is stored in quantity in the intestine and body wall. Analyses indicate 
that as much as 29% of the dry weight of the gut may consist of lipid, but only 
about 2% of dry weight of the test and its tissues consists of lipid. 

While the per cent lipid content of the test appears small, it must be remembered 
that the test forms a considerable part of the entire dry weight of the urchin (Table 
III). An urchin which when wet weighs 96.6 grams, weighs only 11.38 grams 
when dry, including the body fluid salts, indicating that 88.2% of its wet weight is 
water. The dried gonads weigh 0.51 gram and the intestines, washed free of gut 
contents and dried, about 0.66 gram. The dry test and lantern weigh 9.5 grams. 
According to the data in Table II about 1/20 of the test (5.66%) is protein, which 
is probably an approximate measure of the amount of tissue present. Therefore, 



146 ARTHUR C. GIESE 

about 5.66% X 9.5 grams, or 0.54 gram of tissue, is probably present in the test. 
The amount of tissue in the test is thus probably equal to, or greater than that in 
the gut (no account was taken of the other organic constituents in the above cal- 
culations). It is possible that some protein forms a network in the test, in the 
interstices of which the salts are deposited, since the echinoderm test is supposedly 
a mesodermal structure like vertebrate bone (Hyman, 1955). 

To compare the relative stores of lipids in gonad, test tissue, and gut, it is only 
necessary to multiply the amount of each tissue by its content of lipid. In this 
respect it appears that 9.5 grams X 1.79% or 170 mg. are stored in the test and 
lantern, 0.5 grams X 15.5% or 77 mg. are stored in the gonad, and 0.66 grams X 
26.8% or 176 mg. are stored in the gut of an animal which weighs 96.6 grams wet 
weight. This shows that in regard to its storage of lipid, the test and intestine 
may be of equal importance in an animal of intermediate gonad index (2.63) such 
as the one tested here. For an animal with an index of 5 the amount of lipid stored 
in the gonad would be increased by a factor of almost 2, making it about equal to the 
gut or test. For an animal of low index (1.5 to 0.8) the amount of lipid stored 
in the gonad would be reduced by a factor of 1/2 or 1/3, and the stores in the 
intestine and the tissues of the body wall would then be of even greater importance. 

Since so much lipid is stored in the intestinal walls, it seems most likely that 
the food is either the source of the lipid or that the lipid is manufactured from 
carbohydrates or protein in the diet. The gut contents vary in lipid, a relatively 
fresh meal of diatoms containing 8.28% of lipid, whereas a well-digested mass of 
material contains only 1.87% of lipid (Table I). Pease (1932) lists the lipid 
content of diatoms as 8% of the dry weight. a It therefore seems likely that the 
lipid probably is obtained from the diatoms and other food eaten by the urchin. 
The lipid is probably digested and stored, some of it in the gut, some in the other 
tissues. 

In this respect it is interesting that another animal feeding upon diatoms, the 
sipunculid worm Phascolosoma agassisi, also has large stores of lipid in its gut, 
about 25% of the dry weight (A. Towle, personal communication). On the other 
hand, the data in Table II show that the purple sea urchin, Strongylocentrotus 
purpuratus, which feeds upon larger algae, stores only half as much lipid in its gut 
as AllocentrotHs. Nonetheless, lipid is prominent and a fairly large amount is 
present in the intestinal pellets, 3.88% of the dry weight in a fresh meal in the 
intestine. It is surprising in this regard that 6\ franciscanus, which has a diet 
much like 5\ purpuratus, has much more lipid stored in its intestine (Table II). 
Algae may at times also accumulate considerable lipid (Milner, 1953; Fogg and 
Collyer, 1954). Perhaps S. franciscanus has a diet richer in lipids than S. pur- 
puratus. 

How the nutrient gets from the gut to the other tissues is not known at present. 
Lipid may pass out as small droplets of fatty material like the chylomicrons of 
mammals or it might be carried out and distributed by wandering cells. Lipid is 
present in the body fluid but the exact amount has not been determined for lack 
of adequate methods. 

3 Diatoms in culture do not always have this much lipid. According to Barker (1935) 
diatoms in a culture in the laboratory first synthesize carbohydrates during photosynthesis, the 
ratio of oxygen production to carbon dioxide consumption being unity. However, as the diatoms 
age they accumulate oil which is visible in droplets. 



STUDIES ON A DEEP-SEA URCHIN 



147 



Upon starvation, stored nutrients are utilized. This was most evident in the 
shrinkage of the gonads of starved animals. Seven Allocentrotus starved for a 
month in an aquarium supplied with running sea water showed somewhat shrunken 
gonads at least the index for the animals at the time of collection on November 6 
was 3.2 a month later one would expect it to have risen to about 4.7 ; instead the 
gonad index for the starved animals was 2.5 on December 7. Similar shrinkage 
had previously been noted for the purple shore urchin (Lasker and Giese, 1954). 
It is probable that the nutrients stored in the gonads had been resorbed. While it 
seemed likely that the lipids were preferentially utilized, biochemical analysis 
revealed that per unit weight lipid increased in amount in the gonad, although it 
decreased both in total quantity in the animal and per unit weight in the body wall 
and the intestine. While the increase in lipid content per unit weight in the gonad 
may appear paradoxical, it is to be expected if one or more of the other nutrients 
in the gonad are utilized at a more rapid rate than the lipids. Since the total bulk 
of the gonad shrinks and the only other major organic material present in the gonad 

TABLE IV 
Respiration of tissues of Allocentrotus* 



Tissue 


Qo^liS 
/il./mg./hr. 


R.Q. 


Water content 
per cent 


Number of 
experiments 


Test 


0.076 


0.58 


65.6 


2 


Testis 


0.570 


0.92 


80.4 


3 


Ovary 
Gut 


0.088 
0.383 


0.65 
0.57 


80.8 
79.2 


8 
10 



* Note the high respiratory rate for the testis and gut as compared to the ovary and test. 

is protein, proteins are probably being selectively metabolized in the gonads during 
the period of starvation. Wilber (1947) has described similar results after starva- 
tion of Phascolosoma gouldii. 

RESPIRATION 

A few studies were made of the respiration of Allocentrotus tissues, primarily 
with a view of determining how it compared with other marine animals. The 
respiratory quotient was determined to ascertain, if possible, what types of foods 
were being used by the urchins. 

The data are given in Table IV. It is at once apparent that the rate of tissue 
respiration is comparable, per unit wet weight, to that for other sea animals (Nicol, 
1960, p. 152). Of greater interest is the respiratory quotient characterizing the 
respiration of the sea urchin. If lipid is of importance in the economy of the sea 
urchin, a respiratory quotient of about 0.7 might be expected. If the urchin uses 
carbohydrates or mixtures of these with proteins and lipids, the respiratory quotient 
should be higher, approaching 1.0 when only carbohydrate is utilized. 

Determination of the respiratory quotient of entire animals proves difficult 
because at the end of an experiment it is necessary to liberate the carbon dioxide 
which is trapped in the buffering system of the sea water bathing the urchins. Since 
to do this the sea urchins must be removed from the vessel, the extra manipulations 



148 ARTHUR C. GIESE 

may permit the sea water to equilibrate with the air. Furthermore, the urchins 
shed some spines, pedicellariae or other skeletal pieces containing lime salts. Con- 
sequently, it is necessary to filter such water through bolting cloth to get rid of the 
calcareous materials. This involves still another step during which equilibration 
of the bathing water can occur with air, further vitiating the correction. The R.Q. 
for an entire animal small enough to fit into a Warburg flask was 0.7, suggesting 
lipid utilization. However, if any carbon dioxide had accumulated in the sea water 
during respiration, the manipulations preceding addition of acid and measurement 
might have liberated it, favoring a lower R.Q. value. Therefore, the data cannot 
be considered satisfactory since the method is unsatisfactory. 

To by-pass this difficulty the gonad and gut tissues of the urchins were removed 
from animals, washed in sterile sea water, and the pressure changes measured 
manometrically in the presence of KOH in one series, and in the absence of KOH 
in another. In the latter case 3 N sulfuric acid was contained in the side arms, 
and at the end of the experiment the acid was added to liberate the excess carbon 
dioxide contained in the sea water surrounding the tissues. The data for respira- 
tion of the tissues in Table IV are therefore more satisfactory than those for the 
entire animal. Excepting the testes, the R.Q. for the tissues is between 0.6 and 
0.7, definitely suggesting lipid metabolism. Since the experimental deficiencies 
mentioned for the studies on the entire animal do not apply, the data on tissues are 
more convincing than those for the entire animal. Presumably the respiration of 
the entire animal is the sum of the respiration of its various parts (Field ct ol., 
1939). Consequently, one might suppose that the data for the tissues are appli- 
cable to the entire sea urchin. The high R.Q. for the testes could result from 
utilization of carbohydrate along with some other nutrients. 

DISCUSSION 

The present study suggests that lipid may play a significant role in the economy 
of Allocentrotus. The sea urchin has a considerable supply of lipid in its usual 
diet of diatoms. It stores considerable lipid in its intestine and gonad and some 
even in the tissues adherent to the skeleton. Furthermore, the stores of lipids 
decrease in amount when the sea urchin is starved for a month. The amount of 
lipid is thus closely related to the nutritive state of the animal. This has proven 
to be the case in other echinoderms from this area (Giese, 1959) and in other 
regions as well (unpublished). 

The small amount of glycogen found in tissues of Allocentrotus suggests that 
either some other kind of carbohydrate is stored in this urchin or else that carbo- 
hydrate plays a minor role here. Glucose does not increase the respiration of gut 
or gonadal tissue here, just as it failed to do in tissues of a purple sea urchin. 

The respiratory quotient for the tissues studied gut, test and ovary is about 
0.6 to 0.7. This indicates that some lipid is being used for respiration ; that is, it is 
being metabolized. Addition of glucose does not change the R.Q. It thus appears 
possible that lipid is being used preferentially for metabolism although it is more 
likely that added sugar fails to stimulate respiration because it fails to enter the 
tissues. 

In view of the apparent reliance of Allocentrotus on lipids, its occasional eating 
of large algae red, brown and green which have little lipid, but much polysac- 



STUDIES ON A DEEP-SEA URCHIN 149 

charide, is interesting. Either Allocentrotus uses only the readily digestible ma- 
terials in the algae or it has enzymes to utilize some polysaccharides, as does the 
purple sea urchin (Huang and Giese, 1958; Eppley and Lasker, 1959). The algae 
in the fecal pellets of Allocentrotus feeding on large algae were rather completely 
digested, suggesting that more than the lipids are being utilized. It would be 
interesting to know whether the urchin itself has such enzymes, and secondly, 
whether the monosaccharides obtained are stored as polysaccharides in the urchin 
or are converted to lipid. 

The observation that Allocentrotus takes in large algae indicates that it is more 
resourceful than had been previously considered (Boolootian ct al., 1959). In the 
laboratory it failed to eat the large algae but apparently it does so when it gets them 
in nature. 

While Allocentrotus can withstand starvation at least a month, and probably 
longer, judging from the healthy appearance of the specimens starved (in the dark) 
for over a month, in nature this is probably not the rule since dissected animals 
usually had at least a few pellets in the gut. Specimens choked with pellets of algae 
at the time they were collected had nothing whatsoever in the gut. 

No clear-cut correlation between the breeding season and the availability of 
food has been observed in the monthly collections. An annual reproductive cycle 
is suggested by the present study but the stimulus to the development of the gonads 
still remains elusive. 

SUMMARY 

1. Additional data on the size of the gonads relative to the body and the 
presence and ripeness of gametes and their maturity were gathered for a year on 
the deep-sea echinoid, Allocentrotus jragilis. 

2. The data indicate that the breeding season is an annual cycle with a maximum 
gonadal size in January and February, accompanied by the presence of mature eggs 
and sperm. 

3. The sea urchins in one collection were found to have fed on large algae, the 
pellets resembling those of the intertidal sea urchin, Strongylocentrotiis purpuratus. 

4. A considerable quantity of lipid is found to be stored in the wall of the gut, 
less in the gonad, and still less in the body wall (per unit dry weight). Total 
amount of stored lipid is largest in the gut, next in the test, and least in the gonad. 

5. The usual diatom diet of the sea urchin contains much fat; the algae tested 
in one series contain considerably less. 

6. The respiratory quotient of the gut, ovary and test of the sea urchin was 
found to be about 0.6 to 0.7, suggesting utilization of lipids. The R.O. for the testis 
was 0.92. 

7. Some comparisons are made between Allocentrotus and the purple intertidal 
urchin, Strongylocentrotus purpuratus. 

LITERATURE CITED 

BARKER, H. A., 1935. Photosynthesis in diatoms. Arch. Mikrobiol., 6: 141-156. 
BOOLOOTIAN, R. A., A. C. GIESE, J. S. TUCKER AND A. FARMANFARMAIAN, 1959. A contribution 

to the biology of a deep-sea echinoid Allocentrotus fragilis (Jackson). Biol. Bull., 

116: 362-372. 



150 ARTHUR C. GIESE 

EPPLEY, R. W., AND R. LASKER, 1959. Alginase in the sea urchin, Strongylocentrotus pur- 

puratus. Science, 129: 214-215. 
FIELD, J., II, H. S. BELDING AND A. W. MARTIN, 1939. An analysis of the relation between 

basal metabolism and summated tissue respiration in the rat. /. Cell. Comp. Physiol, 

14: 143-157. 
FOGG, C. E., AND D. M. COLLYER, 1954. Accumulation of fats as a characteristic of certain 

classes of algae. Congr. intern, hot. P/u is, !\'<ipps. et communs., 28 : 12530a. Chem. 

Abstr., 48: 11568. 

GIESE, A. C., 1959. Comparative physiology : annual reproductive cycles of marine inverte- 
brates. Ann. Rev. Physiol, 21 : 547-576. 
GIESE, A. C., L. GREENFIELD, H. HUANG, A. FARMANFARMAIAN, R. BOOLOOTIAN AND R. 

LASKER, 1958. Organic productivity in the reproductive cycle of the purple sea urchin. 

Biol. Bull, 116: 49-58. 
HUANG, H., AND A. C. GIESE, 1958. Tests for digestion of algal polysaccharides by some 

marine herbivores. Science, 127 : 475. 

