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Full text of "The Biological bulletin"

THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 

HAROLD C. BOLD, University of Texas ARTHUR W. POLLISTER, Columbia University 

FRANK A. BROWN, JR., Northwestern University c L PROSSER) University of Illinois 

JOHN B. BUCK, National Institutes of Health . T ... .. 

L __ _ . x , _ ,., MARY E. RAWLES, Carnegie Institution or 

T. H. BULLOCK, University of California, . , . 

Los Angeles Washington 

LIBBIE H. HYMAN, American Museum of FRANZ SCHRADER, Duke University 

^?' u ! r Hlstory WM. RANDOLPH TAYLOR, University of Michigan 
V. L. LOOSANOFF, U. S. Fish and Wildlife 

Service CARROLL M. WILLIAMS, Harvard University 

DONALD P. COSTELLO, University of North Carolina 
Managing Editor 



VOLUME 117 

AUGUST TO DECEMBER, 1959 




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 PRF.SS, INC., LANCASTER, PA. 



CONTENTS 



No. 1. AUGUST, 1959 

PAGE 
Annual Report of the Marine Biological Laboratory 1 

BISCHOFF, HARRY W. 

Some observations on Chlamydomonas microhalophila sp. nov 54 

BROOKBANK, JOHN W. 

The presence of fertilizin haptens within the unfertilized sea urchin egg . . 63 
BRYAN, JOHN H. D., AND JOHN W. GOWEN 

X-ray effects on mitotic activity of the accessory sex organs of castrate 

rats stimulated by testosterone propionate 68 

GIESE, A. C., J. S. TUCKER AND R. A. BOOLOOTIAN 

Annual reproductive cycles of the chitons, Katherina tunicata and 

Mopalia hindsii 81 

HARRIS, PATRICIA J. 

A study of thyroid function in Fundulus heteroclitus 89 

JAHN, THEODORE L., AND ROBERT A. RINALDI 

Protoplasmic movement in the foraminiferan Allogromia laticollaris ; 

and a theory of its mechanism 100 

JENNINGS, J. B. ( 

Observations on the nutrition of the land planarian Orthodemus ter- 
restris (O. F. Miiller) 119 

KAWAI, KIYOZO 

The cytochrome system in marine lamellibranch tissues 125 

LlTCHFIELD, J. B., AND A. H. WHITELEY 

Studies on the mechanism of phosphate accumulation by sea urchin 
embryos 133 

MOORE, A. R. 

Some effects of temperature on development in the sea urchin Allocen- 
trotus fragilis 150 

RIEGEL, J. A. 

A revision in the sphaeromid genus Gnorimosphaeroma Menzies 
(Crustacea :Isopoda) on the basis of morphological, physiological and 
ecological studies on two of its "subspecies" 154 

VERNBERG, F. JOHN 

Studies on the physiological variation between tropical and temperate 
zone fiddler crabs of the genus Uca. II. Oxygen consumption of whole 
organisms 163 

a 7G . y 



iv CONTENTS 

No. 2. OCTOBER, 1959 

ANDERSON, JOHN MAXWELL 

Studies on the cardiac stomach of a starfish, Patiria miniata (Brandt) . . 185 

CHUANG, S. H. 

The breeding season of the brachiopod, Lingula unguis (L.) 202 

CONNELL, MARY GRACE 

Nuclear sizes in Rana mesonephroi 208 

DAVIES, J. T., AND F. H. TAYLOR 

The role of adsorption and molecular morphology in olfaction : the 
calculation of olfactory thresholds 222 

GOREAU, THOMAS F., AND NORA I. GOREAU 

The physiology of skeleton formation in corals. II. Calcium deposition 

by hermatypic corals under various conditions in the reef 239 

GREIF, ROGER L., AND MARILYN DU VIGNEAUD 

Distribution of a radiomercury-labelled diuretic (chlormerodrin) in 
tissues of marine fish 251 

GUILLARD, ROBERT R. L. 

Further evidence of the destruction of bivalve larvae by bacteria 258 

GELDIAY, SEMAHAT 

Neurosecretory cells in ganglia of the roach, Blaberus craniifer 267 

HODGSON, E. S., AND S. GELDIAY 

Experimentally induced release of neurosecretory materials from roach 
corpora cardiaca 275 

HENLEY, CATHERINE 

Exaggerated elevation of the fertilization membrane of Chaetopterus 
eggs, resulting from cold-treatment 284 

KRISHNAN, G. 

Histochemical studies on the nature and formation of egg capsules of 
the shark Chiloscyllium griseum 298 

LOOSANOFF, VICTOR L. 

The size and shape of metamorphosing larvae of Venus (Mercenaria) 
mercenaria grown at different temperatures 308 

MORRILL, JOHN B., JR. 

Antigenic differences between stem and hydranth in Tubularia 319 

NELSON, LEONARD 

Adenosinetriphosphatase of Mytilus spermatozoa. II. Effects of sulfhy- 
dryl reagents, temperature and inorganic pyrophosphate 327 

OKA, HlDEMITI, AND HlROSHI WATANABE 

Vascular budding in Botrylloides 340 

PROVASOLI, LUIGI, AND KAGEHIDE SHIRAISHI 

Axenic cultivation of the brine shrimp Artemia salina 347 

REES, GEORGE H. 

Larval development of the sand crab Emerita talpoida (Say) in the 

laboratory 356 

SIMPSON, JOHN W., KENNETH ALLEN AND JORGE A\YAPARA 

Free amino acids in some aquatic invertebrates 371 

Abstracts of papers presented at the Marine Biological Laboratory 382 



CONTENTS v 

No. 3. DKCKMUKR, 1959 

ABOUL-ELA, JMKAHIM AHMED 

( )n the food of nudibranchs 439 

BLACK, ROBERT E., AND ALBERT TYLER 

Effects of fertilization and development on the oxidation of carbon 
monoxide by eggs of Strongylocentrotus and Urechis as determined by 
use of C 13 443 

BLACK, ROBERT E., AND ALBERT TYLER 

Cytochrome oxidase and oxidation of CO in eggs of the sea urchin 
Strongylocentrotus purpuratus 454 

DE LUQUE, ORLANDO, AND F. R. HUNTER 

Osmotic studies of amphibian eggs. I. Preliminary survey of volume 
changes 458 

HUNTER, F. R., AND ORLANDO DE LUQUE 

Osmotic studies of amphibian eggs. II. Ovarian eggs 468 

JONES, BRVN M., AND RONALD S. WILSON 

Studies on the action of phenylthiourea on the respiratory metabolism 
and spinning behaviour of the Cynthia silkworm 482 

MOORE, A. R. 

On the embryonic development of the sea urchin Allocentrotus fragilis. 492 

NOVICK, ALVIN 

Acoustic orientation in the cave swiftlet 497 

PERSON, PHILIP, JAMES W. LASH AND ALBERT FINE 

On the presence of myoglobin and cytochrome oxidase in the cartilagi- 
nous odontophore of the marine snail, Busycon 504 

PRESS, NEWTOL 

Electron microscope study of the distal portion of a planarian retinular cell . 511 

REBHUN, LIONEL I. 

Studies of early cleavage in the surf clam, Spisula solidissima, using 
methylene blue and toluidine blue as vital stains 518 

SCHOTTE, OSCAR E., AND CHARLES B. SMITH 

Wound healing processes in amputated mouse digits 546 

STUNKARD, HORACE W. 

The morphology and life-history of the digenetic trematode, Asymphyl- 
odora amnicolae n. sp. ; possible significance of progenesis for the phy- 
togeny of the Digenea 562 

VERNBERG, F. JOHN 

Studies on the physiological variation between tropical and temperate 
zone fiddler crabs of the genus Uca. III. The influence of temperature 
acclimation on oxygen consumption of whole organisms 582 

WIESER, WOLFGANG, AND JOHN KANWISHER 

Respiration and anaerobic survival in some sea weed-inhabiting in- 
vertebrates 594 

WOLFSON, ALBERT 

Role of light in the progressive phase of the photoperiodic responses of 
migratory birds 601 

CESKA, MIROSLAV, AND JAMES R. FISHER 

Arginase inhibition in chick embryos 611 



Vol. 117, No. 1 August, 1959 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



THE MARINE BIOLOGICAL LABORATORY 
SIXTY-FIRST REPORT, FOR THE YEAR 1958 SEVENTY-FIRST YEAR 

I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 15, 1958) .... 1 

STANDING COMMITTEES 

II. ACT OF INCORPORATION 4 

III. BY-LAWS OF THE CORPORATION 4 

IV. REPORT OF THE DIRECTOR 6 

Statement 7 

Memorial 9 

Addenda : 

1. The Staff 9 

2. Investigators, Lalor and Lillie Fellows, and Students 12 

3. Fellowships and Scholarships 23 

4. Tabular View of Attendance, 1954-1958 24 

5. Institutions Represented 24 

6. Evening Lectures 26 

1 ' . Shorter Scientific Papers (Seminars) 26 

8. Members of the Corporation 28 

V. REPORT OF THE LIBRARIAN 47 

VI. REPORT OF THE TREASURER , 48 



I. TRUSTEES 

EX OFFICIO 

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

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

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

Syracuse 

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

EMERITI 

EUGENE DuBois, Cornell University Medical College 
G. H. A. CLOWES, Lilly Research Laboratory 

1 



2 MARINE BIOLOGICAL LABORATORY 

W. C. CURTIS, University of Missouri 

PAUL S. GALTSOFF, Woods Hole, Mass. 

Ross G. HARRISON, Yale University 

E. B. HARVEY, 48 Cleveland Lane, Princeton, N. J. 

E. NEWTON HARVEY, Princeton University 

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

F. P. KNOWLTON, Syracuse University 
W. J. V. OSTERHOUT, Rockefeller Institute 
CHARLES PACKARD, Woods Hole, Mass. 
LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y. 

TO SERVE UNTIL 1962 

FRANK A. BROWN, JR., Northwestern University 

SEARS CROWELL, Indiana University 

ALBERT I. LANSING, University of Pittsburgh Medical School 

WILLIAM D. MCELROY, 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 

D. W. BRONK, Rockefeller Institute 

G. FAILLA, Columbia University, College of Physicians & Surgeons 
ERIC BALL, Harvard University Medical School 

R. T. KEMPTON, Vassar College 

L. H. KLEINHOLZ, Reed College 

IRVING M. KLOTZ, Northwestern University 

ALBERT SZENT-GYORGYI, Marine Biological Laboratory 

WM. RANDOLPH TAYLOR, University of Michigan 

TO SERVE UNTIL 1960 

H. F. BLUM, Princeton University 

K. S. COLE, National Institutes of Health 

L. V. HEILBRUNN, University of Pennsylvania 

S. W. KUFFLER, Johns Hopkins Hospital 

C. B. METZ, Florida State University 

G. T. SCOTT, Oberlin College 

A. H. STURTEVANT, California Institute of Technology 

E. ZWILLING, University of Connecticut 

TO SERVE UNTIL 1959 

E. G. BUTLER, Princeton University 

C. LALOR BURDICK, The Lalor Foundation, Wilmington, Delaware 

D. P. COSTELLO, University of North Carolina 
H. HIBBARD, Oberlin College 

M. KRAHL, University of Chicago 

D. MARSLAND, New York University, Washington Square College 
R. RUGH, Columbia University, College of Physicians and Surgeons 
H. B. STEINBACH, University of Chicago 



TRUSTEES 

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES 

GERARD SWOPE, JR., ex officio, Chairman 

JAMES H. WICKERSHAM, ex officio RUDOLF KEMPTON, 1960 

ARTHUR K. PARPART, ex officio EDGAR ZWILLING, 1960 

P. B. ARMSTRONG, ex officio W. D. MCELROY, 1961 

E. G. BUTLER, 1959 F. A. BROWN, JR., 1961 
K. S. COLE, 1959 



THE LIBRARY COMMITTEE 

MARY SEARS, Chairman E. G. BUTLER 

R. E. TAYLOR RALPH H. CHENEY 

GROVER STEPHENS SEYMOUR COHEN 



THE APPARATUS COMMITTEE 

C. LLOYD CLAFF, Chairman W. D. MCELROY 

ALBERT I. LANSING 



THE SUPPLY DEPARTMENT COMMITTEE 

RUDOLF KEMPTON, Chairman ROBERT D. ALLEN 

C. B. METZ L. V. HEILBRUNN , 



THE EVENING LECTURE COMMITTEE 

PHILIP B. ARMSTRONG, Chairman L. V. HEILBRUNN 

ERIC G. BALL W. D. MCELROY 



THE INSTRUCTION COMMITTEE 

S. MERYL ROSE, Chairman IRVING KLOTZ 

L. H. KLEINHOLZ C. LADD PROSSER 

BOSTWICK H. KETCHUM 



THE BUILDINGS AND GROUNDS COMMITTEE 

RALPH WICHTERMAN, Chairman HARRY GRUNDFEST 

SEARS CROWELL EDGAR ZWILLING 



THE RADIATION COMMITTEE 

G. FAILLA, Chairman MONES BERMAN 

WALTER L. WILSON ROGER GREIF 

ROBERTS RUGH WALTER VINCENT 



THE RESEARCH SPACE COMMITTEE 

P. B. ARMSTRONG, Chairman FRANK A. BROWN, JR. 

ARTHUR K. PARPART MAC V. EDDS 



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, Di- 
rector, Treasurer, and Clerk. 

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

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

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

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



BY-LAWS OF THE CORPORATION o 

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

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

VIII. There shall be three groups of Trustees : 

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

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

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

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

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

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



6 MARINE BIOLOGICAL LABORATORY 

XL 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 
Gentlemen : 

I submit herewith the report of the seventy-first session of the Marine Biological 
Laboratory. 

1. Attendance 

Attendance has continued at a fairly constant level through the past several 
years as shown in the Tabular View of Attendance. Only minor differences in 
attendance occur from year to year since the laboratory space is fully occupied 
and the classes are at capacity. Recently there have been qualified investigators 
with problems appropriate to the Laboratory's research materials and facilities who 
could not be accommodated. This has posed a difficult problem for the Research 
Space Committee in the assignment of space. It frequently has been difficult for 
the Committee to avoid rather arbitrary decisions. It is anticipated that this prob- 
lem will be eased with the completion of the new research laboratory in the spring 
of 1960. 

2. Crane B nil ding 

Renovation of the Crane Building carried out under a grant by the National 
Science Foundation was completed on schedule, permitting occupancy on June 1, 
1958. Space utilization has been greatly improved by the rearrangement of facili- 
ties within the laboratories. Also the addition of new utilities has resulted in a 
real improvement in scientific working conditions. 

3. New Laboratory Building 

During the past winter the Building Committee has had several meetings with 
the architects for the new building (Sheply, Bulfinch, Richardson and Abbott) and, 
with the plans already well developed, construction will start in May, 1959, the 
building to be completed and ready for summer occupancy in 1960. The building 
will have a ground level basement and three floors. Half of the basement will be 
given over to laboratories, the other half to utilities and services. There will be 
radiobiological service laboratories on the top floor. A caesium 137 radiation unit, 
a new departure in radiobiological technique, is being planned and will be constructed 
under the direction of Dr. G. Failla and installed in one of the radiobiological service 
laboratories. 



REPORT OF THE DIRECTOR 7 

Attached to the new building will be a lecture room seating 150. The experience 
gained in renovating the Crane Wing is being used to advantage by the Building 
Committee. 

4. Devil's Lane Housing Project 

The attendance of younger investigators at the Laboratory, particularly those 
with families, has frequently been limited by the cost of housing rental. The 
Laboratory applied to the National Science Foundation and received a grant for 
housing construction on the Laboratory's Devil's Lane property. Twenty-four 
cottages are being erected and will be ready for 1959 summer occupancy. Through 
the generosity of the Grass Foundation another cottage is being added to those 
above. Through the efforts of Mr. Smith, General Manager, the Laboratory has a 
tax exemption on this new housing project which will permit modest rental changes. 

The recently completed and the projected construction at the Laboratory has 
been made possible by the grants listed below : 

National Science Foundation Crane Wing $415,000 

National Science Foundation New laboratory and housing 544,250 

National Institutes of Health New laboratory 369,250 

Rockefeller Foundation New laboratory 738,500 

Grass Foundation Housing 10,000 

5. Grants, Contracts and Contributions 

The total income to the laboratory from these sources of support amounted to 
$184,445.23 in 1958. This represents 34.3% of the total income and is made up 
of the following accounts : 

American Cancer Society RC4A( + ) Studies in Radiobiology .... $ 6,600.00 
AEC 1343 Program of Research on the Physiology of Marine 

Organisms Using Radioisotopes 10,775.00 

NIH 4359 Biological Research on the Morphology, Ecology, 

Physiology, Biochemistry and Biophysics of Marine Organisms . . . 40,000.00 

NIH C3813 (A) Bio-Equipment 1,600.00 

NSF 5143 Training Program in Nerve Muscle Physiology 51,593.00 

ONR 1497 Studies in Marine Biology 15,000.00 

ONR 09701 Studies on Isolated Nerve Fibers 6,712.23 

ONR 09702 Studies in Ecology 6,936.00 

M.B.L. Associates 3,150.00 

Abbott Laboratories 1 ,000.00 

American Philosophical Society 2,500.00' 

Carter Products, Inc 1,000.00 

Ciba Pharmaceutical Products, Inc 1,000.00 

Josephine B. Crane Foundation 2,000.00 

Eli Lilly and Company 5,000.00 

Hoffman-LaRoche, Inc 1 ,000.00 

Merck Company Foundation 1,000.00 

Pfizer Foundation, Inc. 1,000.00 



MARINE BIOLOGICAL LABORATORY 

Rockefeller Foundation 20,000.00 

Sobering Foundation, Inc 1,000.00 

Smith, Kline, and French Foundation 3,000.00 

The Upjohn Company 1,000.00 

Wyeth Laboratories 1,000.00 

Miscellaneous Individuals 579.00 



$184,445.23 

It is gratifying that such a diverse group of agencies, foundations, companies 
and individuals as listed above are interested in the support and development of 
the Laboratory and its research programs. 

6. Courses 

The course in Physiology will be modified and operate in 1959 as a Physiology 
Training Program under the direction of Dr. William D. McElroy with financial 
support from the National Institutes of Health. The additional support will permit 
well-qualified doctoral and post-doctoral students to participate in the program 
who otherwise might not be able to do so. Selected students will continue on in 
the program doing research through the last half of the summer following the 
completion of the earlier formal part of the program. 

With the loss of certain dormitory facilities, the Old Rockefeller Building is 
being converted into a dormitory for some of the summer service personnel. The 
course in Marine Ecology which formerly occupied this building is being moved 
into the Old Main Building. 

7. Boats 

Two new collecting boats were purchased and were received by the Laboratory 
in May, 1958. These are 24-foot open-cockpit boats of rugged construction and 
have proved very sea-worthy. One is used for the daily trips to the fish traps and, 
together wdth the second boat, for inshore collecting. They replace two old boats, 
the Sagitta and Tern. A diesel engine was installed in the Arbacia resulting in 
real economies in operation. 

8. Survey 

During the summer of 1958 the firm of Shurcliff and Merrill, Landscape Archi- 
tects and Town Planners, was retained jointly by the Marine Biological Laboratory 
and the Woods Hole Oceanographic Institution to survey Woods Hole, including 
the physical lay-out of each institution on its own campus and also problems of the 
institutions as they relate to the community. The resulting report emphasized the 
present wasteful utilization of the six acres of land which make up the central 
campus of the M.B.L. The six frame houses, formerly private dwellings, which 
are used as dormitories occupy an excessive amount of land for the number of 
people they accommodate. They are old buildings, ill adapted for dormitory 
purposes and should be replaced. The old Mess Hall, adapted to self-service, 
could advantageously be replaced by a new building in combination with 



REPORT OF THE DIRECTOR 

dormitory facilities. Consolidation of these facilities will open up areas urgently 
needed for parking. With the development of additional training programs, there 
will develop a need for training facilities space. The Old Lecture Hall can readily 
be converted to such temporary use but it will not be entirely satisfactory for 
a program involving the new techniques required for a modern training program. 
Only a new building will adequately serve this purpose. 

Respectfully submitted, 
PHILIP B. ARMSTRONG, 

Director 

MEMORIAL 

ALLEN R. MEMHARD 

by 
Donald P. Costello 

Allen R. Memhard had a lifelong interest in biology, but, by reason of training 
for the Bar and practicing international and corporation law, was able to indulge 
this interest only in his later life. 

Mr. Memhard was born in Chicago, and was graduated from the New York Law 
School in 1915. Soon after his graduation, he began an independent law practice in 
New York City. This was interrupted briefly while he served in U. S. Army Intel- 
ligence during World War I, after which he returned to his own practice. Later 
he became a member of the law firm of O'Brien, Boardman, Fox, Memhard and Early. 
In 1933 he was a delegate to a conference on international law at The Hague. From 
1939 to 1950 he was counsel for the Geological Society of America. He was also very 
active in town affairs in his home community of Greenwich, Connecticut. Here he 
and Mrs. Memhard raised their fine family of two daughters and three sons. 

In 1941, to learn more about the field that had interested him since childhood, he 
enrolled in a course in biology at New York University, where he came into contact 
with the late Professor Robert Chambers. By this time, Mr. Memhard was in a 
position to take time from his professional work to pursue his interest in marine 
biology, and Dr. Chambers was so impressed with his sincerity and abilities that he 
encouraged him to apply for space at the Marine Biological Laboratory. During 
the summer of 1942, Mr. Memhard first took the Embryology Course, which was 
under the direction of Professor Viktor Hamburger, and then was engaged in research 
during the remainder of the summer. He continued his research work in marine 
embryology during the summers of 1943, 1944, 1945, and part of 1947. He was 
elected a member of the Corporation in 1945. 

In 1946, Mr. Memhard established a scholarship fund at the Marine Biological 
Laboratory, the income of which is awarded to qualified students who complete the 
course in embryology and wish to return or stay on for further work. 

Mr. Memhard was a charming gentleman, a diligent scholar, and an interested 
observer of marine embryological phenomena. He was a fine example of that group 
of enlightened laymen who deserve the title, "Friend of the Marine Biological 
Laboratory." 

1. THE STAFF, 1958 

PHILIP B. ARMSTRONG, Director, State University of New York, School of Medicine. 
Syracuse 



10 MARINE BIOLOGICAL LABORATORY 

ZOOLOGY 

I. CONSULTANTS 

F. A. BROWN, JR., Morrison Professor of Zoology, Northwestern University 
LIBBIE H. HYMAN, American Museum of Natural History 
A. C. REDFIELD, Woods Hole Oceanographic Institution 

II. INSTRUCTORS 

GROVER C. STEPHENS, Assistant Professor of Zoology, University of Minnesota ; in 

charge of course. 

JOHN B. BUCK, Senior Biologist, National Institutes of Health 
DEMOREST DAVENPORT, Associate Professor of Biology, Santa Barbara College 
PETER W. FRANK, Associate Professor of Biology, University of Oregon 
CLARK P. READ, Associate Professor, School of Hygiene and Public Health, Johns 

Hopkins University 
MORRIS ROCKSTEIN, Associate Professor of Physiology, New York University College 

of Medicine 

HOWARD A. SCHNEIDERMAN, Associate Professor of Zoology, Cornell University 
MILTON FINGERMAN, Assistant Professor of Zoology, Tulane University 

III. LABORATORY ASSISTANTS 

FRANK E. FRIEDL, University of Minnesota 
IRWIN W. SHERMAN, Northwestern University 

EMBRYOLOGY 
I. INSTRUCTORS 

MAC V. EDDS, JR., Professor of Biology, Brown University; in charge of course 

PHILIP GRANT, Assistant Professor of Pathobiology, Johns Hopkins University 

JOHN W. SAUNDERS, JR., Professor of Zoology, Marquette University 

NELSON T. SPRATT, JR., Professor of Zoology, University of Minnesota 

MAURICE SUSSMAN, Associate Professor of Biological Sciences, Northwestern University 

LIONEL REBHUN, Assistant Professor of Anatomy, University of Illinois 

II. LABORATORY ASSISTANTS 

CHANDLER M. FULTON, Rockefeller Institute for Medical Research 
DAVID S. LOVE, University of Colorado 

PHYSIOLOGY 
I. CONSULTANTS 

MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania 
ARTHUR K. PARPART, Professor of Biology, Princeton University 
ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Marine Biological 
Laboratory 



REPORT OF THE DIRECTOR 11 

II. INSTRUCTORS 

W. D. McELROY, Professor of Biology, Johns Hopkins University; in charge of 

course 

FRANCIS D. CARLSON, Assistant Professor of Biophysics, Johns Hopkins University 
BERNARD D. DAVIS, Professor of Bacteriology, Harvard Medical School 
DONALD GRIFFIN, Professor of Zoology, Harvard University 
HOWARD SCHACHMAN, Virus Laboratory, University of California 
ALBERT W. FRENKEL, University of Minnesota 

III. LABORATORY ASSISTANT 
Louis OTERO, University of Puerto Rico, Rio Piedras 

BOTANY 

I. CONSULTANT 

WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan 

II. INSTRUCTORS 

HAROLD C. BOLD, Professor of Botany, University of Texas; in charge of course 
JOHN M. KINGSBURY, Assistant Professor of Botany, Cornell University 
RICHARD C. STARR, Associate Professor of Botany, Indiana University 

III. LECTURER 
RUTH PATRICK, Curator of Limnology, Academy of Natural Sciences of Philadelphia 

IV. LABORATORY ASSISTANTS 

TEMD R. DEASON, University of Texas 
HARRY W. BISCHOFF, Texas Lutheran College 

ECOLOGY 

I. CONSULTANTS 

PAUL GALTSOFF, U. S. Fish and Wildlife Service, Woods Hole 

ALFRED C. REDFIELD, Woods Hole Oceanographic Institution 

BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution 

EDWIN T. MOUL, Rutgers University 

CHARLES E. JENNER, University of North Carolina 

HOWARD L. SANDERS, Woods Hole Oceanographic Institution 

II. INSTRUCTORS 

HOWARD T. ODUM, Director, Institute of Marine Science, University of Texas 
JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution 
ALBERT J. BERNATOWICZ, Chairman, Department of Botany, University of Hawaii 

III. LABORATORY ASSISTANT 
CAMERON E. GIFFORD, Harvard University 



12 



MARINE BIOLOGICAL LABORATORY 



THE LABORATORY STAFF, 1958 
HOMER P. SMITH, General Manager 



MRS. DEBORAH LAWRENCE HARLOW, 

Librarian 
CARL O. SCHWEIDENBACK, Manager, 

Supply Department 



ROBERT KAHLER, Superintendent, 
Buildings and Grounds 

ROBERT B. MILLS, Manager, De- 
partment of Research Service 



GENERAL OFFICE 
IRVINE L. BROADBENT, Office Manager 



MRS. LILA S. MYERS 

MRS. VIVIEN R. BROWN 

MRS. VIRGINIA M. MOREHOUSE 



MRS. EMILY B. MERRILL 
MRS. VIVIAN I. MANSON 
MRS. SHIRLEY ELDER 



LIBRARY 



MARY ROHAN 

MRS. M. VERNA HANKS 



MRS. NAOMI BOTELHO 
ALBERT K. NEAL 



MAINTENANCE OF BUILDINGS AND GROUNDS 



ROBERT ADAMS 
ELDON P. ALLEN 
EDMOND BOTELHO 
ARTHUR D. CALLAHAN 
ROBERT GUNNING 
WALTER J. JASKUN 



DONALD B. LEHY 
RALPH H. LEWIS 
RUSSELL F. LEWIS 
ALAN G. LUNN 
ALTON J. PIERCE 
JAMES S. THAYER 



DEPARTMENT OF RESEARCH SERVICE 



GAIL M. CAVANAUGH 
JOHN P. HARLOW 



SEAVER R. HARLOW 
LUDIE A. JOHNSON 



SUPPLY DEPARTMENT 



DONALD P. BURNHAM 
MILTON B. GRAY 
GEOFFREY J. LEHY 
ROBERT O. LEHY 
MRS. MILDRED MIXSON 



ROBERT M. PERRY 
BRUNO TRAPASSO 
JARED L. VINCENT 
SAMUEL W. VINCENT 
HALLETT S. WAGSTAFF 



2. INVESTIGATORS, LALOR AND LILLIE FELLOWS, AND STUDENTS 
Independent Investigators, 1958 

AFZELIUS, BJORN, Assistant Professor of Biophysics, Johns Hopkins University 

ALLEN, M. JEAN, Associate Professor of Biology, Wilson College 

ALLEN, ROBERT D., Assistant Professor of Biology, Princeton University 

ARMSTRONG, PHILIP B., Professor and Chairman, Department of Anatomy, State University 

of New York, College of Medicine at Syracuse 

ARNOLD, WILLIAM, Principal Biologist, Division of Biology, Oak Ridge National Laboratory 
BAIRD, SPENCER, Associate Researcher, Institute for Muscle Research, Marine Biological 

Laboratory 



REPORT OF THE DIRECTOR 13 

BANG, FREDERIK B., Professor of Pathobiology, Johns Hopkins University School of Medicine 

BARTH, LESTER G., Professor of Zoology, Columbia University 

BATTLEY, EDWIN H., Instructor in Biochemistry, The Medical Center, Jersey City 

BENESCH, REINHOLD, Investigator, Marine Biological Laboratory 

BENNETT, MICHAEL V. L., Research Associate, Columbia University, College of Physicians & 

Surgeons 

BERMAN, MONES, Assistant Physicist, Sloan Kettering Institute 
BERNATOWICZ, ALBERT J., Chairman, Department of Botany, University of Hawaii 
BETTELHEIM, FREDERICK A., Assistant Professor in Chemistry, Adelphi College 
BISHOP, DAVID W., Staff Member, Department of Embryology, Carnegie Inst. of Washington 
BOLD, HAROLD C., Professor of Botany, University of Texas 

BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University 
BRYANT, SHIRLEY H., Assistant Professor of Pharmacology, University of Cincinnati 
BUCK, JOHN B., Physiologist, National Institutes of Health 
BURBANCK, W. D., Professor of Biology, Emory University 

CARLSON, FRANCIS D., Associate Professor of Biophysics, Johns Hopkins University 
CASE, JAMES F., Assistant Professor of Zoology, State University of Iowa 
DEL CASTILLO, JOSE, Head, Section of Clinical Neurophysiology, National Institutes of Health 
CHAET, ALFRED B., Assistant Professor of Physiology, Boston University School of Medicine 
CHANG, JOSEPH J., Visiting Scientist, National Institutes of Health 
CHASE, AURIN M., Associate Professor of Biology, Princeton University 
CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College 
CHILD, FRANK M., Instructor in Zoology, The University of Chicago 
CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School 
CLAUDATUS, JOHN C., Head, Department of Biochemistry, The Cancer Institute at Miami 
CLEMENT, ANTHONY C., Professor of Biology, Emory University 
CLOWES, G. H. A., Research Director Emeritus, Lilly Research Laboratories 
COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health 
COLLIER, JACK R., Assistant Professor of Zoology, Louisiana State University 
COLWIN, ARTHUR L., Associate Professor, Queens College 
COLWIN, LAURA HUNTER, Lecturer, Queens College 
COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve University, 

School of Medicine 

COSTELLO, DONALD P., Kenan Professor of Zoology, University of North Carolina 
COWGILL, ROBERT W., Assistant Professor of Biochemistry, University of Colorado, School 

of Medicine 
CRANE, ROBERT K., Associate Professor of Biological Chemistry, Washington University 

Medical School 

CROWELL, SEARS, Associate Professor of Zoology, Indiana University 
CSAPO, ARPAD, Associate Professor, Rockefeller Institute for Medical Research 
DAVENPORT, DEMOREST, Associate Professor in Zoology, University of California, Santa 

Barbara College 

DAVIS, BERNARD D., Professor of Bacteriology, Harvard Medical School 
ECCLES, ROSAMOND, Research Fellow, Australian National University 
EDDS, MAC V., JR., Professor of Biology, Brown University 
FAILLA, G., Professor, Columbia University 

FANKUCHEN, ISIDOR, Professor and Head, Division of Applied Physics, Polytechnic Institute 
FAWCETT, DON W., Professor of Anatomy, Cornell University Medical College 
FINGERMAN, MILTON, Assistant Professor of Zoology, Newcomb College, Tulane University 
FISHMAN, Louis, Research Associate, New York University College of Dentistry 
FRANK, PETER W., Associate Professor of Biology, University of Oregon 
FRENKEL, ALBERT W., Professor of Botany, University of Minnesota 
FRIEDL, FRANK E., Teaching Assistant, University of Minnesota 

GALTSOFF, PAUL S., Director, U. S. Fish & Wildlife Service, Department of the Interior 
GOLDBERG, IRVING H., Post-Doctoral Student, Rockefeller Institute for Medical Research 
GOTO, MASAYOSI, Investigator, Rockefeller Institute for Medical Research 
GRANT, PHILIP, Assistant Professor of Pathobiology, Johns Hopkins University 
GREEN, JAMES W., Associate Professor of Physiology, Rutgers University 



14 MARINE BIOLOGICAL LABORATORY 

GREIF, ROGER L., Associate Professor of Physiology, Cornell University Medical College 

GRIFFIN, DONALD R., Professor of Zoology, Harvard University 

GRODZINSKI, F., Professor of Comparative Anatomy, University of Krakow, Poland 

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, Columbia University, College of 

Physicians and Surgeons 

GUTTMAN, RITA, Assistant Professor of Biology, Brooklyn College 
HAGINS, WILLIAM A., Research Medical Officer, Naval Medical Research Institute 
HARDING, CLIFFORD V., Visiting Assistant Professor of Zoology, University of Pennsylvania 
HARVEY, E. NEWTON, Professor of Biology emeritus, Princeton University 
HARVEY, ETHEL BROWNE, Investigator in Biological Research, Princeton University 
HAYASHI, TERU, Professor of Zoology, Columbia University 
HEGYELI, ANDREW, Associate Researcher, Institute for Muscle Research, Marine Biological 

Laboratory 

HEILBRUNN, L. V., Professor of General Physiology, University of Pennsylvania 
HENLEY, CATHERINE, Research Associate, University of North Carolina 
HERVEY, JOHN P., Senior Electronic Engineer, Rockefeller Institute for Medical Research 
HIATT, HOWARD, Associate in Medicine, Harvard Medical School 
HICKSON, ANNA KELTCH, Independent Investigator, Lilly Research Laboratories 
HOLZ, GEORGE G., JR., Associate Professor of Zoology, Syracuse University 
HUGHES, GEORGE M., University Lecturer, University of Cambridge, England 
HUSSEY, KATHLEEN L., Assistant Professor of Parasitology, Columbia University 
HYDE, BEAL B., Assistant Professor of Plant Sciences, University of Oklahoma 
ISENBERG, IRVIN, Associate Researcher, Institute for Muscle Research, Marine Biological 

Laboratory 

ITO, SUSUMU, Instructor in Anatomy, Cornell University Medical College 
JACKSON, SYLVIA FITTON, Assistant Professor, Kings College, London 
JENNER, CHARLES E., Associate Professor of Zoology, University of North Carolina 
JOHNSON, FRANK H., Professor of Biology, Princeton University 
KAJI, AKIRA, Fellow in Ophthalmology, Johns Hopkins University 
KANE, ROBERT E., Postdoctoral Research Fellow, Johns Hopkins University 
KEMPTON, RUDOLF T., Professor of Zoology, Vassar College 
KENNEDY, DONALD, Assistant Professor of Zoology, Syracuse University 
KINGSBURY, JOHN M., Assistant Professor of Botany, Cornell University 
KLEINHOLZ, L. H., Professor of Biology, Reed College 
KLOTZ, IRVING M., Professor of Chemistry, Northwestern University 
KLUSS, BYRON C, Instructor, Albion College 
KOHLER, KURT, Research Associate, Florida State University 
KRAMER, SOL, Special Research Fellow, Marine Biological Laboratory 
KRANE, STEPHEN M., Instructor in Medicine, Massachusetts General Hospital 
KCJFFLER, STEPHEN W., Professor of Ophthalmic Physiology, Johns Hopkins University 
KURY, LIVIA REV, Institute for Muscle Research, Marine Biological Laboratory 
LANSING, ALBERT L, Professor of Anatomy, University of Pittsburgh 
LASH, JAMES W., Instructor in Anatomy, University of Pennsylvania 
LENHOFF, HOWARD M., Investigator in Biochemistry, Carnegie Institution 
LEVY, MILTON, Professor of Biochemistry, New York University College of Dentistry 
LEWIN, RALPH A., Independent Investigator, Marine Biological Laboratory 
LITT, MORTIMER, Instructor in Bacteriology, Harvard Medical School 

LOWENHAUPT, BENJAMIN, Research Associate, Rockefeller Institute for Medical Research 
LUBIN, MARTIN, Assistant Professor of Pharmacology, Harvard Medical School 
MCELROY, W. D., Chairman, Department of Biology, Johns Hopkins University 
MARSLAND, DOUGLAS, Professor of Biology, Washington Square College, New York University 
MATEYKO, G. M., Assistant Professor of Biology, Washington Square College, New York 

University 

MERRIAM, ROBERT W., Associate in Zoology, University of Pennsylvania 
METZ, CHARLES B., Professor, Florida State University 
METZ, CHARLES W., Research Professor, University of Pennsylvania 






REPORT OF THE DIRECTOR 15 

MIDDLEBROOK, ROBERT, Associate Researcher, Institute for Muscle Research, Marine Biological 

Laboratory 

MONROY, ALBERTO, Professor of Comparative Anatomy, University of Palermo 
MOORE, JOHN W., Associate Chief, Laboratory of Biophysics, National Institutes of Health 
MUELLER, HELMUT, Associate Researcher, Institute for Muscle Research, Marine Biological 

Laboratory 

MULLINS, L. J., Associate Professor of Biophysics, Purdue University 
NACE, PAUL FOLEY, Associate Professor of Zoology, McMaster University 
NELSON, LEONARD, Assistant Professor of Anatomy, University of Chicago 
NICKERSON, NORTON H., Instructor in Botany, Cornell University 

NORRIS, WILLIAM ELMORE, JR., Professor of Biology, Southwest Texas State Teachers College 
ODUM, HOWARD T., Director, Institute of Marine Science, The University of Texas 
OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research 
PADAWER, JACQUES, Assistant Professor of Biochemistry, Albert Einstein College of Medicine 
PARKER, JOHNSON, Assistant Professor in Plant Physiology, Yale School of Forestry 
PARPART, ARTHUR K., Professor and Chairman of Biology, Princeton University 
PATERSON, MABEL C, Assistant Professor of Zoology, Vassar College 

PERSON, PHILIP, Chief, Special Dental Research Program, Veterans Administration Hospital 
POTTER, DAVID D., Johns Hopkins University School of Medicine 
PRESTON, JAMES B., Assistant Professor of Physiology, State University of New York at 

Syracuse 

PROSSER, C. LADD, Professor of Physiology, University of Illinois 
READ, CLARK P., Associate Professor of Parasitology, Johns Hopkins University 
REBHUN, LIONEL I., Assistant Professor of Anatomy, University of Illinois 
RIESER, PETER, Research Associate, University of Pennsylvania 
ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University-Bellevue Medical 

Center 

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

ROSENBERG, EVELYN K., Associate Professor, New York University-Bellevue Medical Center 
ROSENTHAL, THEODORE B., Assistant Professor of Anatomy, University of Pittsburgh 
ROSLANSKY, JOHN D., Research Associate, Princeton University 
RUGH, ROBERTS, Associate Professor of Radiology, Columbia University 
RYTHER, J. H., Marine Biologist, Woods Hole Oceanographic Institution 
SAKAI, TOSHIO, Research Fellow, Rockefeller Institute for Medical Research 
SANBORN, RICHARD C., Assistant Professor of Zoology, Purdue University 
SANDEEN, MURIEL I., Assistant Professor of Zoology, Duke University 
SAUNDERS, JOHN W., JR., Professor of Zoology, Marquette University 
SCHECHTER, VICTOR, Associate Professor of Biology, City College of New York 
SCHNEIDERMAN, HOWARD A., Associate Professor of Zoology, Cornell University 
SCHNELLER, SISTER MARY BEATRICE, Professor of Biology, St. Joseph College for Women 
SCHUH, REV. JOSEPH E., Chairman, Biology Department, Saint Peter's College 
SCOTT, ALLAN, Chairman, Department of Biology, Colby College 
SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College 
SCOTT, GEORGE T., Professor of Zoology, Oberlin College 
SEGAL, JOHN R., Graduate Student, Massachusetts Institute of Technology 
SENFT, ALFRED W., Woods Hole, Massachusetts 

SHAW, EVELYN, Research Associate, American Museum of Natural History 
SLIFER, ELEANOR H., Professor of Zoology, State University of Iowa 
SMITH, PAUL FERRIS, Electronics Engineer, Rockefeller Institute for Medical Research 
SPEIDEL, CARL C., Professor of Anatomy, University of Virginia 
SPIEGEL, MELVIN, Assistant Professor of Biology, Colby College 
SPRATT, NELSON T., Professor of Zoology, University of Minnesota 
SPYROPOULOS, CONSTANTINE S., Neurophysiologist, National Institutes of Health 
STARR, RICHARD C., Associate Professor of Botany, Indiana University 
STEINBACH, H. BURR, Chairman, Department of Zoology, University of Chicago 
STEIN HARDT, JACINTO, Director, Operations Evaluation Group, Massachusetts Institute of 

Technology 
STEPHENS, GROVER C., Assistant Professor of Zoology, University of Minnesota 



16 MARINE BIOLOGICAL LABORATORY 

STOKEY, ALMA G., Professor Emeritus Mount Holyoke College 

STONE, WILLIAM, Director, Ophthalmic Plastics Laboratory, Massachusetts Eye and Ear 

Infirmary 

STROHMAN, RICHARD C, Assistant Professor of Zoology, University of California 
STUNKARD, HORACE W., Research Scientist, U. S. Fish and Wildlife Service 
STURTEVANT, A. H., Thomas Hunt Morgan Professor of Genetics, California Institute of 

Technology 

SUDAK, FREDERICK N., Instructor, Albert Einstein College of Medicine 
SUSSMAN, MAURICE, Associate Professor, Biology Department, Northwestern University 
SZENT-GYORGYI, Albert, Chief Investigator, Institute for Muscle Research, Marine Biological 

Laboratory 
SZENT-GYORGYI, Andrew G., Investigator, Institute for Muscle Research, Marine Biological 

Laboratory 
SZENT-GYORGYI, Eva, Associate Researcher, Institute for Muscle Research, Marine Biological 

Laboratory 

TALEPOROS, PLATO, Postdoctoral Research Fellow, University of California 
TASAKI, ICHIJI, Chief, Special Senses Section, Laboratory of Neurophysiology, National 

Institutes of Health 

TAYLOR, ROBERT E., Physiologist, National Institutes of Health 
TAYLOR, WILLIAM RANDOLPH, Professor of Botany, University of Michigan 
TOBIAS, JULIAN M., Professor of Physiology, University of Chicago 
TODD, ROBERT E., Professor of Zoology, Colgate University 

TRAUTWEIN, WOLFGANG, Associate Professor of Physiology, Johns Hopkins Hospital 
TRENDELENBURG, ULLRICH, Associate in Pharmacology, Harvard Medical School 
TRINKAUS, J. P., Associate Professor of Zoology, Yale University 

TROLL, WALTER, Assistant Professor of Zoology, New York University-Bellevue Medical Center 
TWEEDELL, KENYON S., Assistant Professor of Zoology, University of Maine 
TYLER, ALBERT, Professor of Embryology, California Institute of Technology 
DEViLLAFRANCA, GEORGE W., Assistant Professor of Zoology, Smith College 
VINCENT, WALTER S., Assistant Professor of Anatomy, State University of New York, Upstate 

Medical Center, at Syracuse 

WAGNER, CAPTAIN HENRY G., Head, Physiology Division, Naval Medical Research Institute 
WAINIO, WALTER W., Associate Professor of Biochemistry, Rutgers University 
WEBB, H. MARGUERITE, Research Associate, Northwestern University 
WHITING, P. W., Professor of Zoology Emeritus, University of Pennsylvania 
WICHTERMAN, RALPH, Professor of Biology, Temple University 

WILBER, CHARLES G., Chief, Comparative Physiology Branch, Army Chemical Center 
WILLEY, C. H., Professor and Chairman of Biology, New York University, University College 
WILSON, WALTER L., Assistant Professor of Physiology and Biophysics, College of Medicine, 

University of Vermont 
WITTENBERG, JONATHAN B., Assistant Professor of Physiology and Biochemistry, Albert 

Einstein College of Medicine 

WRIGHT, PAUL A., Associate Professor of Zoology, University of Michigan 
WURZEL, MENACHEM, Research Worker, College of Physicians & Surgeons, Columbia University 
ZWEIFACH, BENJAMIN W., Associate Professor of Pathology, New York University Bellevue 

Medical Center 
ZWILLING, EDGAR, Associate Professor of Genetics, University of Connecticut 

LALOR FELLOWS, 1958 

AFZELIUS, B., Johns Hopkins University 

BETTELHEIM, F., Adelphi College 

CZERLINSKI, G., Max-Planck Inst. Physikal Chemie 

ECCLES, ROSAMOND, Australian National University 

HARDING, CLIFFORD V., University of Pennsylvania 

KLUSS, BYRON C., Albion College 

LASH, JAMES, University of Pennsylvania 

LUBIN, MARTIN, Harvard Medical School 

NELSON, LEONARD, University of Chicago 

WITTENBERG, JONATHAN B., Albert Einstein College of Medicine 



REPORT OF THE DIRECTOR 17 

Lillie Fellow, 1958 

MONROY, ALBERTO, University of Palermo, Italy 

Grass Fellows, 1958 

BENNETT, MICHAEL V. L., Columbia University 

REUBEN, JOHN, University of Florida 

RICKLES, WILLIAM H., JR., Baylor University, College of Medicine 

American Philosophical Fellows 

CHOI, Ki-CnuL, Seoul National University, Korea 
GRODZINSKI, F., University of Krakow, Poland 
MONROY, ALBERTO, University of Palermo, Italy 

Beginning Investigators, 1958 

ASHTON, FRANCIS T., University of Pennsylvania 

BRAVERMAN, MAXWELL H., University of Illinois 

BRETT, WILLIAM J., Indiana State Teachers College 

CAGLE, JULIEN, Princeton University 

DUBNAU, DAVID A., Columbia University 

ELLIOTT, PAUL R., University of Michigan 

FELDHERR, CARL, University of Pennsylvania 

FIELDEN, ANN, University of Illinois 

FRANKEL, JOSEPH, Yale University 

FRIZ, CARL T., University of Minnesota 

FUJIMORI, EIJI, University of Tokyo 

GRIFFIN, JOE L., Princeton University 

HATHAWAY, RALPH R., Florida State University 

JACKSON, JAMES A., Western Reserve University 

JOHNSON, SISTER MARIA BENIGNA, Saint Joseph College 

KAHLBROCK, MARGIT M., Columbia University 

KERENYI, THOMAS, Cornell Medical College 

KRASSNER, STUART, Johns Hopkins University 

LAMBERT, LORETTA, Harvard University 

MCCLUSKEY, ROBERT T., New York University College of Medicine 

MATURANA, HUMBERTO R., Harvard University 

MOORE, RICHARD DAVIS, Purdue University 

NAGLER, ARNOLD L., New York University-Bellevue Medical Center 

RALPH, CHARLES L., U. S. Dept. of Agriculture 

RHODES, WILLIAM C, Johns Hopkins University 

RICKLES, WILLIAM H., JR., Baylor University College of Medicine 

Ross, SAMUEL M., State University of New York at Brooklyn 

ROTHMAN, ALVIN H., Johns Hopkins School of Hygiene and Public Health 

SHIRODKAR, MANOHAR V., Johns Hopkins School of Hygiene and Public Health 

SJODIN, RAYMOND A., Purdue University 

STEINBERGER, WILLIAM W., University of Michigan 

STREHLER, B. L., National Institutes of Health 

SWETT, JOHN EMERY, University of California 

VILLEGAS, RAIMUNDO, Harvard Medical School 

WALLACE, ROBIN A., Columbia University 

WERMAN, ROBERT, Columbia University, College of Physicians and Surgeons 

WERNTZ, HENRY O., Harvard University 

WYLIE, RICHARD M., Harvard University 

YOUNG, ROBERT R., Harvard Medical School 



18 MARINE BIOLOGICAL LABORATORY 

Research Assistants, 1958 

ADLER, HOWARD, Albert Einstein Medical School 

ADLER, JULIUS, Washington University School of Medicine 

ALLEN, ARCHIE, University of North Carolina 

ALSUP, PEGGY ANN, University of Pennsylvania 

ARNOLD, ELIZABETH, Indiana University 

AUCLAIR, WALTER, New York University 

BARNWELL, FRANKLIN, Northwestern University 

BEDFORD, BONNIE V., Vassar College 

BENSAM, BERTRAND, State University of New York Medical College at Syracuse 

BIRKY, CARL WILLIAM, JR., Indiana University 

BISCHOFF, HARRY W., Texas Lutheran College 

BORGESE, THOMAS A., Rutgers University 

BOSLER, ROBERT B., Johns Hopkins University School of Medicine 

BRISKOW, CORNELIA, Barnard College 

BUNIM, LESLEY S., Barnard College 

CAMOUGIS, GEORGE, University of California 

CANTOR, MARVIN H., Massachusetts Institute of Technology 

CARANASOS, GEORGE J., St. Peter's College 

CHESEBROUGH, CAROLYN, Mount Holyoke College 

CLARK, ELOISE E., University of California 

CLARK, LENORA M., Lilly Research Laboratories 

CLARK, LYNNE G., Queens College 

CLOSE, RUSSELL I., University of Illinois 

COHEN, JANICE, New York University, College of Medicine 

COHEN, STAFFORD I., Boston University Medical School 

COLE, ELLEN L., University of Pennsylvania 

CONWAY, DOROTHY M., Rockefeller Institute for Medical Research 

COUTINHO, ELSIMAR M., Faculdade de Medicina Universidade de Bahia, Brazil 

DAVIS, ANN, Columbia University 

DEASON, TEMD R., University of Texas 

DINGLE, AL D., McMaster University 

DOOLITTLE, RUSSELL F., Harvard University 

EIN, DANIEL, New York University-Bellevue Medical Center 

ERDMAN, HOWARD E., North Carolina State College 

ESPER, HILDEGARD, Reed College 

FINE, ALBERT S., Veterans Administration Hospital 

FRANK, COLIN H., Belmont High School 

FRIEDMAN, LEONARD, Rutgers University 

FULTON, CHANDLER M., Rockefeller Institute 

GEBHART, JOHN H., National Institutes of Health 

GIFFORD, CAMERON E., Harvard University 

GOULD, EDWIN, Harvard University 

GREEN, JONATHAN, University of Minnesota 

GREENBERG, ALAN, Albert Einstein College of Medicine 

GREENLEES, JANET, Rutgers University 

GROSS, MARCIE, Yale University 

GRUPP, ERICA, Columbia University 

GUTTMAN, BURTON S., University of Minnesota 

HAMBURGER, CAROLA L., St. Louis 

HICKS, MARY, Rockefeller Institute 

HOLTZMAN, ERIC, Columbia University 

HUMPHREYS, TOM D., University of Chicago 

JONES, RAYMOND, England 

KAGEY, KAREN, New York University College of Medicine 

KANUNGO, MADHU S., University of Illinois 

KENT, JOAN L., Columbia University 



REPORT OF THE DIRECTOR 19 

DELANNURIEN, ANNE, Smith College 

LAURIE, JOHN S., Tulane University 

LEHV, JANE W., Vassar College 

LORENZO, MICHAEL A., St. Louis University 

LOVE, DAVID S., University of Chicago 

LOWE, MILDRED E., Tulane University 

LOWE, RUTH N., New York University-Bellevue Medical Center 

McCANN, FRANCES V., Columbia University 

McCANN, MARJORIE, Louisiana State University 

MCLAUGHLIN, JANE, Institute for Muscle Research, Marine Biological Laboratory 

MACMULLEN, JOYCE A., Cornell University 

MALKOFF, DONALD B., University of Pittsburgh School of Medicine 

MARGOLIS, PHYLLIS, Barnard College 

MINGIOLI, ELIZABETH S., Harvard Medical School 

MORGAN, MIRIAM, Smith College 

MOSHER, CARTER G., Boston University School of Medicine 

MOULE, MARGARET, McMaster University 

MURRELL, LEONARD R., McMaster University 

NASS, SYLVAN, New York University 

NOEL, ELISABETH S., Seton Hill College 

OBERPRILLER, JOHN, University of Illinois 

OTERO, Luis R., University of Puerto Rico 

PALIWAL, RIPUSUDAN L., University of Oklahoma 

PALMIERI, AMELIA, Cornell University Medical College 

PEARSE, JOHN S., University of Chicago 

PHILPOTT, DELBERT, Institute for Muscle Research, Marine Biological Laboratory 

PLUMB, MARY E., North Carolina State College 

ROBERTSON, LOLA, Fish and Wildlife Service 

RODENBERG, JEANNETTE M., New York University-Bellevue Medical Center 

ROSENBLUM, WILLIAM, New York University-Bellevue Medical Center 

ROSENBLUTH, RAJA, Columbia University 

ROSILLO, LUDWIG, St. Peter's College 

RUBINOFF, IRA, American Museum of Natural History 

SALACH, JAMES I., University of Chicago 

SATUREN, JANICE R., Syracuse Medical School 

SCHINSKI, ROBERT A., University of Minnesota 

SCHUEL, HERBERT, University of Pennsylvania 

SHEPARD, DAVID, University of Chicago 

SHERMAN, IRWIN W., Northwestern University 

SIGER, ALVIN, Johns Hopkins University 

SIMMONS, JOHN E., Johns Hopkins University 

SPYRIDES, GEORGE J., Johns Hopkins University 

STAUB, HERBERT W., Rutgers University 

SUNDARARAJ, B. I., Tulane University 

SWOPE, JULIA C, Massachusetts General Hospital 

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

TSUK, MARIANNE, Smith College 

WAHL, ROSEMARIE, University of Chicago 

WALTERS, C. PATRICIA, Lilly Research Laboratories 

WARWICK, ANNE C., Johns Hopkins School of Hygiene 

WHITCOMB, ERNEST R., National Institutes of Health 

WILBER, JOHN F., Harvard Medical School 

WILBOIS, ANNETTE, Indiana University 

WONG, EDWARD T., University of Minnesota 

WOOD, ROBERT W., Sloan-Kettering Institute 

WOODS, B. LOUISE, Lilly Research Laboratories 

WYTTENBACH, CHARLES R., Carnegie Institution of Washington 

YIP, CECIL, McMaster University 



20 MARINE BIOLOGICAL LABORATORY 

Library Readers, 1958 

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

BATHAM, ELIZABETH J., Lecturer, University of Otago, New Zealand 

BAYLOR, MARTHA B., Investigator, Marine Biological Laboratory 

BEIDLER, LLOYD M., Professor of Physiology, Florida State University 

BODANSKY, OSCAR, Professor of Biochemistry, Sloan-Kettering Institute 

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

BROBERG, PATRICIA L., Postdoctoral Fellow, Brandeis University 

BROWNE, L. BARTON, Johns Hopkins University 

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

CHANUTIN, ALFRED, Professor of Biochemistry, University of Virginia 

CLARK, ELIOT R., Professor Emeritus of Anatomy, University of Pennsylvania 

CLAUDATUS, JOHN C, Head, Department of Biochemistry, The Cancer Institute at Miami 

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

DuBois, ARTHUR, Associate Professor of Physiology, University of Pennsylvania 

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

FEENBERG, EUGENE, Professor of Physics, Washington University 

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

GABRIEL, MORDECAI L., Associate Professor of Biology, Brooklyn College 

GAFFRON, HANS, Professor of Biochemistry, University of Chicago 

GINSBERG, HAROLD S., Associate Professor of Preventive Medicine, Western Reserve University 

GUDERNATSCH, FREDERICK, Director, Cornell University Medical College 

HACKETT, DAVID P., Associate Professor of Biology, University of Buffalo 

HIMMELFARB, SYLVIA, Instructor in Physiology, University of Maryland Medical School 

HOBERMAN, H. D., Professor of Biochemistry, Albert Einstein College of Medicine 

HORSFALL, FRANK L., Vice-President and Physician-in-Chief, Rockefeller Institute 

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

JONES, SARAH R., Instructor in Zoology, Connecticut College 

KAAN, HELEN W., Indexer, National Academy of Sciences 

KABAT, ELVIN A., Professor of Microbiology, Columbia University 

KARUSH, FRED, Professor of Immunochemistry, University of Pennsylvania School of Medicine 

KEOSIAN, JOHN, Professor of Biology, Rutgers University 

KINDRED, JAMES E., Professor of Anatomy, University of Virginia 

KLEIN, MORTON, Professor of Microbiology, Temple University School of Medicine 

LAZZARINI, ABEL A., Associate Professor of Research Surgery, New York University 

LIONETTI, FABIAN J., Associate Professor of Biochemistry, Boston University School of Medicine 

LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont 

LOWENSTEIN, OTTO, Research Associate in Ophthalmology, Columbia University 

MCDONALD, SISTER ELIZABETH SETON, Chairman, Department of Biology, College of Mt. St. 

Joseph 

MARFEY, S. PETER, Research Associate, Princeton University 
MARSHAK, ALFRED, Marine Biological Laboratory 
MAVOR, JAMES, Professor Emeritus, Union College 
MOUL, EDWIN T., Associate Professor of Botany, Rutgers University 
OVERTON, JANE H., Assistant Professor of Natural Sciences, University of Chicago 
PRICE, WINSTON H., Associate Professor of Epidemiology and Biochemistry, Johns Hopkins 

University, School of Hygiene and Public Health 

PULLMAN, BERNARD, Professor of Theoretical Chemistry, University of Paris 
ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College 
SONNENBLICK, B. P., Professor of Biology, Rutgers University 
SULKIN, S. EDWARD, Professor and Chairman, University of Texas Southwestern Medical 

School 

SWANSON, CARL P., Gill Professor in Biology, Johns Hopkins University 
TRURNIT, HANS J., Principal Scientist, Research Institute for Advanced Studies 
UZIEL, MAYO, Instructor in Biochemistry, Tufts University Medical School 
WHEELER, GEORGE EDWARD, Instructor in Biology, Brooklyn College 



REPORT OF THE DIRECTOR 21 

YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate Medical 

Center 
ZINN, DOXALD J., Associate Professor of Zoology, University of Rhode Island 

Students, 1958 
BOTANY 

BIEBEL, PAUL J., Indiana University 

GUMMING, KENNETH B., Harvard University Graduate School 

DAWSON, WILLIAM A., Harvard University 

FARQUHARSON, Lois I., Franklin College 

FLETCHER, JOYCE V., Cornell University 

GARNETT, ELLEN M., Indiana University 

GIBBS, SARAH P., Woods Hole 

GOLDSTEIN, MELVIN E., Indiana University 

GOODWIN. MARY LINDER, Radcliffe College 

GRILLO, RAMON S., Fordham University 

HANCOCK, KENNETH F., University of Alabama 

HOFFMAN, LARRY RONALD, Iowa State College 

KAUSHIK, NILIMA, Vassar College 

MIDDLETON, KATHERINE, Vassar College 

MORAN, MARIUS R., Fordham University 

MORRILL, JOY F., University of Alabama 

MUMFORD. F. JOYCE, Smith College 

ROPES, MARIAN C, Radcliffe College 

EMBRYOLOGY 

BLANCHARD, ANN M., State University of Iowa 

BOASS, AGNA, Radcliffe College 

CAHN, ROBERT D., Rockefeller Institute 

CORLETTE, SALLY L., University of Pennsylvania, Institute for Cancer Research 

CROWELL, JANE, Radcliffe College 

DIBERARDINO, MARIE A., University of Pennsylvania, Institute for Cancer Research 

FINCH, CYNTHIA L., Oberlin College 

HUNTER, ROY, JR., Brown University 

KAIGHN, MORRIS E., Massachusetts Institute of Technology 

MINDICH, LEONARD E., Rockefeller Institute 

ROBERTS, B. DE\VAYNE, Roswell Park Memorial Institute 

ROTH, WILLARD D., Harvard Medical School 

SERGENT, DOROTHY J., Mount Holyoke College 

SIEGEL, PAULA H., University of Rochester 

SONNEBORN. DAVID R., Rockefeller Institute 

THOMPSON-UPHAM, A. E., Amherst College 

TROFFKIN, WALTER H., Brooklyn College 

TUMASONIS, REV. CASIMIR, Fordham University 

VANABLE, JOSEPH W., Brown University 

VINING, GEORGE JOSEPH, Yale University 

WEISS, LEON P., Harvard Medical School 

WESTON, CHARLES R., Princeton University 

PHYSIOLOGY 

ASCHEIM, EMIL, New York University 
AXELROD. DAVID, Harvard Medical School 
BEAULNES, AURELE, University of Montreal 



MARINE BIOLOGICAL LABORATORY 

CARDELL, ROBERT R., JR., University of Virginia 

CEGLER, ANNELIESE M., Marquette University 

CURTIS, BRIAN A., University of Rochester 

CZERLINSKI, GEORG H., Max Planck Inst. physikal Chemie 

DEMOVSKY, RONALD A., University of Illinois, College of Medicine 

ELLIOTT, PAUL R., University of Michigan 

FAUST, ROBERT G., Princeton University 

FERRANS, VICTOR J., Tulane Medical School 

FRANZEN, JAMES S., University of Illinois 

GOLDBERG, EDWARD, Johns Hopkins University 

GOYER, ROBERT A., St. Louis University 

GRIFFIN, JOE LEE, Princeton University 

HASELKORN, ROBERT, Harvard University 

HOEBEL, BART, Rockefeller Institute 

HUTTON, KENNETH, C, San Jose State College 

KRAUSE, ROBERT L., Haverford College 

MARKS, WILLIAM B., Massachusetts Institute of Technology 

NOVICK, RICHARD P., New York University 

ROLLER, ANN, California Institute of Technology 

ROSENKRANZ, HERBERT, Sloan-Kettering Institute 

RUECKERT, ROLAND R., McArdle Memorial Institute, University of Wisconsin 

TRYGSTAD, CARL W., University of Florida 

WEISBERG, ROBERT A., Harvard College 

WYLIE, RICHARD M., Harvard University 

INVERTEBRATE ZOOLOGY 

ASHMAN, ROBERT F., Wabash College 

BARRY, CORNELIUS, University of Maryland 

BAY, ERNEST C., Cornell University 

BIRKY, CARL WILLIAM, JR., Indiana University 

BLANK, FENJA, City College of New York 

BROSEGHINI, ALBERT L., Iowa State College 

CARDELL, ROBERT R., JR., University of Virginia 

CLARK, JAMES M., Franklin and Marshall College 

COOPER, EDWIN L., Atlanta University 

DAVIS, ROBERT P., Cornell University 

DAWSON, RICHARD G., Shawnee-Mission High School 

DRAINVILLE, FATHER GERARD, Universite de Montreal 

DUNAGAN, TOMMY T., Purdue University 

ELDRIDGE, PETER J., University of Massachusetts 

FERNOW, LEONARD R., Cornell University 

FIGGE, ROSALIE A., Oberlin College 

FISKE, TIMOTHY, University of Minnesota 

FORREST, HELEN F., Rutgers University 

Fox, SISTER M. ALICE MARIE, Saint Louis University 

GOLD, KENNETH, New York University 

GOULD, EDWIN, Tulane University 

GRANT, DAVID C., College of Wooster 

GUSSIN, ARNOLD ELY, Tulane University 

GUTTMAN, BURTON S., University of Minnesota 

HALL, DONALD J., University of Michigan 

HARRER, LORA, Marquette University 

HENNEN, SALLY H., Indiana University 

HILTY, CAROL R., Oberlin College 

KELSO, JUDITH I., Brown University 



REPORT OF THE DIRECTOR 23 



KROECKEL, REV. CLARENCE J., St. Joseph's College 
LEARY, DONALD E., Notre Dame 
LEET, ROSEMARY, Chatham College 
LIGHT, PAUL, Washington University 
LINDSAY, DAVID T., Johns Hopkins University 
LUNING, ANNE, Vassar College 
MACIOR, FR. LAZARUS, University of Wisconsin 
MACMULLEN, JOYCE, Cornell University 
McCREASH, ARTHUR H., Temple University 
McRrrcHiE, ROBERT G., Vanderbilt University 
PAINE, ROBERT T., Ill, University of Michigan 
PEEL, ROSEMARY E., Drew University 
PERNAA, JUDITH E., Cornell University 
ROBINSON, MARTHA A., Oberlin College 
ROTH, THOMAS F., Harvard University 
SANCHEZ, PATRICIO, Rockefeller Foundation 
SAVAGE, ALICE M., Brown University 
SCHMIDT, REV. MATTHIAS, St. Benedict's College 
SMITH, WILLIE R., Fordham University 
SNOW, ISABEL W., Hunter College 
STILWELL, SHIRLEY E., Wheaton College 
TUNNOCK, SHEILA M., Colby College 
VAHARU, TIIU, Syracuse University 
WESTON, JAMES A., Yale University 
WETZEL, BRUCE K., Harvard University 
WILLIAMS, DEBORAH C, Tufts University 

ECOLOGY 

BEYERS, ROBERT J., University of Texas 

CHOI, Ki-CnuL, Seoul National University 

CUMMING, KENNETH B., Harvard University 

FONDA, SHIRLEY L., Oberlin College 

GOUDSMIT, ESTHER M., University of Michigan 

HOCHMAN, ROBERT A., Lafayette College 

MOLL, CAROLYN J., Mount Holyoke College 

MUELLER, WAYNE PAUL, Indiana University 

PATCHEN, JOAN D., Drew University 

ROSEN, DONN ERIC, American Museum of National History 

SCULLY, MARGARET A., Framingham State Teachers College 

SEWALL, JANE, Goucher College 

SIMCOX, RICHARD F., Los Angeles State College 

STAGNER, MARILYN L., Duke University 

WILLIAMS, RICHARD B., Harvard University 

WILSON, RONALD F., Dartmouth College 

3. FELLOWSHIPS AND SCHOLARSHIPS, 1958 

Lucretia Crocker Scholorship : 

LARRY R. HOFFMAN, Botany Course 

Calkins Scholarship : 

DONALD J. HALL, Invertebrate Zoology Course 
PAULA SIEGEL, Embryology Course 
WALTER TROFFKIN, Embryology Course 

Bio Club Scholarship : 

FENJA BLANK, Invertebrate Zoology 



24 



MARINE BIOLOGICAL LABORATORY 



4. TABULAR VIEW OF ATTENDANCE, 1954-58 



1954 

INVESTIGATORS TOTAL 298 

Independent 180 

Under Instruction 20 

Library Readers 52 

Research Assistants 46 

STUDENTS TOTAL 134 

Invertebrate Zoology 56 

Embryology 29 

Physiology 28 

Botany 12 

Ecology 9 

TOTAL ATTENDANCE 432 

Less persons represented as both investigators and 

students 5 



427 



7955 1956 1957 



250 

162 

9 

54 

25 

148 

56 

30 

30 

19 

13 

398 



398 



304 

184 

20 

50 

50 

140 

55 

28 

30 

18 

9 

444 



442 



326 

186 

23 

42 

75 

139 

55 

27 

30 

IS 

9 

465 



462 



1958 

410 

203 

39 

54 

114 

138 

55 

22 

27 

18 

16 

548 

5 

543 



INSTITUTIONS REPRESENTED TOTAL 136 129 130 129 142 

By Investigators 104 95 97 94 110 

By Students 32 34 33 35 74 

SCHOOLS AND ACADEMIES REPRESENTED 

By Investigators 2 3 3 

By Students 1 2 1 1 2 

FOREIGN INSTITUTIONS REPRESENTED 

By Investigators 11 8 9 11 20 

By Students 13 6 6 5 6 



5. INSTITUTIONS REPRESENTED, 1958 



Adelphi College 

Agnes Scott College 

Agricultural Research Center 

Alabama, University of 

Albert Einstein Medical School 

Albion College 

American Heart Association 

American Museum of Natural History 

Amherst College 

Army Chemical Center 

Atlanta University 

Barnard College 

Baylor University 

Boston University School of Medicine 

Brandeis University 

Brooklyn College 

Brown University 

Bryn Mawr College 

Buffalo, University of 

California Institute of Technology 

California, University of 

Cancer Institute of Miami 

Carnegie Institute of Technology 

Carnegie Institution of Washington 



Chatham College 

Chicago, University of 

Cincinnati, University of 

City College of New York 

Colby College 

Colgate University 

Colorado, University of 

Columbia University 

Columbia University College of Physicians 

and Surgeons 
Connecticut College 
Connecticut, University of 
Cornell University 
Cornell University Medical School 
Dartmouth College 
Drew University 
Duke University 
Eli Lilly and Company 
Emory University 
Florida State University 
Fordham University 
Framingham State Teachers College 
Franklin College 
Franklin and Marshall College 



HAPTENS OF UNFERTILIZED EGGS 



25 



Goucher College 

Hahnemann Medical School 

Harvard University 

Harvard University Medical School 

Haverford College 

Howard Hughes Medical Institute 

Hunter College 

Illinois, University of 

Indiana State Teachers College 

Indiana University 

Institute for Muscle Research 

Iowa State University 

Johns Hopkins University 

Lafayette College 

Los Angeles State College 

Louisiana State University 

Maine, University of 

Marquette University 

Maryland, University of 

Mass. Eye and Ear Infirmary 

Mass. General Hospital 

Mass. Institute of Technology 

Massachusetts, University of 

Michigan, University of 

Minnesota, University of 

Mount Holyoke College 

Mt. St. Joseph, College of 

National Academy of Sciences 

National Institutes of Health 

Naval Medical Research Institute 

New York, State University of, Medical 

School at Syracuse 

New York, State University of, at Brooklyn 
New York University, Bellevue Medical 

Center 

New York University, School of Dentistry 
New York University, Washington Square 

College 

North Carolina State College 
North Carolina, University of 
Northwestern University 
Notre Dame University 
Oak Ridge National Laboratory 
Oberlin College 
Oklahoma, University of 
Oregon, University of 
Pennsylvania, University of 
Pennsylvania Medical School, University of 
Pittsburgh, University of 
Polytechnic Institute of Brooklyn 



Princeton University 

Purdue University 

Queens College 

Radcliffe College 

Reed College 

Research Institute for Advanced Studies 

Rhode Island, University of 

Rochester, University of 

Rockefeller Institute for Medical Research 

Roswell Park Memorial Institute 

Rutgers University 

St. Benedict's College 

St. Joseph College for Women 

St. Joseph's College 

St. Louis University 

St. Peter's College 

San Jose State College 

Seton Hall College 

Seton Hill College 

Sloan-Kettering Institute 

Smith College 

Southwest Texas State Teachers College 

Syracuse University 

Temple University 

Texas Lutheran College 

Texas, University of 

Texas, University of, Southwestern Medical 

School 

Tufts University 

Tufts University Medical School 
Tulane University 
Tulane University Medical School 
Union College 

U. S. Fish and Wildlife Service 
U. S. Public Health Service 
Vanderbilt University 
Vassar College 
Vermont, University of 
Veterans' Administration Hospital 
Virginia, University of 
Washington University 
Washington University Medical School 
Western Reserve University 
Wheaton College 
Wilson College 
Wisconsin, University of 
Wooster, College of 
Yale University 
Yeshiva University 



FOREIGN INSTITUTIONS REPRESENTED, 1958 



Australian National University, Australia 
Universidade de Bahia, Brazil 
Atomic Energy Authority, British Isles 
Kings College, British Isles 
University of Cambridge, British Isles 



University of Reading, British Isles 

McMaster University, Canada 

University of Montreal, Canada 

University of Chile, Chile 

Ecole Scientia et Faculty de Mecidna, France 



26 



MARINE BIOLOGICAL LABORATORY 



Max Planck Institut fur physikalische Chemie, 
Germany 

Physiological Institute of Heidelberg, Ger- 
many 

University of Tubingen, Germany 

University of Hawaii 

Central College, University of Mysore, India 

University of Palermo, Italy 

Hebrew University, Hadassah Medical School, 
Jordan 



Kogoshima University, Japan 
Tokyo Jikei-kai School of Medicine, Japan 
University of Tokyo, Japan 
Seoul National Institute, Korea 
University of Otago, New Zealand 
Fagellonian University, Poland 
University of Puerto Rico, Puerto Rico 
Wenner Grens Institute, Sweden 
Institute de Investigaciones Medicas, Vene- 
zuela 



SUPPORTING INSTITUTIONS AND AGENCIES, 1958 



Abbott Laboratories 

American Cancer Society 

American Philosophical Society 

Associates of the Marine Biological Laboratory 

Atomic Energy Commission 

Ciba Pharmaceutical Products, Inc. 