HYMAN, L. H., 1955. The Invertebrates, Vol. 4. Echinodermata. McGraw Hill, New York. 
LASKER, R., AND A. C. GIESE, 1954. Nutrition of the sea urchin, Strongyloccntrotus purpuratus. 

Biol Bull, 106 : 328-340. 
MILNER, H. W., 1953. Chemical composition of algae. Carnegie Inst. Wash. Publ #60: 

285-302. 

NICOL, J. A. C., 1960. The Biology of Marine Animals. Interscience Publ. Inc., New York. 
PEASE, H. D., 1932. The oyster: modern science comes to the support of an ancient food. 

/. Chcm. Ed., 9: 1673-1712. 
WILBER, C., 1947. The effect of prolonged starvation on the lipids in Phascolosoma gouldii. 

J. Cell. Comp. Physiol, 29: 179-183. 



A DUAL EFFECT OF CARBON DIOXIDE ON INSECTS 
POISONED BY OXYGEN x 

MARY HELEN M. GOLDSMITH AND HOWARD A. SCHNEIDERMAN 

Botany Department, Yale University, New Haven, Conn., and Department of Biology, 
Western Reserve University, Cleveland, Ohio 

The presence of small amounts of carbon dioxide during exposure to high pres- 
sure of oxygen accelerates the appearance of convulsions and death in oxygen 
poisoning of vertebrates (Hill, 1933; Shaw et al., 1934). Several explanations 
have been offered to account for this phenomenon. For example, it was early 
proposed that during exposure to oxygen, carbon dioxide accumulates in the tissues, 
and that carbon dioxide is the effective agent in poisoning (Gesell, 1923). Other 
workers claimed that carbon dioxide contributes only secondarily to the lethal effect 
of oxygen itself (Shaw et al., 1934), and the effect of carbon dioxide on oxygen 
poisoning of vertebrates is no doubt a complex one (Lambertsen et al., 1953). 
Williams and Beecher (1944), in a study of oxygen poisoning in Drosophila, found 
that this sensitizing effect of carbon dioxide on oxygen poisoning was not restricted 
to vertebrates. They reported that the presence of small amounts of carbon dioxide 
increased the sensitivity of adult Drosophila azteca to 10 atmospheres (atms.) of 
oxygen. In view of these findings, we did not anticipate a curious result that we 
encountered while investigating oxygen poisoning in the parasitic wasp, Mor- 
moniella vitripcnnis: carbon dioxide appeared to protect these wasps from oxygen 
poisoning (Goldsmith, 1955). This discovery, which found no parallel in the 
literature on oxygen poisoning, prompted a detailed study of the effects of carbon 
dioxide on oxygen poisoning of adult and developing insects. 

MATERIALS AND METHODS 
1. Experimental animals 

The chalcid wasp, Marmoniella ritripennis Walker, was used for these ex- 
periments. Procedures for rearing and handling this insect have already been 
described, along with detailed accounts of its life history and postembryonic de- 
velopment (Tiegs, 1922; Schneiderman and Horwitz. 1958; Goldsmith and 
Schneiderman, 1960). The present experiments utilized animals at three stages 
of development: (1) "pink stage" developing adults (24- hours after ecdysis from 
the final larval cuticle) ; (2) "black stage" developing adults (12 to 24 hours prior 
to adult emergence) ; and (3) adults (various ages, both males and females). 
Details of the method used to select insects of a uniform age and stage of develop- 
ment are given in a previous paper (Goldsmith and Schneiderman, 1960). 

In most experiments with adult insects, 10 males or 10 females were placed in 
a one-dram shell vial, loosely plugged with cotton ; a single vial was compressed 

1 This investigation was supported by a research grant from the National Heart Institute 
of the Public Health Service. 

151 



152 MARY HELEN M. GOLDSMITH AND HOWARD A. SCHNEIDERMAN 

in each pressure chamber. The compression chambers were constructed of trans- 
parent polymethylmethacrylate (Incite), and the activity of adult wasps could be 
observed during exposure. Observations were begun when full pressure was 
reached and continued at ten-minute intervals until decompression. 

Characteristically, normal wasps quickly right themselves if they fall or are 
knocked over. During the exposure to various gas mixtures, the times at which 
the first and last adults in a group became unable to right themselves, as well as 
the times at which the first and last adult ceased moving, were noted. In experi- 
ments in which the recovery of adults was to be studied, the chambers containing 
the experimental insects were decompressed 10 minutes after all the adults ceased 
moving. The activity of the wasps was observed immediately and then at inter- 
vals of 12 to 24 hours for several days. The reappearance of the righting behavior 
and the ability to walk were convenient measures of the extent of recovery. In 
observations during the recovery period, the wasps were knocked on their backs 
and the number which righted themselves as quickly as the control wasps was 
recorded. Most of the adults that could right themselves attempted to walk, but 
not all regained the agility of normal adults. Wasps that had recovered enough 
to run normally could, when tapped to the bottom of the glass vial, walk or run 
up the vertical side (30 mm.) within one minute. This last test was used as a 
convenient measure of full recovery. The activity and co-ordination of wasps 
which walked normally was not otherwise distinguishable from the control wasps 
which had not been exposed. 

The responses of the sexes were similar, and results for males and females 
were averaged together. The data from each treatment were compared with those 
from other treatments in the same experiment. Each experiment was repeated 
at least once and usually several times. 

2. Compression and decompression 

Most experiments were carried out at 10 atms. of oxygen at 20 C., the same 
conditions used by Williams and Beecher (1944). The compression chambers 
enclosing the experimental wasps were equilibrated at the desired temperature for 
one-half to one hour prior to compression. In experiments with carbon dioxide, 
the desired pressure of carbon dioxide was superimposed on the atmosphere of 
air present in the compression chamber following equilibration at the experimental 
temperature. The amount of carbon dioxide added was monitored on a sensitive 
Bourdon-type gauge which had a capacity of one atmosphere (gauge pressure). 
After the addition of carbon dioxide, the compression chamber was sealed and 
the 1-atm. gauge was replaced by a gauge with a capacity of 20 atms. Following 
this, ten atms. of oxygen were added to the chamber within one minute. The 
procedure of adding the desired gases to the atmosphere of air already present 
in the tank insured that the insects were never subjected to oxygen tensions which 
were below normal. Throughout this paper all pressures are reported as gauge 
pressures. 

Decompression was performed step-wise over a period of five minutes. Further 
details of these compression and decompression procedures have been given in 
an earlier paper (Goldsmith and Schneiderman, 1960). 



CO, EFFECTS ON O 2 POISONING 153 

RESULTS 
1. Effects of carbon dioxide at normal oxygen tension 

Before appraising the influence of carbon dioxide on oxygen poisoning, it was 
necessary to assess the effects of carbon dioxide in the presence of a normal oxygen 
tension (0.2 atm.). The desired pressure of carbon dioxide was added to the at- 
mosphere of air initially present in the compression chamber. Studies with nitrogen 
and helium have shown that small increases in ambient pressure, as would result 
from the added carbon dioxide, do not affect the insect's behavior. (Goldsmith, 
1955). 

As is well known carbon dioxide is commonly used as an anesthetic for insects. 
Ordinarily the movement and co-ordination of adult wasps were not conspicu- 
ously affected by one hour of exposure to 0.1 or 0.2 atm. of carbon dioxide. 
Occasionally, the wasps appeared less active in 0.2 atm. of carbon dioxide than 
in air, but they always maintained a standing posture, and they resumed normal 
activity as soon as they were returned to air. In 0.5 atm. of carbon dioxide, the 
wasps ceased moving after approximately 10 minutes. 

The effectiveness of carbon dioxide was only slightly increased when 10 atms. 
of nitrogen were superimposed upon the carbon dioxide. Adults compressed 
with 10 atms. of nitrogen plus 0.2 atm. of carbon dioxide were only slightly less 
active than normal. However, when 10 atms. of nitrogen were superimposed upon 
0.5 atm. of carbon dioxide, the insects ceased to move almost immediately. Since 
one atmosphere of air was present in all experiments, these results cannot be 
attributed to lack of oxygen. After four hours, the insects in 0.5 atm. of carbon 
dioxide and 10 atms. of nitrogen were decompressed. These wasps rapidly re- 
gained normal activity as did wasps decompressed after four hours in 0.5 atm. of 
carbon dioxide. From these results, it appears that in Monnoniella carbon dioxide 
at 0.1 atm. has no detectable effect, at 0.2 atm., a very slight effect, and at 0.5 atm., 
it is an effective anesthetic which has no detectable after-effects. 



TABLE I 

Duration of exposure to cause loss of spontaneous movements in adult Mormoniella 

exposed to various gas mixtures 









Time at which first insect lost spontaneous movement to time at which 








entire population succumbed (minutes) 


Exp't 


Number of 


Age of 




No. 


wasps 
















10 atms. O2 


10 atms. Oi and 
0.2 atm. CO 2 


10 atms. Oi and 
0.5 atm. CO2 


26 


200 


0-1 


70-80 


55-65 


not determined 


33 


95 


0-1 


40-50 


35-45 


0* 


31 


155 


1-2 


40-50 


40-50 


not determined 


25 


60 


1-2 


50-60 


50-60 





23 


50 


1-2 


40-50 


35-45 


not determined 


24 


60 


0-2 


40-50 


40-50 


not determined 


32 


75 


4-5 


40-50 


30-40 






* Activity disappeared immediately. 



154 MARY HELEN M. GOLDSMITH AND HOWARD A. SCHNEIDERMAN 

2. Effect of carbon dioxide on the onset of oxygen poisoning 

To appraise the effects of carbon dioxide on the onset of oxygen poisoning 
in adult wasps, insects in 10 atms. oxygen and 10 atms. oxygen plus 0.2 or 0.5 atm. 
carbon dioxide were observed at 10-minute intervals. Table I shows that complete 
loss of movement in all individuals consistently occurred within 10 minutes after 
the first wasp succumbed. In a few experiments (numbers 26 and 32), adults 
in oxygen plus 0.2 atm. of carbon dioxide appeared to lose spontaneous movement 
somewhat sooner than wasps in oxygen. This effect, however, was neither as 
marked nor as consistent (cj., numbers 24, 25, 31) as that described by Williams 




10 20 30 40 50 60 70 

MINUTES EXPOSURE TO 10 ATM. 2 + 0.2 ATM. C0 2 

FIGURE 1. The loss of normal activity (as shown by loss of the righting reflex) during 
exposure to oxygen. Observations were made at 10-minute intervals on 80 adult wasps com- 
pressed in 0.2 atm. of carbon dioxide plus 10 atms. of oxygen. In this experiment, the activity 
of almost all the wasps was depressed initially, but there was a considerable return of activity 
before the paralytic symptoms of oxygen poisoning commenced (30 minutes). 



and Beecher (1944) for 10- to 11-day-old Drosophila. They found that adults 
lost spontaneous activity almost twice as fast in oxygen mixed with 0.16 atm. 
carbon dioxide as in oxygen alone. 

Invariably 0.5 atm. carbon dioxide plus 10 atms. oxygen immediately paralyzed 
adults, and they remained motionless for the duration of exposure. This was to 
be expected, since in control experiments 0.5 atm. carbon dioxide and 10 atms. 
nitrogen also had an immediate anesthetic effect. 

In some experiments wasps in 0.2 atm. of carbon dioxide plus 10 atms. of 
oxygen ceased moving almost immediately; however, wasps that were affected in 



CO, EFFECTS ON O 3 POISONING 155 

this way often recovered while still under compression.- This can he seen in the 
experiment in Figure 1. Immediately after compression, all but 1% of the 
wasps had lost their righting behavior. After 10 minutes about 30% had regained 
the ability to right themselves, and after 20 minutes more than 50% could right 
themselves. Thereafter anesthesia set in again, and after 60 minutes of compression 
none were able to right themselves. 

This initial loss of activity and subsequent recovery was not invariable. In 
eight different experiments involving a total of 500 adults, 37% of the insects 
exhibited this initial paralysis. In three of these experiments none of the adults 
was initially affected, while in one experiment 99% became motionless imme- 
diately. It seems likely that this variability in the effect of 0.2 atm. carbon dioxide 
administered with 10 atms. oxygen can be attributed to variation in the sensitivity 
of different groups of wasps and to the fact that 0.2 atm. was approximately the 
threshold pressure of carbon dioxide for producing an immediate loss of co-ordina- 
tion in 10 atms. oxygen. In the presence of 10 atms. oxygen, the initial depression 
of activity was never observed at pressures of carbon dioxide less than 0.2 atm., 
but at higher pressure (0.25, 0.5 atm.) of carbon dioxide, activity ceased almost 
immediately. Apparently in some cases, an insect's activity can be immediately, 
albeit temporarily, depressed by a mixture of carbon dioxide and oxygen at a 
carbon dioxide concentration which by itself is not anesthetic (0.2 atm.). 

3. Effect of the presence of carbon dioxide during oxygen poisoning on recovery 

Although the previous experiments revealed that under some conditions simul- 
taneous exposure to carbon dioxide and oxygen resulted in almost immediate, 
but temporary, loss of co-ordination, the presence of carbon dioxide also prevented 
permanent injury by oxygen. Thus, wasps exposed to oxygen plus carbon dioxide 
showed striking recovery from the effects of oxygen poisoning. 

Groups of wasps were kept compressed until 10 minutes after all visible move- 
ments had ceased. They were then decompressed, and their behavior observed 
for several days. Maximum recovery occurred within 48 hours. In a typical 
experiment (Fig. 2, Curve 1) at the time of maximum recovery, only 33% of 
the wasps that had been exposed to oxygen behaved normally. By contrast, 
most of those that had been exposed to oxygen and carbon dioxide recovered; 
in fact, all of those exposed to oxygen plus 0.2 or 0.5 atm. of carbon dioxide (Fig. 2, 
Curve 1) regained normal activity. Clearly, carbon dioxide has a marked pro- 
tective effect. 

The protective action of carbon dioxide was even more conspicuous when wasps 
were kept compressed for periods one and one-half times and twice that required 
to render them motionless (i.e., up to 2% hours). The results are recorded in 
Figure 2 (Curves 1.5 and 2). When the wasps were returned to air, none exposed 
to oxygen for 2% hours survived; however, 27% of the wasps exposed for 2% 
hours to oxygen and 0.2 atm. carbon dioxide, and 53% of the wasps exposed for 
the same period to oxygen plus 0.5 atm. carbon dioxide recovered their righting 
ability. The presence of small amounts of nitrogen, instead of the carbon dioxide, 

2 In these cases, the time given in Table I does not refer to this initial temporary paralysis, 
but to the time at which spontaneous movement ceased permanently under compression. 