Josephine B. Crane Foundation 

Carter Products Inc. 

The Grass Foundation 

Hoffman-LaRoche, Inc. 

The Lalor Foundation 



Eli Lilly and Company 

Merck and Company, Inc. 

National Institutes of Health 

National Science Foundation 

Office of Naval Research 

The Pfizer Foundation, Inc. 

The Rockefeller Foundation 

Schering Corporation 

Smith, Kline and French Foundation 

Wyeth Laboratories 

The Upjohn Company 



6. FRIDAY EVENING LECTURES, 1958 

July 4 

ALBERT SZENT-GYORGYI "Muscle and energetics" 

July 11 

COLIN S. PITTENDRIGH "A coupled oscillator scheme for the daily 

rhythms of organisms" 
July 18 

BERNARD D. DAVIS "Bacterial mutants and the study of cell 

physiology" 
July 25 

WARREN O. NELSON "The physiological control of fertility" 

August 1 

ROLLIN D. HOTCHKISS "The dissemination of genetic substance" 

August 8 

STEPHEN W. KUFFLER "A single nerve cell looks at neurophysiol- 

ogy" 
August 15 

DON W. FAWCETT "The submicroscopic structure and func- 
tional behavior of the membranous com- 
ponents of the cytoplasm" 
August 22 

BERNARD PULLMAN "Electronic structure and activity in cancer 

chemotherapy of purine antimetabolites" 

7. TUESDAY EVENING SEMINARS, 1958 

July 1 

EDGAR ZWILLING "Reconstitution from one germ layer in 

Cordylophora ( hydroid ) " 



REPORT OF THE DIRECTOR 27 

MAXWELL BRAVERMAN "Neural and mesodermal hierarchies in 

chick development" 

S. MERYL ROSE "Mutual growth inhibition in frog tadpoles" 

July 8 

FREDERICK A. BETTELHEIM "The nature of chromatographic amylose 

and amylopectin fractions" 

PAUL S. GALTSOFF "Coordination of ciliary motion and muscu- 
lar activity in Ostrea virginica" 

BENJAMIN LOWENHAUPT "The carrier for calcium transport in aquatic 

leaves" 
July 15 

P. W. WHITING "Factors and genes in Mormoniella" 

SEARS CROWELL "Tail regeneration in experimentally short- 
ened and lengthened earthworms" 

C. C. SPEIDEL "Motion pictures showing some changes in 

cells induced by x-ray treatments of tad- 
poles and tetrahymenae" 
July 22 

JAMES LASH "The uptake of radiosulphur during the in- 
duction of cartilage" 

MENACHEM WURZEL "Mode of action of choline esters, substrate 

specificity of their 'receptor protein' ' 

IRVIN ISENBERG "Free radical formation in riboflavin com- 
plexes" 
July 29 

A. J. BERNATOWICZ "Ecological isolation of alternate generations 

of plants" 

DEMOREST DAVENPORT "A technique of investigating the effect of 

host-factor on the behavior of polychaete 
and crustacean commensals" 

EVELYN SHAW "The development of schooling behavior in 

the silverside fish, Menidia menidia" 

CHARLES JENNER "Schooling behavior in the marine snail, 

Nassarius obsoletus" (with colored movie) 
August 5 

ARTHUR L. COLWIN and 

LAURA H. COLWIN "Effects of sperm extract and other agents 

on the egg membranes, in relation to 
sperm entry in Hydroides" 

CHARLES B. METZ "Fertilization and agglutination inhibitors 

from Arbacia" 

LIONEL I. REBHUN "Behavior of metachromatic granules during 

cleavage in Spisula" 
August 12 

G. W. DE VlLLAFRANCA, T. S. 

SCHEINBLUM and D. E. PHILPOTT ...."The a-band of muscle from Limulus poly* 

phemus" 

D. W. BISHOP "Sperm cell models and the question of 

ATP-induced rhythmic motility" 

L. NELSON "ATP An energy source for sperm mo- 
tility" 



MARINE BIOLOGICAL LABORATORY 

R. D. ALLEN "Polarized optical studies on Ameba" 

F. CHILD "Isolation and analysis of cilia" 

August 19 

L. V. HEILBRUNN "A physical study of the ground substance 

of the Spisula egg" 
CARL FELDHERR "Physical properties of lobster nerve axo- 

plasm" 
ROBERT W. MERRIAM "Some aspects of the nuclear membrane in 

developing sand dollar eggs" 

8. MEMBERS OF THE CORPORATION, 1958 
1. LIFE MEMBERS 

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

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

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

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

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

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

DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut 

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

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

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

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

MACNAUGHT, MR. FRANK M., Woods Hole, Massachusetts 

MACKLIN, DR. CHARLES C., 37 Gerard Street, London, Ontario 

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

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

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

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

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

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

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

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

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

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

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

YOUNG, DR. B. P., Cornell University, Ithaca, New York 

2. REGULAR MEMBERS 

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

Dentistry, Rochester, New York 

ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota 
ALLEN, DR. M. JEAN, Department of Biology, Wilson College, Chambersburg, 

Pennsylvania 



REPORT OF THE DIRECTOR 

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

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

AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland 
School of Medicine, Baltimore, Maryland 

ANDERSON, DR. J. M., Department of Zoology, Cornell University, Ithaca, New 
York 

ANDERSON, DR. RUBERT S., Medical Laboratories, Army Chemical Center, Mary- 
land (Box 632 Edgewood, Maryland) 

ANDERSON, DR. T. F., c/o Dr. A. Lurff, Institut Pasteur, 28 Rue du Dr. Roux, 
Paris 15e, France 

ARMSTRONG, DR. PHILIP B., State University of New York College of Medicine, 
Syracuse 10, New York 

ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory, 
Oak Ridge, Tennessee 

ATWOOD, DR. KIMBALL C, 6029 University Avenue, Chicago 37, Illinois 

AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts 

AYERS, DR. JOHN C., Department of Zoology, University of Michigan, Ann Arbor, 
Michigan 

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

BAKER, DR. H. B., Department of Zoology, University of Pennsylvania, Philadel- 
phia 4, Pennsylvania 

BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University 
Medical School, Boston 15, Massachusetts 

BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire 

BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School 
of Hygiene, Baltimore 5, Maryland 

BARD, DR. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland 

EARTH, DR. L. G., Department of Zoology, Columbia University, New York 27, 
New York 

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

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

BECK, DR. L. V., Department of Physiology and Pharmacology, University of Pitts- 
burgh School of Medicine, Pittsburgh 13, Pennsylvania 

BEERS, DR. C. D., Department of Zoology, University of North Carolina, Chapel 
Hill, North Carolina 

BEHRE, DR. ELINOR H., Black Mountain, North Carolina 

BENESCH, DR. REINHOLD, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 

BENESCH, DR. RUTH, Marine Biological Laboratory, Woods Hole, Massachusetts 

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

BERG, DR. WILLIAM E., Department of Zoology, University of California, Berke- 
ley 4, California 



30 MARINE BIOLOGICAL LABORATORY 

BERMAN, DR. MONES, Institute for Arthritis and Metabolic Diseases, National Insti- 
tutes of Health, Bethesda 14, Maryland 
BERNHEIMER, DR. ALAN W., New York University College of Medicine, New York 

16, New York 
BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne University College of 

Medicine, Detroit 7, Michigan 

BERTHOLF, DR. LLOYD, Illinois Wesleyan University, Bloomington, Illinois 
BEVELANDER, DR. GERRIT, New York University School of Medicine, New York 

16, New York 
BIGELOW, DR. HENRY B., Museum of Comparative Zoology, Harvard University, 

Cambridge 38, Massachusetts 

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

BLANCHARD, DR. K. C., Johns Hopkins Medical School, Baltimore, Maryland 
BLOCK, DR. ROBERT, 518 South 42nd Street, Apt. C 7, Philadelphia 4, Pennsylvania 
BLUM, DR. HAROLD F., Department of Biology, Princeton University, Princeton, 

New Jersey 
BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center, 444 

East 68th Street, New York 21, New York 
BODIAN, DR. DAVID, Department of Anatomy, Johns Hopkins University, 709 

North Wolfe Street, Baltimore 5, Maryland 
BOELL, DR. EDGAR J., Osborn Zoological Laboratories, Yale University, New 

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

Storrs, Connecticut 

BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin, Texas 
BOREI, DR. HANS, Department of Zoology, University of Pennsylvania, Philadel- 
phia 4, Pennsylvania 
BOWEN, DR. VAUGHAN T., Woods Hole Oceanographic Institution, Woods Hole, 

Massachusetts 

BRADLEY, DR. HAROLD C., 2639 Durant Avenue, Berkeley 4, California 
BRIDGMAN, DR. ANNA J., Department of Biology, Agnes Scott College, Decatur, 

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

York 21, New York 
BROOKS, DR. MATILDA M., Department of Physiology, University of California, 

Berkeley 4, California 
BROWN, DR. DUGALD E. S., Department of Zoology, University of Michigan, Ann 

Arbor, Michigan 
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 



REPORT OF THE DIRECTOR 31 

BULLOCK, DR. T. H., Department of Zoology, University of California, Los An- 
geles 24, California 

BURBANCK, DR. WILLIAM D., Box 721, Woods Hole, Massachusetts 

BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilmington, 
Delaware 

BURKENROAD, DR. M. D., c/o Lab. Nal. de Pesca, Apartado 3318, Estofeta #1, 
Olindania, Republic of Panama 

BUTLER, DR. E. G., Department of Biology, P.O. Box 704, Princeton University, 
Princeton, New Jersey 

CAMERON, DR. J. A., Baylor College of Dentistry, Dallas, Texas 

CANTONI, DR. GIULIO, National Institutes of Health, Mental Health, Bethesda 14, 
Maryland 

CARLSON, DR. FRANCIS D., Department of Biophysics, Johns Hopkins University, 
Baltimore 18, Maryland 

CARPENTER, DR. RUSSELL L., Tufts University, Medford 55, Massachusetts 

CARSON, Miss RACHEL, 11701 Berwick Road, Silver Spring, Maryland 

CASE, DR. JAMES, Department of Zoology, State University of Iowa, Iowa City, 
Iowa 

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

CATTELL, MR. WARE, Cosmos Club, Washington 5, D. C. 

CHAET, DR. ALFRED B., Department of Biology, American University, Washing- 
ton 16, D. C. 

CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical 
School, Coral Gables, Florida 

CHANG, DR. JOSEPH J., National Institute of Neurological Diseases and Blindness, 
National Institutes of Health, Bethesda 14, Maryland 

CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton, 
New Jersey 

CHENEY, DR. RALPH H., Biology Department, Brooklyn College, Brooklyn 10, 
New York 

CLAFF, DR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts 

CLARK, DR. A. M., Department of Biology, University of Delaware, Newark, 
Delaware 

CLARK, DR. E. R., The Wistar Institute, Woodland Avenue and 36th Street, Phila- 
delphia 4, Pennsylvania 

CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, 
New York 

CLARKE, DR. GEORGE L., Harvard University, Biological Laboratories, Cambridge 
38, Massachusetts 

CLELAND, DR. RALPH E., Indiana University, Bloomington, Indiana 

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

CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana 

COE, DR. W. R., 183 Third Avenue, Chula Vista, California 

COHEN, DR. SEYMOUR S., Department of Physiological Chemistry, University of 
Pennsylvania, Philadelphia 4, Pennsylvania 



MARINE BIOLOGICAL LABORATORY 

COLE, DR. KENNETH S., National Institutes of Health (NINDB), Bethesda 14, 

Maryland 

COLLETT, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts 
COLLIER, DR. JACK R., Department of Zoology, Louisiana State University, Baton 

Rouge, Louisiana 

COLTON, DR. H. S., Box 601, Flagstaff, Arizona 
COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, New 

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

York 

COOPER, DR. KENNETH W., Department of Zoology, University of Florida, Gaines- 
ville, Florida 

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

COPELAND, DR. D. E., 8705 Susanna Lane, Chevy Chase 15, Maryland 
COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine 
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., Goucher College, Towson, Baltimore 4, Maryland 
CROWELL, DR. P. S., JR., Department of Zoology, Indiana University, Blooming- 
ton, Indiana 
CSAPO, DR. ARPAD I., Rockefeller Institute for Medical Research, 66th Street and 

York Avenue, New York 21, New York 

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

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

Massachusetts 

DAWSON, DR. A. J., College of the City of New York, New York City, New York 
DEANE, DR. HELEN W., Albert Einstein College of Medicine, New York 61, New 

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

Pennsylvania 

DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania 
DIXON, DR. FRANK J., Department of Pathology, University of Pittsburgh School 
of Medicine, Pittsburgh 13, Pennsylvania 



REPORT OF THE DIRECTOR 33 

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

West Virginia 
DOLLEY, DR. WILLIAM L., Department of Biology, Randolph- Macon College, 

Ashland, Virginia 

DONALDSON, DR. JOHN C., University of Pittsburgh School of Medicine, Pitts- 
burgh, Pennsylvania 
DOTY, DR. MAXWELL S., Department of Biology, University of Hawaii, Honolulu, 

T. H. 

DuBois, DR. EUGENE F., 200 East End Avenue, New York 28, New York 
DURYEE. DR. WILLIAM R., George Washington University School of Medicine, 

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

Rhode Island 

EDWARDS, DR. CHARLES, University of Utah, Salt Lake City, Utah 
EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania 
ELLIOT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann 

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

Medicine, New York 61, New York 

EVANS, DR. TITUS C., State University of Iowa, Iowa City, Iowa 
FAILLA, DR. G., Columbia University, College of Physicians and Surgeons, New 

York 32, New York 

FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France 
FERGUSON, DR. F. P., Department of Physiology, University of Maryland Medical 

School, Baltimore 1, Maryland 
FERGUSON, DR. JAMES K. W., Connought Laboratories, University of Toronto, 

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

Streets, Baltimore 1, Maryland 
FINGERMAN, DR. MILTON, Department of Zoology, Newcomb College, Tulane 

University, New Orleans 18, Louisiana 
FISCHER. DR. ERNST, Department of Physiology, Medical College of Virginia, 

Richmond 19, Virginia 
FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, 

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

Canada 
FORBES, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge 

38, Massachusetts 
FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois, 

Urbana, Illinois 

FREYGANG, DR. WALTER H., JR., P. O. Box 11, Poolesville, Maryland 
FRIES, DR. ERIK F. B., Box 605, Woods Hole, Massachusetts 
FRISCH, DR. JOHN A., Canisius College, Buffalo, New York 
FURTH, DR. JACOB, 18 Springdale Road, Wellesley Farms, Massachusetts 
FYE, DR. PAUL M., Director, Woods Hole Oceanographic Institution, Woods 

Hole, Massachusetts 



34 MARINE BIOLOGICAL LABORATORY 

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

GAFFRON, DR. HANS, Research Institutes, University of Chicago, 5650 Ellis 
Avenue, Chicago 37, Illinois 

GALL, DR. JOSEPH G., Department of Zoology, University of Minnesota, Minneapolis 
14, Minnesota 

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

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

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

GINSBERG, DR. HAROLD S., Western Reserve University School of Medicine, Cleve- 
land, Ohio 

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

GOODRICH, DR. H. B., Wesleyan University, Middletown, Connecticut 

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

GOULD, DR. H. N., Biological Sciences Information Exchange, 1113 Dupont Circle 
Building, Washington, D. C. 

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

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

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

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

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

GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New 
Brunswick, New Jersey 

GREEN, DR. MAURICE, Microbiology Department, St. Louis University Medical 
School, St. Louis, Missouri 

GREGG, DR. JAMES H., Department of Biological Sciences, University of Florida, 
Gainesville, Florida 

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

GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical Col- 
lege, New York 21, New York 

GRIFFIN, DR. DONALD R., Harvard University, Biological Laboratories, Cam- 
bridge 38, Massachusetts 

GROSCH, DR. DANIEL S., Department of Genetics, Gardner Hall, North Carolina 
State College, Raleigh, North Carolina 

GROSS, DR. PAUL, Department of Biology, New York University, University 
Heights, New York 53, New York 

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



REPORT OF THE DIRECTOR 35 

GUDERNATSCH, DR. FREDERICK, 41 Fifth Avenue, New York 3, New York 
GUTHRIE, DR. MARY J., Detroit Institute for Cancer Research, 4811 John R. Street, 

Detroit, Michigan 
GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn 10, 

New York 

GUYER, DR. MICHAEL F., University of Wisconsin, Madison, Wisconsin 
HAJDU, DR. STEPHEN, U. S. Public Health Institute, Bethesda 14, Maryland 
HALL, DR. FRANK G., Department of Physiology, Duke University Medical School, 

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

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

Iowa 

HANCE, DR. ROBERT T., Box R.R. # 3, Loveland, Ohio 
HARDING, DR. CLIFFORD V., JR., Columbia University Medical School, New York 

City, New York 
HARNLY. DR. MORRIS H., Washington Square College, New York University, 

New York 3. New York 
HARRISON, DR. Ross G., Osborn Zoological Laboratories, Yale University, New 

Haven, Connecticut 
HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research. 66th Street 

and York Avenue, New York 21, New York 

HARTMAN, DR. FRANK A., Hamilton Hall, Ohio State University, Columbus, Ohio 
HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New 

Jersey 

HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey 
HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo 

3, New York 

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

27, New York 

HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesly 81, Massachusetts 
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts 
HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, 

Philadelphia 4, Penna. 
HENDLEY, DR. CHARLES D., 615 South Second Avenue, Highland Park, New 

Jersey 
HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 

HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts 
HESS, DR. WALTER N., Hamilton College, Clinton, New York 
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 
HINRICHS, DR. MARIE, 344E Quincy Street, Riverside. Illinois 



36 MARINE BIOLOGICAL LABORATORY 

HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge 38, 

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

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

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

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

Pennsylvania 

HOLLAENDER, DR. ALEXANDER, Biology Division, O.R.N.L., Oak Ridge, Tennessee 
HOPKINS, DR. HOYT S., New York University College of Dentistry, New York 

City, New York 
HUNTER, DR. FRANCIS R., University of the Andes, Calle 18-a Carreral-E, Bogata, 

Colombia, South America 
HUTCHENS, DR. JOHN O., Department of Physiology, University of Chicago, 

Chicago 37, Illinois 

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

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

IRVING, DR. LAURENCE, U. S. Public Health Service, Anchorage, Alaska 
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts 
JACOBS, DR. M. H., University of Pennsylvania School of Medicine, Philadelphia 4, 

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

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

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

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

Florida, Gainesville, Florida 

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

New York City, New York 

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

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

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

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

New Jersey 






REPORT OF THE DIRECTOR 37 

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

Massachusetts 

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

Connecticut 

KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia 
KING, DR. JOHN W., Morgan State College, Baltimore 12, Maryland 
KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa 
KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York 
KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland, Oregon 
KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston, 

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

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

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

Illinois 
KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Baltimore 5, 

Maryland 

KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois 
KUFFLER, DR. STEPHEN, Department of Ophthalmology, Johns Hopkins Hospital, 

Baltimore 5, Maryland 
KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 

LACKEY, DR. JAMES B., Box 497, Melrose, Florida 
LANCEFIELD, DR. D. E., Queens College, Flushing, New York 
LANCEFIELD, DR. REBECCA C., Rockefeller Institute, 66th Street and York Avenue, 

New York 21, New York 

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

School, Pittsburgh 13, Pennsylvania 

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

LAVIN, DR. GEORGE I., 3714 Springdale Avenue, Baltimore, Maryland 
LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota Medical 

School, Minneapolis 14, Minnesota 
LEDERBERG, DR. JOSHUA, Department of Genetics, University of Wisconsin^ 

Madison 6, Wisconsin 
LEE, DR. RICHARD E., Cornell University College of Medicine, New York City,, 

New York 

LEFEVRE, DR. PAUL G., Brookhaven Apartments, Upton, Long Island, New York 
LEHMANN, DR. FRITZ, Zoologische Institut, University of Berne, Berne, Switzerland 
LESSLER, DR. MILTON A., Department of Physiology, Ohio State University, 

Columbus, Ohio 
LEVINE, DR. RACHMIEL, Michael Rees Hospital, Chicago, 16, Illinois 



38 MARINE BIOLOGICAL LABORATORY 

LEVY, DR. MILTON, Biochemistry Department, New York University School of 

Dentistry, New York 10, New York 

LEWIN, DR. RALPH A., Marine Biological Laboratory, Woods Hole, Massachusetts 
LEWIS, DR. IVEY F., 1110 Rugby Road, Charlottesville, Virginia 
LING, DR. GILBERT, 307 Berkely Road, Merion, Pennsylvania 
LITTLE, DR. E. P., 216 High Street, West Newton, Massachusetts 
LLOYD, DR. DAVID P. C, Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 

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

LOEB, DR. LEO, 40 Crestwood Drive, St. Louis 5, Missouri 
LOEB, DR. R. F., Presbyterian Hospital, 620 West 168th Street, New York 32, 

New York 

LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York 
LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans- 
ton, Illinois 

LOVE, DR. Lois H., 4233 Regent Street, Philadelphia 4, Pennsylvania 
LOVE, DR. WARNER E., 1043 Marlau Drive, Baltimore 12, Maryland 
LUBIN, DR. MARTIN, Department of Pharmacology, Harvard Medical School, 

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

York 21, New York 
LYNCH, DR. RUTH STOCKING, Department of Botany, University of California, Los 

Angeles 24, California 
LYNCH, DR. WILLIAM, Department of Biology, St. Ambrose College, Davenport, 

Iowa 
LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America, 

Washington, D. C. 
McCoucH, DR. MARGARET SUM WALT, University of Pennsylvania Medical School, 

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

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

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

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

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

Decatur, Georgia 
MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 136 

Harrison Avenue, Boston, Massachusetts 

MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York 
MARSHAK, DR. ALFRED, Department of Biology, University of Notre Dame, Notre 

Dame, Indiana 
MARSLAND, DR. DOUGLAS A., New York University, Washington Square College, 

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

New York 



REPORT OF THE DIRECTOR 39 

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

Williamstown, Massachusetts 

MAYOR, DR. JAMES W., 8 Gracewood Park, Cambridge 58, Massachusetts 
MAZIA, DR. DANIEL, Department of Zoology, University of California, Berkeley 4, 

California 

MEDES. DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania 
MEINKOTH, DR. Norman A., Department of Biology, Swarthmore College, Swarth- 

more, Pennsylvania 
MEN KIN, DR. VALV, Agnes Barr Chase Foundation for Cancer Research, Temple 

University Medical School, Philadelphia, Pennsylvania 
METZ, DR. C. B., Oceanographic Institute, Florida State University, Tallahassee, 

Florida 
METZ. DR. CHARLES W., Department of Zoology, University of Pennsylvania, 

Philadelphia 4, Pennsylvania 
MIDDLEBROOK, DR. ROBERT, Institute for Muscle Research, Marine Biological 

Laboratory, Woods Hole, Massachusetts 
MILLER, DR. J. A., JR., Department of Anatomy, Emory University, Atlanta 22, 

Georgia 
MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire, 

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

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

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

Durham, New Hampshire 
MOORE, DR. JOHN A., Department of Zoology, Columbia University, New York 27, 

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

Health, Bethesda 14, Maryland 
MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick, 

New Jersey 

MOUNTAIN, MRS. J. D., 8 Coolidge Avenue, White Plains, New York 
MULLER, DR. H. J., Department of Zoology, Indiana University, Bloomington, 

Indiana 
MULLINS, DR. LORIN J., Biophysical Laboratory, Purdue University, Lafayette, 

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

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

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

University, Hamilton, Ontario, Canada 

NACHMANSOHN, DR. DAVID, Columbia University, College of Physicians and Sur- 
geons, New York City, New York 

NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts 
NELSON, DR. LEONARD, Department of Anatomy, University of Chicago, Chicago, 

Illinois 



40 MARINE BIOLOGICAL LABORATORY 

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

5, Washington 
NICOLL, DR. PAUL A., Indiana Contract, Box K, A. P.O. 474, San Francisco, 

California 
Niu, DR. MAN-CHIANG, Rockefeller Institute for Medical Research, 66th Street 

and York Avenue, New York 21, New York 
OCHOA, DR. SEVERO, New York University College of Medicine, New York 16, 

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

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

Mawr, Pennsylvania 
OSTER. DR. ROBERT H., University of Maryland School of Medicine, Baltimore 1, 

Maryland 
OSTERHOUT, MRS. MARION IRWIN, Rockefeller Institute, 66th Street and York 

Avenue, New- York 21, New York 
OSTERHOUT, DR. W. J. V., Rockefeller Institute, 66th Street and York Avenue, 

New York 21, New York 

PACKARD, DR. CHARLES, Woods Hole, Massachusetts 
PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio 
PARM ENTER, DR. CHARLES L., Department of Zoology, University of Pennsylvania, 

Philadelphia 4, Pennsylvania 
PARPART, DR. ARTHUR K., Department of Biology, Princeton University, Princeton, 

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

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

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

Chicago 37, Illinois 

PERSON, DR. PHILIP, Chief, Special Dental Research Program, Veterans Adminis- 
tration Hospital, Brooklyn 9, New York 

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

PHILPOTT, MR. DELBERT E., 496 Palmer Avenue, Falmouth, Massachusetts 
PICK, DR. JOSEPH, Department of Anatomy, New York University, Bellevue 

Medical Center, New York City, New York 

PIERCE, DR. MADELENE E., Vassar College, Poughkeepsie, New York 
PLOUGH, DR. HAROLD H., Department of Biology, Amherst College, Amherst, 

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

27, New York 

POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut 
PRATT, DR. FREDERICK H., 105 Hundreds Road, Wellesley Hills 82, Massachusetts 
PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti- 
more 12, Maryland 
PROSSER, DR. C. LADD, 401 Natural History Building, University of Illinois, Urbana, 

Illinois 



REPORT OF THE DIRECTOR 41 

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

New York 

RAMSEY, DR. ROBERT W., Medical College of Virginia, Richmond, Virginia 
RAND, DR. HERBERT W., 7 Siders Pond Road, Falmouth, Massachusetts 
RANKIN, DR. JOHN S., Department of Zoology, University of Connecticut, Storrs, 

Connecticut 
RATNER, DR. SARAH, Public Health Research Institute of the City of New York, 

Foot East 15th Street, New York 9, New York 
RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta 22, 

Georgia 

READ, DR. CLARK P., Johns Hopkins University, Baltimore, Maryland 
REBHUN, DR. LIONEL I., Department of Biology, Box 704, Princeton University, 

Princeton, New Jersey 
RECHNAGEL, DR. R. O., Department of Physiology, Western Reserve University, 

Cleveland, Ohio 

REDFIELD, DR. ALFRED C., Woods Hole, Massachusetts 
REINER, DR. J. M., V. A. Hospital, Albany, New York 
RENN, DR. CHARLES E., 509 Ames Hall, Johns Hopkins University, Baltimore 18, 

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

New York City, New York 

RICE, DR. E. L., 2241 Seneca Avenue, Alliance, Ohio 
RICHARDS, DR. A., 2950E Mabel Street, Tucson, Arizona 
RICHARDS, DR. A. GLENN, Entomology Department, University of Minnesota, St. 

Paul 1, Minnesota 

RICHARDS, DR. OSCAR W., American Optical Company, Research Center, South- 
bridge, Massachusetts 
ROCKSTEIN, DR. MORRIS, Department of Physiology, New York University College 

of Medicine, New York 16, New York 

ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York 
ROMER, DR. ALFRED S., Harvard University, Museum of Comparative Zoology, 

Cambridge, Massachusetts 
RONKIN, DR. RAPHAEL R., Department of Physiology, University of Delaware, 

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

York City, New York 

ROOT, DR. W. S., Columbia University, College of Physicians and Surgeons, De- 
partment of Physiology, New York City, New York 
ROSE, DR. S. MERYL, Department of Zoology, University of Illinois, Champaign, 

Illinois 
ROSENBERG, DR. EVELYN K., Department of Pathology, New York University, 

Bellevue Medical Center, New York 16, New York 
ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pittsburgh 

Medical School, Pittsburgh 13, Pennsylvania 
Rossi, DR. HAROLD H., Department of Radiology, Columbia University, 630 West 

168th Street, New York 32, New York 
ROTH, DR. JAY S., Department of Biochemistry, Hahnemann Medical College, 

Philadelphia 2, Pennsylvania 



42 MARINE BIOLOGICAL LABORATORY 

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., Department of Zoology, University of Pennsylvania, 

Philadelphia 4, Pennsylvania 
RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole, 

Massachusetts 
SANDEEN, DR. MURIEL L, Department of Zoology, Duke University, Durham, 

North Carolina 

SAUNDERS, MR. LAWRENCE, R. D. 7, Bryn Mawr, Pennsylvania 
SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of Cali- 
fornia, Berkeley 4, California 
SCHARRER, DR. ERNST A., Albert Einstein College of Medicine, 1710 Newport 

Avenue, New York 61, New York 
SCHECHTER, DR. VICTOR, College of the City of New York, New York City, New 

York 
SCHLESINGER, DR. R. WALTER, Department of Microbiology. St. Louis University 

School of Medicine, 1402 South Grand Boulevard, St. Louis 4, Missouri 
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio 
SCHMITT, DR. FRANCIS O., Department of Biology, Massachusetts Institute of 

Technology, Cambridge, Massachusetts 
SCHMITT, DR. O. H., Department of Physics, University of Minnesota, Minneapolis 

14, Minnesota 
SCHNEIDERMAN, DR. HOWARD A., Department of Zoology, Cornell University, 

Ithaca, New York 

SCHOLANDER, DR. P. F., Scripps Institute of Oceanography, La Jolla, California 
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, 

Massachusetts 
SCHRADER, DR. FRANZ, Department of Zoology, Columbia University, New York 

27, New York 
SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia University, 

New York 27, New York 
SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington, 

Indiana 

SCOTT, DR. ALLAN C., Colby College, Waterville, Maine 
SCOTT, DR. D. B. McNAiR, Botany Annex, Cancer Chemotherapy Laboratory, 

University of Pennsylvania, Philadelphia, Pennsylvania 

SCOTT, SISTER FLORENCE MARIE, Seton Hill College, Greensburg, Pennsylvania 
SCOTT, DR. GEORGE T., Department of Zoology, Oberlin College, Oberlin, Ohio 
SEARS, DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massachu- 
setts 

SENFT, DR. ALFRED W., Woods Hole, Massachusetts 
SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and 

Surgeons, New York City, New York 
SHANES, DR. ABRAHAM M., Experimental Biology and Medicine Institute, National 

Institutes of Health, Bethesda 14, Maryland 



REPORT OF THE DIRECTOR 43 

SHAPIRO, DR. HERBERT, 5800 North Camac Street, Philadelphia 41, Pennsylvania 
SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East 

Lansing, Michigan 
SHEDLOVSKY, DR. THEODORE, Rockefeller Institute, 66th Street and York Avenue, 

New York 21, New York 

SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont 
SICHEL, MRS. F. J. M., 35 Henderson Terrace, Burlington, Vermont 
SILVA, DR. PAUL, Department of Botany, University of Illinois, Urbana, Illinois 
SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa 

City, Iowa 
SMITH, DR. DIETRICH C, Department of Physiology, University of Maryland 

School of Medicine, Baltimore, Maryland 
SMITH, MR. HOMER P., General Manager, Marine Biological Laboratory, Woods 

Hole, Massachusetts 

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

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

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

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

Virginia 

SPIEGEL, DR. MELVIN, Department of Biology, Colby College, Waterville, Maine 
SPRATT, DR. NELSON T., JR., Department of Zoology, University of Minnesota, 

Minneapolis 14, Minnesota 
SPYROPOULOS, DR. C. S., Department of Neurophysiology, National Institutes of 

Health, Bethesda 14, Maryland 
STARR, DR. RICHARD C., Department of Botany, Indiana University, Bloomington, 

Indiana 
STEINBACH, DR. H. BURR, Department of Zoology, University of Chicago, Chicago 

15, Illinois 
STEINBERG, DR. MALCOLM S., Department of Biology, Johns Hopkins University, 

Baltimore 18, Maryland 
STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota, 

Minneapolis 14, Minnesota 

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 

Medical School, Baltimore 5, Maryland 
STUNKARD, DR. HORACE W., American Museum of Natural History, New York 

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

California 



44 MARINE BIOLOGICAL LABORATORY 

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

SWOPE, MR. GERARD, JR., 570 Lexington Avenue, New York 22, New York 
SZENT-GYORGYI, DR. ALBERT, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 
SZENT-GYORGYI, DR. ANDREW G., Marine Biological Laboratory, Woods Hole, 

Massachusetts 
TASAKI, DR. ICHIJI, Laboratory of Neurophysiology, National Institute of 

Neurological Diseases and Blindness, Bethesda 14, Maryland 
TASHIRO, DR. SHIRO, University of Cincinnati, Medical College, Cincinnati, Ohio 
TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology, National Institute of 

Neurological Diseases and Blindness, Bethesda 14, Maryland 
TAYLOR, DR. WM. RANDOLPH, Department of Botany, University of Michigan, 

Ann Arbor, Michigan 
TEWINKEL, DR. Lois E., Department of Zoology, Smith College, Northampton, 

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

Illinois 

TRACY, DR. HENRY C, General Delivery, Oxford, Mississippi 
TRACER, DR. WILLIAM, Rockefeller Institute, 66th Street and York Avenue, New 

York 21, New York 
TRINKAUS, DR. J. PHILIP, Osborn Zoological Laboratories, Yale University, New 

Haven, Connecticut 
TROLL, DR. WALTER, Department of Industrial Medicine, New York University 

College of Medicine, New York City, New York 
TWEEDELL, DR. KENYON S., Department of Biology, University of Notre Dame, 

Notre Dame, Indiana 
TYLER, DR. ALBERT, Division of Biology, California Institute of Technology, 

Pasadena 4, California 
UHLENHUTH, DR. EDWARD, University of Maryland School of Medicine, Baltimore, 

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

Illinois 

DEViLLAFRANCA, DR. GEORGE M., Department of Zoology, Smith College, North- 
ampton, Massachusetts 
VILLEE, DR. CLAUDE A., Department of Biological Chemistry, Harvard Medical 

School, Boston 15, Massachusetts 
VINCENT, DR. WALTER S., Department of Anatomy, State University of New 

York School of Medicine, Syracuse 10, New York 
WAINIO, DR. W. W., Bureau of Biological Reserch, Rutgers University. New 

Brunswick, New Jersey 
WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge 38, 

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

of Medicine. New York 16, New York 
WATERMAN, DR. T. H., Osborn Zoological Laboratories, Yale University, New 

Haven. Connecticut 
WEBB, DR. MARGUERITE, Department of Physiology and Bacteriology, Goucher 

College, Towson, Baltimore 4, Maryland 






REPORT OF THE DIRECTOR 45 

WEISS, DR. PAUL A., Laboratory of Developmental Biology, Rockefeller Institute, 

66th Street and York Avenue, New York 21, New York 

WENRICH, DR. D. H., University of Pennsylvania, Philadelphia 4, Pennsylvania 
WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut 
WHITAKER, DR. DOUGLAS M., Rockefeller Institute for Medical Research, 66th 

Street and York Avenue, New York 21, New York 
WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania 
WHITING, DR. ANNA R., University of Pennsylvania, Philadelphia 4, Pennsylvania 
W'HITING, DR. PHINEAS W r ., Zoological Laboratory, University of Pennsylvania, 

Philadelphia 4, Pennsylvania 
WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, 

Pennsylvania 

W T ICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York 
WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts 
WIERCINSKI, DR. FLOYD J., Department of Biological Sciences, Drexel Institute of 

Technology, 32nd and Chestnut Streets, Philadelphia 4, Pennsylvania 
W r iLBER, DR. C. G., Medical Laboratories, Applied Physiology Branch, Army 

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

18, Maryland 
WILSON, DR. J. WALTER, Department of Biology, Brown University, Providence 

12, Rhode Island 
WILSON, DR. WALTER L., Department of Physiology, University of Vermont 

College of Medicine, Burlington, Vermont 
W T ITSCHI, 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, Pendleton Hall, Wellesley College, Wellesley, Massachusetts 
WOODWARD. DR. ARTHUR A., Army Chemical Center, Maryland (Applied Physi- 
ology Branch, Army Chemical Corps, Medical Laboratory) 
WRIGHT, DR. PAUL A., Department of Zoology, University of New Hampshire, 

Durham. New Hampshire 
WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton, 

Massachusetts 
YNTEMA, DR. C. L., Department of Anatomy, State University of New York 

College of Medicine, Syracuse 10, New York 
YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts 
ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings- 
ton, Rhode Island 
ZIRKLE, DR. RAYMOND E., Department of Radiobiology, University of Chicago. 