156 MARY HELEN M. GOLDSMITH AND HOWARD A. SCHNEIDERMAN 



during the period of oxygen poisoning had no noticeable effect on the subsequent 
recovery of oxygen-poisoned insects. 

4. Effect of carbon dioxide during oxygen poisoning on the development of "pink" 
and "black stage" -wasps 

Having demonstrated that carbon dixoide protects adult wasps from oxygen 
poisoning, our attention turned to developing wasps. Wasps in the "pink stage" 
of adult development were subjected to oxygen or oxygen and carbon dioxide for 
several hours, then decompressed, and their subsequent development recorded. An 
animal which attains the '"black stage" has completed all the externally visible signs 
of development. During the period of four days which intervenes between the 



100 - 




O (I) - 



(2) 



O.I 



0.2 



0.5 



C0 2 (ATM) 



FIGURE 2. The effect of carbon dioxide on the recovery from oxygen poisoning (10 atms. 
at 20 C). The ordinate gives the percentage of wasps exposed which had recovered the 
ability to right themselves 48 hours after decompression. Each curve shows the effect of a 
different duration of exposure. The basic exposure (1) continued for 10 minutes after spon- 
taneous movements ceased, a total of 75 minutes. Other wasps were given 1.5 times and twice 
the basic exposure (Curves 1.5 and 2). Results obtained from 150 wasps. 

"pink" and "black stages," external development is mainly concerned with epidermal 
pigmentation (Goldsmith and Schneiderman, 1960). Figure 3 reveals that all 
but about 5% of the wasps exposed to oxygen for 12 hours at the "pink stage" 
became black but only 13% of the exposed wasps were ever able to emerge fully; 
the rest remained completely or partially within the pupal cuticle. Although epi- 
dermal pigmentation was not affected, a system necessary for emergence was. These 
results agree with earlier experiments (Goldsmith and Schneiderman, 1960). By 
contrast, when carbon dioxide was present during exposure to oxygen, five times 
as many wasps (65%) emerged fully. Here again carbon dioxide exerted a 
protective action. 

In another series of experiments, wasps were exposed to oxygen and carbon 



C0 2 EFFECTS ON O* POISONING 



157 



100 



Crt 
Q- 
IS) 

< 80 

o 

U 
</) 

o 60 
x 



LJ_ 

40 



LJ 
O 

cr 
LU 

0. 



20 



12 HOURS 



10 atm. Oe 

10 atm. 2 + 0.2 atm. C02 




Early "Black stage" Partly Fully emerged 

developing emerged adult 

adult 

FINAL DEVELOPMENTAL STAGE REACHED 

FIGURE 3. The final stage of development attained by "pink stage" developing adults 
after they were removed from 12 hours of 10 atms. of oxygen (open bars) or 12 hours of 10 
atms. of oxygen plus 0.2 atm. of carbon dioxide (solid bars). Forty wasps were exposed to 
oxygen and 40 to oxygen and carbon dioxide. 

dioxide during the "black stage" and again their ability to emerge was recorded. 
After 6 hours' exposure to oxygen, none of these wasps emerged completely and 
only 15% were active enough to free themselves partially from their pupal cuticle. 
The wasps that had been exposed to carbon dioxide plus oxygen were less severely 
affected; 5% emerged normally and 45% managed to emerge partially (Fig. 4). 

5. Effect o] carbon dioxide on oxygen poisoning in Drosophila 

Experiments were also performed on a modest number of adults of Drosophila 
melanogastcr (128 adults, to 2 days old). When carbon dioxide was present 
during the exposure to oxygen, the flies lost their ability to make co-ordinated 
movements far sooner than in oxygen. Thus, it usually required 40 to 60 minutes 
of exposure to 10 atms. oxygen at 20 C. to abolish movement. By contrast, in 
seven separate compressions, all flies compressed with 0.2 atm. carbon dioxide 
plus 10 atms. oxygen ceased moving during the first 30 minutes of exposure. Such 
inactive flies did not recover while still compressed. Thus carbon dioxide admin- 
istered with oxygen causes an initial loss of activity in Drosophila which is more 
marked than that observed in Mormoniella. 

We may ask if the flies which became motionless with half an hour's or less 
exposure to carbon dioxide and 10 atms. oxygen were as severely affected as 
those subjected to oxygen until they were motionless. It is clear that they were 



158 MARY HELEN M. GOLDSMITH AND HOWARD A. SCHNEIDERMAN 



ffi 

</) 



100 



UJ 

o. 



80 



Q 
UJ 

cn 

2 60 
x 

UJ 

O 40 

f- 
z 

UJ 

20 







6 HOURS 



I | 10 otm. 02 

H 10 atm. 02+0.5 atm. C02 




Black stage 



Partly Fully emerged 
emerged adult 



FINAL DEVELOPMENTAL STAGE REACHED 

FIGURE 4. The emergence of "black stage" developing adults after they were removed 
from 6 hours of 10 atms. of oxygen (open bars) or 6 hours of 10 atms. of oxygen plus 0.5 
atm. of carbon dioxide (solid bars). Forty wasps were used in each exposure. 

not. When decompressed at the time they first ceased moving (i.e., 10-30 minutes 
in oxygen and carbon dioxide, 40-60 minutes in oxygen), none of the former 
exhibited permanent injury; whereas, all but about 10% of the latter remained 
inactive (Table II). 

Flies in oxygen and carbon dioxide were also subjected to the same length 
exposures that caused flies in oxygen without carbon dioxide to cease moving. 

TABLE II 
Survival of Drosophila melanogaster after oxygen poisoning 







10 atms. O2 


10 atms. Oa and 0.2 atm. CO 2 


Exp't No. 


Exposure 
(minutes) 






Activity prior to 
removal 


% Uninjured or 
recovered* 


Activity prior to 
removal 


% Uninjured or 
recovered* 


21 


10 


Normal 


100 


None 


100 


40 


30 


Normal 


100 


None 


100 


21 


40 


None 





Not determined 


43 


50 


None 


20 


None 


50 


40 


60 


None 


10 


None 


40 



* Per cent of adults uninjured is given for flies whose activity was normal on removal. Per cent 
of adults which recovered fully is given for flies who had lost all activity during exposure. 



CO, EFFECTS ON O, POISONING 159 

Under these conditions, it is evident that, as with Mormoniella, the presence of 
carbon dioxide during compression with oxygen promotes recovery of the flies. 
Thus while on the average only 10% recovered after an hour's exposure to oxygen, 
40% recovered when 0.2 atm. of carbon dioxide was simultaneously present. 

DISCUSSION 

From observations on adult Mormoniella and Drosophila, it seems clear that 
the presence of carbon dioxide during oxygen poisoning has at least two effects. 
First, it may hasten loss of activity without producing permanent toxic effects. 
Second, it promotes recovery and prevents permanent injury from oxygen poison- 
ing. Oxygen poisoning in adult insects can be conveniently divided into two phases. 
In the first or reversible phase, the effects of poisoning are evident soon after com- 
pression, but if the insects are decompressed promptly there is no permanent dam- 
age. If decompression is delayed, the second or irreversible phase ensues ; co- 
ordination becomes increasingly impaired, and the effects of poisoning persist indefi- 
nitely after decompression (cf. Williams and Beecher, 1944). Two interpretations 
of the effect of carbon dioxide in promoting the initial loss of activity are possible. 
Perhaps the presence of small amounts of carbon dioxide hastens the appearance 
of the reversible phase of oxygen poisoning or, alternatively, it may be that high 
pressures, particularly of oxygen, in some way potentiate carbon dioxide anesthesia. 
The present experiments do not allow us to choose between these two possibilities. 

The protective action of carbon dioxide is exhibited with developing insects as 
well as adults. It has previously been shown that if the proper exposure is 
selected, wasps exposed to oxygen in the "pink stage" will develop to the "black 
stage" but fail to emerge. This inability to emerge results from failure of muscle 
development caused by oxygen poisoning (Goldsmith and Schneiderman, 1960). 
In the present study, many more wasps emerged after exposure to oxygen and 
carbon dioxide than after exposure to oxygen alone. Thus, "pink stage" wasps 
exposed to carbon dioxide and oxygen must have developed muscles. 

In their study, Williams and Beecher (1944) concluded that the presence of 
carbon dioxide sensitized insects to oxygen poisoning. They examined the effects 
of carbon dioxide on 53 adults of Drosophila azteca. They found 10- to 11-day-old 
flies had lost all their activity when they were decompressed after 21 minutes in 
10 atms. of oxygen at 20 C. The rate of poisoning (on the basis of the activity 
of the adults) was a linear function of the carbon dioxide tension (Williams and 
Beecher, 1944; Fig. 5 ). They reported that Drosophila in 0.16 atm. carbon dioxide 
plus 10 atms. oxygen ceased moving after 10 minutes. It is important to note 
that in this particular experiment they did not directly observe the flies during the 
compression period and did not look for recovery of the motionless flies after 
decompression. On the basis of our results, we suggest that the flies which 
Williams and Beecher exposed for brief periods to oxygen and carbon dioxide 
were not permanently injured, while at least some of the flies subjected to 20 min- 
utes in oxygen alone probably suffered permanent damage. According to this 
view, there are no real differences between the results of the present experiment 
and those of Williams and Beecher. The conclusion of the present study is that 
although carbon dioxide may exert either a direct anesthetic effect in the presence 
of high pressures of oxygen or facilitate the appearance of the reversible phase 



160 MARY HELEN M. GOLDSMITH AND HOWARD A. SCHNEIDERMAN 

of oxygen poisoning, the most striking effect of carbon dioxide is its effectiveness 
in preventing permanent damage from oxygen poisoning. It is provocative to 
speculate that the effectiveness of carbon dioxide in protecting insects from oxygen 
poisoning may be the result of its anesthetic properties, which could render the 
particular system that is sensitive to oxygen less susceptible to injury. Whether 
other narcotic agents similarly render protection from oxygen toxicity remains 
to be seen. 

We wish to thank Professor C. M. Williams for his help in discussing this 
work, and in reading the manuscript. 

SUMMARY 

1. The activity of adults of M onnoniella vitripcunis and Drosophila melano- 
gaster during and after compression in 10 atms. of oxygen plus small amounts of 
carbon dioxide (0.1, 0.2, and 0.5 atm.) was studied. Addition of carbon dioxide 
at pressures above 0.2 atm. to the atmosphere of air in the compression chamber 
anesthetized Mormoniella adults ; on decompression all adults rapidly regained 
normal activity. The effects of carbon dioxide administered along with 10 atms. 
of nitrogen were similar except that with 0.5 atm. carbon dioxide, anesthesia 
occurred more rapidly. 

2. Although in some experiments with adults of Mormoniella the presence of 
carbon dioxide during exposure to oxygen accelerated the onset of paralysis, carbon 
dioxide actually protected the adults from permanent injury caused by exposure to 
oxygen. After 2 l / 2 hours at 10 atms. about half of the adult wasps which had 
been in oxygen plus 0.5 atm. carbon dioxide completely recovered while none which 
had been in oxygen without carbon dioxide survived. The number of wasps which 
recovered increased as the amount of carbon dioxide present during oxygen expo- 
sure increased from 0.1 to 0.5 atm. 

3. Although the presence of carbon dioxide did not totally prevent oxygen 
poisoning, permanent injury to "black stage" and "pink stage" developing adults 
as well as adult Mormoniella was significantly reduced. 

4. In the presence of 10 atms. oxygen, adult Drosophila became motionless more 
quickly when 0.2 atm. of carbon dioxide was also present. These motionless flies 
recovered fully when decompressed. Furthermore, significantly fewer flies recov- 
ered following paralyzing exposures to oxygen without carbon dioxide. 

LITERATURE CITED 

GESELL, R., 1923. On chemical regulation of respiration. I. Regulation of respiration with 
special reference to the metabolism of the respiratory center and the coordination of 
the dual function of hemoglobin. Amcr. J. Physiol., 66 : 5-49. 

GOLDSMITH, M. H. M. (nee M. H. Martin), 1955. Studies on the mechanism of oxygen poison- 
ing in insects and the effect of carbon dioxide on recovery. Thesis, Cornell University. 

GOLDSMITH, M. H. M., AND H. A. SCHNEIDERMAN, 1960. The effects of oxygen poisoning on 
the post-embryonic development and behavior of a chalcid wasp. Biol. Bull., 118: 
269-288. 

HILL, L., 1933. The influence of carbon dioxide in production of oxygen poisoning. Quart. 
J. Exp. Physiol., 23 : 49-50. 



CO, EFFECTS ON O 2 POISONING 161 

LAMBERTSEN, C. J., R. H. KOUGH, D. Y. COOPER, G. L. EMMEL, H. H. LOESCHKE AND C. F. 
SCHMIDT, 1953. Oxygen toxicity. Effects in man of oxygen inhalation at 1 and 3.5 
atmospheres upon blood gas transport, cerebral circulation, and cerebral metabolism. 
/. Appl. PhysioL, 5: 471-486. 

SCHNEIDERMAN, H. A., AND J. HORWITZ, 1958. The induction and termination of facultative 
diapause in the chalcid wasps MormonlcHa I'itripcnnis (Walker) and Tritncptis klugii 
(Ratzeburg). /. E.rp. Bio!.. 35: 520-551. 

SHAW, L. A., A. R. BEHNKE AND A. C. MESSER, 1934. The role of carbon dioxide in pro- 
ducing the symptoms of oxygen poisoning. Amcr. J. Pliysiol., 108: 652-661. 

TIEGS, O. W., 1922. Researches on the insect metamorphosis. Part I : On the structure and 
post-embryonic development of a chalcid wasp Nasonia. Trans. Roy. Soc. S. Aitst., 
46: 319-527. 

WILLIAMS, C. M., AND H. K. BEECHER, 1944. Sensitivity of Drosophila to poisoning by 
oxygen. Amcr. J. PhysioL, 140 : 566-573. 



METABOLIC ANTAGONISTS AND PROLONGED SURVIVAL OF 
SCALE HOMOGRAFTS IN FUNDULUS HETEROCLITUS 1 

RICHARD J. GOSS - 

Department of Biology, Broivn University, Providence, R. I., and Mount Desert Island 
Biological Laboratory, Salisbury Cove, Maine 

The immunological competence of an animal depends upon its ability to syn- 
thesize antibodies, the specificity of which appears to be regulated by nucleic acids. 
The exact mechanism whereby antigenic information is translated into antibody 
specificity in the course of protein synthesis, however, remains more speculated 
about than understood. In order to investigate this, attempts have been made to 
interrupt the hypothetical pathway along which such information might be expected 
to be transferred. Accordingly, the syntheses of nucleic acids and proteins have 
been interfered with by means of various antimetabolites, and the subsequent 
capacity for antibody formation studied. 

As an experimental system, the homograft reaction against foreign scale trans- 
plants in Fnndulus heteroclitns has been adopted. The usefulness of this technique 
in analyzing the homograft reaction has been demonstrated by Hildemann (1956, 
1957a. 1957b, 1958) and Hildemann and Haas (1960). These investigators 
recognized the rejection of foreign scale grafts as a temperature-dependent inflam- 
matory response leading to the destruction of the graft in 4.3 days at 32 C. to 
40.5 days at 10 C. A further innovation was recently reported by Triplett and 
Barrymore (1960) who utilized the time of disintegration of melanophores on trans- 
planted scales as the criterion for estimating the time of onset of the homograft reac- 
tion. According to this method, foreign scale grafts survived an average of 5.8 
days at 20 C. and 7.0 days at 17 C. This technique of observing the duration 
of melanophore survival after grafting has made it possible to judge easily, quickly, 
and accurately the time required for the host to react against the presence of 
immunogenically foreign cells. The relatively abrupt nature of the overt response 
represents a distinct advantage over the more subjective methods of estimating 
homograft survival times in higher vertebrates. 

There is a measure of uncertainty concerning the exact nature of the homograft 
response, particularly as regards the cellular vs. humoral location of factors alleged 
to be responsible for reaction against foreign tissues (cf. Brent, Brown and 
Medawar, 1959; Lawrence, 1960; Gorer, 1960). The demonstration by Triplett 
and Barrymore (1960) that homograft sensitivity can be transferred via intra- 
ovarian fluid from pregnant females to their embryos argues in favor of the possible 
existence of circulating antibodies, at least in fish. Since the present account, 
unhappily, cannot further resolve this problem, the author elects to assume that 

1 This research was supported by a grant (B-923) from the National Institutes of Health. 

2 Fellow of the Carnegie Institution of Washington (Department of Embryology), 1960-61. 

162 



SCALE HOMOGRAFT SURVIVAL 163 

the rejection of homografts is an immunological phenomenon attributable to anti- 
bodies, without reference to their disposition or mode of action, but very much 
concerned with their genesis. As assayed by the prolonged survival of scale 
homografts on treated hosts, it has been found in the present experiments that 
under conditions of restricted protein or nucleic acid synthesis, antibody produc- 
tion has been inhibited. 

MATERIALS AND METHODS 

Experiments were performed on male Fundiilus hcteroclitiis weighing an 
average of 6 to 8 grams each. Fish were maintained in running salt water aquaria 
at a temperature of 28 1 C. except where otherwise stated. All operations 
were performed on fish under chloretone (1 : 1000) anesthesia. Scale transplanta- 
tions \vere achieved by inserting scales in the dermal scale pockets from which 
the original scales had been plucked. If care is taken to graft scales of the same 
size as those being replaced, the transplants are rarely lost. All transplants were 
made to the unpigmented ventral region of the fish. In each animal three auto- 
grafts and three homografts were inserted in parallel rows on either side of the 
linea alba. Pigmented scales derived from the posterior region of the fish proved 
to be of a size commensurate with those of the ventral transplantation site. Each 
experiment comprised four fish in which homografts were made reciprocally 
between pairs. 

Circulation in the scale graft was re-established on the day following operation, 
at which time there was usually observed a slight nonspecific degeneration of a 
few pigment cells in both autografts and homografts (attributable to injury attend- 
ing the transplantation procedures ) . Animals bearing scale grafts were examined 
daily under the dissecting microscope to determine the condition of melanophores 
in the transplants. In control fish, foreign melanophore breakdown was invariably 
complete on the third day after grafting at 28 C. (Figs. 1-5). The reliability 
of this was such that the survival of pigment cells only one day beyond this time 
constituted an unequivocal and statistically significant indication of the efficacy 
of the treatment being tested. The number of days designated as the survival 
time refers to the day on which complete or very extensive destruction of pigment 
cells was observed to occur. As a record of the experiments, photomicrographs 
were taken through the dissecting microscope of scale transplants at critical times. 

In addition to the effects of temperature change, splenectomy, hypophysectomy, 
and trypan blue, the following substances, occasionally in varying doses, were 
administered to hosts by daily intraperitoneal injection in 0.1 ml. distilled water. 
Materials which interfere with nucleic acid synthesis were 5-fluorouracil, 5-fluoro- 
deoxyuridine (FUDR) and 6-mercaptopurine (6-MP). Adrenal cortical hor- 
mones included cortisone acetate, 6-fluorohydrocortisone acetate and delta- 1 -hydro- 
cortisone sodium succinate (Delta-Cortef : The Upjohn Co.). Two antibiotics 
were tested: chloramphenicol sodium succinate (Chloromycetin : Parke, Davis & 
Co.) and tetracycline hydrochloride (Achromycin: Lederle). The amino acid 
analogues used were /3-2-thienylserine and DL-/?-phenylserine (serine analogues), 
DL-a-CH.3 phenylalanine, /?-2-thienylalanine and DL-/?-phenyllactic acid (phenyla- 
lanine analogues), and ethionine (methionine analogue). 



164 



RICHARD J. GOSS 



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I 



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FIGURES 1-5. Daily photographic sequence of autograft (left) and homograft (right) 
scales on the first through fifth days after transplantation at 28 C. The row of three homo- 



SCALE HOMOGRAFT SURVIVAL 165 

RESULTS 
Temperature effect 

Groups of fish each bearing autografts and homografts were maintained at 
temperatures of 7, 14, 21, and 28 C. All except the 7 C. group, which was 
kept in a refrigerator, were in circulating sea water. The scale grafts were inspected 
daily and photographed at frequent intervals to determine as precisely as possible 
the time of melanophore fragmentation. The lower the temperature the greater 
variation there was in the end point. At 28 C., incipient breakdown of pigment 
cells was detectable two days after operation, but not until the third day had all 
melanophores been destroyed. At this temperature, the reaction is relatively abrupt. 
Homotransplants of fish at 21 C. underwent pigment cell disintegration on the 
fifth and sixth days after grafting. Those maintained at 14 C. required 14 to 16 
days to break down. At 7 C. the pigment cells of the homografts remained intact 
for 26 days at which time it was necessary to terminate the experiment. In all 
groups of fish, the autograft scales remained healthy indefinitely. 

Sf>lcncctoin\' 

In four fish, the spleens were removed via a ventral incision on the day prior 
to scale grafting. Four controls were subjected to sham operations. In all fish, 
controls and experimentals, the homografts broke down on the third day. The 
results are consistent with those of Vogel (1940) who noted that splenectomy 
failed to protect skin homografts in Rana pipiens from destruction. 

Hypophysectoiny 

Animals were deprived of their pituitaries, or subjected to sham operations, 
two days before scales were grafted. This operation did not enhance the survival 
of homografts. In 12 hypophysectomized fish and 13 controls, the homografts 
exhibited breakdown of pigment cells on the third day. 

Trypan blue 

Four experimental animals received scale grafts on the day of the first intra- 
peritoneal injection of 0.1 ml. of \% trypan blue in distilled water. Injections 
were repeated daily. Control fish similarly grafted were injected intraperitoneally 
with 0.1 ml. distilled water daily. On the third day there was complete break- 
down of pigment cells in the homografts of both control and experimental groups, 
despite the fact that the treated fish had become intensely stained with dye. 

Xitclcic acid antagonists 

The injection of substances which inhibit nucleic acid synthesis proved to be 
very successful in protecting the homograft scales from the antibody response of 

graft scales exhibited pigment cell breakdown on the third day, with subsequent disappearance 
of the pigment granules. Autografts remained intact throughout. 10 X. 

FIGURE 6. A normal, expanded, scale melanophore showing typical binucleate condi- 
tion. 1000X. 

FIGURES 7-9. Appearance of scales before, during and after onset of homograft re- 
action. 100 X. 



166 RICHARD J. GOSS 

the host. In two separate experimental series in which a total of eight fish were 
given 1 mg. of 5-fluorouracil each daily, starting on the day of operation, the 
homografts showed no signs of pigment cell breakdown as long as the fish sur- 
vived. This dose, although effective, was at the same time toxic and resulted in 
the deaths of animals after two to six days. Nevertheless, four fish still alive on 
the fifth day possessed intact scale homografts, and it would appear that their 
transplants would have survived longer had the hosts lived. Similar experimental 
series on eight fish injected with 2 mg. of 5-fluorodeoxyuridine (FUDR) likewise 
resulted in protection of the homografts for up to eight days, by which time five 
of the animals had died as a result of the toxic effects of the drug. Injection of 
2 mg. of 6-mercaptopurine into each of four fish bearing homograft scales afforded 
protection for four days at which time the injections were discontinued. Thereafter, 
melanophores were gradually destroyed until only one intact homograft scale re- 
mained on the seventh day, when the experiment was terminated. From these 
results it is clear that near-lethal doses of these drugs effectively prolong the survival 
time of homografts. In the cases of all three substances, when the doses were 
reduced to 1/100 of the above levels, no protection whatever was observed. 

Adrenal cortical hormones 

Daily intraperitoneal injection of a 1 mg. suspension of cortisone acetate to 
grafted fish starting on the day of operation had no beneficial effect on the survival 
of the homografts. On the third day there was complete breakdown of all scale 
pigment cells, probably due to inadequate doses of cortisone. More potent prepa- 
rations of cortical hormones, however, proved to be more effective. Injections 
of 2 mgf. of 6-fluorohvdrocortisone actetate on the day of transplantation and on 

o ^ J r 

the two days thereafter resulted in the survival of homografts for four days, at 
which time about half of all the pigment cells were undergoing fragmentation. 
This nevertheless marks a definite delay in the destruction of the grafts. In a 
third experiment, 2 mg. of delta- 1 -by clrocortisone sodium succinate were injected 
daily through the second day after operation. In these fish, no pigment cell break- 
down was observed on the third day, an incipient destruction was noted on the 
fourth, and, after five days, disintegration was well progressed in all except one 
animal in which the homografts remained intact. This compound, therefore, exerted 
a distinct protective effect. 

Antibiotics 

Chloramphenicol sodium succinate was tested at two dose levels. Daily injection 
of 1 mg. per animal intraperitoneally had no detectable effect on the survival time 
of the homografts. Similar injections of 10 mg. chloramphenicol, however, resulted 
in survival of homograft pigment cells beyond the third day. On the fourth day 
there was breakdown of melanophores in the grafts of one fish, and on the next 
day extensive, but still not complete, disintegration of foreign pigment cells had 
occurred. All were destroyed by the sixth day. 

Tetracycline hydrochloride was likewise tested at two different dosages. Injec- 
tion of 0.1 mg. per fish gave no protective effect. Administration of 1 mg. of tetra- 
cycline per day through the third day resulted in prolonged survival of homograft 



SCALE HOMOGRAFT SURVIVAL 167 

pigment cells. In three fish the grafts were destroyed on the fourth day, and in 
three others they broke down on the fifth day. In two animals the pigment cells 
of homograft scales were still intact on the seventh day when the experiment was 
terminated. These antibiotics, therefore, interfere with the immunological response 
of the host against foreign grafts. 

Amino acid analogues 

Six different analogues of amino acids were tested for possible interferences 
with the homograft reaction. In general, they proved to be rather toxic and not 
very effective in protecting the foreign scale grafts from destruction by the host. 
In all cases, at least two dose levels were tried, the larger usually representing 
the limits of solubility in 0.1 ml. distilled water. Two analogues of serine were 

TABLE I 

Summary of effects of antimetabolites, administered to hosts, on survival of 

scale homografts at 28 C. 

Dose Survival time 

Substance injected (IP) (mg. /fish/day) (days) 

Controls 3 

5-fluorouracil 0.01 3 

1.0* 5 + 

5-fluorodeoxyuridine 0.02 3 

2.0* 6-8 

6-mercaptopurine 0.2 3 

2.0 5-7 

Cortisone 1.0 3 

6-fluorohydrocortisone 2.0 4-5 

Delta-1-hydrocortisone 2.0 5 

Chloramphenicol 1.0 3 

10.0 4-6 

Tetracycline 0.1 3 

1.0 4-7 

/3-2-thienylserine 1.0 3 

5.0 3-4 

DL-/3-phenylserine 1.0 3 

5.0* 3-4 

DL-a-CH'j phenylserine 1.0 3-4 

3.3 3-4 

/3-2-thienylalanine 1.0 3 

5.0 4 

DL-|S-phenyllactic acid 1.0 3 

2.5* 3 

5.0* 4 
10.0* 

Ethionine 1.0* 3-4 

3.3* 5 

* Lethal dose. 