Chicago 37, Illinois 
ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie, 

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

York City, New York 
ZWILLING, DR. EDGAR, Department of Genetics, University of Connecticut, Storrs. 

Connecticut 



46 



MARINE BIOLOGICAL LABORATORY 



3. ASSOCIATE MEMBERS 



ALDRICH, Miss AMY 

ALTON, DR. AND MRS. BENJAMIN H. 

ARMSTRONG, DR. AND MRS. P. B. 

BACON, MRS. ROBERT 

BAITSELL, MRS. GEORGE 

BALL, MRS. ERIC 

BARBOUR, MR. Lucius H. 

BARTOW, MR. AND MRS. CLARENCE 

BARTOW, MRS. FRANCIS D. 

BARTOW, MR. AND MRS. PHILIP K. 

BELL, MRS. ARTHUR W. 

BRADLEY, MR. AND MRS. ALBERT L. 

BRADLEY, MR. AND MRS. CHARLES 

BROWN, MRS. THORNTON 

BURDICK, DR. C. LALOR 

BURLINGAME, MRS. F. A. 

CAHOON, MRS. SAMUEL, SR. 

CALKINS, MRS. GARY N. 

CALKINS, MRS. G. NATHAN, JR. 

CALKINS, MR. AND MRS. SAMUEL W. 

CARLTON, MR. AND MRS. WINSLOW 

CLAFF, DR. AND MRS. C. LLOYD 

CLARK, DR. AND MRS. ALFRED HULL 

CLARK, MRS. LEROY 

CLARK, MR. AND MRS. W. VAN ALAN 

CLOWES, MR. ALLEN W. 

CLOWES. MRS. G. H. A. 

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

COLTON, MR. AND MRS. H. SEYMOUR 

CRANE, MR. AND MRS. BRUCE 
CRANE, MR. JOHN 
CRANE, Miss LOUISE 
CRANE, MRS. MURRAY 
CRANE, MR. STEPHEN 
CRANE, MRS. W. CAREY 

COWDRY, DR. AND MRS. E. V. 

CROSSLEY, MR. AND MRS. ARCHIBALD M. 
CROWELL, MR. AND MRS. PRINCE S. 
CURTIS, DR. AND MRS. W. D. 
DANIELS, MR. AND MRS. F. HAROLD 
DAY, MR. AND MRS. POMEROY 
DRAPER, MRS. MARY C. 
DREYER, MR. AND MRS. FRANK A. 
ELSMITH, MRS. DOROTHY 
ENDERS, MR. AND MRS. FREDERICK 
EWING, MR. AND MRS. FREDERIC 
FAY, MR. AND MRS. HENRY H. 
FISHER, MR. AND MRS. B. C. 



FRANCIS, MRS. LEWIS H., JR. 
FROST, MRS. FRANK J. 
GALTSOFF, MRS. PAUL S. 

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

GREEN, Miss GLADYS M. 

HAIG, MRS. R. H. 

HAMLEN, MR. AND MRS. J. MONROE 

HARRELL, MR. AND MRS. JOEL E. 

HARRINGTON, MR. AND MRS. ROBERT 

HERRINGTON, MRS. A. W. S. 

HERVEY, DR. AND MRS. JOHN P. 

HlRSCHFELD, MRS. NATHAN B. 

HOUSTON, MR. AND MRS. HOWARD 

JEWETT, MRS. G. F. 

KEITH, MR. AND MRS. HAROLD C. 

KING, MR. AND MRS. FRANKLIN 

KOLLER, MR. AND MRS. LEWIS 

LEMANN, MRS. BENJAMIN 

LOBB, MRS. JOHN 

LOEB, DR. AND MRS. ROBERT F. 

McCuSKER, MR. AND MRS. PAUL J. 

MCKELVY, MR. JOHN E. 

MARSLAND, MRS. DOUGLAS A. 

MARVIN, MRS. WALTER T. 

MAST, MRS. S. O. 

MEIGS, DR. AND MRS. J. WISTER 

MITCHELL, MRS. JAMES McC. 

MIXTER, MRS. W. JASON 

MOSSER, MRS. BENJAMIN D. 

MOTLEY, MRS. THOMAS 

NEWTON, Miss HELEN 

NICHOLS, MRS. GEORGE 

NICHOLSON, REV. ROBERT W. 

NIMS, MRS. E. D. 

NORMAN FUND, INC., AARON E. 

PACKARD, DR. AND MRS. CHARLES 

PARK, MR. AND MRS. M. S. 

PENNINGTON, Miss ANNE H. 

REDFIELD, DR. AND MRS. ALFRED C. 

REZNIKOFF, DR. AND MRS. PAUL 

RIGGS, MR. AND MRS. LAWRASON 

RIVINUS, MRS. F. M., JR. 

ROOT, MRS. WALTER S. 

ROZENDAAL, DR. H. M. 

RUDD, MR. AND MRS. H. W. DWIGHT 

SANDS, Miss ADELAIDE G. 



REPORT OF THE LIBRARIAN 



47 



SAUNDERS, MR. AND MRS. LAWRENCE 
SHIVERICK, MRS. ARTHUR 
SINCLAIR, MR. AND MRS. W. RICHARD- 
SON 

SPEIDEL, DR. AND MRS. CARL 
STOCKARD, MRS. CHARLES R. 
STONE, MR. AND MRS. LEO 
STONE, MR. AND MRS. S. M. 
STRAUS, DR. AND MRS. DONALD B. 
SWIFT, MR. E. KENT 



SWOPE, MR. AND MRS. GERARD, JR. 
SWOPE, Miss HENRIETTA 
TOMPKINS, MR. AND MRS. B. A. 
WEBSTER, MRS. EDWIN S. 
WHITELEY, Miss MABEL W. 

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

WILLISTON, Miss EMILY 
WILSON, MRS. EDMUND B. 

WOLFINSOHN, MRS. WOLFE 



V. REPORT OF THE LIBRARIAN 

In 1958, fifty-eight new journals were acquired, bringing the total number of 
currently received titles to 1654. Of these, there were 494 (11 new) Marine 
Biological Laboratory subscriptions, 621 (18 new) exchanges and 188 (9 new) 
gifts; 95 (5 new) were Woods Hole Oceanographic Institution subscriptions; 195 
(4 new) were exchanges and 61 (11 new) were gifts. 

The Laboratory purchased 145 books, received 84 complimentary copies (5 
from authors and 79 from publishers) and accepted 38 miscellaneous gifts. The 
Institution purchased 75 titles and received 12 gifts. The total number of books 
accessioned totalled 354. Many books in the physical sciences were purchased, 
thus filling a demand that has been apparent for many years. 

Through purchase, exchange and gift the Laboratory completed 8 journal sets 
and partially completed 32. The Institution completed 3 sets and partially com- 
pleted 5. There were 3873 reprints added to the collection of which 1722 were 
of current issue. 

At the close of the year, the Library contained 74,590 bound volumes and 
209,998 reprints. 

The Library sent out on inter-library loan 332 volumes and borrowed 112, a 
decided increase over 1957. Several copying machines were tried out, none of 
which met the necessary requirements. About 970 volumes and 198 pamphlets 
were bound. 

A large reprint collection was presented by Dr. J. Percy Moore of which about 
1000 papers were added to the shelves. A large percentage of the duplicate ma- 
terial was presented to the U. S. Fish and Wildlife Service Library and to the 
Library of the Narragansett Laboratory. Many zoological papers were stored for 
future replacement copies of articles used in the Invertebrate Course. Through 
Dr. Arnold Lazarow a collection was received from the University of Minnesota 
containing hundreds of papers published during the 19th century. 

Smaller pamphlet collections were received from the Dept. of Biological Chem- 
istry, Harvard Medical School ; Dr. Benj. P. Sonnenblick, and the estate of the late 
Dr. Chas. R. Stockard. Mrs. A. R. Memhard presented fourteen books belonging 
to her late husband. Two books were received from Dr. Alfred G. Marshak ; four 
from Dr. Henry Stommel ; two from Dr. Albert Szent-Gyorgyi ; several early 
Reports of the Division of Fish and Game, State of Massachusetts, from Dr. David 
Belding; and early numbers of the "Biological Bulletin" were returned to stock 
by Dr. Carl Cans and by Dr. P. W. Whiting. 



48 MARINE BIOLOGICAL LABORATORY 

The Library extends grateful acknowledgement to all of its friends who have 
so generously made the donations mentioned above. 

During the year, some of the visiting scientists from foreign countries selected 
duplicate material which was shipped to the following institutions : Caribbean 
Marine Biological Institute, Curacao ; Faculty of Fisheries, Hokkaido University, 
Japan ; Chulalongkorn University, Bangkok ; and the New Zealand Oceanographic 
Institution, Wellington. 

The willing assistance given by the members of the Library Committee and 
the Book Committee has made the year a progressive one. 

Respectfully submitted, 
DEBORAH L. HARLOW, 

Librarian 



VI. REPORT OF THE TREASURER 

The market value of both the General Endowment Fund and the fund for the 
Library at December 31, 1958, amounted to $1,719,105 as compared with the total 
of $1,461,278 as of December 31, 1957. The average yield on the Securities was 
3.48% of market value and 5.90% of book value. The total uninvested principal 
cash in the above accounts as of December 31, 1958, was $2,840. 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, 1958, was 
$297,441 with uninvested principal cash of $469; the value at December 31, 1957, 
being $247,629. The book value of the securities in this account was $243,958 on 
December 31, 1958, compared with $236,735 a year earlier. The average yield 
on market value was 3.64% and 4.44% of book value. 

The proportionate interest in the Pool Fund Account of the various Funds as 
of December 31, 1958, is as follows: 

Pension Funds 19.495% 

General Laboratory Investment 56.540 

Other : 

Bio Club Scholarship Fund 1.648 

Rev. Arsenious Boyer Scholarship Fund 2.017 

Gary N. Calkins Fund 1.889 

Allen R. Memhard Fund 366 

F. R. Lillie Memorial Fund 6.366 

Lucretia Crocker Fund 6.892 

E. G. Conklin Fund 1.166 

M. H. Jacobs Scholarship Fund 831 

Jewett Memorial Fund 612 

Anonymous Gift 2.178 

The Jewett Memorial Fund and the Anonymous Gift Fund are included in the 
Pool Fund Account; however, it has not been determined how these funds are to 
be used. 



REPORT OF THE TREASURER 49 

The special Custodian account which was used to activate available funds of 
a temporary nature last year yielded income of $5,957, plus $1,487 interest from 
Savings Bank deposits of a similar nature. This income is being reserved for 
capital improvements. 

The Securities pledged to cover the M.B.L. Club Loan matured in May, 1958. 
These Securities were converted into cash on deposit in a savings account with 
the Falmouth National Bank in the amount of $3,046. The amount of the Loan 
is approximately $2,000. 

Donations from the M.B.L. Associates for 1958 were $3,150 as compared with 
$3,481 for 1957. Unrestricted gifts from foundations, societies and companies 
amounted to $38,579. 

We are administering 21 grants for investigators in addition to those directly 
to the M.B.L. The amounts of the grants vary in accordance with the investi- 
gator's project of research. An amount of 15% based on the amount expended is 
allowed the Laboratory as overhead. 

Major new construction grants awarded during 1958 are as follows : 

New Laboratory Building: 

Rockefeller Institute $ 738,500 

National Institutes of Health 369,250 

National Science Foundation 369,250 



$1,477,000 
Devil's Lane Housing : 

National Science Foundation $ 175,000 

Grass Foundation 10,000 



$ 185,000 

Lybrand, Ross Bros. & Montgomery have examined our books and submitted 
financial statements for examination. 

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

We have examined the balance sheets of Marine Biological Laboratory as at 
December 31, 1958 and 1957, the related statements of operation expenditures and 
income for the years then ended, and statement of current fund for the year ended 
December 31, 1958. Our examination was made in accordance with generally 
accepted auditing standards, and accordingly included such tests of the accounting 
records and such other auditing procedures as we consider necessary in the 
circumstances. 

In our opinion, the accompanying financial statements present fairly the assets, 
liabilities and funds of Marine Biological Laboratory at December 31, 1958 and 
1957, and the expenditures and income for the years then ended. 

LYBRAND, Ross BROS. & MONTGOMERY 
Boston, Massachusetts 
June 3, 1959 

JAMES H. WICKERSHAM, 

Treasurer 



50 MARINE BIOLOGICAL LABORATORY 

MARINE BIOLOGICAL LABORATORY 

BALANCE SHEETS 

December 31, 1958 

Investments 

Investments held by Trustee : 

Securities, at cost (approximate market quotation 1958 $1,719,105; 1957 

$1,461,278) $1,014,460 

Cash 2,840 



1,017,300 



Investments of other endowment and unrestricted funds : 



Pooled investments, at cost (approximate market quotation 1958 $297,441 ; 1957 

-$247,629) 243,958 

Less temporary investment of current fund cash 5,728 



238,230 

Other investments 69,756 

Cash (note A) 13,401 

Accounts receivable . 2,435 



323,822 



Plant Assets 

Land, buildings, library and equipment (note B) 2,970,655 

Less allowance for depreciation (note B) 1,063,577 



1,907,078 

Construction in progress 156,301 

Cash 9,255 

Accounts receivable 11 ,757 

U. S. Government obligations, at cost : 

$720,000 Treasury notes, 3*s, 11/15/59 722,025 



2,806,416 

Current Assets 

Cash 118,480 

U. S. Government obligations, at cost: 

$30,000 Treasury notes, 3H 11/15/59 30,020 

Temporary investment in pooled securities 5,728 

Accounts receivable (U. S. Government, 1958 $32,808 ; 1957 $19,605) 52,379 

Inventories of specimens and Bulletins 71,110 

Prepaid insurance and other 15,383 



$4,440,638 



REPORT OF THE TREASURER 51 

MARINE BIOLOGICAL LABORATORY 

BALANCE SHEETS 

December 31, 1958 

Endowment Funds 
Endowment funds given in trust for benefit of the Marine Biological Laboratory .. $1,017,300 



Endowment funds for awards and scholarships : 

Principal 64,415 

Unexpended income 3,144 



67,559 

Unrestricted funds functioning as endowment 206,378 

Retirement fund 52,759 

Pooled investments accumulated gain or (loss) (2,874) 



323,822 



Plant Liability and Funds 

Funds expended for plant, less retirements 3,103,854 

Less allowance for depreciation charged thereto 1,063,577 



2,040,277 
Unexpended plant funds 743,037 



2,783,314 
Accounts payable 23,102 



2,806,416 



Current Liabilities and Funds 



Accounts payable 33,155 

Unexpended balances of gifts for designated purposes 9,767 

Advance payments on research contracts 109,792 

Current fund . 140,386 



$4,440,638 

Notes : 

A The Laboratory has guaranteed a note of approximately $2,000 of the M.B.L. 
Club and has deposited as security therefor the passbook for a savings account 
deposit of $3,046, included in cash. 

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



52 MARINE BIOLOGICAL LABORATORY 

MARINE BIOLOGICAL LABORATORY 

STATEMENTS OF OPERATING EXPENDITURES AND INCOME 

Year Ended December 31, 1958 



Operating Expenditures 
Direct expenditures of departments : 

Research and accessory services $162,01 1 

Instruction 39,190 

Library, including book purchases 35,270 

Biological Bulletin 22,050 



258,521 

Direct costs on research contracts 134,566 

Administration and general 61,140 

Plant operation and maintenance 93,823 

Dormitories and dining services 146,526 

Plant additions from current funds 14,867 



709,443 
Less depreciation included in plant operation and dormitories and dining services 

above but charged to plant funds 38,884 



670,559 

Income 
Direct income of departments : 

Research fees 47,269 

Accessory services (including sales of biological specimens, 1958 $73,354 .... 116,366 

Instruction fees 16,865 

Library fees and income 9,586 

Biological Bulletin, subscriptions and sales 16,071 



206,157 

Reimbursement and allowance for direct and indirect costs on research contracts 146,394 

Dormitories and dining services income 108,419 



460,970 
Investment income used for current expenses : 

Endowment funds 88,106 

Current fund investments 7,781 

Gifts used for current expenses 105,829 

Sundry income 5 



Total current income 662,691 



Excess of income or (operating expenditures) ($ 7,868) 



MARINE BIOLOGICAL LABORATORY 
STATEMENT OF CURRENT FUND 

Year Ended December 31, 1958 

Balance January 1, 1958 $148,254 

Excess of operating expenditures over income 1958 (7,868) 



Balance December 31, 1958 $140,386 



REPORT OF THE TREASURER 53 

MARINE BIOLOGICAL LABORATORY 
SUMMARY OF INVESTMENTS OF ENDOWMENT FUNDS 

December 31, 1958 

Approximate Investment 

% of Market % of Income 
Cost Total Quotations Total 1958 
Securities held by Trustee : 
General endowment fund : 



TJ S Government bonds 










$ 387 


Other bonds 


$ 499,403 


58.7 


$ 479,575 


33.9 


15,072 














Preferred stocks 


499,403 
85,788 


58.7 
10.1 


479,575 
71,025 


33.9 
5.0 


15,459 
3,370 


Common stocks 


265,979 


31.2 


862,808 


61.1 


30,824 
















851,170 


100.0 


1,413,408 


100.0 


49,653 


General Educational Board endowment ft 
U S Government bonds 


ind: 








312 


Other bonds 


95,004 


58.2 


89,525 


29.3 


2,963 














Preferred stocks 


95,004 
27,281 


58.2 
16.7 


89,525 
24,268 


29.3 
7.9 


3,275 
1,130 


Common stocks 


41,005 


25.1 


191,904 


62.8 


5,839 
















163,290 


100.0 


305,697 


100.0 


10,244 


Total securities held by Trustee 


$1,014,460 




$1,719,105 




$59,897 


Investments of other endowment and unre- 
stricted funds : 
Pooled investments : 
Other bonds 


135,666 


55.6 


137,816 


46.3 


5,201 


Preferred stocks . . . . 


10,531 


4.3 


11,350 


3.8 


73 


Common stocks 


97,761 


40.1 


148,275 


49.9 


5,547 
















243,958 


100.0 


$ 297,441 


100.0 


10,821 


Other investments : 
U S Government bonds 










103 


Common stocks 


43,600 








22,470 


Real estate 


26,156 








167 
















69,756 








22,740 


Total investments of other en- 
dowment and unrestricted 
funds 


$ 313,714 








$33,561 














Total investment income 










93,458 


Custodian's fees charged thereto 










(885) 


Income of current funds temporarily invested 


in pooled 


securities 






(220) 















Investment income distributed to funds $92,353 



SOME OBSERVATIONS ON CHLAMYDOMONAS MICROHALOPHILA 

SP. NOV. 1 

HARRY W. BISCHOFF 

Department of Biology, Texas Lutheran College, Seguin, Texas 

During the summer of 1958 the writer became interested in the profuse blooms 
of a species of Chlamydomonas in some barrels of water stored at the head of the 
Supply Department dock, Marine Biological Laboratory, Woods Hole, Mas- 
sachusetts. 2 Previous morphological and cytological work on various species of 
Chlamydomonas, especially that of Bold (1949), and Buff aloe (1958), the second 
of which summarized discrepancies in chromosome numbers in several species, 
impelled the writer to investigate this cytologically favorable organism. 

MATERIALS AND METHODS 

Samples of water containing the organism were inoculated into sterile soil- 
water tubes (Pringsheim, 1946) and tubes containing an inorganic solution (Bold, 
1949) fortified with 5% supernatant from soil- water medium. 

Twenty clonal cultures were started by introducing single cells in sterile soil- 
water tubes. In the soil-water tubes and inorganic salt medium with soil-water 
supernatant, the growth of the organism was never as good as that observed in 
the natural habitat. Only a very delicate green phototactic ring appeared at the 
surface of the culture solutions. A sample of water from the barrel in which the 
organisms occurred was filtered several times and analyzed for salt content. The 
latter was found to be approximately 0.022 N in NaCl after titrating for the CT ion 
by the Mohr method. Sea water from the coast of Texas taken from the Port 
Aransas area proved to be approximately 0.66 N in NaCl. To obtain the same 
number of moles of NaCl for a culture solution, as present in the original habitat, 
33 ml. of the Gulf sea water were diluted to one liter by adding inorganic medium 
and soil-water supernatant. After considerable experimentation excellent growth 
was obtained in a medium of the following composition : 

inorganic medium (Bold, 1949) 917 ml. 

soil-water supernatant 50 ml. 

Gulf sea water 33 ml. 

This medium supported good growth both in the liquid state and when solidified 
with agar. 

The addition of sea water to the medium, although not essential for growth, 

1 Investigation initiated while the author was Assistant in the Marine Botany Course, 
summer of 1958. The author wishes to acknowledge gratefully the friendly help and suggestions 
given by Dr. Harold C. Bold in the development of this paper. 

2 The same organism was present in these barrels as long ago as 1948 and seems to recur 
every year. 

54 



A NEW SPECIES OF CHLAMYDOMONAS 



55 



proved very stimulatory to the algal cultures. It was also of interest to note that 
six of the more vigorous clones would grow in media with much higher concentra- 
tions of sea water. 

With this evidence that the organism grows best in relatively high concentra- 
tions of salts, as compared with that in the more commonly employed algal culture 
media, a modified Knop's solution was compounded as follows : 



10% Ca(-N(X), 10 ml. 

5% KNO, 5 ml. 

5% MgSO 4 -7H.,O 5 ml. 

5% KH.,PO 4 5 ml. 



Gulf sea water 33 ml. 

(approx. 4.3% salts by weight) 

Sterile rain water 892 ml. 

Soil-water supernatant 50 ml. 



This solution has a salt concentration more than five times that of Bold's (1949) 
inorganic medium. This solution, with the sea water substituted for 33 ml. of 
sterile water, contains approximately 0.32% salts by weight, and was employed as 
the culture medium throughout the remainder of the observations. One clonal 
culture was inoculated into four sets of tubes containing the modified Knop's 



TABLE I 

Growth of Chlamydomonas microhalophila after two weeks in Knop's medium with 5% soil-water 
supernatant and varying concentrations of Gulf sea -water. Approximate salt 

content shown in parentheses 



Concentration of sea water 
and salt content 


Clone H-l 


Clone H-2 


Clone H-3 


Clone H-4 


4.3% (.36%) 


+ +* 


+ + 


+ + 


+ + 


5.3%, (.40' , ) 


+ + 


+ + 


+ + 


+ + 


6.3% (.45%) 


+ 


+ + 


+ + 


+ + 


7.3% (.49%) 


+ + 


+ 


+ + 


+ + 


8.3% (.53%) 


+ 


+ 


+ + 


+ 


9.3% (.57%) 


+ 


+ 


+ 


+ 


10.3% (.62%) 


+ - 


+ - 


+ - 


+ - 


11.3% (.66%) 


+ - 


+ - 


_i 


+ - 


12.3% (.70%) 


+ - 


+ - 


+ - 


+ - 


13.3% (.75%) 


+ - 


+ - 


+ - 


+ - 


14.3% (.79%) 


+ - 


+ - 


+ - 


+ - 


15.3% (.83%) 


+ - 


+ - 


+ - 


+ - 


16.3% (.88', ) 


+ - 


+ - 


+ - 


+ - 


17.3% (.92',! 


+ - 


+ - 


+ - 


+ - 


18.3% (.96%) 


+ - 


+ - 


+ - 


+ - 


19.3%, (1.00%) 


+ - 


+ - 


+ - 


+ - 


20.3% (1.04%) 


+ - 








+ - 


21.3% (1.09%) 


+ - 











22.3% (1.13%) 














23.3% (1.18%) 















* Explanation of the symbols : 

+ + abundant growth (cultures dark-green), 
+ moderate growth, 

H scant growth, 

no growth apparent macroscopically. 



56 



HARRY W. BISCHOFF 




FIGURES 1-14. 



A NEW SPECIES OF CHLAMYDOMONAS 57 

solution with various concentrations of Gulf sea water. Scant growth occurred in 
the solutions containing as much as 1.0% salts (Table I). 

Hanging-drop preparations were used for observing living cells. All cultures 
were kept under constant fluorescent illumination at an intensity of 600 to 800 
foot-candles and at a temperature of 15-17 C. Flagella were observed by staining 
motile cells with fixative described below, modified by increasing its iodine content 
to the point of saturation. 

The following cytological methods were employed. Motile cells were taken 
from the surface of densely-populated stock cultures growing in liquid media and 
spread over the surface of sterile agar media in Petri dishes. These were illuminated 
at an intensity of 800 foot-candles for about six hours. The cultures were then ob- 
served, from time to time, under a stereoscopic binocular microscope for evidences 
of cell division. The maximum number of dividing cells and nuclear division 
figures was obtained at approximately 10 to 12 hours. Cells were fixed to glass 
slides and these were placed in Coplin jars containing fixative, as described by 
Bufifaloe (1958). The writer also used the fixative which Cave and Pocock (1951) 
had modified from Johansen (1940). except that he reduced the iodine from 5 to 
2.5 grams. The stained chromosomes were seen better when the starch granules 
were not as heavily stained, as in the case when a more concentrated iodine fixative 
is used. Slides were allowed to remain in the fixative for approximately three 
hours, and then were drained of excess fluid. The preparations were then flooded 
with aceto-carmine, prepared according to the method of Cave and Pocock (1951), 
and placed upon a hot plate with the thermostat set at 300 F. In a very 
short time vapors arose from the stain. After \%-2 minutes steaming, during 
which the stain turned a deep red color, the slides were removed from the hot 
plate, drained and destained in 45% acetic acid for approximately 10 seconds. 
The slides next were placed in a mixture of equal volumes of 45% acetic acid and 
95 % alcohol for two minutes, and, then, 95 % alcohol for 5 minutes. Finally, after 
the alcohol bath, a drop of Euparal was placed upon the area occupied by the 
fixed cells and covered with a cover glass. 

All figures were drawn with the aid of a Spencer camera lucida, and reduced % in repro- 
duction. The magnifications are : for Figures 1-4 and 15-18, 2000 X ; for Figures 5-9 and 
19-24, 1750 X ; for Figures 10-14. 1500 X. All figures were drawn from living material except 
Figure 12, which was stained with Io-KI solution, and Figures 19-24 which were stained with 
aceto-carmine. 

FIGURES 1-14. Chlninydoinonas microhalophila. FIGURE 1, vegetative cell in median longi- 
tudinal optical section, showing nucleus, pyrenoid, stigma and form of chloroplast. FIGURE 2,, 
vegetative cell in surface view. FIGURE 3, vegetative cell in anterior polar view showing the 
incised apex of the chloroplast and the relationship of the contractile vacuoles to the plane of 
attachment of the flagella. FIGURE 4, vegetative cell in transverse optical section at the level 
of the pyrenoid. FIGURES 5-7, asexual reproduction ; in this case, parental flagella functional 
during division. FIGURE 5, note 90 rotation, protoplast rotated 90 from longitudinal axis of 
cell wall ; cell is still motile due to incomplete withdrawal of flagella. FIGURE 6, first cleavage 
completed. FIGURE 7, second cleavage completed, with the four daughter cells rotated in line 
with the longitudinal axis of mother cell wall. FIGURES 8-18, sexual reproduction. FIGURE 8, 
gametes after initial entanglement of flagella which are now completely separated; gametes 
probably attached by a protoplasmic thread. FIGURE 9, fusion in progress ; flagella still slightly 
motile; note discarded gamete walls. FIGURE 10, pseudoheterogamous pair. FIGURE 11, the 
same, somewhat later. FIGURE 12, zygote stained with I 2 -KI, showing two distinct nuclei and 
pyrenoids 48 hours after plasmogamy. FIGURES 13, 14, dormant zygotes. 



58 HARRY W. BISCHOFF 

OBSERVATIONS 
Morphology and reproduction 

The organism is ellipsoidal. The anterior and posterior poles are broadly 
rounded, but the anterior one is more acuminate than the posterior. Cell size ranges 
from 8.5/x-20yu, in length and 5 /x-12 /A in width with the population averaging 
14 yu, in length and 8.5 /j, in width. Young cells from germinating zygospores may 
be as small as 6 /x in length and 3 /* in width. The variation in cell size is a 
reflection of phases of development after liberation of daughter cells from the 
parent cell walls. The papilla, w r hich is most clearly visible in small, young cells, 
is truncate, and it becomes obscured with increase in cell size and wall thickness 
(Figs. 1 and 2). Two small flagellar orifices may be observed when the protoplast 
contracts from the wall during cell division (Fig. 5). 

The chloroplast in median, optical section is a relatively thick-walled, hollow, 
ovoidal structure open at the anterior end. Here the chloroplast displays an 
irregularly scalloped margin (Figs. 2 and 3). The inner surface of the plastid is 
slightly undulate. The single spherical pyrenoid always lies in a lateral position, 
in a thickening of the chloroplast, in the posterior third of the cell (Fig. 1). The 
elliptical, disc-shaped stigma is embedded in the periphery of the anterior third of 
the chloroplast. The cell wall protrudes slightly at the region of the stigma 
(Fig. 1). The size and form of the stigma were constant in all cells observed. 

The nucleus is anterior in the colorless cytoplasm. Both in living and stained cells 
a large nucleolus is visible, but a centrosome could not be demonstrated even with 
Heidenhain's iron haematoxylin, as reported for C. tcrricola. Two contractile vacu- 
oles occupy the anterior portion of the protoplast and always lie in a plane perpendic- 
ular to that of the attachment of the flagella (Figs. 3 and 25). The latter are con- 
siderably longer than the cell. Flagella nearly twice the cell length are very 
common among the smaller cells. 

The presence of zygotes in the twenty clonal cultures proved the organism is 
homothallic. The gametes are not distinguishable from the vegetative cells, 
except for their smaller size, an indication that, as in most species of Chlamydomonas, 
only young cells are sexually active. The gametes are isogamous ; although fusion 
between gametes of unlike size was observed, this is explained by the fact that they 
are in various stages of maturation (Figs. 10 and 11). 

In sexual union two gametes, with free flagella, repeatedly and vigorously 
approach each other in the region of the papillae as observed by Bold (1949) in 
C. chlamydogama. After one to two hours of this behavior, they become almost 
motionless. During this process the pairs do not move very far from a given 
point, another similarity to C. chlamydogama. A protoplasmic thread, as described 
by Bold (1949) and Lewin and Meinhart (1953), probably unites the two cells at 
this stage, but because of the constant movement of the cells this thread was not 
observed with certainty. The space between the papillae and four free-moving 
flagella is very good evidence that this thread does exist in this species (Fig. 8). 