168 RICHARD J. GOSS 

used. /?-2-thienylserine and DL-/3-phenylserine were injected in doses of 1 mg. 
and 5 mg. into groups of four fish. The smaller dose in all cases was ineffectual ; 
the larger dose resulted in survival of homograft pigment cells for four days. None 
of these doses was lethal except in one fish which died on the third day following 
injections with 5 mg. of DL-/?-phenylserine. Analogues of phenylalanine included 
DL-a-CH 3 phenylalanine which was administered in doses of 1 mg. and 3.3 mg. 
per day to groups of four fish. In all cases, foreign pigment cell breakdown was 
initiated on the third day but was not complete until the fourth. There was no 
detectable difference between the effects of the two doses utilized, nor did either 
dose prove to be lethal. /3-2-thienylalanine was injected in doses of 1 mg. and 5 mg. 
daily. Only the larger dose prolonged the survival of scale homografts (to the 
fourth day). DL-/3-phenyllactic acid, also an analogue of phenylalanine, was given 
in four different doses to four groups of fish. Doses of 1 mg. and 2.5 mg. failed 
to protect the homografts from destruction on the third day. A single injection of 
5 mg. to another group of fish on the day of transplantation killed two fish the 
next day, but enabled the homografts of the remaining two animals to survive 
to the fourth clay before being destroyed. Administration of 10 mg. of this com- 
pound proved lethal to all fish within one day. A final analogue, ethionine, was 
tested at levels of 1 mg. and 3.3 mg. The lesser dose permitted homografts to 
survive until the fourth day ; 3.3 mg. per day through the second day after trans- 
plantation resulted in survival of homografts for two days beyond the controls. 
The latter dose, however, was lethal to three out of four fish by the fourth day 
after operation, at which time the foreign pigment cells were still intact. The one 
fish still alive on the fifth day exhibited complete breakdown of its homograft 
melanophores at that time. Ethionine therefore was considerably more effective 
in enhancing the survival of homografts than were the other five amino acid ana- 
logues tested. 

DISCUSSION 

The immunological reaction leading to homograft destruction has been divided 
into three phases (Billingham, Brent and Medawar, 1956), involving the release 
of graft antigens (afferent phase), the production of antibodies (central phase), 
and the reaction of antibodies with the graft (efferent phase). Although inter- 
ruption at any one of these levels would insure homograft survival, it is the central 
phase which is most amenable to experimentation. The process of antibody pro- 
duction may in turn be partitioned into subsidiary processes, leading from the 
initiating influence of the antigen on antibody-producing cells (induction or adapta- 
tion phase) to the eventual fabrication of antigen-specific antibodies (production 
phase). The method by which antibodies are formed is essentially a problem of 
protein synthesis with the added prerequisite that the protein antibody be capable 
of reacting specifically with the antigen originally responsible for initiating its 
formation. The synthesis of such specific proteins necessarily involves the participa- 
tion of a system by which the nature of the specificity can be communicated from 
the antigen to the molecular architecture of the antibody concomitant with its 
synthesis. There is reason to believe that nucleic acids constitute such a communi- 
cation system. This is substantiated by the dependent relationships of protein 
synthesis on RNA and of specific RNA synthesis on DNA. If this system is to 
remain sufficiently labile to adapt to new modes of protein synthesis (e.g., specific 



SCALE HOMOGRAFT SURVIVAL 169 

antigen-stimulated antibody production) it is necessary to assume that new specific 
types of RNA molecules can be synthesized on demand. This requirement may 
be taken to indicate that RNA synthesis is necessary for the production of specific 
proteins. Schweet and Owen (1957) have postulated that antigen reacts with 
BNA which in turn makes specific RNA, and that the RNA acts as template in 
giving rise to specific antibodies. 

It is not surprising, therefore, that analogues of purines (6-MP) and pyrimi- 
dines ( 5-fluorouracil and 5-fluorodeoxyuridine), which interfere with nucleic acid 
synthesis, likewise arrest antibody production and thus result in the prolonged 
survival of homografts on treated hosts. Studies on 8-azaguanine. a purine ana- 
logue, have shown that it also inhibits nucleic acid synthesis (Skipper et a!., 1951) 
and antibody production ( Malmgren, Bennison and McKinley, 1952; Button, 
Button and George. 1958). Berenbaum (1960) demonstrated that 6-MP also 
inhibits the production of antibodies, and Schwartz and Bameshek (1960) and 
Meeker ct al. (1960) have reported the protection of skin homografts in rabbits 
by the administration of 6-MP. 

It is generally acknowledged that many antibiotics exert their growth-limiting 
effects by inhibiting protein synthesis, either directly or indirectly. Chloram- 
phenicol, for example, has been noted to prevent the synthesis of BNA (Brakulic 
and Errera, 1959; Schneider, Cassir and Chordikian, 1960), RNA (Gros and 
Gros, 1956; Webster, 1957), and protein (LePage, 1953; Smith, 1953; Pardee 
and Prestidge, 1956; Webster, 1957; Gale, 1958) in bacteria and mammalian 
tissues. Because of such manifold effects of chloramphenicol, and probably other 
antibiotics as well, their interference with antibody production and the homograft 
reaction is not unreasonable. 

Amino acid analogues, in so far as they have been tested, were generally less 
effective in protecting homografts from destruction than were the other agents 
already discussed. There is evidence that [3-2- and /?-3-thienylalanine inhibit anti- 
body formation in the rat (Ferger and du Vigneaud, 1949; Wissler et al., 1956). 
and thymidine uptake in BNA is inhibited by /3-2-thienylalanine and ethionine 
(Schneider, Cassir and Chordikian, 1960). Amino acid analogues are generally 
agreed (Matthews, 1958; Shive and Skinner, 1958) to act either by preventing 
protein synthesis via interference with the utilization of natural ammo acids or 
by becoming incorporated themselves into proteins, thus displacing their normal 
counterparts. Of the amino acid analogues studied in the present investigation, at 
least ethionine and /3-2-thienylalanine have been shown to act in the latter fashion 
(Levine and Tarver, 1957; Munro and Clark, 1958; Munier and Cohen, 1959). 
Structurally defective proteins would be expected not to be biologically inactive 
unless the incorporated analogues occupied an indispensable position. Since con- 
siderable portions of protein molecules are known to be functionally superfluous, 
the relative ineffectiveness of amino acid analogues in promoting homograft sur- 
vival may find an explanation along these lines of reasoning. 

The ability of cortisone to protect homografts from immunological destruction 
is too well known to require elaboration (Morgan, 1951; Billingham, Krohn and 
Medawar, 1951; Krohn, 1954; Medawar and Sparrow, 1956; Scothorne, 1956; 
Hamer and Krohn, 1959). This hormone also depresses antibody production 
(Germuth and Ottinger, 1950; Kass and Finland, 1953; Berglund, 1956) and 



170 RICHARD J. GOSS 

inhibits nucleic acid synthesis (Skipper ct al., 1951). It has been claimed that 
these effects of cortisone are augmented by its interference with the release of 
antigens during the afferent phase of the homograft reaction (Billingham, Krohn, 
and Medawar, 1951; Medawar and Sparrow, 1956; Scothorne, 1956). In view of 
the well documented evidence in favor of the efficacy of cortisone in suppressing 
the homograft reaction, plus the demonstrated effectiveness of the more potent 
preparations (6-fluorohydrocortisone and Delta- 1-hydrocortisone), the failure of 
cortisone to enhance the survival of scale homografts in the present experiments 
may reasonably be ascribed to insufficient dosages. 

With reference to the mode of action of the various agents found effective in 
promoting extended survival of homografts, it could be argued that such results 
might be attributed to nonspecific toxicities rather than to effects directly related 
to the inhibition of antibody synthesis. Although some of the drugs tested proved 
to be fatal at effective doses, there is little reason to conclude that their efficacy 
resulted directly from their lethality per sc. The majority of the compounds which 
prolonged homograft survival manifested no other toxic effects during the period 
of treatment. Moreover, in the case of DL-/3-phenyllactic acid, a dose of 2.5 nig. 
was lethal without being effective in precluding the homograft reaction. Additional 
treatments not reported here have also failed to interfere with foreign tissue rejec- 
tion at otherwise lethal doses. Thus, while inhibition of nucleic acid or protein 
synthesis may be fatal, other kinds of toxicity need not interfere with immunological 
mechanisms. 

The accumulated evidence supports the contention that homograft rejection may 
be subject to a moratorium in the absence of the successful synthesis of nucleic 
acids and/or proteins. On the basis of the limited number of compounds tested, 
there is reason to expect that numerous other agents with comparable physiological 
properties might exert similar influences. Granted that there are numerous factors 
which inhibit antibody production and thus actually or potentially interfere with 
the homograft reaction, it remains to be demonstrated conclusively whether such 
effects are permanent or temporary. In their investigations of the beneficial effects 
of 6-MP on skin homograft survival in rabbits, Meeker ct al. (1960) noted that 
sustained treatment was necessary to insure continued survival of the grafts. In 
the present experiments, a comparable conclusion seems to be indicated, for despite 
the survival of scale homografts in treated hosts beyond the control period, eventual 
though dilatory breakdown was the rule. Notwithstanding these preliminary 
observations, it remains as a theoretical possibility that a specific tolerance might 
be conferred upon an adult host exposed to a foreign antigen by selectively inacti- 
vating those antibody-synthesizing pathways specifically stimulated by the antigen. 
If, as Burnet (1959) contends, antibody-producing clones are descended from 
specific cells stimulated to proliferate by exposure to antigen, then the application 
of treatments designed to render such cells vulnerable to destruction or inactivation 
at this critical period should, perforce, result in an animal subsequently tolerant 
to the original antigen. Alternatively, if antibody production can continue irrespec- 
tive of whether or not the involved cells are stimulated to proliferate, specific 
tolerance could be realized only by permanently and selectively incapacitating the 
biochemical pathways by which the specifically stimulated antibodies are synthe- 
sized. To achieve this without doing violence to any other mechanism of protein 
synthesis will be a challenging enterprise. 



SCALE HOMOGRAFT SURVIVAL 171 

The author extends his thanks to Miss Marsha Rankin for technical assistance, 
and to Drs. David A. Karnofsky and Charles E. Wilde, Jr. for their generous 
donations of base analogues and amino acid analogues, respectively. 

SUMMARY 

1. At 28 C., the melanocytes on scale homografts in Funditlus are destroyed 
in three days by the immunological response of the host. This reaction is slower 
to occur at progressively lower temperatures, but is not adversely affected by 
splenectomy or hypophysectomy of the host, nor by daily injections of trypan blue. 

2. Survival of homografts was enhanced by daily intraperitoneal injections of 
base analogues, potent preparations of adrenal cortical hormones, antibiotics and 
amino acid analogues. 

3. These results are taken to indicate that the inductive and productive phases 
of antibody formation are particularly vulnerable to agents which interfere with 
protein and/or nucleic acid synthesis. 

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THE LIFE-CYCLE OF PORPHYRA TENERA IN VITRO 

HIDEO IWASAKIi 
Haskins Laboratories, 305 East 43rd Street, Nczc York 17, N. Y. 

Cultivation of the red sea- weed Porpliyra tcncra was started in Japan several 
centuries ago. It is now the largest industrial cultivation of any marine product. 
Despite this success, more knowledge of the life-cycle and innate potencies of 
Porpliyra is needed to improve methods of cultivation to bring them under a 
control comparable to that achieved in land agriculture. At present this goal is 
unrealizable to its fullest extent. Mass-scale use of artificial fertilizers on 50 square 
miles of bays is uneconomical. But improvements in production and control of 
seeding, genetic improvement of the plant in respect to greater production and 
resistance to parasites, and perhaps extension of the growth period of the thallus, 
seem attainable goals. 

Several obstacles have slowed research on the life-cycle and potencies of Por- 
pliyra. Until a few years ago only one part of the life-cycle was known : mysteri- 
ously, the bays abound in monospores in the autumn ; these monospores, collected 
on bamboo or cord nets, develop into the edible, leafy thallus which is periodically 
harvested until March, when it fruits and disintegrates while producing carpospores. 
What was happening to the carpospores, and the origin of the monospores, were 
unknown. Those mysteries were solved after Drew (1949) discovered that the 
carpospores of another species, Porph\ra uuibilicalis, germinated into a filament 
which, in enriched sea water, produced a flimsy, sickly mat. The fact that the 
germ tubes produced by the carpospores are very similar to those of fungal spores, 
and that the older filaments of the mat are generally abnormal in appearance sug- 
gested to her that a specific host or a special substrate are needed for normal 
growth. Indeed several molluscan shells and even egg shells proved an excellent 
substrate. The filamentous thallus grows well in the shells, forming colonies 
identical with Concliocclis rosca ; C. rosca is obviously merely a phase of the life- 
cycle of Porpliyra. Kurogi (1953) and Tseng and Chang (1954) found that the 
carpospores of Porpliyra tcncra behaved similarly. Kurogi (1953) studied the 
growth of the carpospores of Porpliyra uuibilicalis pro.v., P. snborbicnlata, P. 
pscudolincaris and P. tcncra ; these form "'ConcJwcclis" colonies in the shells which 
can hardly be told from one another. In Kurogi's cultures the "ConcJwcclis" phase 
cultured on glass slides produced monosporangiate branches but not free mono- 
spores. However, from ConcJwcclis in oyster shells Kurogi (1953) obtained 
monospores which produced germlings of the leafy thallus. 

The complete life-cycle of Porpliyra was now known. This discovery renewed 
interest in the biology of Porpliyra, especially the conditions for growth (Iwasaki 
and Matsudaira, 1958) and production of monospores (Kurogi and Hirano, 1956). 

1 Present Address : Tohoku University, Faculty of Agriculture, Department of Fisheries, 
Sendai, Japan. 

173 



174 HIDEO IWASAKI 

Obstacles to speedy progress were: (a) inability to grow the Conchocelis phase 
outside of the shells in free conditions; and (b) inability to cultivate in the labora- 
tory, out of season, the two growth phases of Porphyra which in nature are strictly 
seasonal (autumn- winter for the thallus phase and spring-summer for the Concho- 
celis phase). 

As mentioned, Drew and Kurogi had grown the Conclwcclis phase in enriched 
sea water on glass slides. Although the growth of Conchocelis was poor, Drew 
(1954) mentioned (p. 193) ". . . . that such free-living filamentous growths can 
be maintained and continue to grow indefinitely provided the culture solution is 
renewed regularly"; this seemed promising. Drew obtained with P. uinbilicalis 
only filamentous Conchocelis growth on glass slides and no monosporangia were 
formed, while the four species of Porphyra (including P. umbilicalis pro.v.) studied 
by Kurogi produced monosporangia. The discrepancy between these results implied 
that good growth and fruiting of the Conchocelis phase in the free-living conditions 
might be obtained under different cultural conditions and with better media. 
Another reason for trying again to grow the Conchocelis phase of P. tenera in vitro 
was the success of Hollenberg ( 1958 ) in obtaining on glass slides in liquid media 
minute filamentous Conclwcelis-\ike plants of P. perjorata which produced "spo- 
rangia branchlets" and fertile "conchospores." 