After two to three hours, a permanent union of the gametes occurs at the region 
of the papillae, as the flagella continue to beat very feebly. The gamete walls are 
shed (Fig. 9) as in C. chlaniydogama. The union of the gamete protoplasts is 
very slow, sometimes covering a period of 5 to 6 hours. The flagella disappear 



A NEW SPECIES OF CHLAMYDOMONAS 

and within 24 hours a zygote is formed. If the gametes are of equal size a 
spherical zygote results (Fig. 13) ; if the gametes are of unequal size, a "pear- 
shaped" zygote is formed, resulting possibly from the denser consistency of the 
larger cell (Fig. 14). A thick wall is secreted around the zygote. Both pyrenoids 
and nuclei are visible in the zygotes for as long as 48 hours (Fig. 12). In some 




FIGURES 15-24. Chlatnydonwnas inicrolialopliila. FIGURE IS, six-month-old zygote in me- 
dian optical section with large oil droplets concentrated at the periphery. FIGURES 16-18, zygote 
germination. FIGURE 19, early prophase of nuclear division showing some of the chromatin 
bodies before condensation into chromosomes, pyrenoid elongating. FIGURE 20, pyrenoid has 
divided and cytokinesis is being initiated in the region of the nucleus ; the latter in late pro- 
phase showing 16 chromosomes. FIGURE 21, anaphase stage with approximately 16 chromo- 
somes moving toward the opposite poles ; the pyrenoid has not divided nor has cytokinesis 
been initiated. FIGURE 22, first cleavage is completed, and nuclei are in prophase stage for 
second division ; pyrenoid in one of the cells is elongating. FIGURE 23, second cleavage, pyre- 
noids divided, and chromosomes at metaphase. FIGURE 24, division of pyrenoids preceding 
second cleavage. 

instances, the pyrenoids were visible after several weeks. The zygote enlarges to 
as much as 22 /j. in diameter as it matures. Dormant zygotes several weeks old 
accumulate droplets of colorless oil in the periphery, with the chlorophyll con- 
centrated in the center (Figs. 15 and 30). With increasing age the oil droplets 
enlarge. Very large, reddish-orange colored oil droplets, as confirmed by Sudan 
III, were observed in dormant zygotes 6 months old. 

To effect germination, zygotes which had dried on agar, under illumination 



60 



HARRY W. BISCHOFF 



of 800 foot-candles for a period of over six months, were flooded with distilled 
water and kept in darkness for three days. After this, a small volume of soil- 
water supernatant was added and the tubes illuminated. The first germination 
occurred within 48 hours. At the end of six days most of the zygotes had liberated 
four small, motile daughter cells approximately 6 p. in length (Figs. 16, 17 and 18). 




jfef 27 




29 






* 
'> 



26 



t 

^ 



28 



30 



FIGURES 25-30. Chlamydomonas microhalophila. Photomicrographs of living cells, except 
Figure 27. FIGURE 25, mature cell just prior to division; note contractile vacuoles. FIGURE 
26, immature cell, median optical section ; note unilateral pyrenoid and chromatophore thick- 
ness. FIGURE 27, cell treated with I 2 -KI, showing flagella. FIGURE 28, rotation of protoplast 
prior to cleavage. FIGURE 29, beginning of second cleavage. FIGURE 30, maturing zygote. 

Cytology 

As in many species of Chlamydomonas, the protoplast rotates 90 within the 
wall prior to cell division. Cytokinesis in this species is initiated by the appearance 
of a furrow in the region of the nucleus, and opposite the position of the pyrenoids 
or dividing pyrenoid ( Fig. 20 ) . The first division occurs perpendicular to the 
longitudinal axis of the cell. This coincides with the description given by Kater 
(1929) for C. nasuta, Akins (1941) for Carteria cnicifcm. Bold (1949) for C. 
chlamydogama, and by Buffaloe (1958) for four species of Chlamydomonas. 

The two daughter cells usually undergo one more division which takes place 
in the same manner (Figs. 23 and 29). Cytokinesis is unilateral in the cytoplasm 
surrounding the nucleus (Figs. 20 and 23). Buffaloe (1958) reported that for 
four species of Chlamydomonas he studied, there was no exact synchrony between 
the division of the nucleus and the division of the pyrenoid. The same is true for 
the present organism, but there is synchrony between division of the pyrenoid and 



A NEW SPFXIES OF CHLAMYDOMONAS 61 

cytokinesis. It was observed many times that cytokinesis is initiated while the 
nucleus is still in the prophase stage (Fig. 20). The division of the pyrenoid 
appears always to signal the inception of cytokinesis. The division of the pyrenoid 
follows its elongation (Figs. 19 and 24). Nuclear division may begin before or 
after the initiation of cytokinesis (Figs. 20 and 21). 

Each dividing mother cell usually gives rise to two or four daughter cells 
(Figs. 7, 22 and 23). Sometimes 8 and in exceptional cases 16 and even 32 cells 
were observed. Cell division generally takes place in non-motile cells, the flagella 
of which have been withdrawn. Occasionally, the mother cell does not become 
entirely non-motile, and following one or two successive bipartitions, two or four 
daughter cells propelled by the original flagella may be observed swimming about 
slowly and in cumbersome fashion (Figs. 5, 6 and 7). 

The interphase nucleus is approximately 4//, in diameter and possesses one large 
nucleolus. During early prophase 30 or more irregularly shaped, darkly-stained 
bodies may be observed, scattered about the nucleolus (Fig. 19). In later pro- 
phases, the nucleolus disappears and the darkly-stained chromosomes number ap- 
proximately 16 (Fig. 22). These 16 spherical and slightly oblong bodies were 
never observed to form a ring in the metaphase as reported by Buff aloe (1958) 
for C. reinhardti. The polar view of the metaphase stage appears rather as a 
solid disc consisting of irregularly scattered chromosomes. Fewer than 16 1 
chromosomes were never observed. Early anaphase stages with spindle fibers 
clearly visible also exhibited two sets of approximately 16 chromosomes moving 
toward opposite poles (Fig. 21). 

DISCUSSION 

On the basis of the morphological and cytological data reported in this paper, 
the writer attempted to ascertain the specific identity of the Chlamydomonas studied 
by consulting the literature. The organism is somewhat suggestive of C. tcrricola 
Gerloff (1940) but differs from it clearly in a number of respects such as position 
of the stigma, nuclear position, chromosome number, behavior of gamete walls at 
copulation and nature of the zygote wall, among others. It differs from C. inter- 
media Klebs in the anterior position of the nucleus and posterior position of the 
pyrenoid. Further search of the literature has failed to reveal an organism with a 
combination of attributes like those of the organism studied in the investigation 
here reported. Therefore, it is described as a new taxon, C. microhalophila sp. 
nov., the specific name an allusion to its tolerance of a relatively high concentration 
of salt, as compared to other species. The specific diagnosis follows : 

Chlamydomonas microhalophila 3 

Cellulae ellipsoideae, ad polum anteriorem paululum attenuatae ; magnitude 
cellularum, secundum aetatem, 8. 5-20 ^ long, atque 5-12 //. lat. Chromatophorus 
cavus urceolatus, paululum infra polum anteriorem abrupte terminatus atque 
incisus, incrassatione unilateral} prope basim, pyrenoideum prominens continente, 
praeditus. Stigma anterius protuberans ; nucleus anterior. Duae vacuolae 
pulsantes atque duo flagella longitudine corpori aequa aut longiora. Numerus 

:! The writer is grateful to Dr. Hannah T. Croasdale for preparing the Latin diagnosis. 



62 HARRY W. BISCHOFF 

chromatosomatum (n)=16. Planta homothallica, in reproductione sexuali 
isogamica, membranis gametarum tempore coniunctionis sexualis, zygotum ef- 
ferentis, omnino adiectis. Zygota matura usque ad 22 /JL cliam., membranan levem 
habentia, in quattuor cellulas filias plerumque germinantia. 

Origo : In doliis magnis aquae plenis in loco Supply Department clock, M.B.L., 
Woods Hole, Mass, dicto. 

SUMMARY 

1. Morpbological and cytological observations of a microhalophilic alga are 
described and illustrated. 

2. Clonal cultures isolated from a barrel of water at the Marine Biological Lab- 
oratory, Woods Hole, Massachusetts, proved to be members of an undescribed 
species of Chlamydoinonas. 

3. The organism is described as C. microhalophila sp. nov., a member of the 
Chlamydella section of the genus. 

4. The organism tolerates concentrations of salts (predominantly NaCl) up 
to approximately l.Q ( /c, and responds by marked increase in growth in concentra- 
tions up to approximately 0.5%. 

5. Its homothallic sexual reproduction and zygote germination are described 
and figured. 

6. Its chromosome number has been determined as n == 16 1. 

7. Cultures of the organism have been deposited in the Culture Collection of 
Algae, Department of Botany, Indiana University, and herbarium specimens have 
been sent to the Chicago Natural History Museum. 

LITERATURE CITED 

AKINS, V., 1941. A cytological study of Cartcria crucifcra. Bull. Torrc\ Bot. Club, 68: 

429-445. 
BOLD, H. C., 1949. The morphology of Chlamydomonas chlamydogama, sp. nov. Bull. Torres 

Bot. Club, 76: 101-108. 
BUFFALOE, X. D., 1958. A comparative cytological study of four species of Chlamydomonas. 

Bull. Torrey Bot. Club, 85: 157-178. 
CAVE, M. S., AND M. A. POCOCK, 1951. The aceto-carmine technic applied to the colonial 

Volvocales. Stain Tech., 26: 173-174. 

GERLOFF, J., 1940. Beitrage zur Kenntnis der Variabilitat und Systematik der Gattung Chlamy- 
domonas. Arch. f. Protistcnk., 94: 347-352. 

JOHANSEN, D. A., 1940. Plant Microtechnique. McGraw-Hill Book Co., Inc., New York. 
KATER, J. Me., 1929. Morphology and division of Chlamydomonas with reference to the 

phylogeny of the flagellate neuromotor system. Univ. Calif. Publ. Zoo/., 33: 125-168. 
LEWIN, R. A., AND J. O. MEINHART, 1953. Studies on the flagella of algae III. Electron 

micrographs of Chlamydomonas innci^iisii. Canad. J. Bot., 31 : 711-717. 
PRINGSHEIM, E. G., 1946. Pure Cultures of Algae, Their Preparation and Maintenance. 

Cambridge Univ. Press. 



THE PRESENCE OF FERTILIZIN HAPTENS WITHIN THE 
UNFERTILIZED SEA URCHIN EGG 1 

JOHN W. BROOKBANK 

Department of Biology, University of Florida, Gainesville, Florida, and The Friday Harbor 
Laboratories, University of Washington,- Friday Harbor, Washington 

Tyler and Brookbank (1956a) have shown that rabbit antisera against purified 
fertilizin are capable of reacting with the hyaline layer of fertilized eggs, and of 
inhibiting the mitotic division of these eggs. This indicates a similarity between 
the hyaline layer combining groups or haptens and fertilizin haptens. Absorption 
of anti-fertilizin sera with sperm or coelomic fluid ("blood") does not remove the 
reaction of anti-fertilizin sera with fertilizin or hyaline layer material (Tyler and 
Brookbank, 1956b), indicating that species antigens are not involved. In addition, 
antisera against extracts of jelly-free unfertilized and fertilized eggs also possess 
properties of antisera against fertilizin (Tyler and Brookbank, 1956a). The 
possibility therefore exists that fertilizin haptens may be present within the eggs. 
The present report is concerned with the presence of fertilizin haptens within 
a granular fraction of the unfertilized egg. 

MATERIALS AND METHODS 

Fertilizins of Strongylocentrotus purpuratus (Friday Harbor, Washington) 
and Lyt echinus variegatus (Sea Horse Key, Florida) were prepared from acid- 
(pH 3.5) treated unfertilized eggs by the method of Tyler (1949). After a pre- 
injection control bleeding, rabbits were injected on alternate days, over a three- 
week period, with ca. 50 /ug of fertilizin. Two intravenous injections alternated 
with a single intraperitoneal injection. The sera were recovered 4-5 days after the 
final injection, and thoroughly dialyzed against sea water. Reaction of the sera 
with fertilized eggs (cleavage block), fertilizin (ring precipitin test), and sperm 
(agglutination) was recorded. 

Preparations of adenosine-triphosphatase-bearing granules (ATPase-granules) 
were made according to a method devised by Whiteley (unpublished data). Un- 
fertilized eggs were deprived of soluble fertilizin by acid (pH 3.5) treatment, and 
thoroughly washed. Since acid-treated eggs are fertilizable, and are agglutinated 
by solutions of antifertilizin, it is apparent that some fertilizin remains on the sur- 
face after acid treatment. In order to reduce possible contamination of the 
ATPase-granules with this remaining fertilizin, the eggs were treated for 20 

1 This investigation was supported in part by a research grant (RG 4659) from the National 
Institutes of Health of the Public Health Service. 

2 The author wishes to express his gratitude to the Friday Harbor Laboratories of the 
University of Washington for the use of space and equipment during the summer of 1957, to 
Professor A. H. Whiteley, of the University of Washington, for permission to include some of 
his unpublished results in this report, and to Dr. Ruth Cooper, for a critical reading of the 
original manuscript. 

63 



64 



JOHN W. BROOKBANK 



minutes with 10 mg.% trypsin in sea water (crystalline, lyophillized trypsin, 
Worthington Biochemical Company, Freehold, New Jersey) prior to homogeniza- 
tion. Eggs so treated were found to have reduced (no greater than 25% cleavage 
in 0.25% sperm suspension) fertilizability as compared with controls (ca. 90% 
cleavage in 0.25% sperm suspension), and also a reduced capacity to absorb anti- 
bodies against fertilizin (Table I). In order to minimize the amount of fertilizin 
present on the surface, the eggs were, therefore, routinely trypsinized in this manner 
prior to homogenization. One ml. of the washed, settled eggs was homogenized 
with 9 ml. of cold KCl-citrate solution (1 part .35 M Na citrate: 9 parts .55 M 
KC1, pH 6.8). Following two low speed centrifugations to remove unbroken eggs 
etc., the ATPase-granules were recovered, as a yellow pellet, by two successive 
centrifugations of 15 minutes duration (10 C.) at 10,000 X gravity. The re- 
suspended particles were spherical, of uniform size (ca. 2 microns in diameter) 
and readily distinguishable from the larger yolk granules which remain, for the most 
part, in the supernatant. Such small-granule preparations have 85% of the total 
ATPase activity of the whole egg homogenate (Whiteley, unpublished data). Prep- 

TABLE I 

The effect of trypsin treatment on the capacity of unfertilized eggs 
to absorb antibodies against fertilizin 



Serum 

#10 (pre-injection serum) 

10' (antiserum) unabsorbed 

10' trypsinized egg absorbed 

10' egg absorbed (no trypsin) 



Ring with 
fertilizin 



2nd absorption + 
2nd absorption 



arations of this sort, with no more than an estimated 10% contamination by yolk 
granules, were used as absorbing antigens (preparations which contained little or 
no discernible yolk were also successfully utilized). Prior to use in absorptions, 
the yellow pellets were rinsed with sea water to remove the KCl-citrate mixture. 

Sperm which were used as absorbing antigens were centrifuged and washed 
three times to remove seminal fluid. "Blood" was obtained from KCl-injected 
adult animals through a puncture in the peristomial membrane, and examined for 
contamination with eggs or sperm. This material was then allowed to clot at room 
temperature. The clot was recovered by centrifugation, washed with sea water, 
and used as an absorbing antigen. Jelly-free unfertilized eggs, deprived of the 
soluble portion of their fertilizin coat by acid treatment (pH 3.5), were also used 
for absorptions. For each experiment, approximately equal volumes of serum and 
absorbing material were mixed for 5 minutes at room temperature, and then 
centrifuged to recover the serum. 

Clear supernatants of homogenized blood clots and homogenized ATPase- 
granule supernatant were used as test antigens in ring precipitin tests, as were 
fertilizin solutions. Visible precipitin reactions occurring within 2-5 minutes were 
scored as + + + , while those appearing after 30 minutes are indicated as +. Re- 
actions appearing after 90 minutes are indicated by . Tests were carried out 
with undiluted sera in tubes of ca. 1.5 mm. internal diameter. 

Tests for cleavage blocking activity involved mixing one drop of egg suspension 
(about 100 eggs) and one drop undiluted serum (sea water dialyzed). The sera 



HAPTENS OF UNFERTILIZED EGGS 65 

were scored as blocking ( + + + ) if no more than one cell division of the membrane- 
less fertilized eggs occurred following the addition of the serum. Retardations of 
development without inhibition of cleavage are indicated by a plus-minus sign (). 

RESULTS AND DISCUSSION 

The results of experiments with the two species of sea urchin are summarized 
in Table II. From this table it can be concluded that absorption with eggs or 
ATPase-granules removes most of the cleavage-blocking activity of the immune 
sera, as well as the majority of the fertilizin precipitins ; sperm absorption is not 
effective, nor is absorption with blood. Absorption with blood removes virtually 
all antibodies against this material, though sperm absorption leaves antibodies, 
capable of precipitating soluble blood antigens, in solution. Since the unabsorbed 
Lytechinus antisera against fertilizin do not react with sperm, it is reasonable that 
sperm absorption should fail to abolish any reactions exhibited by these sera. The 
reaction of L. variegatus anti-fertilizin sera with ATPase-granule supernatant may 
be due to the presence of unsedimented ATPase-granules in this material, or to 
the presence of fertilizin haptens associated with some other cytoplasmic fraction. 

Concerning contamination of the ATPase-granules with surface fertilizin, all 
that can be said, at present, is that attempts have been made to hold this to a 
minimum. As can be seen in Table I, the trypsinized eggs show only a reduction 
in ability to absorb antibodies against fertilizin, indicating that some fertilizin 
remains after this treatment (further trypsin treatment tends to make the eggs 
excessively fragile, and therefore difficult to handle). Quantitative studies on the 
amount of granular material necessary to absorb a given volume of antiserum are 
necessary before the effectiveness of trypsin treatment in reducing possible con- 
tamination of the ATPase-granules can be evaluated. On the other hand, the 
acid-treated eggs were washed three times with sea water, and, following trypsin 
treatment, an additional five times with sea water and three times with the 
homogenization medium. It is therefore unlikely that any soluble fertilizin is 
available, for adsorption to internal constituents, at the onset of homogenization. 
Small particles of fertilizin, in an insoluble complex of some sort, may be present 
and able to combine with ATPase-granules or other fractions. 

These results are of value in interpreting the similarity of action of antisera 
against purified fertilizin and antisera against extracts of "jelly-free" unfertilized and 
fertilized eggs. Both types of antisera block cleavage, precipitate fertilizin, and 
may fail to agglutinate sperm (Tyler and Brookbank, 1956a, 1956b). In addition, 
antisera against Lytechinus fertilizin, and against "jelly-free" unfertilized and 
fertilized Lytechinus eggs, increase the respiration rate of unfertilized and fertilized 
eggs (Tyler and Brookbank 1956b ; Brookbank, 1959). 

The presence of fertilizin haptens within the eggs may be of importance in 
the chemical "architecture" of the eggs. Tyler (1940) discovered that unfertilized 
sea urchin eggs contain a substance complementary to the surface fertilizin (termed 
antifertilizin from eggs). This discovery and other investigations (Tyler, 1946) 
led Tyler (1947) to propose an auto-antibody concept of cell structure and cell 
adhesion, involving a system of interlocking, mutually complementary substances 
extending from sites of synthesis to the external boundary of the cell. The 
demonstration of fertilizin haptens within the eggs would be consistent with this 



66 



JOHN W. BROOKBANK 

TABLE II 

Ring tests, sperm agglutination, and cleavage inhibition 
tests on antisera against fertilizin 





Ring with 
fertilizin 


Agglutina- 
tion of 
sperm 


Cleavage 
block 


Ring with 
"blood" 


Ring with 
ATPase 
granule 
supernatant 


S. purpuratus 












Pre-injection sera 












d. unabsorbed 


o 


O 


O 






d. sperm absorbed 


o 


O 


O 






d. ATPase-granule absorbed 


o 


o 


O 






e. unabsorbed 





o 









e. sperm absorbed 


db 


o 









f. unabsorbed 





o 


O 






f. sperm absorbed 





o 









Immune sera 












d. unabsorbed 


+ + + 


+ 


+ + + 






d. sperm absorbed 


+++ 





+ + + 






d. ATPase-granule absorbed 





+ 









e. unabsorbed 


+++ 





+ + + 






e. sperm absorbed 


+++ 





+ + + 






f. unabsorbed 


+++ 





+ + + 






f. sperm absorbed 


+ + + 





+ + + 






L. variegatus 












Pre-injection sera 












#4. unabsorbed 





o 


O 








#4. sperm absorbed 





o 


o 








#4. blood absorbed 


o 


o 











#4. unfert. egg absorbed 








o 








#4. ATPase-granule absorbed 


o 





o 





o 


#10. unabsorbed 


db 


o 


o 








#10. sperm absorbed 





o 


o 


o 





# 10. unfert. egg absorbed 


o 














# 10. ATPase-granule absorbed 

















Immune sera 












4. unabsorbed 


++ + 


o 


++ + 


+ 


+ 


4. sperm absorbed 


+++ 


o 


+ + + 


+ 





4. blood absorbed 


+++ 


o 


+++ 








4. unfert. egg absorbed 








o 





db 


4. ATPase-granule absorbed 








o 





o 


10. unabsorbed 


+ + + 


o 


++ + 


+ 





10. sperm absorbed 


+++ 


o 


+ + + 


+ 





10. unfert. egg absorbed 

















10. ATPase-granule absorbed 


+ 











1 



- indicates test not performed. 

theory. However, the presence of fertilizin haptens, as demonstrated by serological 
techniques, does not, of course, necessarily imply that the substances bearing these 
haptens are able to combine specifically with antifertilizin of eggs of sperm. Or, 
to rephrase the foregoing sentence, there is no reason to assume that the rabbit 



HAPTENS OF UNFERTILIZED EGGS 67 

antibodies against fertilizin are directed, exclusively or in part, against the fertilizin- 
antifertilizin combining sites. 

SUMMARY 

1 . ATPase-bearing granules of unfertilized sea urchin eggs were shown, through 
absorptions of antisera against fertilizin, to possess fertilizin-like combining groups. 

2. These granules were also capable of neutralizing the cleavage-blocking action 
of these antisera. 

3. These results are discussed in light of the similarity of action of antisera 
against purified fertilizin to the action of antisera against extracts of jelly-free (acid 
treated) unfertilized eggs, and washed, demembranated fertilized eggs. 

LITERATURE CITED 

BROOKBANK, J. W., 1959. The respiration of unfertilized sea urchin eggs in the presence of 

antisera against fertilizin. Biol. Bull., 116: 217-225. 
TYLER, A., 1940. Agglutination of sea urchin eggs by means of a substance extracted from the 

eggs. Proc. Nat. Acad. Sci., 26: 249-256. 
TYLER, A., 1946. On natural auto-antibodies as evidenced by antivenin in serum and liver 

extract of the Gila monster. Proc. Nat. Acad. Sci., 32 : 195-201. 
TYLER, A., 1947. An auto-antibody concept of cell structure, growth and differentiation. 

Growth, 10 (Suppl.) : 7-19. 
TYLER, A., 1949. Properties of fertilizin and related substances of eggs and sperm of marine 

animals. Amer. Natnr., 83: 195-219. 
TYLER, A., AND J. W. BROOKBANK, 1956a. Antisera that block cell division in developing sea 

urchin eggs. Proc. Nat. Acad. Sci., 42: 304-308. 
TYLER, A., AND J. W. BROOKBANK, 1956b. Inhibition of division and development of sea 

urchin eggs by antisera against fertilizin. Proc. Nat. Acad. Sci., 42: 308-313. 



X-RAY EFFECTS ON MITOTIC ACTIVITY OF THE ACCESSORY 

SEX ORGANS OF CASTRATE RATS STIMULATED BY 

TESTOSTERONE PROPIONATE 1 

JOHN H. D. BRYAN AND JOHN W. GOWEN 

Department of Genetics, Iowa State College, Ames, Iowa 

The effects of irradiation of the mouse testis with 320 r of x-rays have been 
discussed in a previous paper (Bryan and Gowen, 1956). The testis is character- 
ized by a relatively high level of mitosis and in this respect is quite different from 
most other organs. Our findings indicate that irradiation markedly inhibited 
mitotic activity in spermatogonia. In addition data were obtained which suggested 
that irradiation-induced inhibition of desoxyribose nucleic acid (DNA) synthesis 
contributed to this suppression of mitotic activity. Observations on other tissue 
cells would broaden the significance of these findings both to the normal mitotic 
behavior of these cells and to the effects of radiation on them. 

The mitotic behavior of the accessory organs of the castrate rat, the seminal 
vesicle and the dorsal prostate, is of significance to this problem. These organs 
may have high mitotic rates. They have the further advantage that the rates may 
be controlled through castration which reduces mitotic activity through removal of 
hormonal stimulation and/or by testosterone injections which enhance the mitotic 
activity (Moore et al., 1930; Burrows, 1940; Cavazos and Melampy, 1954; 
Melampy et al., 1956 and others). 

The response of these tissues to irradiation and/or hormone treatments as 
described herein was measured by mitotic counts coupled with cytophotometric 
measurements of the DNA-Feulgen content of interphase nuclei. This approach 
has the advantage that the methods complement one another. Together they 
provide information with respect to the relations between the visual manifestations 
of mitotic activity and certain underlying biochemical activities. 

MATERIALS AND METHODS 

Male rats of Fischer line 344 were used in the present experiments. The 
animals were castrated when nine weeks old and the accessory organs allowed to 
regress for twenty days. All the experimental animals were then given daily 
subcutaneous injections of testosterone propionate (500 jug) in oil. 2 Half the 
animals were anesthetized and exposed to 320 r of x-rays (130 pkv; 10 ma; filtra- 
tion 0.25 mm. Al; anode-target distance 20 cm. in air; dose rate 320 r/min.). 
The irradiation was delivered to the pelvic region only, the rest of the body being 
shielded with lead. The irradiation was given coincident with the initial hormone 
injection. 

1 Journal Paper No. J-3512 of the Iowa Agricultural and Home Economics Experiment 
Station, Ames, Iowa. Project No. 1187. This work has received assistance from Contract 
No. AT (11-1) 107 from the Atomic Energy Commission. 

2 Peranderan, Ciba Pharmaceutical Products, Inc., Summit, N. J. 

68 



X-RAY EFFECTS ON CASTRATE RATS 



69 



Pairs of animals were killed at 24, 48, 60 and 72 hours following irradiation 
and/or initial hormone injection. The seminal vesicles and dorsal prostate were 
rapidly removed, cut into small pieces, blotted to remove any secretion and dropped 
into the fixative. Tissues were fixed overnight in 10% neutral formalin, washed 
for 24 hours in running water and then divided into two portions. One was de- 

TABLE I 

The effects of irradiation and of androgen injection on the mitotic activity of 
the secretory epithelium of the accessory organs of castrate male rats 





Seminal vesicle 


Dorsal prostate 


Treatment 


No. cells 
counted 


% 

Mitosis 


Mitosis 
as % of 
control 


No. cells 
counted 


c/ 
/o 
Mitosis 


Mitosis 
as % of 
control 


Intact controls 


7,697 


0.25 





7,733 


0.52 





20 day castrates 


8,753 


0.10 


40 


8,861 


0.06 


11.5 


20 day castrates : 














(a) 24 hrs. after 1st hormone in- 














jection 


9,280 


0.38 


152 


7,326 


0.38 


73.1 


24 hrs. after 1st hormone injection 














and x-rays 


7,637 


0.41 


164 


6,796 


0.43 


82.7 


(b) 48 hrs. after 1st hormone in- 














jection 


7,575 


1.49 


596 


8.171 


0.71 


136.5 


48 hrs. after 1st hormone injection 














and x-rays 


13,375 


1.30 


520 


8,752 


0.63 


121.1 


(c) 60 hrs. after 1st hormone in- 














jection 


7,162 


2.76 


1,104 


11,901 


2.05 


394.2 


60 hrs. after 1st hormone injection 














and x-rays 


7,832 


3.12 


1,248 


7,570 


0.99 


190.4 


(d) 72 hrs. after 1st hormone in- 














jection 


9,901 


3.53 


1,412 


11,742 


1.97 


378.8 


72 hrs. after 1st hormone injection 














and x-rays 


7,254 


2.25 


900 


7,603 


1.82 


350.0 



hydrated, cleared in benzene and embedded in 56-58 C. Tissuemat ; the other was 
taken up to 70% alcohol and stored in the refrigerator. Kidney tissue from control 
rats also was fixed and processed in the above manner. 

The embedded material was sectioned at 6 /x, and the slides therefrom were used 
for mitotic index determinations. The material stored in alcohol was used to pre- 
pare isolated nuclei for photometric purposes since examination of sectioned material 
indicated that most nuclei were badly overlapped. 

Small pieces of tissue were run down to water and hydrolyzed in 1 N HC1 for 
12 minutes at 60 C., washed, and stained in toto by means of the Feulgen reaction 
for two hours at room temperature (Stowell, 1945). After passing through the 
bleach baths the tissue pieces were washed in distilled water, passed into 45% 
acetic acid and left for 10 minutes. Small fragments were then removed into 



70 



JOHN H. D. BRYAN AND JOHN W. GOWEN 



TABLE II 

An analysis of the variation in the mitotic index data 







Between organs or rats 


Among counts 


Binomial error 


Treatment 














M.S. 


df 


FI 


M.S. 


df 


F 




df 




Rat A S.V. & D.P. 


.000 


1 




.004 


2 




.003 


6903 




Rat B S.V. & D.P. 


.042 


1 


8.9** 


.002 


4 




.005 


8458 


Control 


Rats A & B S.V. 


.000 


1 




.003 


4 




.002 


7673 




Rats A & B D.P. 


.029 


1 




.003 


3 




.005 


7688 




Rat A S.V. & D.P. 


.001 


1 




.000 


1 




.001 


7503 


20-day castrate 


Rat B S.V. & D.P. 
Rats A & B S.V. 


.000 
.000 


1 

1 




.000 
.000 


2 

2 




.001 
.001 


10,090 
8740 




Rats A & B D.P. 


.000 


1 




.000 


1 




.001 


8853 




Rat A S.V. & D.P. 


.001 


1 




.001 


5 




.004 


8851 


24-hr, hormone 


Rat B S.V. & D.P. 
Rats A & B S.V. 


.001 
.000 


1 
1 




.004 
.003 


4 
5 




.003 
.004 


7707 
9238 




Rats A & B D.P. 


.003 


1 




.002 


4 




.004 


7320 




Rat A S.V. & D.P. 


.000 


1 




.002 


3 




.004 


7121 


24-hr, hormone 


Rat B S.V. & D.P. 


.001 


1 




.001 


3 




.004 


7242 


+ x-rays 


Rats A & B S.V. 


.001 


1 




.001 


4 




.004 


7600 




Rats A & B D.P. 


.000 


1 




.003 


2 




.004 


6963 




Rat A S.V. & D.P. 


.040 


1 


4.4* 


.009 


3 




.009 


7054 


48-hr, hormone 


Rat B S.V. & D. P. 
Rats A & B S.V. 


.281 
.118 


1 
1 


23.4** 
7.9** 


.003 
.006 


4 

4 




.012 
.015 


8510 
7450 




Rats A & B D.P. 


.002 


1 




.006 


3 




.007 


8108 




Rat A S.V. & D.P. 


.005 


1 




.008 


7 




.007 


11,591 


48-hr, hormone 


Rat B S.V. & D.P. 


.348 


1 


24.8** 


.016 


6 




.014 


10,290 


+ x-rays 


Rats A & B S.V. 


.404 


1 


30.3** 


.014 


8 




.013 


13,191 




Rats A & B D.P. 


.000 


1 




.008 


5 




.006 


8690 




Rat A S.V. & D.P. 


.010 


1 




.097 


4 


4.0** 


.024 


9304 


60-hr, hormone 


Rat B S.V. & D.P. 
Rats A & B S.V. 


.358 
.044 


1 
1 


15.5** 


.054 
.093 


5 
3 


2.4* 
3.4* 


.023 
.026 


9304 
6959 




Rats A & B D.P. 


.084 


1 


4.2* 


.063 


6 


3.1** 


.020 


11,649 




Rat A S.V. & D.P. 


1.191 


1 


56.7** 


.025 


4 




.021 


7617 


60-hr, hormone 


Rat B S.V. & D.P. 


.689 


1 


34.4** 


.009 


4 




.020 


7454 


+ x-rays 


Rats A & B S.V. 


.027 


1 




.029 


4 




.031 


7582 




Rats A & B D.P. 


.007 


1 




.005 


4 




.010 


7489 




Rat A S.V. & D.P. 


1.190 


1 


37.0** 


.064 


6 


2.0 


.032 


11,593 


72-hr, hormone 


Rat B S.V. & D.P. 
Rats A & B S.V. 


.185 

.744 


1 
1 


9.4** 
21.2** 


.009 
.074 


5 
5 


2.1 


.020 
.035 


9454 
9544 




Rats A & B D.P. 


.136 


1 


6.8* 


.010 


6 




.020 


11,503 




Rat A S.V. & D.P. 


.072 


1 


3.5 


.061 


4 


3.0* 


.021 


7246 


72-hr, hormone 


Rat B S.V. & D.P. 


.011 


1 




.081 


4 


4.0** 


.020 


7298 


+ x-rays 


Rats A & B S.V. 


0.012 


1 




.061 


4 


2.7 


.022 


7085 




Rats A & B D.P. 


.003 


1 




.081 


4 


4.5** 


.018 


7459 



1 F values marked * shows significance at the 0.05 level and ** at the 0.01 level. 



X-RAY EFFECTS ON CASTRATE RATS 



71 



drops of 45% acetic acid on microscope slides, covered with a coverslip and 
macerated with the aid of a Burgess Vibro-graver equipped with a plastic tip. By 
this means epithelial nuclei were isolated in a manner suitable for photometric 
purposes. 