PRELIMINARY EXPERIMENTS 

The original materials brought from Japan were a few sterilized oyster shells 
which had been inoculated in March, 1959, with carpospores produced by natural- 
grown thalli. Colonies of Conchocelis developed normally in the shells kept in 
Woods Hole sea water enriched with nitrate, phosphate, and EDTA, (medium 
SWI, Table I) indicating the suitability of Atlantic sea water for Porphyra tenera. 

TABLE I 
Enriched sea water media 

SWI SWI I 

Filtered sea water 1000 ml. 1000 ml. 

KNO 3 72.2 mg. ( = 10 mg. N) 72.2 mg. ( = 10 mg. N) 

KH 2 PO 4 8.8 mg. (=2 mg. P) 4.5 mg. ( = 1 mg. P) 

Na 2 -glycerophosphate .5H 2 O 10.5 mg. ( = 1 mg. P) 

Fe-EDTA (1:1 chelation) 0.5 mg. (as Fe) 0.5 mg. (as Fe) 

"Tris" buffer* 500 mg. 500 mg. 

pH 8.0-8.2 8.0-8.2 

* Tris (hydroxymethyl) amino methane (Sigma Company). 

The nutrient solution was changed fortnightly ; the shells were periodically cleaned 
with cotton to eliminate epiphytic diatom growth and kept in subdued, continuous 
fluorescent light 2 (10-30 foot-candles) at 13-15 C. In September, 1959, some 
shells were broken in pieces and thin flakes containing one Conchocelis colony were 
thoroughly wiped clean of epiphytes with cotton and also by repeated dipping 
into 1.5% agarized enriched sea water containing antibiotics. The flakes were 

2 "Cool white." 



LIFE-CYCLE OF PORPHYRA TENERA 175 

then inoculated into various artificial marine media and in enriched sea water, 
and kept in continuous subdued light at 13-15 C. At the end of December, 1959, 
in two tubes of medium ASP 2 NTA and in a tube of SWI + 5 ng.% indolacetic acid, 
a few young thalli appeared at the bottom of the test tube and on the shell flakes. 
Simultaneously in two tubes of SWI. tufts of free Conchocclis were growing out 
from the shell flakes. Later on (February-April, 1960), free Conchocclis colonies, 
attached to the bottom of the test tube or to the shells, appeared in the two 
ASP 2 NTA tubes and the SWI + IAA. The young thalli and the free Conchocelis 
employed in the subsequent experiments were derived from these 5 original cul- 
tures, which are unialgal, but accompanied by bacteria and yeasts. Microbial con- 
tamination, though permanent, was minimal in all the media employed because 
of the lack of organic substrates and because aseptic techniques were employed 
throughout. 

IN VITRO CULTURE OF FREE-LIVING CONCHOCELIS PHASE 
Origin of free Conchocclis 

Strains of free-living Conchocclis were obtained in several ways: (1) from the 
free Conchocelis growing out of the shell flakes in SWI medium; (2) directly 
from carpospores released by mature thalli collected in Japan, shipped to New York 
(March, 1960), and germinated in liquid media; (3) from carpospores produced 
by thalli grown in artificial media /;; vitro. 

At first, on the assumption that a substrate might be somehow advantageous 
to the Conchocclis phase, tufts of Conchocclis filaments, cut from the free growth 
on shell flakes in SWI, were transferred into biphasic media. To simulate the 
conditions in shells, the solid phase (10 ml. ASPI medium + agar 1.5%) was 
enriched with 0.1 % CaCCX, 0.01 % chondroitin, or both; the liquid phase consisted 
of 5 ml. either of ASP7 or SWI; the Conchocclis tufts were implanted in the 
agar at the interphase. All these combinations allowed good growth at 13-15 C. 
and continuous subdued light and at 18-20 C. and 10 hours daily. In 2-3 months, 
from an initial tuft 1 mm. in length, spherical colonies of 0.5-1 cm. were obtained; 
later, new colonies formed at the interphase or on the glass wall. Growth was 
almost entirely in the liquid phase and in all the different combinations, indicating 
that a solid substratum rich in CaCO., or protein is unnecessary. Further experi- 
ments were done in liquid media to determine the best cultural conditions for free 
growth in liquid media. 

Once some of these conditions were known, it became possible to germinate 
directly in liquid media carpospores collected from thalli grown either in nature or 
in vitro. Thalli of P. tcncra collected in Matsukawa-ura inlet were shipped to 
Xew York in March, 1960. Following the method suggested by Professor Y. 
Yamada of Hokkaido University, the thalli were put between pads of absorbent 
cotton wet with sea water and shipped in Thermos bottles; this method avoids 
rotting and gives good survival. Upon their arrival in New York, the thalli were 
placed in enriched sea water and produced carpospores. The collected carpospores 
were washed several times in sterile sea water by means of capillary pipettes, and 
3-5 carpospores were inoculated in test tubes containing 10 ml. of 3 types of 
enriched sea water (AS\Y8 ; SWI, SWII) and 9 artificial media (ASM, ASPI, 



176 



HIDEO IWASAKI 



TABLE II 

Artificial media composition (w./v.) 





ASPl 


ASP2(NTA) 


ASP6 


ASP7 


ASP12(NTA)* 


Distilled water 


100 ml. 


100 ml. 


100 ml. 


100 ml. 


100 ml. 


NaCl 


2.4 g. 


1.8 g. 


2.4 g. 


2.5 g. 


2.8 g. 


MgSO 4 -7H,O 


0.6 g. 


0.5 g. 


0.8 g. 


0.9 g. 


0.7 g. 


MgCl 8 -6H.O 


0.45 g. 








0.4 g. 


KC1 


0.06 g. 


0.06 g. 


0.07 g. 


0.07 g. 


0.07 g. 


Ca (as C1-) 


40 mg. 


10 mg. 


15 mg. 


30 mg. 


40 mg. 


NaNO, 


10 mg. 


5 mg. 


30 mg. 


5 mg. 


10 mg. 


K 2 HPO 4 


2 mg. 


0.5 mg. 








K 3 PO, 










1.0 mg. 


Na 2 -glycerophosphate 






10 mg. 


2 mg. 


1.0 mg. 


Na 2 SiO 3 -9H 2 O 


2.5 mg. 


15 mg. 


7 mg. 


7 mg. 


15 mg. 


Na 2 CO 3 




3 mg. 








Fe (as Cl) 




0.05 mg. 








BIJ 


0.02 M g. 


0.02 Mg- 


0.05 /xg. 


0.1 Mg- 


0.02 Mg- 


Biotin 










0.1 Mg- 


Thiamine 










10 Mg- 


Vitamin mix 8** 


0.05 ml. 




0.1 ml. 






Vitamin mix S3** 




1 ml. 




1 ml. 




PII Metals**** 


1.0 ml. 


3 ml. 




3 ml. 


1 ml. 


SI I Metalsf 










1 ml. 


P8 Metalsff 






1 ml. 






Tris buffer 


0.1 g. 


0.1 g. 


0.1 g. 


0.1 g. 


o.i g. 


Nitrilotriacetic acid 




(10 nig.) 




7 mg. 


(10 mg.) 


pH 


7.6 


7.8 


7.4-7.6 


7.8-8.0 


7.8-8.0 



* Developed by L. Provasoli for tropical species of dinoflagellates. 

** One ml. of Vitamin mix 8 contains: thiamine HC1, 0.2 mg. ; nicotinic acid, 0.1 mg. ; 
putrescine 2HC1, 0.04 mg. ; Ca pantothenate, 0.1 mg. ; riboflavin, 5 Mg- ; pyridoxine 2HC1, 0.04 
mg. ; pyridoxamine 2HC1, 0.02 mg. ; />-aminobenzoic acid, 0.01 mg. ; biotin, 0.5 Mg- ; choline H 
citrate, 0.5 mg. ; inositol, 1.0 mg. ; thymine, 0.8 mg. ; orotic acid, 0.26 mg. ; Bi 2 , 0.05 Mg- ; folic acid, 
2.5 Mg- ; folinic acid, 0.2 Mg- 

*** One ml. of Vitamin mix S3 contains: thiamine HC1, 0.05 mg. ; nicotinic acid, 0.01 mg. ; 
Ca pantothenate, 0.01 mg. ; />-aminobenzoic acid, 1 Mg- ; biotin, 0.1 Mg- ; inositol, 0.5 mg. ; folic acid, 
0.2 Mg- ; thymine 0.3 mg. 

**** One ml. of PII metal contains: ethylenediamine tetracetic acid, 1 mg. ; Fe (as Cl), 
0.01 mg. ; B (as H 3 BO 3 ), 0.2 mg. ; Mn (as Cl) 0.04 mg. ; Zn (as Cl), 0.005 mg. ; Co (as Cl), 0.001 mg. 
f One ml. of SI I metals contains: Br (as Na), 1.0 mg. ; Sr (as Cl), 0.2 mg. ; Rb (as Cl), 
0.02 mg. ; Li (as Cl), 0.02 mg. ; I (as K), 0.001 mg. ; Mo (as Na), 0.05 mg. 

ft One ml. of P8 metal contains: Na 3 versenol, 3 mg. ; Fe (as Cl), 0.2 mg. ; Mn (as CD, 
0.1 mg. ; Zn (as Cl), 0.05 mg. ; Co (as Cl) 0.001 mg. ; Cu (as Cl), 0.002 mg. ; Mo (as Na), 0.05mg. ; 
B (as H 3 BO 4 ), 0.2 mg. Versenol =hydroxyethyl-ethylenediamine triacetic acid. 

ASP2, ASP2NTA, ASP6, ASP7, ASP12, ASP12NTA and D; Table II). 
Conchocelis growth was obtained in most of these media except ASW9 and ASP2. 
ASPl, ASP6, ASP12NTA, ASP12 and ASW8 gave very good growth; ASP7, 
D, and SWII were less good ; SWI very poor. 

Young germlings of P. tenera (5 mm. long) cultured at 14-16 C., and illumi- 
nated 13 hours a day with 400-500 foot-candles of incandescent light, did not grow 
normally (see later) and produced carpospores from which Conchocelis colonies 
developed. 



LIFE-CYCLE OF PORPHYRA TENERA 177 

Suitable media and cultural conditions for free-living growth of Conclioeelis 

Several artificial media and enriched sea waters permit continued growth of 
the Conclioeelis phase. In decreasing order, ASP12NTA, ASP2NTA, ASP12, 
ASP6, and ASP7 are the most suitable artificial media, and SWII and SWI the 
enriched sea waters. Conclioeelis cultures easily last 6 months ; the color of the 
colonies varies in different media : pinkish-red in ASP12NTA, ASP12, and ASP6; 
pale brown in ASP2NTA, dark brown in SWII, and pinkish-grey in ASM. The 
color is more intense in the center of the colony, probably because of the presence 
there of intensely pigmented monosporangial branches. The type of medium in- 
fluences growth rate and monosporangia formation. In decreasing order, growth 
was fastest in ASP12NTA, SWII and ASP7 and slower in ASP2, ASP12, 
and MEC3. Monosporangia were formed and monospores liberated earlier in 
ASP12NTA, and in decreasing order in ASP12, ASP7, ASP2, SWII, SWI. 
The temperature range is between 10 and 26 C. ; the optimum between 13 and 
20. Single pieces of the filament of the Conclioeelis phase ("-'I mm.) transferred 
in new media grew into new Conclioeelis colonies vegetatively. It was possible in 
this way to subculture the Conclioeelis phase : 5 serial transfers (one every 2-3 
months from February to December, 1960) resulted in good growth. Quite likely 
the Conclioeelis phase can be grown indefinitely as free-floating colonies in liquid 
media. 

The Conclioeelis colonies in test tubes of liquid media generally grew at the 
bottom of the tube attached to the glass wall and appeared as fuzzy balls 4-10 mm. 
in diameter (Fig. 1). In larger containers, where they grow free-floating in 
the medium, they were stellate, often reaching a diameter of 10-15 mm. (Fig. 2). 
The Conclioeelis phase can be grown, but poorly, also on agar slants in screw- 
cap tubes. 

At the beginning of this work the free-living Conclioeelis colonies were grown 
in subdued light (20-40 ft.c. ) to simulate natural conditions; under these conditions 
growth was quite slow. Later, in surveying the effect of light intensity, it was 
found that growth was greatly increased by higher light intensities the higher, 
the better (maximum tried, 350 ft. c.). Incandescent and fluorescent light were 
equally effective ; however, the color of the Conclioeelis was different : reddish in 
fluorescent light and cool brown-black in incandescent light. Continuous illumina- 
tion also favored growth. Under these conditions (350 ft. c. continuous fluorescent 
light) mass cultures were obtained in 2-liter Erlenmeyer flasks and in tall, 4-liter 
bottles (Fig. 3) by gradual transfer in increasingly larger containers (10 ml. 
inoculated into 100 ml.; 100 ml. in 1 liter, etc.). 

Effect of fihotoperiodisni on monosporangia and monospore production 

Kurogi's experiments (1959) indicated that photoperiodism may govern mono- 
sporangia production and monospore liberation in Conclioeelis grown in shells. 
The following experiment was set to test the effect of photoperiodism on free- 
living Conclioeelis. With fluorescent light of 150-250 ft. c., a daily photoperiod 
of 8-11 hours induced formation of monosporangia in 2-3 weeks and young thalli 
in 3-8 weeks from the time of inoculation into new media of pieces of Conclioeelis 
filaments (Figs. 5, 6, 7). No substantial difference was found in cultures grown 



178 



HIDEO IWASAKI 



















FIGURES 1-7, 12. 



LIFE-CYCLE OF PORPHYRA TENERA 179 

at 13-15 C. or 18-20 C. in high light (150-250 ft. c.) for a daily photoperiod 
of 8-11 hours. When the light intensity was reduced to 30-50 ft. c., the appearance 
of young thallus germlings was greatly retarded in the 8-hour photoperiod ( > 96 
and < 184 days) and apparently prevented in the 11 -hour photoperiod (no leafy 
thalli in 180-240 days). 

Under continuous fluorescent light at intensities of 150-250, 60-100, and 
10-20 ft. c., both at 13-15 and 20-26, neither spores nor thallus germlings were 
found during the two experiments which lasted, respectively, 180 and 240 days. 
Growth of Conchocelis filaments and colonies proceeded normally, and may, indeed, 
be favored by continuous light ; very good mass cultures were obtained in con- 
tinuous light. Intensely purple, inflated portions similar to monosporangia, 
appeared after a month or more in cultures of 60-250 ft. c. At that time these 
structures were thought to be small monosporangia. Unfortunately the obser- 
vations of these experiments were done at great intervals (one month or more), 
and through the walls of the test tubes using a dissecting microscope. Only 
later, when it became evident that these structures were not producing spores, 
was a simple experiment tried : a Conchocelis colony grown for two months in 
continuous light at 13-15 C. was transferred to new medium and illuminated 8 
hours a day ; after 5 weeks many thallus germlings ( 5-8 mm. long ) were growing 
alongside the Conchocelis colony. 

Evidently, maturation of monosporangia, release of monospores, or both, are 
induced by a short photoperiod and prevented by continuous light. 

The sporangia produced in continuous light (250350 ft. c. ) in the mass cultures 
seemed to be morphologically different from the monosporangia produced under 
short-day conditions. Sporangia cells in continuous light have thicker walls and 
length of the cells is usually about half their diameter ; some cells are quadrate 
(Fig. 4). They are very similar to the ''plantlets" described for P. iiinbilicalis var. 
laciniata by Drew (1954, p. 203, Fig. 4c). 

On the contrary, the cells of the short-day monosporangia often have elongated 
cells and the appearance of monosporangia is much more twisted (Figs. 5, 6, 7) 
because of the lateral branches. Are the continuous-light sporangia undeveloped 
or abnormal monosporangia, or are they a new type of sporangium ? The evidence 
at hand does not exclude either possibility. 

The aforementioned experiment of transferring a Conchocelis colony from con- 
tinuous light to short-day is indicative but not conclusive ; only one observation 
was made 38 days after the transfer : young thalli of 5-8 mm. were found. The 
formation dc noz'o of true monosporangia is not excluded because under the same 
light and temperature conditions (exp. //, Table III) young thalli appeared between 
22 and 31 days in a culture started with pieces of Conchocelis filaments (no length 
was noted in the protocols; they were probably 2-3 mm. long). 

FIGURE 1. Colonies of free-living Conchocelis in artificial medium. 

FIGURE 2. Free-floating Conchocclis (detail of Figure 3). 

FIGURE 3. Mass culture of Conchocelis in aerated 3-liter bottle, continuous light. 

FIGURE 4. Sporangia formed in continuous illumination. 

FIGURE 5. Typical monosporangia formed in short day conditions (8-11 hours daily). 

FIGURES 6, 7. Same detail. 

FIGURE 12. Young thallus germlings, and monospores. 



180 



HIDEO IWASAKI 



The hypothesis that the Conchocelis phase can produce other sporangia besides 
the monosporangia has already been advanced by Drew for Porphyra umbilicalis 
var. laciniata (1954) and Bangia juscopiirpiirea (1958) to explain Conchocelis 
infections in sterile shell derived from other Conchocelis-miected shells. This 
possibility is greatly reinforced by our experiments with P. tenera. More than 5 
serial transfers were done directly from Conchocelis to Conchocelis in test tubes 
or in mass culture without passing through the thallus phase or carpospores : in 
every case, inoculating pieces (in test tubes) of Conchocelis filaments or entire 
colonies (in mass cultures ) led to numerous new colonies. These experiments 
also do not prove conclusively that the increase in the number of Conchocelis colonies 
is due to the production of special spores developing into new Conchocelis colonies, 

TABLE III 

Effects of short-day and continuous light conditions on Conchocelis phase* 



Temperature 


Light** 
period 


Light 
intensity** 


Appearance of 
sporangiaf 


Appearance of 
foliaceous thallif 


Remarks 


13-15 C. 


8 hr. 


150-250 ft. c. 


/ 19 days M. 
II <22 days M. 


<46 days 
>22-<3l days 


good growth of thalli 
(1-1.5 cm.) 
3 cm. thalli in 96 days 


30-50 ft. c. 


I 27 davs M. 
11 <48 days M. 


none up to 84 days 
>96- <184 days 


discontinued at 84 days 
small thalli 


18-20 C. 


11 hr. 


150-250 ft. c. 


1 19 days M. 
11 23 days M. 


>56- <84 days 
31 days 


good growth of Concho- 
celis-tha\\i soon 
bleached 


30-50 ft. c. 


I 19 days S2 
II 23-31 daysS.? 


none up to 240 days 
none up to 184 days 


large Conchocelis 
colonies 


13-15 C. 


continuous 


150-250 ft. c. 


/ 35 days Sl-SZ 
11 31 days S1-S2 


none up to 240 days 
none up to 184 days 


tt 


30-50 ft. c. 


I 84 days SZ 
II 72-96 days S2 


none up to 240 days 
none up to 184 days 




20-26 C. 


continuous 


60-100 ft. c. 


I 35 days SZ 
II 31 days S3 


none up to 240 days 
none up to 184 days 




10-20 ft. c. 


/ 56 davs S2 
// S? 


none up to 240 days 
none up to 184 days 


(Figs. 8-11) 



* Media: ASP7 and SWII. Results of two separate experiments (/ and //). M = monosporangia; SI, see 
Figure 4; 5.?, see Figures 8-11. 
** Fluorescent : "cool white." 

t Days from date of inoculation. Inoculum = a small piece of Conchocelis filament. 

ft At 50 days a Conchocelis colony was transferred to new medium and to 8 hours of light. In a month monospores 
and thalli appeared. 

because even small pieces of Conchocelis filament, which can grow into a full new 
colony, could have been present. However, the short-celled sporangia produced by 
P. tenera in continuous light (Fig. 4) or the sporangia-like swollen cells described 
by Drew for Bangia (1958, Fig. 3, p. 366) may be a new type of sporangium 
whose spores produce a new Conchocelis colony. 

Other very strange structures are produced in continuous (10100 ft. c.) or 
11 hours subdued fluorescent light (30-50 ft. c.) at 13-15 C. and 20-26 C. 
(Table III). The similarity of the latter sporangia with fungal structures is striking 
(Figs. 8, 9, 10, 11 ; sporangia (?) 52 of Table III). 

The variety of structures created in different lights and temperatures shows 
that P. tenera has unusual powers of adaptation. The Conchocelis phase can now 



LIFE-CYCLE OF PORPHYRA TENERA 181 

be grown free, making the morphological observations easy. This permits a wider 
analysis of the unusual morphological versatility of P. tcnera as well as of the 
possible deviations from the normal life-cycle induced by various lights and 
temperatures. 

THE LEAFY THALLUS PHASE 

Some cultural conditions for the growth of the thallus had been determined 
previously (Iwasaki and Matsudaira. 1958, and unpublished). 

(1) Leafy thalli grow normally in enriched sea water (Miquel's sea water), 
while they are short and unhealthy when grown in filtered, unenriched inshore 
sea water. 

(2) High-intensity, incandescent light is required for normal continued growth; 
growth is, however, slower than in natural sunlight. Young plants grown in 
fluorescent light die in a few days. 

(3) Young plants grow normally when illuminated 8-10 hours daily but die 
quickly when grown in continuous light. 

These results were on the whole confirmed by the present investigation. 

Effect of media on leafy thallus t/ro'^'th 

Thalli (1-2 mm.) derived from monospores produced by free-living Conchocelis 
(Fig. 12) were grown in enriched sea water and artificial media at 1416 C., 

TABLE IV 

Thallus growth (two-month) 
Media Growth Color 

ASP1 10 X 40 mm. brown 

ASP2 8 X 50 mm. 

ASP12 20 X 40 mm. red-brown 

ASP12NTA 8 X 20 mm. 

SWI 8 X 80 mm. reddish 

SWII 20 X 35 mm. pale brown 

and illuminated 9 hours daily with 400 ft. c. of incandescent light. The experiment 
was done in test tubes (20 X 120 mm.) containing 10 ml. of medium ; once a month 
the medium was replaced aseptically with 10 ml. of fresh medium. Good normal 
growth was obtained in two months in some artificial media and in enriched sea 
water (Table IV and Fig. 13). Narrow long thalli were obtained in SWI and 
most artificial media, broader thalli in SWII and ASP12. The 1957 experiments 
were done during the season in which the thalli grow in nature (fall- winter). On 
the contrary, the new experiment was done between May and August. 1960, indi- 
cating that normal thalli can be grown out of season if the light period is suitable 
(8-11 hours daily ). 

Effect of long-day conditions on leafy thalli 

In retrospect, the importance of the photoperiod for Porphyra tenera might 
have been suspected because the two phases of the life-cycle of P. tcncra correspond 
so sharply to the seasons. The leafy thallus grows in the short-day seasons 



182 



HIDEO IWASAKI 








FIGURES 8, 9, 10, 11. Inflated cells (sporangia?) produced in subdued light. 

FIGURE 13. Two-month-old thalli grown in test tube. From left, medium SWI, SWII, 
ASP12, ASP1. 

FIGURE 14. Young thallus degenerated under long-day conditions (13 hours daily). Lower 
part bleached; large pigmented cells at top; Conchocclis filaments germinating from "spores." 

FIGURE 15. Root-like projections growing out of a young thallus grown in SWII under 
long-day conditions. 



LIFE-CYCLE OF PORPHYRA TENERA 



183 



(autumn-winter), the Conchocelis phase in the long-day seasons (spring-summer). 
Furthermore, the transition between the two phases of the life-cycle coincides with 
the equinox (Fig. 16). On the contrary a great part of the temperature range 
(7-21 C.) is common to the two phases: normal thalli grow in nature between 
3-21 C. and the Conchocelis between 7 and 25 C. Therefore, only the lower 

j 

zone (3-7 C.) may be suspected to affect Conchocelis growth and the upper zone 
(21-25 C. ) thallus growth. These considerations, and the already known effects 
of continuous light on thallus growth (Iwasaki and Matsudaira, 1958) and short 
day on monosporangia formation (Kurogi, 1959) suggested trial of growth under 
long-day conditions. 

Five young germlings (0.5 mm.), derived from monospores of free-living 
Conchocelis, were inoculated in each tube of the following media: SWI, SWII, 
ASP1, ASP2, and ASP12. They were incubated at 14-16 C. and illuminated 
13 hours daily with 400-500 ft. c. of incandescent light. The controls were grown 



o 

.c 



o> 

c 



o 

Q 



15 
14 
13 
12 

I I 

10 

9 




30 



o 

o 



0> 

CL 


o> 



10 



o 

0> 

CO 



Jon. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan. 
FIGURE 16. Day length at Sendai and average sea water temperature in Matsushima Bay. 

under similar conditions but illuminated 8 hours daily. The control gave, as in the 
previous experiment, normal thalli of the narrow shape. 

The thalli in long-day conditions grew very slowly and very soon became thick 
and irregular in shape. After 20 days or more, the thalli became pale, leaving 
numerous scattered big reddish-colored cells. The thallus around the edges as- 
sumed the appearance of a callus tissue (Fig. 14). After 40 days (in ASP2) or 
more, spores were released. Since these spores germinated into filaments which 
later formed well-developed Conchocelis colonies, we assume that, at least function- 
ally, they are equivalent to carpospores. These events differ slightly in time and 
amount of growth in the various media except SWII. In SWII the thallus after 
27 days started to produce root-like projections (Fig. 15) which branched out 
into thinner filaments; after two months the thallus became covered with Concho- 
celis colonies. Apparently 13 hours of daylight, which corresponds at the latitude 
of Sendai (39 N) to late April, already inhibits normal thallus growth and in- 



184 HIDEO IWASAKI 

duces the formation of structures functionally equivalent to carpospores. This 
experiment was repeated later with similar results. 

In another experiment in tall covered containers (10 cm. diameter; 7 cm. high) 
containing 200 ml. of ASP1, young thallus buds (1-2 mm.) were grown for one 
month (April 22-May 25) at 14-16 and illuminated 8 hours daily with 400 ft. c. 
of incandescent light : the thalli, which had reached by then an average size of 
3 cm. 2 , were illuminated for 10 days (May 25-June 4) with 100-200 ft. c. of 
incandescent light : on alternate days, 8 hours daily followed by one day in 
continuous light. After this period of alternating photoperiods, the culture was 
grown in 100-200 ft. c. and 8 hours daily of fluorescent light. In a month (July 5) 
big, dark cells appeared, scattered at the edges of the leafy thalli which were 
stunted and curled. Ten days later these cells produced germ tubes which devel- 
oped into Conchocelis colonies. Two months (August 5) after the alternating light 
treatment, many colonies of Conchocelis were growing free on the bottom of the 
dish and covering the stunted disintegrating thalli. After 82 days (August 25) 
mature monosporangia were formed and a few days later the monospores were 
released and produced thallus germlings. These germlings on September 10 had 
already reached an average size of 1 cm. 2 

The complete life-cycle was obtained in 5-6 months. 

It is remarkable that the leafy thalli of the second generation grew normally, 
though slowly, in fluorescent light and at low intensities (100-200 ft. c.). This is 
by no means an isolated case of thallus growth in fluorescent light : all the cultures 
of Conchocelis grown at 13-20 C. for 8-11 hours daily of fluorescent light even- 
tually released monospores (1-4 months, depending upon light intensity). These 
spores gave rise to leafy thalli reaching 2-10 mm. before they became pale and died. 
The arrest in growth of these leafy thalli was probably due to lack of nutrients : 
the medium in these experiments was not changed monthly, as was done for the 
experiment on thallus growth in different media. Ability of the thalli to grow 
in fluorescent light may be an adaptation to utilization of fluorescent light acquired 
during the Conchocelis phase : the Conchocelis phase does not require incandescent 
light. This adaptability, whatever the cause, reflects again the great plasticity and 
versatility of P. tenera. 