TABLE III 

DNA-Feulgen content of seminal vesicle and dorsal prostate nuclei 





Seminal vesicle 


Dorsal prostate 




Class 


N 


DNA-Feulgen 
content 


Class 


N 


DNA-Feulgen 
content 


Intact controls 


II 


50 


3.58 0.07 


II 


49 


3.26 0.06 










Ila 


1 


4.41 




20-day castrates 


II 


50 


3.13 0.06 


II 


49 


3.11 0.07 










III 


1 


6.36 


20-day castrates : 


II 


50 


2.92 0.06 


II 


48 


2.35 0.08 


(a) 24 hrs. after 1st hormone in- 








Ila 


1 


3.51 


jection 








III 


1 


4.27 


24 hrs. after 1st hromone injec- 


II 


49 


2.58 0.07 


II 


50 


2.49 0.06 


tion and x-rays 


Ha 


1 


4.60 








(b) 48 hrs. after 1st hormone in- 


II 


25 


3.05 0.10 


II 


40 


2.74 0.07 


jection 


Ha 


7 


4.35 


Ila 


2 


4.13 




III 


18 


5.73 0.12 


III 


8 


5.71 


48 hrs. after 1st hormone injec- 


II 


38 


3.14 0.08 


II 


43 


2.74 0.08 


tion and x-rays 


Ha 


5 


4.80 


Ila 


3 


4.29 




III 


7 


6.05 


III 


4 


5.35 


(c) 60 hrs. after 1st hormone in- 


II 


25 


2.61 0.11 


II 


37 


1.63 0.06 


jection 


Ha 


9 


3.83 


Ila 


5 


2.65 




III 


15 


4.96 0.27 


III 


8 


3.19 


60 hrs. after 1st hormone injec- 


II 


27 


2.61 0.11 


II 


48 


2.10 0.08 


tion and x-rays 


Ila 


6 


4.18 










III 


17 


4.74 0.11 


III 


2 


3.87 


(d) 72 hrs. after 1st hormone in- 


II 


39 


3.11 0.08 


II 


50 


2.39 0.06 


jection 


Ila 


2 


4.42 










III 


9 


5.88 








72 hrs. after 1st hormone injec- 


II 


26 


2.74 0.11 


II 


49 


2.24 0.04 


tion and x-rays 


Ila 


14 


4.19 0.10 


Ila 


1 


3.67 




III 


10 


5.73 0.46 









The slides were frozen on a block of dry ice and the coverslips removed. 
Slides were passed into 95 % alcohol, further dehydrated and mounted in oil of 
matching refractive index. 

Measurements of the DNA-Feulgen complex were made with the apparatus as 
described previously (Bryan and Gowen, 1956). Transmittance data were ob- 



72 



JOHN H. D. BRYAN AND JOHN W. GOWEN 



1500 



1300 



I 100 



o 
o 



900 



co 700 



CO 
CO 

o 



500 



300 



100 



HORMONE ONLYJ SEM|NAL 

HORMONE AND fVESICLE 



O O 



A A 



X-RAYS 

HORMONE ONLY 

HORMONE AND 
X-RAYS 



DORSAL 
PROSTATE 




24 48 

TIME IN HOURS 



60 



72 



FIGURE 1. The mitotic response of seminal vesicle and dorsal prostate epithelium at different 
times following irradiation and/or hormonal stimulation. 

tained by the "plug" method of Swift (1950); however, since the majority of 
nuclei were ellipsoidal rather than spherical, the formula derived by Kasten (1956) 
was substituted for the one devised by Swift. 

An important factor in the utilization of the Feulgen procedure for cyto- 
photometric purposes is the precise control of the hydrolysis step. When small 
pieces of tissue rather than thin sections are used, the hydrolysis procedure 
becomes subject to more variation. The end result is that DNA-Feulgen values 
for the same tissue processed in two separate procedures may be more variable 



X-RAY EFFECTS ON CASTRATE RATS 73 

than the results obtained with sectioned material processed in a similar manner. 
However, for any single piece of tissue the staining appears to be quite uniform. 
Thus in the case of the kidney, DNA-Feulgen measurements made on fragments 
drawn from different regions of the same piece did not differ significantly from 
each other. This experiment was repeated at a later date with a fresh batch of 
Feulgen reagent and similar results were obtained. However, the mean values for 
the DNA-Feulgen content were about 28% higher. In view of these findings and 
of the fact that the slides were made at different times involving different batches 
of the Feulgen reagent, care must be taken not to misinterpret the variation be- 
tween the mean DNA-Feulgen values reported in Table III. 

Feulgen-stained sections were used for mitotic index determinations. A total 
of 3000-6000 cells was counted and classified per tissue per animal. Fields were 
chosen at random, every cell in the field being classified and recorded. 

RESULTS 
A. Mitotic index determinations 

The data obtained are summarized in Table I. The values in columns 2, 3, 6 
and 7 of the table represent the combined counts from each pair of animals. In 
columns 5 and 9 the mitotic activity is expressed as per cent of control values for 
seminal vesicle and dorsal prostate, respectively. These values are presented in 
graphical form in Figure 1. 

Seminal vesicle 

The data indicate that by 20 days following castration the level of mitotic 
activity has declined to about 40% of the control value. The hormone-treated 
animals show a steady increase in activity reaching a peak at 72 hours after the 
initial injection was given. The level of activity reached at this time represents 
a fourteen-fold increase over the value obtained for the controls. In the case of 
the animals receiving irradiation as well as hormone, the rise in mitotic activity is 
similar over the period through 48 hours. Here, however, the peak of mitotic 
activity is reached at 60 hours and the level then undergoes a marked decline. This 
60-hour value is not very different from the corresponding value of the animals 
which received hormone alone. 

Dorsal prostate 

In this organ the response was found to be quite different from that of the 
seminal vesicle (Fig. 1). At no time during the experiment did the levels of 
mitotic activity of the dorsal prostate reach levels comparable with those deter- 
mined for the epithelium of the seminal vesicle. The level of activity increased 
more slowly over the period through 48 hours. In the case of the animals 
which received hormone alone the mitotic activity reached a peak at 60 hours and 
essentially the same level was found at 72 hours. Unlike the conditions prevailing 
in the seminal vesicle, the mitotic activity in irradiated and hormone-treated animals 
did not reach a peak until the end of the experimental period, the value being 
almost identical with the terminal value for the hormone-only group. 



74 JOHN H. D. BRYAN AND JOHN W. GOWEN 

In certain pairs of animals some variation in the mitotic index is apparent. As 
examples of the magnitude of these variations the most extreme cases are pre- 
sented herewith. In the seminal vesicle, the 48-hour hormone + x-ray data 
range from 0.75% (rat A) to 1.83% (rat B). Similarly in the case of the dorsal 
prostate, mitotic counts for the 72-hour hormone animals range from 1.63% to 
2.29%. A statistical analysis of the variation in the mitotic index data is set forth 
in Table II. The variation between seminal vesicles at 48 and 72 hours following 
the initial hormone injection and at 48 hours after hormone and irradiation is 
significant at the 1% level. The dorsal prostates show some variation between 
rats at 60 and 72 hours after the start of the hormone treatment. These differences 
in response are significant at the 5% level. 

On account of this observed variation between animals in the present work, 
differences in the magnitude of the mitotic response of irradiated versus unirradiated 
animals should be viewed with circumspection. However, the data do establish 
the trend in response to the treatments. Within this framework, the mitotic counts 
when taken together with the DNA-Feulgen data do allow meaningful conclusions 
to be drawn. 

B. DNA-Feulgen content of nuclei 

The data obtained are presented in Table III. Tissue from each member of 
pairs of animals was subjected to the photometric procedure. From each animal 
and tissue, samples of twenty-five nuclei were measured. The means are there- 
fore based on samples of fifty measurements each. The mean amount of DNA- 
Feulgen complex, in arbitrary units, together with their respective standard errors 
are listed in columns 4 and 7 of the table. Nuclei possessing the diploid amount 
of DNA-Feulgen complex are designated as Class II nuclei, those with twice this 
amount Class III, and Class Ila represents nuclei possessing an amount of DNA- 
Feulgen complex intermediate between these levels. 

The range of values for the DNA classes was determined from measurements 
of nuclei isolated from kidney tissue of control animals. The spread of values 
was slightly variable; thus two samples gave highest values of 1.41.5 units higher 
than the lowest (for example, ranging from 2.69 units to 4.17 units) while in 
two samples measured at a later date a range of 1.2 units was obtained (2.15-3.37 
units ) . 

Since the kidney nuclei have a more uniform appearance than the seminal 
vesicle or dorsal prostate nuclei, the larger range of values was chosen as a better 
approximation to the range expected for the diploid class. An approximation to 
the range expected for Class III nuclei was made by doubling the values obtained 
for the Class II nuclei in each set of measurements. Any values falling between 
the upper limits of Class II and the lower limits of Class III were assigned to 
Class Ila. 

Seminal vesicle 

It is evident from Table III that until 48 hours following irradiation and/or 
the initial hormone injection, the DNA-Feulgen values fall almost entirely into 
Class II. At 48 hours significant numbers of Class III nuclei appear but they are 



X-RAY EFFECTS ON CASTRATE RATS 75 

much less frequent in the irradiated material. By 72 hours following the initial 
hormone injection the frequency of Class III nuclei has declined to about half the 
48-hour value. In the case of the irradiated animals the highest number of Class 
III nuclei appears in the 60-hour material. 

This variation in number or lack of Class III and Class Ha nuclei is not due 
primarily to errors of sampling (though the latter may contribute to the variation) 
since duplicate samples have yielded essentially the same results. 

Dorsal prostate 

In the section on mitotic activity it was pointed out that the epithelium of the 
dorsal prostate was much less mitotically active than that of the seminal vesicle. 
This behavior is paralleled by the synthetic activity as judged by the lower 
frequency of Class Ila and Class III nuclei. Not only are these classes almost 
absent until the 48-hour period, but also at 72 hours following the start of the 
hormone treatment. This latter decline is in contrast with the findings with 
respect to the seminal vesicle. 

DISCUSSION 

As pointed out earlier, the testis is characterized by a high rate of spermatogonial 
mitosis. It is this mitotic activity which is markedly inhibited following irradiation. 
Other data indicate that inhibition of DNA synthesis in interphasic spermatogonial 
nuclei contributes to this suppression of mitotic activity. It would therefore seem 
likely that other mitotically active tissues would respond in a similar manner 
following exposure to similar x-ray doses. 

A brief resume of the spermatogonial response is given here to facilitate com- 
parison with the results of the present work. In the normal mouse testis 4 per 
cent of the spermatogonia appeared to be undergoing mitosis at a given time. 
Spermatogonial divisions rapidly declined to less than 5 per cent of the control 
level by 3 days after exposure to 320 r of x-rays. A rise in mitotic activity was 
apparent 5-10 days following irradiation. The highest level of activity was not 
observed until the termination of the experiment, i.e., at 28 days after exposure. 
At this time the level attained was slightly more than twice that of the controls. 

As may be seen from Table I and Figure 1, the mitotic response of the accessory 
organs follows a pattern quite different from that just described. The onset of 
activity occurs in a matter of hours rather than days. This suggests that the 
period of mitotic inhibition is markedly contracted. 

In the case of the seminal vesicle peak mitotic activity is reached at 60 hours 
after irradiation. Moreover, this peak value is approximately 12 times higher than 
the control level. The mitotic response of this organ is -then much stronger and 
more rapid than in the case of the testis. 

With respect to the dorsal prostate the highest level of activity following ir- 
radiation was not reached until 72 hours after exposure. This level was 3.5 times 
higher than the control. The response of the dorsal prostate is clearly on a much 
lower level than that of the seminal vesicle. 

It would appear from the above discussion that the radiation response of 
somatic and germinal tissue is quite different. This may be a reflection of under- 



76 JOHN H. D. BRYAN AND JOHN W. GOWEN 

lying metabolic differences between tissues. This idea receives some support from 
the recent work of Pelc and Howard (1956). These workers, using C 14 -labelled 
adenine, found a difference in incorporation between spermatogonia and somatic 
tissues such as seminal vesicle, skin and intestine. Their data suggest that a DNA 
precursor becomes maximally labelled soon after injection and is drawn upon by 
spermatogonia but not by somatic tissues. There is then the possibility that the 
DNA of reproductive cells may be synthesized in a somewhat different manner 
from that of somatic cells. These biochemical processes may differ in their 
sensitivity to x-rays. 

Certain other factors may also contribute to the observed differences in response. 
As is evident from our present data and from the prior work of Cavazos and 
Melampy (1954), hormonal stimulation of mitotic activity does not, in these 
organs, become markedly effective until 48 hours after the initial injection. It 
has been pointed out by Lea (1955) that the effect of the x-ray dose decays with 
time (see page 289). So it is possible that the effect of the dose has undergone 
some decay before the inhibitory action can become effective. In other words, the 
"effective" dose of x-rays may not be identical for the two experiments. 

The response of the seminal vesicle to androgen and irradiation treatments has 
been studied by several other workers (see for example Cavazos and Melampy, 
1954; Fleischmann and Nimaroff, 1954; Melampy et al, 1956). The results 
reported here differ in some aspects from those of the studies; however, taken as 
a whole they do show similar trends. These previous workers used colchicine to 
facilitate mitotic index determinations whereas colchicine was not used in the 
experiments reported here. Therefore the mitotic indices listed in Table I are 
much lower than those published by the workers cited above. 

The data of Fleischmann and Nimaroff (1954) and of Melampy et al. (1956) 
were interpreted as suggesting that following irradiation there is a delay in the 
onset of mitotic activity. After doses of 640 r or less (Melampy et al., 1956) the 
delay was such that the peak level of mitotic activity was observed at 60 hours. 
Furthermore, Fleischmann and Nimaroff (1954) found that if an interval of 5 
days was allowed between irradiation and hormone injection the delaying effect 
was lost. This latter observation is of interest inasmuch as it clearly demon- 
strates that recovery occurs after a partial-body exposure to 3000 r. 

In the present work, the 60-hour mitotic index following exposure to 320 r was 
only about 12% higher than that of the controls (hormone alone) whereas Melampy 
et al. found corresponding values to differ by a factor of two. Strain differences 
may be involved since the Fischer strain (line 344) appears to be more radio- 
resistant than certain other strains (Dunning, personal communication). On this 
basis a shortening of the delay period would be expected with the consequence that 
the mitotic index would be depressed towards control levels. Also from a statisti- 
cal viewpoint, the numbers of animals per time period are rather small so that errors 
of sampling are of increased significance (see elsewhere in this paper for further 
discussion of sources of variation which bear on this point). 

As shown in Figure 1, the mitotic response of the dorsal prostate following ir- 
radiation and/or hormonal stimulation is on a much lower level than that of the 
seminal vesicle. This is most probably a reflection of innate differences in activity 
of these organs as well as possible differences in magnitude of the response to the 



X-RAY EFFECTS ON CASTRATE RATS 77 

experimental treatments. The shape of the curves for the dorsal prostate would 
suggest that recovery from any induced mitotic delay does not occur until close to the 
end of the experimental period. This is in contrast with the findings for the 
seminal vesicle. 

It has been shown by various workers that the synthesis of chromosomal ma- 
terial in preparation for mitotic division occurs during the interphase. Experiments 
with radio-active precursors by Pelc and Howard (1952), Taylor and McMaster 
(1954) and recently, by Taylor et al. (1957), point clearly in this direction. 
Cytophotometric studies (see for example Swift, 1950; Bryan, 1951; Taylor and 
McMaster, 1954) are in agreement with the results obtained by autoradiographic 
procedures. 

From the above work it should follow that any treatment which suppresses or 
inhibits mitotic activity should result in the absence or reduction in number of 
Class III nuclei. In a sample of nuclei selected at random from a mitotically 
active tissue the relative proportions of the DNA classes will depend upon the 
speed at which DNA synthesis is accomplished and the rate at which Class III 
nuclei enter into prophase. Thus if conditions are such that Class III. nuclei enter 
division without any intervening delay, the chances of encountering such nuclei in 
a sample are rather small. If, on the other hand, chromosomal reduplication is pro- 
ceeding at a rate appreciably faster than that at which such nuclei enter division, 
then Class III nuclei should accumulate and should constitute a significant propor- 
tion of the measured sample. The mitotic activity measured in terms of re- 
duplication of the DNA content of interphase nuclei will then be a measure of 
the balance between these mechanisms. It should also follow that, within certain 
limits, a comparison of the DNA-Feulgen data and the mitotic counts should 
afford some insight with respect to the nature of the response evoked by agents 
which inhibit or enhance mitotic activity. 

In the case of the intact controls, where mitotic activity was found to be rather 
low and most probably represented replacement of dead cells rather than tissue 
growth. 98-100% of the nuclei measured fell into the diploid DNA class (Class 
II). Similar results were obtained with respect to the 20-day castrates where the 
mitotic index was still lower. Not until the 48-hour period were significant 
numbers of Class III (and Ha) nuclei encountered. With respect to the seminal 
vesicle material, the proportion of Class III nuclei at this time was 63% in the 
case of the unirradiated animals, whereas following irradiation 14% of th^. sample 
was composed of such nuclei. At 60 hours after the initial hormone injection the 
proportion of Class III nuclei remained about the same. In the case oi the 60-hour 
irradiation and hormone-treated material, the proportion of Class III nuclei had 
increased to 34% (from 14% at 48 hours). At 72 hours after the start of the 
treatment, the proportion of Class III nuclei had fallen to 18% of the sample from 
animals receiving hormone alone and to 20% in the case of the irradiated animals. 

These cytophotometric data indicate that the tissue response evoked by the ir- 
radiation treatment is rather similar to the response to hormone injection but that 
a difference in timing is apparent. In the case of the animals receiving only 
hormone, the highest frequency of Class III nuclei is reached at 48 hours following 
the initial injection. After irradiation, on the other hand, the peak frequency is 
not attained until 60 hours after exposure. Moreover, following irradiation, the 



78 JOHN H. D. BRYAN AND JOHN W. GOWEN 

60- and 72-hour frequencies are rather similar to the corresponding values for 
animals receiving hormone alone. 

It should be recalled that the frequency of Class III nuclei most probably 
represents a balance between the rate of chromosomal reduplication and the rate 
at which nuclei enter into prophase of mitosis. From this it would follow that 
the rise in frequency of Class III nuclei very probably is an indication of increased 
synthesis following hormonal stimulation. By the same token, the slower rise in 
frequency noted in the irradiated material suggests that in such material the 
synthetic rate responds less rapidly to the hormonal stimulus. The present data 
then may be interpreted to mean that irradiation does interfere with the synthetic 
mechanism of these hormonally stimulated tissues. 

The data pertaining to the dorsal prostate showed little change from the control 
level until the 48-hour period. At this time the frequencies of Class III nuclei 
following irradiation and/or hormone were 8% and 16%, respectively. At 60 
hours, the frequency of such nuclei in the sample from irradiated material had 
fallen to 4%, while the corresponding value for unirradiated material remained un- 
changed from the 48-hour level. At the termination of the experimental period 
(72 hours) the samples measured contained no Class III nuclei regardless of 
treatment. 

These cytophotometric data show that the response of the dorsal prostate is 
on a much lower level than that determined for the seminal vesicle. Just as in 
the case of the mitotic counts, the DNA-Feulgen data point up a response to the 
experimental treatments which is different from that shown by the seminal vesicle. 

As is the case with the mitotic counts, the DNA-Feulgen data show that the 
response of these somatic tissues to irradiation and/or hormonal stimulation is 
much more rapid than in the case of the spermatogonia. In the latter the frequency 
of Class III nuclei reaches its highest level (51%) at about 10 days following ir- 
radiation and thereafter declines rapidly to about 5% of the sample at 28 days. 
These data when considered along with the corresponding mitotic counts indicate 
a rapid utilization of Class III nuclei during the regenerative period. From the 
present DNA-Feulgen data it may be seen again that a situation analogous to the 
spermatogonial one presents itself but over a much shorter period of time. 

The mitotic index determinations and the DNA-Feulgen data taken together 
allow the interpretation that hormonal stimulation of mitotic activity does not, in 
these organs, become markedly effective until 48 hours after the initial injection. 
Furthermore, the data prior to the 48-hour period lend themselves to the conclusion 
that mitotic activity is very closely correlated with the rate of chromosomal re- 
duplication (formation of Class III nuclei). It would also appear that by 48 hours 
the rate of chromosomal reduplication is beginning to run ahead of mitotic activity 
and, with respect to the seminal vesicle, that this "imbalance" is maintained to a 
lesser degree for the duration of the experimental period. With respect to the 
dorsal prostate similar conclusions may be drawn with the notable distinction that 
over the period of 48 through 72 hours the "balance" has again changed in a manner 
such that, at the termination of the experiment, the close correlation evident 
earlier has been restored. 

The results reported here lead to the conclusion that exposure to a similar dose 
(320 r) of x-rays is much less effective in inhibition of mitosis in these tissues 



X-RAY EFFECTS ON CASTRATE RATS 79 

(under conditions of hormonal stimulation) than in the case of the spermatogonia. 
The DNA-Feulgen data may be interpreted to mean that, following irradiation, 
DNA synthesis suffers some impairment but recovers in a relatively short period 
of time. The demonstrated mitogenic action of testosterone propionate may have 
in some manner interfered with, or masked, the inhibiting action of the irradiation. 
Such an idea receives some support from the work of Rugh and Clugston (1954). 
These authors found that the stage of the oestrus cycle (at the time of irradiation) 
affected the radiation sensitivity of female mice. They found that females in 
dioestrus were about twice as sensitive (in terms of the male LD 50/30 days 625 r) 
to the irradiation as were females in oestrus. Thus an increased level of oestrogen 
appears to be correlated with increased resistance to x-irradiation. The same may 
be true also for androgens, especially so since both classes of steroid hormones 
elicit similar responses in experimental animals (see Bullough, 1952). 

The present results, together with our earlier data (mouse testis), not only 
imply that the male germ line may differ from somatic tissues in its response to 
irradiation, but also that somatic tissues may differ from one another in this 
respect. At the present time it would appear that differences between the tissues 
studied are mainly ones of degree and rate of response. However, the findings of 
Pelc and Howard (1956) point out the possibility that somatic and germinal tissues 
may also differ in their metabolic pathways. Until more biochemical and cytological 
information is at hand the precise nature of these differences must remain an open 
question. 

SUMMARY 

1. The accessory organs of castrated male rats have been used in a study of 
the effects of x-rays on mitosis in somatic tissues. 

2. Animals nine weeks old were castrated and the organs allowed to regress 
for 20 days prior to use. Half the animals received daily injections of 500 fj.g of 
testosterone propionate (in oil) until death; the others, in addition to the hormone 
injections, w r ere exposed to a single dose of 320 r of x-rays, the irradiation being 
given at the time of the first hormone injection. Pairs of animals were killed at 
24, 48, 60 and 72 hours following the irradiation and/or initial hormone treatment. 

3. The response to the treatments was followed by means of mitotic index 
determinations and cytophotometric measurements of the DNA-Feulgen content 
of interphase nuclei. 

4. The cytological data indicate the existence of a difference in response be- 
tween the epithelium of the seminal vesicle and of the dorsal prostate. At no time 
during the experiment did the mitotic activity of the latter rise to levels char- 
acteristic of the former. In addition, the time-response curves for the two organs 
indicate that the dorsal prostate responds more slowly than the seminal vesicle. 

5. The DNA-Feulgen measurements together with the mitotic index data 
indicate that in the controls and in the experimental animals killed prior to 48 
hours there is a close correspondence between the level of mitotic activity and the 
rate of chromosomal reduplication. Over the period of 48-72 hours in the case of 
the dorsal prostate the data show that, during the time of maximal hormonal stimula- 
tion, DNA synthesis is proceeding at a rate appreciably faster than the rate at 
which nuclei enter into prophase. 



80 JOHN H. D. BRYAN AND JOHN W. GOWEN 

6. The results obtained have been compared with those obtained from a similar 
experiment involving the mouse testis. The accessory organs appear to be less 
sensitive to the irradiation than the testis. Factors bearing on this point are 
discussed. 

LITERATURE CITED 

BRYAN, J. H. D., 1951. DNA-protein relations during microsporogenesis of Tradescantia. 

Chromosoma, 4: 369-392. 
BRYAN, J. H. D., AND J. W. GOWEN, 1956. A histological and cytophotometric study of the 

effects of x-rays on the mouse testis. Biol. Bull., 110: 229-242. 

BULLOUGH, W. S., 1952. The energy relations of mitotic activity. Biol. Rev., 27: 133-168. 
BURROWS, H., 1949. Biological Actions of Sex Hormones. Second ed., The University Press, 

Cambridge, England. 
CAVAZOS, L. F., AND R. M. MELAMPY, 1954. Cytological effects of testosterone propionate on 

epithelium of rat seminal vesicles. Endocrinology, 54: 640-648. 
FLEISCHMANN, W., AND M. NIMAROFF, 1954. Combined effects of x-irradiation and testosterone 

propionate on accessory sex organs. Proc. Soc. Exp. Biol. Med., 85: 655-658. 
KASTEN, F. H., 1956. Cytophotometric studies of deoxyribose nucleic acid in several strains 

of mice. Physiol. Zool, 29: 1-20. 
LEA, D. E., 1955. Actions of Radiations on Living Cells. Second Ed., edited by L. H. Gray. 

The University Press, Cambridge, England. 
MELAMPY, R. M., J. W. GOWEN AND J. W. WACASEY, 1956. Effects of x-irradiation on andro- 

genic response of seminal vesicles of the castrate rat. Proc. Soc. Exp. Biol. Med., 

92: 313-315. 
MOORE, C. R., W. HUGHES AND T. F. GALLAGHER, 1930. Rat seminal vesicle cytology as a 

testis-hormone indicator and the prevention of castration changes by testis-extract 

injection. Amer. J. Anat., 45: 109-135. 
PELC, S. R., AND A. HOWARD, 1952. Chromosome metabolism as shown by autoradiographs. 

Exp. Cell Res., Suppl, 2: 269-278. 
PELC, S. R., AND A. HOWARD, 1956. A difference between spermatogonia and somatic tissues 

of mice in the incorporation of (8-"C)-adenine into deoxyribonucleic acid. Exp. Cell 

Res., 11: 128-134. 
RUGH, R., AND H. CLUGSTON, 1954. Radiosensitivity with respect to the oestrus cycle in the 

mouse. Biol. Bull, 107: 289-290. 

STOWELL, R. E., 1945. Feulgen reaction for thymonucleic acid. Stain Technol., 20: 45-56. 
SWIFT, H. H., 1950. The desoxypentose nucleic acid content of animal nuclei. Physiol. Zool., 

23: 169-198. 
TAYLOR, J. H., AND R. D. McMASTER, 1954. Autoradiographic and microphotometric studies 

of desoxyribose nucleic acid during microgametogenesis in Lilium longifolhim. 

Chromosoma, 6: 489-521. 
TAYLOR, J. H., P. S. WOODS AND W. L. HUGHES, 1957. The organization and duplication of 

chromosomes as revealed by autoradiographic studies using tritium labeled thymidine. 

Proc. Nat. Acad. Sci., 43: 122-127. 



ANNUAL REPRODUCTIVE CYCLES OF THE CHITONS, 
KATHERINA TUNICATA AND MOPALIA HINDSII x 

A. C. GIESE, J. S. TUCKER AND R. A. BOOLOOTIAN 2 

Hopkins Marine Station of Stanford University, California 

The reproductive season of marine invertebrates has been determined in a 
variety of ways: spawning, the appearance of young in plankton, increase in size 
of gonad relative to the body, and development of ripe gametes (see Giese, 1959, 
for reference). In most cases an annual reproductive season has been observed, 
but many species breed more than once during the season, some showing a monthly 
rhythm (Korringa, 1947). Data on reproductive seasons of marine invertebrates 
are still not extensive and the problems posed are numerous. It is desirable that 
data on many more species be gathered to gain a better perspective. This paper 
records observations on reproduction in two species of chitons, Katherina tunicata, 
an intertidal form, and Mopalia hindsii, a pile dweller. 

METHODS 



Ten specimens of Katherina were gathered monthly from the mid-littoral zone' 
of Carmel Point during 1954-55 and from the same zone at Yankee Point during 
1956-58, in each case from rocks in the Postelsia and mussel zone exposed at low 
tide. The specimens at Carmel Point were larger but the population was sparser, 
and perhaps less representative than that at Yankee Point. The sample from 
Yankee Point was therefore selective inasmuch as very small specimens were avoided 
(weights: 1957, 16-44 gms.; 1958, 11-53 gms.) 

Ten specimens of Mopalia were obtained during monthly low tides from the 
pilings at Monterey harbor. The population in this region is unusual in that the 
specimens are preponderantly large and not too numerous, therefore the samples 
may not be truly representative of the species (weights : 1957, 14-73 gms. ; 1958, 
16-65 gms.). However, this species was not found in other accessible environments 
in sufficient numbers for sampling. 

Each chiton was weighed, the foot removed and the animal eviscerated, per- 
mitting easy removal of the gonad. The gonad volume was determined and the 
gonad index (the volume of the gonad is divided by the weight of the animal and 
multiplied by 100) determined. 

To determine whether any striking changes in the content of nutrients oc- 
curred in the blood during the course of the reproductive cycle of Katherina, the 
reducing sugar, protein and non-protein nitrogen were determined monthly in 
samples of blood. For this purpose the blood of from ten to twenty individuals 

1 Supported in part by U.S.P.H. Grant 4578 to A. C. Giese. We are indebted to Mrs. M. 
Lusignan, Mr. G. Araki, Mr. J. Bennett, Mr. A. Farmanfarmaian, Dr. A. T. Giese and Mr. 
John Schmaelzle for cartographic, statistical and sampling help. 

2 Now at the Department of Zoology, University of California at Los Angeles. 

81 



82 



GIESE, TUCKER AND BOOLOOTIAN 



K. tunicata 



GONAD 
INDEX 



PROTEIN 
mg. % 



NPN 
mg. % 



1954 1955 

026 Nil D 7 JI3 F3 M7 All M22 J 7 J5 AI6SI2 03 N22DI5 



REDUCING 

SUGAR 

mg.% 



16 

14 

12 

10 

8 

6 

4 

2 



12 

10 

8 

6 

4 

2 



340 

300 

260 

220 

180 

140 

14 

12 

10 

8 

6 

4 

2 







FIGURE 1. Top: Reproductive cycle of Katherina tunicata for 1955 as determined by the 
gonad index. The gonad index is the ratio of gonad volume to wet weight of the entire 
animal times 100. The specimens were collected at Carmel Point, California. The dots 
indicate the means and the lines, the 95 per cent confidence limits. Lower three graphs show 
variation in reducing sugar, protein and non-protein nitrogen in the population samples used 
for gonad analyses. 



REPRODUCTION OF CHITONS 

had to be pooled because a single animal does not have enough for analysis, a 33- 
gram animal giving only several milliliters of blood. Blood samples were obtained 
from the lateral sinuses following puncture through the mantle cavity on either side 
of the animal. The blood is a yellowish fluid which was not observed to coagulate 
although it showed some murkiness on standing, suggesting a feeble clumping of 
the blood corpuscles. 

Reducing sugar was determined by the anthrone method (Seifter et al., 1950) 
after precipitating the proteins with 10 per cent trichloracetic acid. Non-protein 
nitrogen was determined with the micro-Kjeldahl after precipitating the protein, 

Katherinq tunicata ~ l957 

1958 



10 

8 
X 

UJ 

a 
^ 6 













MAMJJASON 



FIGURE 2. Reproductive cycle for Katherina tunicata during 1957 and 1958 as determined 
by gonad index. The specimens were collected at Yankee Point, with the exception of points 
marked C which came from Carmel Point and those marked P which came from Pescadero 
Point. 

and total nitrogen was determined by the same methods, without the preliminary 
addition of protein-precipitating agents. Protein was taken to be the difference 
between these two volumes multiplied by 6.25. 

KATHERINA TUNICATA 

For each of the three years during which Katherina was studied an annual 
reproductive cycle was found. It is most clear-cut for the year 1954-55 (the 
population collected at Carmel Point) as shown in Figure 1. It is less distinct 
for the collections at Yankee Point during the years 1956-57 and 1957-58, the 
gonad index being high for several months preceding the spawnout, as shown in 
Figure 2. The difference in sharpness of the cycle at the two locations may be 



84 GIESE, TUCKER AND BOOLOOTIAN 

the result of a more homogeneous population of large animals at Carmel Point. 
A comparison of the gonadal index for random specimens from both localities 
during the same year is desirable. 

During the height of the season males are easily distinguished from females by 
the color of the gonad, which is greenish in the female (the eggs being green) and 
very light in the male (the sperm being white). However, it is not possible to 
distinguish the sexes of individuals from spawned out gonads, but the shell gland 
of the female is always diagnostic of sex even in a spent female. When minute 
spherical eggs begin to develop as a new cycle comes into prospect, diagnosis from 
gonads is again easy. Statistical analysis of the 195455 data showed no essential 
difference in the time of onset in maturity of testes and ovaries in males and 
females; therefore the data for both sexes were combined. It was, however, 
noted that the gonad index of males appears to be slightly higher than that of fe- 
males at the height of the season and lower than that of the females during the 
decline of the season. The 95 per cent confidence limits for the pooled males and 
females given in Figure 1 indicate that the entire population of animals is relatively 
homogeneous, since the confidence band is never very wide. 

The data for almost two years of the breeding cycle for the same population 
at Yankee Point, given in Figure 2, indicate that the cycle varies somewhat from 
year to year. After slowly spawning during the months of March, April, May 
and June of 1958 the gonad index again rose, suggesting minor ripples in sexual 
activity or a second active period because of favorable conditions in the area. During 
this second cycle the gonad index rose significantly from 5.5 on June 9 to 8.9 on 
July 9 then fell precipitously to 1.9 on July 18 of 1958. 

It is interesting to note that the populations sampled at three stations on July 
17 and 18 showed different average gonad indices : 6.8 at Carmel Beach, 3.6 for 
Pescadero Point, and 1.9 for Yankee Point. This indicates that the exact time of 
spawning is probably in part determined by the geographical and ecological location. 

Two findings are similar for the three years of data available ; 1 ) in all cases the 
chitons have small shrunken gonads late in July, and 2) the gonads remain shrunken 
during the late summer, fall and early winter. In the months after the beginning 
of winter the gonads grow rapidly and gametogenesis occurs. The reproductive 
cycle therefore consists of several months of stasis, several months of growth, 
several months during which the gonads are ready for spawning, and probably 
several periods of spawning. MacGinitie and MacGinitie (1949) report that 
Katherina lays eggs in the Monterey Bay area in July while Hewatt (1938) 
reports it spawning in the same area in May, and Rickets and Calvin (1948) 
say it spawns in Puget Sound a month or two earlier. 

Attempts to study breeding and spawning of Katherina in the laboratory did 
not prove successful since often the animals crawled out of the tanks to dry loca- 
tions, and even though periodically submersed, they deteriorated. They did not 
seem to eat while in the laboratory although red and brown algae were provided 
and a scum of diatoms was always present on the surface of the aquaria. 

In its natural environment Katherina feeds upon diatoms and upon algae, since 
the pellets in the gut of animals sampled consisted of partially digested algae of 
various kinds, including brown and red algae. In other cases the gut was filled 
from end to end with diatoms and skeletons of diatoms. Apparently the chitons 






REPRODUCTION OF CHITONS 85 

eat what is readily available to them and are always found within reach of algae. 
Katherina does not move out of the sunlight, remaining on the upper surfaces of 
the rocks during low tide. It is therefore subjected to and weathers all the changes 
in conditions which obtain during low tide. 3 Most chitons have tegmental aesthetes 
or "shell eyes" which enable them to perceive the light. These are absent in 
Cryptochiton and may be of less importance in Katherina in which much of the 
shell is covered by tissue (Crozier, 1921). A variety of other organisms are 
found growing upon Katherina. These include occasional barnacles, coralline 
algae, and colonial animals such as bryozoans and hydroids, all of which anchor to 
the skeletal plates. The overgrowth by algae and various colonial animals in- 
dicates the sessile nature of the animals and their tendency to remain on the upper 
surface of the rocks. 

The nutrients in the blood of Katherina vary during the year (Fig. 1). The 
most prominent constituent of the blood is protein which is always present in large 
amounts, varying from about 180 to 240 mg. per cent. The lowest values of blood 
protein appear to coincide with the highest gonad index. The non-protein nitrogen 
of the blood is small in amount, reaching a maximal value of about 12 mg. per cent 
and falling to almost zero when the gonad is increasing most rapidly in size. Re- 
ducing sugar is present in rather small amounts from almost zero to about 11 mg. 
per cent. A single determination of the blood constituents therefore has little 
meaning as a value for the species since the constituents vary so much in amount 
from month to month. The variations in blood constituents in Figure 1 may 
possibly be correlated with the breeding season. Similar variations in content of 
organic constituents of the body fluid of echinoderms (Bennett and Giese, 1955), 
and in the blood of a number of species of crabs (Leone, 1953) have also been 
documented. It would be interesting to know whether the blood of marine in- 
vertebrates is generally so variable. 

A certain amount of organic material is stored in the foot, the gonads and the 
hepatic gland. It is clear from preliminary tests that neither the digestive gland 
nor the foot has much glycogen, although almost 0.1 per cent wet weight of the 
soft tissue of the animal consisted of glycogen (0.83 per cent of the dry weight). 
Lipid seems to be more important than glycogen as a storage material. Detailed 
studies on these constituents are under way. 

MOPALIA HINDSII 

Mopalia hindsii is listed by MacGinitie and MacGinitie (1949) as an estuarine 
form which is often found on pilings. They have found some specimens as large 
as 10 centimeters long and 7.5 centimeters wide. The animals used in this study 
were usually smaller than the maximum, being generally about 8.0 centimeters long 
and 5.5 centimeters wide. Specimens from the pilings in Monterey Harbor were 
covered by a forest of bryozoans, hydroids, and a multitude of larval crustaceans, 

3 Heath (1899, 1905) found that many of the chitons are highly sensitive to sunlight and 
creep into crevices, and that one species, Ischnochiton magdalenensis, even buries itself in the sand 
with the approach of day. Katherina tunicata and Cryptochiton stelleri are among the few 
species which remain exposed during the bright daylight. Mopalia hindsii, it is true, remains 
on the inner pilings at Monterey Harbor during the day, but this is an environment of rather 
attenuated light. 



86 



GIESE, TUCKER AND BOOLOOTIAN 



nemerteans, round worms, annelid worms, and protozoans (including the large 
ciliate Condylostoma). This association is common to this species (MacGinitie 
and MacGinitie, 1949). Shells of Mopalia are often weakened by a boring worm 
(Tucker and Giese, 1959), probably of the family Spionidae. 

The intestinal contents of the specimens studied were filled with skeletons of 
bryozoans and other organisms which grow on the pilings which they inhabit. 
Mopalia was generally absent from the outer pilings where the light is sufficient 
for a relatively abundant growth of algae and were more numerous in the darker 
regions where bryozoans, hydroids, anemones, and starfishes occurred. 



Mopaliq hindsii 



O 1957 

1958 



14 
12 

10 

8 



4 
2 




o or 



M 



M 



N 



FIGURE 3. Reproductive cycle of Mopalia hindsii during 1957 and 1958 as determined by 
gonad index. Specimens collected on pilings in Monterey Harbor. 

A definite annual reproductive cycle is observed for Mopalia (Fig. 3). Breed- 
ing, as indicated by enlargement of gonads with development of eggs and sperm, 
occurs in the late fall and winter, approximately from October to February or 
March. That it was somewhat different each year is indicated by the data in 
Figure 3. 

DISCUSSION 

A number of previous workers have described the breeding habits of various 
chitons (Heath, 1899, 1905; Grave, 1922; Stephenson, 1934; Costello et al., 1957). 
Grave (1922) found that Chaetopleura apiciilata spawns at ten-day intervals during 
the months of June, July and August at Woods Hole, the activity declining in the 
intermediate periods (for other references, see Costello et al., 1957). Spawning 
in this species occurred during the night and is possibly associated with phases of 



REPRODUCTION OF CHITONS 87 

the moon (Korringa, 1947). Stephenson (1934) found that Acanthosostera 
gcmmata bred in the Great Barrier Reef of Australia from September to April, 
spawning occurring every four weeks during the breeding period, associated with 
phases of the moon. After April it entered a prolonged resting period. 

Heath (1899, 1905) made many excellent observations of the breeding activities 
of chitons in nature. He observed I. magdalenensis breeding in the Monterey area 
in May and June, in the latter month over a considerable length of the coastline in 
the area. Hewatt (1938) also observed the same species breeding in June, but 
found Mopalia muscosa breeding in September. Heath (1899, 1905) found that 
chitons of the Monterey area do not breed in captivity. 

Heath (1905) also observed that isolated males of /. magdalenensis and Mopalia 
lignosa spawn, but isolated females do not. However, females placed with males in a 
tide pool spawn soon after the males have spawned, suggesting chemical stimulation. 
This observation confirms similar observations by Metcalf (1892) on Chiton 
squamosus and C. marmoratus (see also Crozier, 1922). Heath observed shedding 
and egg laying in Ischnochiton mertensii, I. cooperi, M. muscosa and K. tunicata 
in large pools isolated by low tides. The males began shedding when the waters 
became tranquil after recession of the tide and stopped shedding when disturbed 
by water movements. Shedding sometimes continued for as long as two hours, 
steadily or in spurts. He bred some chitons in isolated pools and grew them to 
maturity. Young K. tunicata isolated in pools near mean tide mark grew to 25 mm. 
in length in one year and reached their average length of 55 mm. in three, a rapid 
rate of growth characteristic of other species of chitons as well. 

The two species of chitons discussed in this paper, K. tunicata and M. hindsii, 
show a distinct breeding season which, while similar from year to year, is sufficiently 
distinctive each year to indicate that it is not closely tied to day-length or some other 
factor invariant from year to year, but rather is subject to action of various local 
factors. The local variation in reproductive state found in one collection at three 
different stations in the Monterey area (Yankee Point, Pescadero Point, and 
Carmel Point) during early July, 1958 indicates how important to the breeding 
cycle are the small differences in the ecology of the three environments. 

The most curious feature of breeding in the two species of chitons studied here 
is the reciprocal nature of their breeding seasons, Katherina breeding in the summer, 
Mopalia in the winter. This resembles the pattern described for the crab Pach- 
ygrapsus crassipes which breeds in the summer, while Hemigrapsus nudus, another 
grapsoid crab of similar habits and nature, breeds in winter (Boolootian et al., 
1959). It would be valuable to ascertain what factors trigger the sweep of the 
reproductive cycles at a period approximately six months apart in the two species 
of chitons. Other cases of this type are known but in no case has an adequate 
experimental analysis of causal mechanism yet been made (Giese, 1959). 

SUMMARY 

1. The reproductive cycles of two species of chitons, Katherina tunicata and 
Mopalia hindsii, collected in Monterey Bay are recorded, the first for three years 
and the second for two. 

2. Katherina breeds in the summer, Mopalia in the fall and winter. 



88 GIESE, TUCKER AND BOOLOOTIAN 

3. In each case the gonad index (ratio of gonad volume to body weight times 
100) rises gradually and falls rather precipitously as spawning occurs. Differences 
in onset of breeding occurred during the years for which records are available, 
suggesting timing of events by local conditions. 

4. Blood protein, non-protein nitrogen, and reducing sugar vary during the 
year but it is not clear whether these variations are significantly correlated with 
reproductive condition. 

LITERATURE CITED 

BENNETT, J., AND A. C. GIESE, 1955. The annual reproductive and nutritional cycles in two 

western sea urchins. Biol Bull, 109: 226-237. 
BOOLOOTIAN, R. A., A. C. GIESE, A. FARMANFARMAIAN AND J. TUCKER, 1959. Reproductive 

cycles of five west coast crabs. Physiol. Zool. (in press). 
COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Methods 

for Obtaining and Handling Marine Eggs and Embryos. Marine Biol. Lab., Woods 

Hole, Mass., 247 pp. 

CROZIER, W. J., 1921. "Homing" behavior in Chiton. Amer. Nat., 55: 276-281. 
CROZIER, W. J., 1922. An observation on cluster formation of sperm of Chiton. Amer. Nat., 

56: 478-480. 

GIESE, A. C., 1959. Comparative physiology: annual reproductive cycles of marine inverte- 
brates. Ann. Rev. Physiol, 21: 547-576. 
GRAVE, B. H., 1922. An analysis of the spawning habits and spawning stimuli of Chaetopleura 

apiculata (Say). Biol. Bull, 42: 234-256. 

HEATH, H., 1899. The development of Ischnochiton. Zool. Jahrb., 12 : 630-720. 
HEATH, H., 1905. The breeding habits of chitons of the California coast. Zool. Anz., 29: 

390-393. 
HEWATT, W. G., 1938. Notes on the breeding seasons of the rock beach fauna of Monterey 

Bay, California. Proc. Calif. Acad. Sci., 23: 283-288. 
KORRINGA, P., 1947. Relations between the moon and periodicity in breeding of marine animals. 

Ecol Mono., 17: 347-381. 
LEONE, C. A., 1953. Preliminary observations on intraspecific variation of total protein in 

sera of some decapod Crustacea. Science, 118: 295-296. 

MACGINITIE, G. E., and N. MACGINITIE, 1949. Natural History of Marine Animals. McGraw- 
Hill Book Co., New York, 473 pp. 
METCALF, M. M., 1892. Preliminary notes on the embryology of Chiton. Johns Hopkins Univ. 

Circular, 11: 79-80. 

RICKETS, E. F., AND J. CALVIN, 1948. Between Pacific Tides. Stanford Univ. Press, 365 pp. 
SEIFTER, S., S. DAYTON, B. Novic AND E. MUNKWYLER, 1950. The estimation of glycogen 

with the anthrone reagent. Arch. Biochem., 25: 191-200. 
STEPHENSON, A., 1934. The breeding of reef animals, Part II. Invertebrates other than 

corals. Great Barrier Reef Exp. 1928-29. Sci. Kept., 3: 247-272. 
TUCKER, J. S., AND A. C. GIESE, 1959. Shell repair in Chitons. Biol Bull, 116: 318-322. 



A STUDY OF THYROID FUNCTION IN FUNDULUS 

HETEROCLITUS * 2 

PATRICIA J. HARRIS 3 

The Bingham Oceano graphic Laboratory and the Department of Zoology, Yale University, 

New Haven, Connecticut 

The role of the thyroid gland in regulating the metabolic rate, and its effect on 
growth and other physiological functions in higher vertebrates have been well 
established, but in fish, despite the many studies using anti-thyroid drugs, thyroxine, 
thyroid extract, and thyrotropin, its function is still obscure. The diffuse nature 
of the thyroid gland of most teleost fishes has rendered the study of its function 
extremely difficult; surgical removal is impossible except in a very few species 
where the gland is encapsulated, and the use of anti-thyroid drugs introduces dis- 
advantageous collateral effects (reviewed by Chambers, 1953). The availability 
of radioactive iodine has now made possible a new technique for the extirpation of 
the thyroid gland, especially suited to one of diffuse nature as found in these fish. 
La Roche and Leblond (1954) were the first to investigate the problem of radiation 
thyroidectomy in fishes. They found that total destruction of the thyroid in salmon 
(Salmo salar L.) required repeated injections of large, but progressively down- 
graded doses of I 131 . Fish weighing 30-32 grams at the start of the experiment 
received 100, 50, 40 and 30 ^C at the rate of one dose per month. Arvy, Fontaine 
and Gabe (1956) also used a series of injections, but only claimed to have achieved 
a state of hypothyroidism in rainbow trout (Salmo gairdneri Richardson). They 
gave a total dose of 260 /*C in three injections at 30-day intervals, to fish weighing 
about fifty grams. More recently, Fromm and Reineke (1957) have reported 
successful destruction of the thyroid after a single injection of 250 ^C to fingerling 
trout weighing 3.8-6 grams. In an abstract, which has not been reported in detail, 
Baker, Berg, Gorbman and Gordon (1955) described the effects of partial or 
complete thyroid destruction in platyfish by the addition of 4.5-7 pC of I 131 to 
200 ml. of aquarium water. The period of exposure was 2448 hours, but more 
complete destruction resulted from the longer treatment. Fontaine, de la Querriere 
and Raffy (1957), in a study of the respiratory metabolism of hypophysectomized 
eels, noted a fall in the oxygen consumption 48 hours after the injection of 334 fiC 
of I 131 into fish weighing about 70 grams. This was followed by a gradual return 
towards normal over a period of several weeks. Olivereau (1957) has discussed 
the problem of dosage, tissue damage, and regeneration of the thyroid in eels 
treated with radioactive iodine. 

In this study Fundulus heteroclitus was chosen as an experimental fish, not 

1 Supported by National Science Foundation Grant No. G941 under the direction of Dr. 
Grace E. Pickford, principal investigator. 

2 Part of a thesis submitted to the Department of Zoology, Yale University, in partial 
fulfillment of the requirements for the degree of Master of Science. 

3 Present address : Department of Zoology, University of California, Berkeley 4, California. 

89 



90 PATRICIA J. HARRIS 

only for reasons of its hardiness and availability, but since it is a euryhaline species, 
it afforded the opportunity to study the possible role of the thyroid in water and 
salt regulation. Following an initial pilot experiment to determine the dosage 
of I 131 necessary for thyroidectomy, the following experiments were designed to 
study the physiological effects of thyroid deficiency on growth and ability to 
osmoregulate, as well as effects on other organs and tissues that might possibly 
be dependent on thyroid function. 

MATERIALS AND METHODS 

The fish used were Fundulus heteroclitus males of about 7.5 cm. in length, 
caught in the New Haven area in August, 1956. They were placed in storage 
tanks kept at 20 C. for two months before use, at which time they were measured, 
numbered, and selected for uniformity of weight of approximately five grams. 
The results of the previous experiment to determine dosage indicated that dosage 
levels somewhat lower than 10 juC/gm. wt. spaced over a period of several months 
would be the most effective procedure for destroying the active as well as the 
initially inactive follicles. Accordingly, in this experiment a series of graded doses 
was given, 25, 15, and 10 p,C, spaced about five weeks apart. Thus, with five-gram 
fish, the doses were approximately 5, 3, and 2 ^C per gram weight of fish. 

On October 23, the thirty-six fish selected for I 131 treatment were given a dose of 
5 fig per fish of thyrotropin (Armour 317-51) ; on the following day they were given 
the first injection of I 131 , 25 pC per fish in a volume of .05 ml. The injected fish were 
placed in five-gallon wide-mouth carboys provided with aeration and means for feed- 
ing and changing water without handling the fish, and after eight days they were 
returned to their regular aquaria. On November 27 the second dose of iodine 
was given, 15 pC per fish, with an injection of 5 /j.g of thyrotropin given two days 
before. The third iodine injection of 10 /xC per fish was given on January 4, 1957, 
following the usual dose of thyrotropin. These fish were subsequently screened by 
the tracer method described below, and those showing the greatest impairment of 
thyroid activity were set aside for further experiments. The fish were anaesthetized 
with tricaine methane sulfonate (MS 222) for injections and later screening opera- 
tions. 

The fish were fed once daily with Aronson's formula fish food, consisting of a 
cooked mixture of ground beef liver, kidney, greens, dried shrimp, and Pablum. 
The temperature in all tanks was kept at 20 C. and illumination was ten hours 
per day. 

Experimental setup for osmoregulation studies 

To study the physiological effects of hypothyroidism following direct transfer 
of fish from sea water to fresh water, it is necessary to maintain tanks with sea 
water of constant salinity as well as several with running fresh water. A small 
circulating sea water system of simple design was used, with a 55-gallon polyethylene 
reservoir drum feeding by gravity into the aquaria, the overflow draining through 
a filter, and subsequently returned to the reservoir by means of a hard rubber 
pump. The salinity of this system was kept at 26 %o, with a pH of 7.5 and 
oxygen content of 4.28 ml. /liter. 



THYROID FUNCTION IN FUNDULUS 91 

A dechlorinating system for tap water similar to that described by Burden 
(1956) was the fresh-water source. The pH was 6.9, oxygen content 5.46 ml. /liter, 
and a flow rate of 350-400 ml. per minute was maintained in each of the tanks. 

Autopsy procedures 

All fish which died during the iodine treatment or prior to the osmoregulation 
experiment were autopsied to determine the possible causes of death, and the 
thyroids were fixed in Bouin's for histological examination. At the termination of 
the osmoregulation experiment, the remaining fish were autopsied, measurements 
of standard length, weight, and testis weight were made, the thyroid was fixed in 
Bouin's. and blood samples were taken for hematology and chloride titration, as 
described below. 

Hematology 

Red and white cell counts, thrombocytes, and per cent hemoglobin were de- 
termined on blood samples which were allowed to drop on siliconized slides from 
the cut end of the tail. Heparin was used on the razor blade. The procedures will 
be described elsewhere by Dr. Anne M. Slicher, who made this study. 

Blood chloride 

Blood chloride determinations were made using a micro-adaptation of the method 
of Schales and Schales (see Hawk, Oser and Summerson, 1954). Whole blood 
was collected from tail cuts, centrifuged in an air-driven "spinning top" rotor, and 
4.8-/X.1 samples of serum were pipetted into titration vessels, closed with paraffined 
corks and frozen. Before titration the samples were diluted with 42.6 pi of distilled 
water to which indicator had been added (3 ml. stock indicator/50 ml. solution). 
It was found necessary to add about six drops of 2.0 N HNO 3 to the 50 ml. of 
diluted indicator in order to release the bound chloride in the sample. 

To check the accuracy of the method, standard human serum (Hyland Lab- 
oratories, Los Angeles, lot 369E6) with known NaCl was titrated, using the same 
amounts and procedures as with the fish preparation. The standard contained 
544 mg% with an acceptable range of 533-555 mg%. The test titrations gave a 
value of 549 mg%, well within the range. 

Screening method for determining degree of thyroidectomy 

Although all fish were ultimately examined histologically to determine the 
degree of thyroidectomy, it was desirable to know how effectively the thyroid had 
been destroyed before using the fish for experimental purposes. Thus a method 
using a tracer dose of I 131 to measure the rate of activity loss in the throat region 
was worked out and a device w r as designed to hold the fish over a Geiger counter 
so that comparison readings could be made between normal and treated fish. 

A lead block was cast around the mold of a fish to serve as shielding, as well 
as a holder for the experimental fish. A hole was drilled through the bottom of 
the block to connect with the thyroid region of the fish, and the block was mounted 
on a wood frame so that the opening coincided with the %-inch end window of a 
Geiger-Muller tube (Mark 1, Model 105. Radiation Counter Laboratories, Inc., 



92 



PATRICIA J. HARRIS 




CAST LEAD 



END WINDOW 
C-M TUBE 

UEAD SHIELD 



SCAU&P. 



J 



FIGURE 1. Counter arrangement for measuring activity of thyroid area. 

Skokie, 111.). Lead shielding was found to be necessary around the tube, and 
its photosensitivity was obviated by cementing a thin plastic film over the opening 
in the bottom of the block and coating with "Aquadag" (see Fig. 1). 

A series of trial injections of I 131 indicated that a dose of 2.5 yu,C per five-gram 
fish gave an adequate counting rate, and since this was also in line with tracer doses 
used by Gorbman and Berg (1955) and others, it was subsequently used in this 
screening procedure. 

RESULTS 
Histological studies of the thyroid 

Following fixation in Bouin's solution at the time of autopsy, the thyroid regions 
of the experimental and control fish were examined histologically to determine the 
degree of thyroidectomy. Serial sections were made through the entire region, 
and an evaluation was made based on the relative number of follicles present, the 
height of the epithelial cells, and amount of colloid, in relation to the control fish. 
Few of the treated fish showed a total lack of follicles, but it was interesting to 
note that these fish, and others with very few follicles, did not survive to the end of 
the experiment. There was a great range in the amount of thyroid tissue present. 
However, while some fish showed evidence of considerable regeneration, in the 
majority of fish regeneration was present to a much lesser extent, and on the 
whole they could be considered markedly hypothyroid. 

Uptake of a tracer dose of 7 131 in hypothyroid and control fish 

The most promising means of evaluating the tracer data appeared to be a simple 
comparison of rate of activity loss in the thyroid region of the treated (hypothyroid) 



THYROID FUNCTION IN FUNDULUS 



93 



fish with that of the controls. In the first of two groups run to test the screening 
method, the initial readings were made four hours following the injection of I 131 . 
In the hypothyroid fish, the counting rate ranged from 193 to 398 counts per 
minute, with a mean of 297 counts per minute. The controls ranged from 231 to 
465 counts per minute, with a mean of 345. Since there was already a drop in the 
activity in the hypothyroid fish, as compared with the controls, a second group of 
fish was screened in the same manner, but taking the initial reading at one and a 
half hours, rather than four. Here the range in the hypothyroid fish was 313 to 



120 



Q. 



lu 
O 



80. 



60- 



40- 



20- 



CROUP I. (fe CONTROLS, 5 TREATED) 

UPTAKE AFTER A- MRS. TAKEN AS 100% 
CORRECTED FOR BACKGROUND AND 
RADIOACTIVE DECAY 




CONTROLS 

TREATED 



120- 



ul 100 - 

X 
f. 
V- 
L 

1 



j 
'. 60-1 



o 

fe? 



40- 



20 H 



10 



TIME IM DAYS 



GROUP H . (FIVE FISH IN EACH GROUP) 

UPTAKE AFTER i.5 MRS. TAKEN AS 100% 
CORRECTED FOR BACKGROUND AND 
RADIOACTIVE DECAY 



CONTROLS 

I 13 ' TREATED 



10 



TIME IN DAYS 

FIGURE 2. Activity loss in control and treated fish following a tracer dose of 2.5 



of I 



94 



PATRICIA J. HARRIS 



447 counts per minute, with a mean of 383. The controls ranged from 315 to 507 
counts per minute with a mean of 385. However, in both groups the subsequent 
readings on each fish were converted to percentage of its initial reading, the best 
way to take into account the variations between individual fish resulting from weight 
differences, thyroid activity, or unavoidable variations in dose. 

While the activity of hypothyroid fish dropped immediately, the activity of the 
controls continued to increase for several hours before beginning to drop off. 
The peak appears to be around three to four hours following injection. The two 
sets of curves show essentially the same pattern : the hypothyroid fish losing 
activity quite rapidly while the controls lose it at a much slower rate (see Fig. 2). 
The mean values of the retained activity in the treated and control fish showed 
the greatest difference three days following the injection of the tracer dose; thus in 
the following screening of treated fish, the activity on the third day, as per cent 
of initial uptake, was the basis on which evaluation of thyroid destruction was made. 



TABLE I 

Blood chloride of hypothyroid and control fish kept in sea water 
and fresh water for 17 days 



Group 


No. 


Range, gm. 


Mean 


s 


Sea water hypothyroid 


7 


.730-862 


.792 


.045 


Sea water controls 


6 


.731-875 


.804 


.055 


Fresh water hypothyroid 


7 


.432-743 


.655 


.113 


Fresh water controls 


6 


.579-.751 


.683 


.068 



Those treated fish whose activity approached or exceeded the mean activity of the 
controls were not used in later physiological experiments. However, all fish were 
subjected to a histological examination of the thyroid region following autopsy, 
and a comparison of the histological picture with the tracer readings was made. 

Survival in fresh water 

The experiment on osmoregulation was to have continued for six weeks following 
the abrupt transfer of the experimental fish from sea water to fresh water. How- 
ever, after two weeks, three of the sea water hypothyroid fish had died, and three of 
the fresh water hypothyroid fish appeared very ill, showing such symptoms as 
tremors and difficulty in breathing. While these reactions occurred only among 
the treated fish and appeared to be a result of hypothyroidism, the occurrence of a 
sick fish among the sea water controls raised the possibility of some sort of infection, 
and the experiment was ended before any more fish succumbed, seventeen days after 
the transfer to fresh water. 

Although the difference in salinity, per sc, did not appear to influence survival, 
there was a definite correlation between the degree of thyroidectomy (based on 
histological evaluation) and survival in either fresh or salt water. Seven fish 
which died either before or during the experiment, had either no visible follicles, 
or extremely few. 



THYROID FUNCTION IN FUNDULUS 



95 



Blood chloride 

All fish in fresh water showed a definite drop in blood chloride (about 24 per 
cent), but within each group (i.e., salt and fresh water) there was no significant 
difference between the hypothyroid and control fish (see Table I). 

TABLE II 

Growth of treated and control fish during nine months following 
the beginning of I 131 treatment 



Group 


Length incr. % 


s 


Weight incr. % 


s 


Hypothyroid 
Controls 


11.71 
13.02 


3.9 

7.0 


54.66 

57.74 


23.1 
29.9 



Grozvth 

Since there was no increase in length, but a significant weight loss during the ex- 
periment on osmoregulation, the readings used for growth measurements were those 
taken at the beginning of the I 131 injections (October 13, 1956) and at the beginning 
of the osmoregulation experiment (July 3, 1957). The results show no significant 
difference in growth rates, either in length or weight, between the hypothyroid fish 
and the controls (see Table II). 

TABLE III 

Gonadosomatic index of hypothyroid and control fish kept in sea 
water and fresh water for 17 days 



Group 


GSI 


s 


GSI (comb.) 


s 


Sea water hypothyroid 


3.09 


1.57 


2.47 


1.62 


Fresh water hypothyroid 


2.14 


1.39 






Sea water controls 


2.51 


1.49 


2.91 


1.20 


Fresh water controls 


3.01 


.69 







Effect on other organs or tissues that may be dependent on thyroid junction 

The gonadosomatic index (GSI = testis weight/body weight X 100), calculated 
for each group separately, as well as for the combined hypothyroid and combined 
controls, showed no significant differences between any of the groups (see 
Table III). 

Differences in the blood picture were to be found in the combined groups of 
sea water and fresh-water fish, rather than between hypothyroid and controls. 
The greatest difference appeared in the hemoglobin, with the sea water fish showing 
somewhat higher values. There was also a slightly higher red cell count in the 
sea water fish. The white cells showed a very puzzling drop in the sea water 
hypothyroid group, which cannot be accounted for. The thrombocytes show no 
significant differences (see Table IV). 



96 



PATRICIA J. HARRIS 



TABLE IV 

Hemoglobin tilers, red cell, white cell and thrombocyte counts of hypothyroid and 
control fish kept in sea water and fresh -water for 17 days 



Group 


Hb% 


s 


RBC* 


s 


WBC 


s 


Thromb. 


s 


S.W. hypothyroid 


60 


11.3 


4.32 


.69 


5.54 


.48 


12.7 


1.9 


F.W. hypothyroid 


55 


11.4 


4.11 


.54 


9.92 


3.5 


12.6 


1.8 


S.W. controls 


63 


7.9 


4.64 


.83 


9.21 


3.5 


14.0 


4.0 


F.W. controls 


50 


13.6 


4.51 


.86 


9.20 


1.5 


14.2 


2.4 



* RBC in millions, WBC in 1000's, Thrombocytes in 10,000's. 

DISCUSSION 
Osmoregulation 

There is good evidence to support the idea that the thyroid gland plays some 
role in the salinity tolerance of fish. It is well known that many species of fish 
show an activated thyroid gland during spawning migration from the sea to fresh 
water; experimental work of Olivereau (1948) and Leloup (1948) on several 
species of marine teleosts has shown that there is a strong activation of the thyroid 
gland with decreasing salinity of the medium; Fontaine and Callamand (quoted by 
Fontaine, 1956) have shown that thyroxine injections increase the survival time 
of several marine fish when transferred to fresh water. Thus it was of interest to 
test the salinity tolerance of hypothyroid fish, in terms of survival time and blood 
chloride concentrations. 

Survival time, however, proved to be a function of degree of thyroidectomy, 
rather than of the salinity of the medium. Almost all of the treated fish which died 
during the course of the experiment were those with no follicles, or extremely few. 

The blood chloride concentrations were obviously a function of the salt con- 
centration of the medium. All fish in fresh water showed a definite drop in blood 
chloride (about 24 per cent) but there was no significant difference between the 
hypothyroid fish and the control fish. Burden (1956), on the other hand, found no 
change in blood chloride concentration of Fundulus kept in fresh water for eight 
days. Since the same method was used for the chloride determinations reported 
here. Burden's higher results might be attributed to the difficulty in determining 
the end point when using non-deproteinized serum. Sex and season were the 
same, and variations attributable to such causes are excluded. However, Burden's 
experiments were made at a lower temperature (15 C.) and this may have con- 
tributed to a slower period of adjustment to the new external environment. It is 
possible that there is a gradual chloride loss which was not detectable during the 
short period of time employed by Burden, although V. S. Black (1948) demon- 
strated a loss of body chloride in Fundulus heteroclitus transferred directly from 
sea water to fresh water, with a stable level of about 60 per cent reached after the 
fourth day. Bergeron (1956) has shown that Fundulus maintains a constant blood 
osmotic pressure in both salt and fresh water, confirming earlier work of Garrard 
(1935). It may be that the osmotic pressure is maintained by an exchange of 
carbonate ions for chloride, since the alkali reserve of the blood of marine fish is 
lower than that of fresh water species, and there is a relative decrease in the 



THYROID FUNCTION IN FUNDULUS 97 

bicarbonate ion when migrating eels are transferred to sea water (Drilhon and 
Florence, 1936; Fontaine and Boucher-Firley, 1934). Work of Koch and Heuts 
(1942) and Heuts (1943) showed that changes in serum osmotic pressure of 
mature sticklebacks transferred to sea water could not be entirely accounted for 
by changes in blood chloride. In this light, further experiments seem to be called 
for to determine the rate of blood chloride loss following transfer from sea water 
to fresh water, and the factors involved in maintaining osmotic pressure, with 
special emphasis on the alkali reserve. 

Other effects 

The hemoglobin and red cell counts, like the blood chlorides, indicate a de- 
pendence on the medium, with no apparent influence by the presence or absence of 
thyroid tissue. While the increased values found in the sea water fish may be 
accounted for by the lower oxygen content of that medium (see Prosser, Barr, 
Pine and Lauer, 1957), or by an increased energy demand for fish in higher 
salinities, as found by Hickman (1958) in Platichthys stcllatus, the starry flounder, 
the differences are small and cannot be considered significant. 

Other than the effect on survival, where the actual cause of death is unknown, 
there was no apparent effect of hypothyroidism on any of the physiological processes 
studied here. Growth rates were not affected by the hypothyroid condition, nor 
was there any effect on gonadosomatic index. 

The literature devoted to thyroid regulation of growth in teleost fishes is con- 
fusing. With the species Lebistes rcticulatus alone, anti-thyroid drugs have been 
reported to retard growth (Hopper, 1950, 1952; Gaiser, 1952; Vivien and Gaiser, 
1952; Smith, Sladek and Kellner, 1953), while Fortune (1955) found no effect 
on growth in either Pho.vinus or Lebistes (see Pickford and Atz, 1957). Possibly 
collateral toxic effects of the anti-thyroid drugs may be responsible for the retarda- 
tion of growth, and it is of interest that in salmon parr thyroidectomized with I 131 
no effect on growth was found (La Roche and Leblond, 1954). Thyrotropin 
injected into hypophysectomized Fundulus was found by Pickford (1954) to have 
no effect in restoring growth, indicating that the thyroid at least has no direct 
effect on growth. However, in such fish, as in hypophysectomized rats, thyroid 
stimulation undoubtedly enhances the response to exogenous growth hormone 
(Pickford and Atz, 1957, p. 99). 

Data concerning the role of the thyroid in sexual maturation are no less 
conflicting than those on growth. However, studies with anti-thyroid drugs 
strongly indicate that the thyroid is instrumental in the maturation of the gonads 
(reviewed by Pickford and Atz, 1957). While Harrington (1954) and Fortune 
(1955) found that Phoxinus could reach sexual maturity despite treatment with 
thiouracil, it is possible that there was not complete inhibition of thyroid function, 
as in the work reported here, and that this minimal amount of hormone was sufficient 
to permit sexual maturation. 

Screening method 

There appears to be a considerable discrepancy between the evaluation obtained 
with the tracer technique and that from the histological examination, based on 



98 PATRICIA J. HARRIS 

apparent number and size of follicles and cell height. Since there was a time lapse 
of approximately three months between the time of tracer screening and autopsy, 
the most plausible explanation for the difference is the regeneration of thyroid 
tissue in that period. Olivereau (1957), in her work on radiothyroidectomy of 
eels, found that in fish of about 40 grams that had received a total of 1000 /*C of 
I 131 in three doses, functional thyroid tissue had already regenerated after two 
months, and seven months later she found complete absence of the thyroid in only 
three out of fifteen fish. It would thus be advisable to repeat the tracer screening 
just prior to autopsy. 

SUMMARY 

Thyroidectomy of Fundulus heteroclitus was attempted with the use of radio- 
active iodine, administered in three doses of 25, 15, and 10 ;u,C per five-gram fish 
at intervals of five weeks. A screening method was developed whereby the de- 
gree of thyroidectomy could be determined by the rate of activity loss in the throat 
region of the fish following a tracer dose of I 131 . Thyroidectomy was not complete, 
and in some cases there was considerable regeneration. However, in general, the 
resulting condition was one of extreme hypothyroidism, and physiological studies 
conducted with these fish yielded the following results : 

1. There was no special effect on the fishes' ability to survive in fresh water, 
although there seemed to be an impairment of their ability to survive at all, in 
either salt or fresh water. Deaths occurring during the experiment in general 
involved fish with very few follicles or no thyroid tissue remaining. 

2. Blood chloride titers were a function of the salinity of the medium and were 
not affected by lack of thyroid. 

3. Hemoglobin titers and red cell counts indicated an effect of the medium, and 
were not influenced by lack of thyroid. 

4. Hypothyroidism had no effect on growth, either in length or weight. 

5. Hypothyroidism had no effect on the gonadosomatic index. 

LITERATURE CITED 

ARVY, L., M. FONTAINE AND M. GABE, 1956. Fonction thyroidienne et complexe hypothalamo- 

hypophysaire. C. R. Soc. BioL, Paris, 150: 625-627. 

BAKER, K. F., O. BERG, A. GORBMAN AND M. GORDON, 1955. Observations on radiothyroidec- 
tomy of juvenile platyfish. Anat. Rec., 122: 453-454. 
BARRINGTON, E. J. W., 1954. Hormones and control of zoological function. Nature, 174: 

720-722. 
BERGERON, J. A., 1956. A histophysiological study of osmotic regulation in the euryhaline 

teleost Fundulus heteroclitus. Diss. Abstr., 16: 2192-2193. 
BLACK, V. S., 1948. Changes in density, weight, chloride, and swimbladder gas in the killifish, 

Fundulus heteroclitus, in fresh water and sea water. Biol. Bull., 95: 83-93. 
BURDEN, C. E., 1956. The failure of hypophysectomized Fundulus heteroclitus to survive in 

fresh water. Biol. Bull, 110: 8-28. 
CHAMBERS, H. A., 1953. Toxic effects of thiourea on the liver of the adult male killifish, 

Fundulus heteroclitus (Linn.). Bull. Bingham Oceanogr. Coll., 14(2) : 69-93. 
DRILHON, A., AND G. FLORENCE, 1936. Nouvelle contribution a 1'etude physico-chimique du 

sang des poissons. Bull. Soc. Chini. Biol., 18: 1055-1073. 
FONTAINE, M., 1956. The hormonal control of water and salt-electrolyte metabolism in fish. 