DISCUSSION 

These results may help solve some of the problems of life-cycle and growth 
potencies of P. tenera. Solutions here, in turn, may improve the farming of this 
sea weed. The first report of the entire life-cycle of a Porphyra obtained in vitro 
is the one of Hollenberg (1958). He obtained from carpospores Conchocelis-\ike 
filaments which formed sporangia and liberated spores (16 days from carpospore 
germination ) . These spores in turn developed into blade-like plantlets (young 
thalli). Since the cultures were grown during the summer in north light, it is 
possible that the very rapid formation of sporangia and the poor growth of the 
Conchocelis phase were due to light conditions. While this paper was being 
written, the paper on the Conchocelis stage of P. umbilicalis by Kornmann (1960) 
appeared. Like us, Kornmann obtained the complete life-cycle in vitro. He started 
in November, 1959, with a "plantlet" (probably an immature, or abnormal mono- 
sporangium) cultured in Erdschreiber. The "plantlet" became fertile and made 



LIFE-CYCLE OF PORPHYRA TENERA 185 

monospores which did not develop. Only a few cells of this structure remained 
vegetative and reproduced in a month another "plantlet" (without "rhizoids") 
which produced many monospores. Of these, only 6 germinated into leafy thalli 
which in a month and a half reached 1.5-2 mm. in length. The thalli formed 
"Ballchen" from which thin filaments grew out; the filaments, by division, produced 
"zweige" (his Figure 5 -- "plantlet" = monosporangia ?). From the "zweige" 
arose as side-branches ("seitliche Verzweigung") thin filaments which in free 
culture produced a confused ball of yarn (verworrene Knauel = free Conchocelis) 
or enveloped the original plantlet. These filaments grow also as a typical Concho- 
celis in calcareous shells. 

Unfortunately, no data are given of the light period under which the cultures 
were grown. From his Figures 2 and 5, the "plantlets" are very similar to mono- 
sporangia. If so, the thin filaments (which are Conchocelis filaments) should 
produce, and not be produced by the monosporangia (as Kornmann states in Figure 
5 and the text). But the structure in Figure 5 could be equivalent to the sporangia 
which were produced in our Conchocelis colonies grown in continuous light (our 
Fig. 4). As mentioned, these sporangia are suspected of producing spores germi- 
nating into a new Conchocelis. Kornmann's light conditions seem also to be inade- 
quate for thallus growth because, as in our thallus cultures under long-day condi- 
tions, Conchocelis filaments arise from the thallus (Kornmann, Fig. 3B) or big 
colored cells are formed (Kornmann's "Ballchen" which can be seen at the base 
of the thallus of Figure 3, C) from which Conclwcelis filaments arise. Kornmann's 
Figure 1C represents, most likely, true monosporangia and Conchocelis filaments. 

Whatever the interpretation, it is seen that, both in Kornmann's and in our 
experiments, the life-cycle can be obtained in vitro. Detailed morphological studies 
are planned to solve some of the many questions ; e.g., what is the typical mor- 
phology of the true monosporangia of Conchocelis grown free how do they differ 
from those produced in shells ? What are the mysterious "plantlets" of Drew, 
and of Figures 1A and 2A of Kornmann are they sporangia whose spores develop 
another Conchocelis phase, or abnormal monosporangia? What are the deviations 
from the natural life-cycle in shells that develop when the Conchocelis phase is 
grown free and in different day-lengths and light-intensities? What are the big, 
dark cells formed in the degenerating thalli under long-day conditions ? 

The present research confirms and extends previous results on the effect of the 
photoperiod on P. tcncra. As mentioned, the Conchocelis phase grows in nature 
during the long-day seasons and the leafy thallus phase in short-day seasons. The 
leafy thallus phase is apparently a short-day plant: growth is arrested and the 
thallus degenerates when exposed to 13 hours of light daily. The Conchocelis 
phase is not strictly a long-day plant : in vitro it grows, but slowly, under short- 
day (8-hour) conditions and in subdued light. However, high light, longer day 
(11-hour), and especially continuous light enhance growth vigorously. The in- 
complete data available indicate that the photoperiod governs the formation of 
monosporangia and the liberation of monospores. Our in vitro experiments confirm 
fully the results of Kurogi (1959) obtained with Conchocelis grown in shells. He 
found that photoperiods of 10 and 12 hours of light (corresponding to conditions 
of winter, spring and autumn, respectively) induce an abundant formation of mono- 
spores, while 15 hours of light daily did not enhance the formation of monosporangia. 



186 HIDEO IWASAKI 

Furthermore, the Conchocelis which were liberating monospores in 10-hour 
photoperiods continued for only a few days, and then stopped liberating mono- 
spores, when transferred to 15 hours of light; conversely the long-day (15-hour) 
Conchocelis began to liberate monospores after they were transferred to short-day 
(10-hour) conditions. Similarly the in vitro experiments on free-living Concho- 
celis show that short-day (8-, 11-hour) induces early formation of monosporangia 
and liberation of monospores. Continuous light, or subdued 11-hour photoperiods, 
induce the formation of interesting and different sporangia, or peculiar inflated 
cells in the Conchocelis filaments, whose fate and origin need further investigation. 

The preliminary experiments on the thallus indicate that the photoperiod also 
governs the formation of carpospores ; 13 hours of light daily induce cessation of 
growth and degeneration of the leafy thallus, followed by formation of carpospores 
or their physiological equivalents. Exposure of full-grown thalli to different photo- 
periods is now needed to define precisely the effect of the photoperiod on carpospore 
production. 

These findings emphasize the need of determining the effect of photoperiods 
on the life-cycle and alternation of generations in sea weeds. Foyn (1955) had 
observed that the northern species of Uh'a (lactuca) can grow normally in con- 
tinuous light, while the southern Mediterranean species (Tliurcti) dies in such 
conditions. 

This work was supported by contract NR 104-202 of the Office of Naval Re- 
search and by Grant G-1198 of the National Science Foundation to Dr. L. Provasoli. 
of Raskins Laboratories. I wish to thank Dr. Provasoli for his hospitality, advice 
and constant interest. 

SUMMARY 

1. The complete life-cycle of Porphyra tcncra was obtained in vitro. 

2. Chemically defined media or enriched sea water permit good growth of these 
unialgal (not bacteria-free) cultures. 

3. Under suitable light and temperature, the complete life-cycle is completed in 
5-6 months. Both the Conchocelis and the thallus phases may be grown out of 
season. 

4. The Conchocelis phase grows well free in liquid media ; a calcareous substrate 
is unnecessary. Conchocelis colonies grown in liquid media when free-floating, 
are stellate and round, but mold-like when attached to glass walls. They are brown- 
black or purple-red, depending on the composition of the medium. Rapid and 
abundant growth of the free Conchocelis is elicited by high-light intensities. Fluor- 
escent light is a good light source. 

5. Monosporangia formation and release of fertile monospores are induced by 
short-day conditions (8-11 hours daily) ; monosporangia and germinating mono- 
spores develop after 1-2 months from the inoculation of the Conchocelis filaments. 
In continuous light, Conchocelis growth is rapid but the sporangia produced are 
somehow different from the ones produced in short-day conditions. 

6. In continuous light, the number of colonies increases rapidly after transfer 
to new media. This could be due to formation of new colonies from small pieces 
of filaments. However, even though free spores were not found, it is not excluded 
that ne\v Conchocelis colonies may have been derived from special spores. 



LIFE-CYCLE OF PORPHYRA TENERA 187 

7. The Conchocelis phase was cultured for one year by transferring free Concho- 
cclis colonies or pieces of filaments every two months in new media. Mass cultures 
with good yields were obtained in continuous fluorescent light. 

8. The leafy thallus, derived from monospores grown in shells, grows well and 
normally in artificial media, at 13-18 C. and in high intensity incandescent light 
of 8-11 hours daily, but not in fluorescent light. 

9. A photoperiod of 13 hours daily inhibits growth of young thalli (1-2 mm.). 
The thalli became thick, curly, degenerate, assume a callus appearance, bleach 
almost completely except for scattered groups of dark-pigmented, big cells which 
produce spores germinating into ConcJwcclis filaments. In one type of enriched sea 
water (SWII), the thalli, after thickening, and while degenerating, produce rhizoid- 
like structures which give rise to Conclwcelis filaments. 

10. In nature, the Conchocelis phase grows in the long-day seasons, the leafy 
thallus phase grows in the short-day seasons ; and the transition between the two 
phases is almost exactly at the equinox. On the contrary, no correlations exist 
between temperature and the phases of the life-cycle : a large temperature zone 
(7-21 C.) is common to the two phases. Similarly, our preliminary experiments 
show that the length of the photoperiod has remarkable effects on the Conchocelis 
and leafy-thallus phases of P. tcncra. The photoperiod governs, besides growth, the 
formation of the spores producing the next phase of the life-cycle. It is reasonable, 
therefore, to suppose that like land plants, some sea w r eeds, or phases of their life- 
cycle, may be long- or short-day plants. 

LITERATURE CITED 

DREW, K. D., 1949. Conchocelis in the life history of Porphvra umbilicalis (L.) Kiitz. Nature , 

164: 748. 
DREW, K. D., 1954. Studies in the Bangioidae III. The life-history of Porphyra umbilicalis 

(L.) Kutz. var. laciniata (Lightf.) J. Ag. Ann. Bot. N. S., 18: 183-211. 
DREW, K. D., 1958. Studies in the Bangiophycidae IV. The Conchocelis-phase of Bangia 

juscopurpurca (Dillw.) Lyngbye in culture. Pubbl. Stas. Zool. Napoli, 30: 358-372. 
FOYN, B., 1955. Specific differences between northern and southern European populations of 

the green alga Uh'a lactnca L. Pubbl. Stas. Zool. Napoli, 27 : 261-270. 
HOLLENBERG, G. J., 1958. Culture studies of marine algae III. Porphvra pcrjorata. Anier. 

J. Bot., 45: 653-656. 
IWASAKI, H., AND C. MATSUDAIRA, 1958. Culture of a laver, Porphyra tcncra Kjellm. I. 

Preliminary research on cultural conditions. Bull. Jap. Soc. Sci. Fish.. 24: 398-401 
KORNMANN, P., 1960. Von Conchocelis zu Porphyra. Hclgolander Wiss. Mccrcsuntcrs., 1 : 

189-193. 
KUROGI, M., 1953. Studies of the life-history of Porphyra. I. The germination and development 

of carpospores. Bull. Tohoku Reg. Fish. Lab., No. 2: 67-103. 
KUROGI, M., 1959. Influences of light on the growth and maturation of Conchocelis-tiiallus of 

Porphyra. I. Effect of photoperiod on the formation of monosporangia and liberation 

of monospores. Bull. Tohoku Reg. Fish. Lab. No. 15 : 33-42. 
KUROGI, M., AND K. HIRANO, 1956. Influence of water temperature on the growth, formation of 

monosporangia and monospore-liberation in the Conchocelis phase of Porphyra tcncra 

Kjellm. Bull. Tohoku Reg. Fish. Res. Lab., No. 8: 45-61. 
TSENG, C. K., AND T. J. CHANG, 1954. Studies on the life history of Porphyra tcnera Kjellm. 

Sci. Sinica, 4: 375-398. 



RESPIRATION RATES IN PLANARIANS. III. THE EFFECT OF 
THYROID COMPOUNDS ON OXYGEN CONSUMPTION 1 

MARIE M. JENKINS 

Department of Zoology, Unii'crsity of Oklahoma, Norman, Oklahoma 

lodinated proteins have been found throughout the invertebrate world (Roche, 
1952; Gorbman ct al., 1954), primarily in the form of mono- and diiodotyrosine, 
although in a number of insects (Limpel and Casida, 1957) and in Miisculium, a 
fresh-water fingernail clam (Gorbman ct al., 1954), a high percentage of the 
protein-bound iodine has been shown to be in the form of thyroxine. None of 
these compounds has been demonstrated unequivocally to take part in physiological 
processes in the invertebrate animal (Goldsmith, 1949; Gorbman ct a!., 1954), but 
recent reports indicate the question is not settled. Wingo and Cameron (1952) 
found that thyroxine hampered the multiplication of a ciliate protozoan, Tetra- 
hyincna gcleii, but increased the rate of oxygen uptake above that of parallel control 
cultures. Thyroxine added to the diet of rice moth (Corcyra cephalonica) larvae 
is reported to have increased the oxygen consumption requirement, although 
thyroglobulin was without effect (Srinivasan ct al., 1955). 

The presence of iodinated proteins in planarians has not been investigated, but 
several workers have reported a positive action of thyroid compounds on physio- 
logical activities in this group. Castle (1928) observed that Phagocata (Planaria) 
vclata was attracted to and fed readily upon macerated sheep thyroid, and sub- 
sequently decreased in size even more rapidly than worms subjected to starvation. 
Goldsmith (1937), studying the effect of endocrine feeding on regeneration and 
growth in Ditgcsia tigrina (Planaria inaculata), noted no significant differences in 
the head regeneration time in the gland-fed animals, but found that thyroid-fed 
individuals increased in size to a lesser extent than the liver- and pituitary-fed 
forms. The influence of thyroxine on eye formation in Phagocata gracilis was 
investigated (Weimer ct al., 1938) in pieces of planarians cut at different levels and 
allowed to regenerate in a saturated thyroxine solution.. Once the reconstitution 
process had begun, the rate of eye formation was reported to be much higher for 
the pieces in thyroxine. 

No reports are available of the effect of thyroid hormones on oxygen con- 
sumption in planarians. Phenylthiourea, an anti-thyroid agent, has been shown, 
however, to exert a depressing effect on planarian respiration (Jenkins, 1961). In 
view of these findings an investigation was undertaken to ascertain the effect of 
certain thyroid compounds on respiration rates in Dugcsia dorotoccphala, a common 
fresh-water planarian. 

1 Supported in part by grants from the National Science Foundation (G-3209), the South- 
ern Fellowship Fund, and the University of Oklahoma Alumni Development Fund. This 
study represents part of a dissertation submitted in partial fulfillment of the requirements for 
the Ph.D. degree at the University of Oklahoma, under the direction of Dr. Harriet Harvey. 

188 



THYROID COMPOUNDS AND PLANARIANS 189 

DESIGN OF EXPERIMENT 

The planarians used in this study were large, sexually mature animals, collected 
from Buckhorn Springs - in Murray County, Oklahoma. They were maintained 
in pans of lake water, provided with an aerator, at a constant temperature of 20 C. 
Experimental animals were taken on the seventh day after feeding and were not 
fed during the course of the experiment. 

Compounds used for this investigation were thyroxine (T 4 ), 3,5,3'-triiodo- 
thyronine (T 3 ), 3 and 3,5-diiodotyrosine (DIT). In order to determine the con- 
centration to be used, groups of cut posterior ends of planarians were allowed to 
regenerate in a graded series of molar s