Mem. Soc. Endocrinol., 5: 69-81. 



THYROID FUNCTION IN FUNDULUS 99 

FONTAINE, M., AND S. BOUCHER-FIRLEY, 1934. La reserve alcaline du sang des poissons ; ses 

variations au cours des changements de salinite. Bull. Inst. Oceanogr. (Monaco) 

No. 646; 1-12. 
FONTAINE, M., F. DE LA QUERRIERE AND A. RAFFY, 1957. Action de 1'hypophysectomie sur le 

metabolisme respiratoire de 1'anguille (Anguilla angnilla L.). C. R. Soc. Biol. Paris 
151 : 232-233. 
FORTUNE, P. Y., 1955. Comparative studies of the thyroid function in teleosts of tropical and 

temperate habits. /. Exp. Bio!., 32: 504-513. 
FROMM, P. O., AND E. P. REINEKE, 1957. Some aspects of thyroid physiology in rainbow 

trout. /. Cell. Comp. Physio!., 48: 393-404. 
GAISER, M. L., 1952. Effets produits par l'administration prolongee de thiouree et de thyroxine 

chez Lcbistes rcticulatus. C. R. Soc. Biol., Paris, 146: 496-498. 
GARRARD, B. M., 1935. The osmotic pressure of the blood of Funduhis living in salt water and 

in fresh water. /. Physio!., 85: 6P-7P. 
GORBMAN, A., AND O. BERG, 1955. Thyroid function in the fishes Funduhis heteroditus, F. 

majalis, and F. diaphanus. Endocrin., 56: 86-92. 
HAWK, P. B., B. L. OSER AND W. H. SUMMERSON, 1954. Practical Physiological Chemistry. 

13th edition. The Blakiston Co., Inc., New York. 
HEUTS, M. J., 1943. La regulation osmotique chez 1'epinochette (Pygostcus pungitius L.). 

Ann. Soc. Zoo!. Bclg., 74: 99-105. 
HICKMAN, C. P., 1958. The osmoregulatory metabolism of the starry flounder, Platichthys 

stcllatus. Ph.D. Thesis, Univ. of British Columbia. 
HOPPER, A. F., 1950. The effect of mammalian thyroid powder and thiouracil on growth rates 

and on the differentiation of the gonopod in Lebistcs rcticulatus. Anat. Rec., 108: 554. 
HOPPER, A. F., 1952. Growth and maturation response of Lcbistes rcticulatus to treatment 

with mammalian thyroid powder and thiouracil. /. E.rp. Zoo/., 119: 205-217. 
KOCH, H. J., AND M. J. HEUTS, 1942. Influence de 1'hormone thyroiidienrie sur la regulation 

osmotique chez Gasterostcus acitlciitits L. forme c/vninunts. Cuv. Ann. Soc. Roy. 

Bclgiquc, 73: 165-172. 
LA ROCHE, G., AND C. P. LEBLOND, 1954. Destruction of the thyroid gland of the Atlantic 

salmon (Salmo salar L.) by means of radio-iodine. Proc. Soc. Exp. Biol. Mcd., 87: 

273-276. 
LELOUP, J., 1948. Influence d'un abaissement de salinite sur la cupremie de deux Teleosteens 

marins : Maurina helcna L., Labnis bcrgylta Asc. C. R. Soc. Biol., Paris, 142: 
178-179. 
OLIVEREAU, M., 1948. Influence d'une diminution de salinite sur 1'activite de la glande thyroide 

de deux Teleosteens marins : Mitracna helcna L. et Labrus bcrgylta Asc. C. R. Soc. 

Biol., Paris, 142: 176-177. 
OLIVEREAU, M., 1957. Radiothyroidectomie chez 1'anguille (Anguilla an g nill a L.). -Arch. 

Anat. Micr. Morph. Exp., 46: 39-59. 

PICKFORD, G. E., 1954. The response of hypophysectomized male killifish to prolonged treat- 
ment with small doses of thyrotropin. Endocrinol., 55 : 589-592. 
PICKFORD, G. E., AND J. W. ATZ, 1957. The Physiology of the Pituitary Gland of Fishes. 

New York Zoological Soc., New York. 613 pp. 
PROSSER, C. L., L. M. BARR, R. D. PINC AND C. Y. LAUER, 1957. Acclimation of goldfish to 

low concentration of oxygen. Physiol. Zool., 30: 137-141. 
SMITH, D. C., S. A. SLADEK AND A. W. KELLNER, 1953. The effect of mammalian thyroid 

extract on the growth rate and sexual differentiation in the fish, Lcbistes reticulatus, 

treated with thiourea. Physiol. Zool., 26: 117-124. 
VIVIEN, J. H., AND M. L. GAISER, 1952. Action d'un traitement de longue duree par la 

thiouree sur la structure et le fonctionnement de la thyroide et de 1'hypophyse chez 

le Lcbistes rcticulatus, modification du rythme de la croissance staturale et de processus 

de la differenciation genetale puberale. C. R. Acad. Sci., Paris, 234: 1643-1645. 



PROTOPLASMIC MOVEMENT IN THE FORAMINIFERAN, 
ALLOGROMIA LATICOLLARIS ; AND A THEORY OF 

ITS MECHANISM 

THEODORE L. JAHN AND ROBERT A. RINALDI 

Department of Zoology, University of California, Los Angeles 24, California 

Protoplasmic streaming in reticulopodia has been described by numerous in- 
vestigators, most of whom have commented on what an impressive phenomenon 
it is. One of the most detailed but condensed descriptions is that of Leidy (1879) 
who stated (pp. 279-280) : 

"In the emission of the pseudopodal filaments of Gromia terricola, the protoplasm 
pours from the mouth of the shell in a slow manner, and gradually envelopes the 
body. . . .From the protoplasmic envelope delicate streams extend outwardly, at 
first emanating from the front ; they more or less rapidly multiply and radiate in 
all directions. Gradually extending, they fork into branches of the utmost tenuity. 
Contiguous branches freely join or anastomose with one another, and thus establish 
an intricate net, which in its full extent covers an area upward of four times the 
diameter of that of the body of Gromia. The pseudopodal net incessantly changes, 
putting forth new branches in any position, while others are withdrawn, diminishing 
and disappearing in one spot, while it spreads and becomes more complex in 
ano'ther spot. 

"Gromia terricola, with its pseudopodal net fully spread, like its near relatives, 
reminds one of a spider occupying the center of a circular web. If we imagine 
every thread of the latter to be a living extension of the animal under the same 
control as its limbs, the spider would be a nearer likeness to the Gromia. Over 
each and every thread of the pseudopodal net, Gromia has a complete control as 
if the threads were permanently differentiated limbs acted on by particular muscles, 
and directed in their movements by nervous agency. Threads dissolve their con- 
nection and are withdrawn ; new ones are formed and establish other con- 
nections ; they bend ; they contract into a spiral ; they occasionally move like the 
lashing of a whip, and indeed produce almost every conceivable variety of motion. 
Not infrequently spindle-like accumulations of protoplasm occur in the course of 
the pseudopodal threads. Sometimes, through the conjunction and spreading of 
several of the latter together, islet-like expansions occur ; and become centres of 
secondary nets. 

"The pseudopodal extensions of Gromia consist of pale granular protoplasm 
with coarser and more defined granules. The latter are observed to be in incessant 
motion along the course of the threads, flowing in opposite directions in all except 
those of greatest delicacy." 

Another excellent description of foraminiferan movement was provided by 
Jepps (1942) for Polystomella. She described more or less the same activity 
described by Leidy for Gromia. In regard to the pseudopodia she states (p. 624) 
that they "wave about like minute feelers, bending, undulating, quivering, and 

100 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 101 

putting out side branches which meet and fuse and so establish the reticulum". . . 
and that (p. 625) "The pseudopodia show a fairly high degree of stiffness; they 
extend in a straight line as a rule, and may stretch unsupported through the 
water for a distance at least two to three times as great as the shell diameter." 
(The greatest shell diameter she mentioned was about 1.8 mm.) 

All of the above statements of Leidy and Jepps concerning streaming in Grotnia 
and Polystomella are equally true of streaming in Allogromia laticollaris, and 
presumably apply to most, if not all, organisms with reticulopodial nets, with the 
exception that some species have been described as being much more active than 
others, but with no real differences described in the general type of protoplasmic 
activity, sometimes described as "filament streaming" (Fadchenstromung, Engel- 
mann, 1879). 

It has been recognized that the theory of flow caused by differential pressure in 
the plasmasol, which is so well accepted for Amoeba proteus and related genera and 
for Mycetozoa (reviews, Seifriz, 1942; De Bruyn, 1947; Bovee, 1952; Noland, 
1957; also Kamaiya and Kurodo, 1958), would not explain the streaming of 
Foraminifera. This was pointed out clearly by Sandon (1934) who recognized the 
significance of the fact that there is no tube of plasmagel and no evidence of sol- 
gel reversibility in the active pseudopodia, and stated that it was time for another 
explanation to be developed. Considerable doubt concerning applicability of the 
pressure differential theory was also expressed by Noland (1957). So far no 
detailed alternative theory has been proposed to explain the streaming in Foram- 
inifera, although the direction that such a theory should take was clearly pointed 
out by Noland (see below). 

The purpose of the present paper is 1) to extend the observations of Leidy, 
Jepps, and others on movement in reticulopodial nets, 2) to postulate an active 
shearing mechanism for this type of movement, based on observations by the 
present authors and by previous investigators and on recently discovered facts, 
concerning the mechanism of muscular contraction, and 3) to discuss briefly the 
taxonomic implications of the existence of two basic types of protoplasmic move- 
ment in the Sarcodina. 

These two basic types of protoplasmic movement are: a) flow of plasmasol 
caused by differential pressure, which in turn is caused by contraction of a plasmagel 
cortex, and b) flow presumed to be caused by the newly postulated active shearing 
force between two adjacent oppositely moving gel-like filaments of protoplasm in 
the same pseudopod and in the absence of a typical plasmagel cortex. 

Allogromia laticollaris was described by Arnold (1948) who has studied its 
movement and dispersal (Arnold, 1953), variation and isomorphism (Arnold, 
1954), and life history and cytology (Arnold, 1955). Arnold's published studies 
on movement have included changes in location of the organism as a function of 
time and in relation to other organisms and in relation to environmental influences,, 
but do not include a study of movement in the sense of protoplasmic flow and the 
mechanism of flow, as used in the present paper. 

MATERIAL AND METHODS 

Allogromia laticollaris, originally described from Florida (Arnold, 1948), is 
a common foraminiferan of the sea coasts of the United States. It is a large 



102 THEODORE L. JAHN AND ROBERT A. RINALDI 

organism, possessing a globular test with an average diameter of 200 to 400 
microns, and a reticulopodial net of several times this diameter. It has been 
maintained continually in laboratory culture by Dr. Zach Arnold, who has kindly 
provided us with the ancestors of the organisms used in this work. The only 
requirements for prolific culture are sea water, nitrate and phosphate, some soil 
humus, a light source to permit growth of algae used as food, and containers, such 
as finger bowls. Allogromia is tolerant of heat and will reproduce within the 
range of 15 to 34 C. 

Observations were made with the aid of ordinary bright field microscopes, a 
Zeiss inverted microscope with phase and bright field equipment, and a Leitz 
variable phase microscope, which also permitted dark field observations. 

Micrurgical experiments were performed on organisms on open slides in a 
drop of sea water surrounded by a vaseline ring. Coverslips were added later for 
critical microscopic observations. 

RESULTS 

1. General protoplasmic arrangement 

The protoplasm of Allogromia,, like that of all Foraminifera, lies 1 ) within the 
test, 2) around the outside of the test so that the test is more or less internal, and 3) 
in a network of pseudopodia, usually called reticulopodia, which may and usually 
do fuse peripherally to form complicated anastomoses, with numerous nodes, of 
various and continually varying sizes, all of which results in the formation of a 
reticulum, sometimes of very great complexity. 

The following discussion applies specifically to the reticulopodia of medium or 
small diameter, i.e., under 5 ju. Near the body of the organism some of the 
pseudopodia are larger, but at a short distance from the body those over 10 //, are rare. 
It appears as if some of the larger pseudopodia are fundamentally bundles of the 
smaller ones. Some of the following statements, e.g., those concerning absence of 
a non-moving central core, and possible absence of a cell membrane, do not neces- 
sarily apply to the pseudopodia of larger diameter and definitely do not apply to 
the main body of protoplasm or even to the larger nodes of the reticulum. 

Attachment to the substratum occurs in some of the more peripheral nodes of 
the reticulum, and presumably in some of the small peripheral masses of protoplasm 
sometimes found near the ends of the pseudopodia and which do not have side 
branches of reticulopodia, and sometimes under the main body of the organism. 
The active portions of pseudopodia under 5 p. are not attached directly to the 
substratum for much, if any, of their length, and many of them certainly are not 
attached to the substratum at all, except indirectly through the nodes or main 
body of the animal. 

Branching and rebranching may occur throughout the length of the reticulopodia, 
i.e., for several millimeters or more. However, a very high degree of branching 
occurs at the region where the mass of protoplasm merges from the opening in 
the test, so that the pseudopodia are seldom more than 10 //, in diameter at the base 
as they emerge from the general protoplasmic mass. In the early stages of 
emergence or the later stages of withdrawal the appearance of the numerous 
pseudopods sometimes resembles a tuft of brush bristles being pushed free end 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 103 

foremost out from the body or being drawn into the body. A similar description is 
given by Dorlein (1916) for Gromia. 

2. Protoplasmic or filament streaming 

Allogromia is able to extend a network of radial granular reticulopodia irom 
its test as far as 15 millimeters in a circular pattern into its environment. In 
addition to the radial pseudopodia there are pseudopodia which form cross-connec- 
tions between one radial pseudopodium and another. Individual pseudopodia have 
an average diameter of 2-5 ^, but some have a diameter of considerably less than 
1 jj.. Structure of a small branched pseudopodium is shown in Figure 1. 

Streaming can be determined by observing the movement of granules. Previous 
investigators of the Foraminifera have pointed out that streaming is usually in two 
directions simultaneously in the same pseudopodium. One important point is 
that in our observations we have found that streaming is always in tivo directions 
simultaneously in every pseudopod as shown in Figure 2. In radial pseudopods 
one stream goes to^vard the body and the other away from the body, and in 
pseudopods that form cross-connections in the reticulum, each stream goes in the 
direction opposite from the other. We have never been able to observe streaming 
in one direction only. In certain of the smaller reticulopods it sometimes may 
appear superficially that movement is unidirectional because one stream may be 
out of focus or has fewer granules. However, upon careful focussing with bright 
field objectives and more easily with phase objectives we have ahvays been able 
to find movement in the opposite direction even in the finest pseudopods. This is 
an observation that has great theoretical importance as far as the proposed mecha- 
nism of filament streaming is concerned. 

In the medium sized and smaller pseudopodia of Allogromia, the protoplasmic 
material consists of two parts, each more or less the shape of a semi-cylinder, but 
also possibly flattened. In radial pseudopodia one semi-cylindrical portion is 
streaming in the outward or distal direction and the other semi-cylindrical portion 
is streaming in the inward or basal direction. We have not been able to detect a 
gel tube in any of the pseudopodia, even in those of large diameters. Neither is it 
possible to see the line of demarcation between the two oppositely moving layers. 




FIGURE 1. The general shape and structure of the distal portion of one of the finer pseu- 
dopodia, with a single bifurcation into branches about one micron in diameter. Arrows show 
movement of the granules (g), and of a small cytoplasmic mass (c), all of which are attached 
to the actively moving filament (f). 



104 



THEODORE L. JAHN AND ROBERT A. RINALDI 




FIGURE 2. Anastomoses of three reticulopodia. Body of organism at left. Arrows 
show direction of streaming. Node (n) mentioned in text. 



Reticulopodia are extended by a greater flow in the outward direction and at 
least usually are retracted by a greater flow in the inward direction. However, 
by simple visual observation it is not possible to say definitely whether greater 
flow is obtained by greater velocity or by greater cross-sectional area in one of 
the two directions. 

The reticulopods are capable of great activity. They can bend and twist or 
even move laterally as they are extended ; they can split, forming Y junctions, with 
base toward the body, and can anastomose, forming inverted Y junctions, with the 
base toward the periphery. One pseudopodium may split to form two pseudopodia, 
which may be parallel to or divergent from each other, with active two-way stream- 
ing in both. Also, new side projections or branches may be pushed out from a 
pseudopodium, and these projections are sometimes carried by the protoplasmic 
stream, and always exhibit two-way internal streaming themselves. The simple 
Y junctions can migrate along the pseudopodium in either direction and combine 
with other junctions to form X's and more complex types of junctions. Activity, 
always meaning double streaming, regardless of whatever else might be included, 
is at least almost continuous, and no pseudopodia appear to be in a condition of 
rest ; those that are not streaming are invariably moribund. This constant state 
of activity, with continual splitting and anastomosing of pseudopodia, with bending, 
twisting, and lateral movement, can readily give the teleological impression, men- 
tioned by Leidy (1879) that protoplasmic flow is under a most delicate if not 
deliberate central control, of the organism. 

It is assumed that the streaming protoplasm of the reticulopodia is in a gel 
rather than a sol state. This assumption arises from the following observations : 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 105 

1) Pseudopodial extensions only a few microns in diameter may be extended 
at almost any angle into the medium, for at least hundreds of microns, in a relatively 
straight line. They are more numerous along or near the substratum, hut they 
are by no means limited to the substratum, and they certainly are not necessarily 
attached to the substratum. 

2) The pseudopodia are not readily bent or shoved aside when bumped by 
ciliates, microcrustacea, or other swimming organisms, and exhibit a certain degree 
of rigidity. 

3) The granules in the pseudopodia of small diameter, at least under simple 
visual observation (and with only occasional exceptions, mentioned below), seem 
to move in the stream without changing their relative positions and at least about 
the same distance apart. 

The terms "gel" and "sol" are relative ones used to describe different ranges 
of viscosity. Consequently, the line of demarcation between them is not a sharp 
one. As used here, the word "gel" denotes a viscosity high enough to permit 
retention of form under a considerable degree of stress, for example, under the 
conditions mentioned above. In order to explain the observed phenomena the 
assumption of a considerable degree of rigidity seems necessary, and this degree of 
rigidity seems far in excess of that of the sol and comparable to that of the gel 
of Amoeba protens. We have planned a cinephotomicrographic analysis in order 
to elucidate this point. The simple criterion of making an estimate of the degree 
of Brownian movement is not applicable because of the continuous streaming. The 
usual methods of measuring viscosity cannot be used for the same reason. 

Another important observation is that at least in all pseudopodia under 5 ^ 
in diameter all of the visible granules are streaming. There is no tube of gel. 

Neither is there any appreciable space, or any other evidence whatever, for a 
hyaline layer outside of the moving stream. The stream consists of a hyaline gel 
material to which the large granules are attached. 

Furthermore, there is no evidence for a central core of refractive non-moving 
protoplasm (stereoplasm), even when the pseudopodia are observed with the aid 
of dark field and phase equipment. This observation is in agreement with those of 
Doflein (1916) on Groinia diijardini in which he was unable to find a central 
core, and are in contrast to the observations of Doflein (1916), Schmidt (1937), 
Jepps (1942) and others who have described central cores in other genera of 
Foraminifera, which are not so closely related to Allogroinia. 

The granules of the reticulopodia are 2 to 4 p. in diameter, which may be 
greater than the average diameter of the pseudopodium in which they are contained. 
Therefore, they seem to be attached to rather than contained within the clear 
streaming material which comprises the actively moving portion of the pseudopodia. 
Sometimes the granules traveling in one direction can be observed to bump into 
granules traveling in the opposite direction, and to hit with such force that they 
become detached and then attached to the oppositely directed thread, thereby re- 
versing their direction of movement. Less vigorous bumping may result in a back- 
ward shift of the position of a granule in relation to others in the same stream, but 
without a shift into the opposite stream. 

Frequently there are small masses of protoplasm, more or less spindle shaped, 
up to several times the diameter of a pseudopodium, which migrate along with 



106 THEODORE L. JAHN AND ROBERT A. RINALDI 

the protoplasmic stream, either outward eventually to fuse with one of the nodes and 
build up a secondary protoplasmic center from which more pseudopodia may radiate, 
or inward toward the body. This has been described by Leidy (quotation above) 
and others for other species, and is shown in text Figure 1 (C). Such spindles 
are few in newly formed reticula but may be numerous in older ones. 

Cross-connections may remain more or less in one position or may move 
laterally, that is, basally or distally, depending upon whether both ends are in- 
volved w r ith outgoing or with ingoing streams, or may be pulled diagonally, with 
one end moving basally and the other end distally, if connected to one outgoing 
and one ingoing stream. Lateral movement of cross-connections, either basally 
or distally, but usually basally, is very useful in the engulfment of food particles. 

Streaming granules can be seen going through the nodes of the reticulum in 
definite pathways, so that a node with a dozen or more or even with only a few 
radiating pseudopodia (as in Figure 2, n) seems a jumble of moving granules. 
These bump into each other continually, and therefore may seem superficially to 
be merely undergoing Brownian movement. However, closer examination under 
high magnification reveals that the pathways are quite definite and that most of 
the granules are moving in single file. Furthermore, under dark field illumination it 
seems at times as if these pathways are traced by very fine clear fibers to which 
the granules are attached, exactly as in the very fine pseudopodia described above. 

Likewise, near the base of the larger pseudopodia which have many branches, the 
streaming is in the form of many narrow pathways, lying side by side, some with 
granules moving single file and some obviously in multiple lines, some directed 
basally and some distally, but usually with the lines well mixed in arrangement and 
not completely segregated according to direction. Similarly the pseudopodia inter- 
mediate in diameter seem to be made up of the same paired filaments of each of its 
branches, so that it is entirely probable that all except the finest pseudopodia are 
fascicles of the finer units, often with a partial fusion of filaments moving in the 
same direction, but certainly often without a complete fusion. This lack of com- 
plete fusion can account for the existence of more than one speed of streaming, 
as sometimes seen in the pseudopodia of intermediate and of larger diameter. 

For these reasons it seems as if the protoplasmic threads to which the single 
rows of granules are attached are continuous, both in at least some of the nodes of 
the reticulum and in the larger pseudopodia. Therefore, in a certain sense and to 
a very considerable degree the paired hyaline filaments of the finer pseudopodia 
may be considered the fundamental structural units of the reticulum. 

These fundamental streaming units, considerably less than a micron in diameter, 
are optically homogeneous as viewed with bright field, phase, and dark field objec- 
tives. Except for the bumps on the pseudopodia caused by the presence of granules 
the pseudopodia seem to be of quite uniform diameter throughout their unbranched 
portions, but they can differ in diameter from each other and are different in 
diameter before and after branching. We assume that this means that two or 
more of the paired fundamental units are fused to form all but the finest of the 
pseudopodia. In the smallest filaments there are fewer granules, but these granules 
can be traced individually as they move completely to the tip of the smallest clear 
filament and then turn 180 around the tip and start back toward the base of the 
filament. 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 107 

Streaming is about 8 to 15 ^ per second under conditions of our observations, 
but the results of modifying these conditions have not been studied. 

The above description of filament streaming applies not only to the large adult 
organisms, but also to the smaller specimens, and even to the smallest ameboid 
forms that we have identified in cultures. Presumably this includes most of the 
stages of the life cycle. 

Occasionally the end of a reticulopod may be turned back upon itself by 
extraneous forces and then begin to roll up into a spiral so the general form of 
the pseudopodium resembles a more or less flattened coil, with each turn in close 
contact and presumably fused with the adjacent turns, and with two-way streaming 
continuing for a number of minutes. The coiling seems to result when the tip of 
a pseudopod is bent so that it comes in contact with the outgoing protoplasmic 
stream. Two-way streaming continues in all parts of the spiral, and the coil 
continues to increase in size as long as contact is maintained only with the out- 
going stream. It is possible that this is what Leidy meant when he stated (quoted 
above) "they contract into a spiral." Coiling requires about a minute, and a few 
minutes later the coil degenerates into a simple protoplasmic mass and then de- 
velops new pseudopodia. 

3. Floiv on the reticulopodial surface 

Streaming of foreign material can easily be demonstrated on the surface of 
reticulopodia in Allogroniia by use of a dye, e.g., Evans brilliant vital red, which is 
insoluble in sea water. The dye particles, which may be ten or more times the 
diameter of the pseudopodia, stick to the protoplasmic surface and flou> along tvitli 
the protoplasm (Fig. 3). Individual dye particles may stick to either the distally 
or the basally directed streams and therefore may pass each other going in opposite 
directions. The same phenomenon can be demonstrated less colorfully by means 
of particles of finely ground glass. This is apparently a non-specific reaction, and 
occurs normally with all materials (primarily algae) that serve as the food source 
for the organisms. Normally food particles are carried by the basally directed 
stream until engulfed by the main body of protoplasm or by the distally directed 
stream until engulfed by one of the major distal masses in the network or until 
it is redirected into a basally directed stream. 

4. Movement of the entire organism 

When the organism is moving there is no apparent contraction of the anterior 
pseudopodia, as is well known for other shelled rhizopods (e.g., Arcclla). The 
anterior pseudopodia, which apparently pull the body and test forward, continue 
to have a rapid, and perhaps have a more rapid, two-directional streaming while the 
body is moving. The most reasonable explanation seems to be that the distal 
portion of the reticulum is attached to the substrate, that the distally streaming 
protoplasm is actually pulling the body forward, and that the motive power is the 
same active shearing process responsible for the streaming. The possibility should not 
be overlooked that movement may also involve some type of rapid contraction of 
the larger pseudopodia, as mentioned by Doflein (1916), Schmidt (1937), Jepps 



108 



THEODORE L. JAHN AND ROBERT A. RINALDI 




e 



FIGURE 3. Photographs showing movement of dye particles attached to reticulopodia. 

a, Note large dye particles at lower right, and small ones scattered; Allogromia at upper left. 

b. Thirty minutes after a. Note movement of large particle toward opening in test of Allo- 
i/rmnia. c. Thirty seconds after b. d and c. Dye particles moving along the pseudopodia. 
Note lack of engulfment of particle. 



(1942), and others for other Foraminifera. However, we have not seen any 
movement which could be interpreted in this manner. 

The tensile strength of active pseudopodia can be demonstrated by means of 
displacement with microneedles or movement of the medium. If a microneedle 
is entangled in some of the pseudopodia. the whole organism can be broken loose 
from the slide, leaving behind some fragments of protoplasm at the points of 
attachment, mostly in the peripheral portions of the net. Then the organism can be 
held by the microneedle attached to the pseudopodia while the slide is moved by 
the mechanical stage of the microscope. The pseudopodia which are not attached 
to the needle are dragged by the medium and trail as loose lines. When the 
direction of the slide is reversed the relative positions of the body and of the 
trailing pseudopodia are also reversed. 

However, under these conditions the protoplasm in the pseudopodia attached 
to the needle, even if only a single pseudopodium, continues to flow, and in both 
directions. 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 

5. Formation and behavior of protoplasmic fragments or satellites 

We have confirmed the experiments of Grell (1956) that if the peripheral 
portion of a pseudopod of Allogroinia is amputated, the fragment can fuse with the 
pseudopodial stump and again become part of the organism. 

Furthermore, by repeated cutting of pseudopodia and removal of the main body 
of the organism we have been able to obtain small fragments of protoplasm. In 
small segments of a pseudopod, about 40 ^ long, cut at both ends, two-way stream- 
ing was observed immediately after the cuts were made. This proves that the 
connection to the main body of the organism is not necessary for two-way stream- 
ing. However, such fragments soon become rounded, forming what we have 
termed "protoplasmic satellites." These satellites can persist for about forty 
minutes under conditions of our experiments. During this time they become 
stellate in appearance by extending several fine pseudopodia which are capable of 
bending, twisting, and anastomosing, and which exhibit two-way streaming. In 
small satellites most of the larger granules often remain in the central mass of the 
satellite and usually are rare in the pseudopodia (Fig. 4). The larger satellites 
have granular pseudopods. Satellites are capable of fusing with the parent organ- 
ism and also with each other. Furthermore, upon disintegration the pseudopods 
of satellites have been seen to split into two filaments, free of granules. 

Satellites also may be formed by rapidly crushing the organism between slide 
and coverslip. A rapid crushing causes most or all of the protoplasm of the body 
to dissolve in the sea water, but this does not necessarily result in solution of the 
uncrushed portion of the network. The larger nodes become the center of 
stellate protoplasmic masses, sometimes with dozens of radiating pseudopodia and 
numerous cross-connections, all of which exhibit two-way streaming. These may 
continue to stream for at least several hours. The nodes and the main body of 
these satellites also exhibit the definite granule pathways described for the intact 
organism. W r hen the organism is crushed some of the pseudopodia may become 
completely isolated, and these also exhibit two-way streaming and may form small 
satellites similar to those obtained by micrurgical methods. 




FIGURE 4, a, b, AXD c. Protoplasmic satellites of Allogroinia. Note anastomoses and 
orientation of pseudopods exactly as in intact organism. 



110 THEODORE L. JAHN AND ROBERT A. RINALDI 

Satellites which behave exactly as those described above can also be formed 
by quickly detaching the organism after it has extended pseudopods and attached 
them to the substrate, so that the attached ends remain attached to the substrate 
and become satellites. 

In general, in so far as the size of the satellite permits, streaming in satellites 
is exactly the same as that in the intact organism. 

DISCUSSION 

1. Inadequacy of flic pressure floiv theory applied to AHogroinia 

The idea that protoplasmic flow in AHogroinia could be caused by differential 
pressure in a sol resulting from contraction of a partially surrounding gel tube 
does not seem to be in accord with the following facts : 

1 ) Absence of a gel tube in the pseudopodia, and even the possible absence of 
a pseudopodial membrane (see below). 

2) Presence of two-way streaming in all active pseudopodia, even those of 
smallest diameter, including all of the radial and all types of cross-connecting 
pseudopodia, both in the intact organism and in even the smallest protoplasmic 
satellites. 

3) Presence of definite criss-crossing pathways for granules through nodes 
of the reticulum. 

4) Presence of numerous parallel granule pathways in the larger pseudopodia, 
without segregation of pathways on the basis of direction, and without any evi- 
dence of gel tubes through which the granules could flow. 

5) Presence of two-way streaming in freshly cut segments of pseudopodia. 

6) Reversal of direction of granules at the tip of each pseudopodium. 

Therefore, it is assumed that the theory of protoplasmic flow caused by dif- 
ferential pressure in a sol cannot apply to the protoplasmic flow of the reticulopodia 
of Allogroniia. Likewise, it seems as if the pressure flow theory can not apply to 
the same general type of streaming in other Foraminifera, which has been described 
by other investigators. Therefore, a new theory is proposed. 

2. Theory of the uiecJianisiu of filament streaming in Allogrouiia 

The mechanism proposed to explain this type of movement is the existence of 
active shearing forces located in the reticulopodia between two paired filaments of 
protoplasmic gel, or rather between two portions of the same filament. These 
forces act longitudinally and oppositely and thereby produce the typical two-way 
streaming. 

This is shown diagrammatically in Figure 5. In its simplest form the pseu- 
dopodium includes only two approximately semi-cylindrical, but possibly flattened, 
filaments of protoplasmic gel, labelled f, and f 2 . Filament f t is the outgoing 
portion, and f 2 is the ingoing portion, as denoted by the large arrows. These are 
continuous at the tip of the pseudopodium and are therefore parts of the same 
filament. There may be some reorganization of the outgoing protoplasm at the 
tip before it becomes ingoing, but we have not been able to determine the degree 
of reorganization by simple visual methods. 




FORAMINIFERAN PROTOPLASMIC MOVEMENT 111 



fl 




c> 



f2 




A B 

FIGURE 5. Arrangement of actively moving filaments (fi and f 2 ) of pseudopod. A, cross- 
section ; B, side view. Direction of movement shown by arrows. The moving material is 
assumed in the diagram to be in the form of a semi-cylindrical filament, turned back upon 
itself at the tip, with the flat surfaces opposed. The shearing force is assumed to be between 
the adjacent surfaces and is designated by the short curved lines. 

The small hook-like structures represent the active shearing mechanism, which 
at present is completely unknown. It must act in the direction indicated by the 
small arrows, and it must be capable of acting against the same mechanism on the 
opposite semi-cylinder. All other properties of the mechanism remain undeter- 
mined. As diagrammed here one could imagine the gel filament with its active 
mechanism to be similar to a millipede of relatively enormous and indeterminate 
length, folded back upon itself at the tip and with the legs of one portion of the 
body pushing against those of another portion throughout the length of the 
pseudopodium. 

The basic idea is not new, but was proposed in somewhat different form by No- 
land (1957), in a general manner and without detailed application. In discussing 
the structure of protoplasm in Amoeba protcus Noland stated (p. 4), ". . . the endo- 
plasmic molecules, some of them at least, must be quite linear in form. If one lets 
his imagination have free play he might compare the molecules of the plasmasol to a 
writhing mass of centipedes, each hanging on to his neighbors with a leg or two, 
but often losing hold and grasping any other one that comes with reach. Thus 
the whole mass, though moving, would maintain a certain coherence, so that a tug 
at one centipede would be communicated some distance into the mass." Further- 
more, in expressing doubt that the pressure differential theory could be applied to 
reticulopodia, he stated (p. 6), "To revert to our centipede similes, what we need 
are molecules that can orient themselves in one direction and crawl forward on any 
solid surface, while others of the same sort crawl forward on the back of the first 
layer." 

The present proposal, aside from substituting millipedes for centipedes, but 
otherwise continuing the simile, assumes that instead of one animal moving on the 
back of another, that the distally bound millipede is merely a more posterior segment 
of the basally bound millipede and that "they" are moving with the feet of one 
segment pushing backward against the feet of the other. One very important de- 
velopment since Noland wrote his paper on this subject is that it has now been defi- 
nitely proven that in striated muscle we do have a mechanism that can "crawl" in the 
sense used by Noland, not on any solid surface, but on a highly specialized surface 
(review, A. F. Huxley, 1957). The existence of such a solid foundation changes 
what might have been considered a rather speculative hypothesis into a well founded 
theory, worthy of considerable development and investigation. 



112 THEODORE L. JAHN AND ROBERT A. RINALDI 

The completely unknown part of the mechanism is what the "millipede" uses for 
legs. In striated muscle the connections from the myosin fiber to the actin fiber, 
that is, the connections which actually exert the force involved in the sliding motion, 
may be seen with the aid of an electron microscope. It is also obvious from chemi- 
cal and cytological studies that ATP is used by the sliding mechanism (review, 
A. F. Huxley, 1957). Otherwise the status of definite knowledge is not much more 
advanced for muscle than for reticulopodia ; the actual mechanism of the "legs" is 
unknown in both. Furthermore, in reticulopodia we can observe this sliding 
of one fiber upon another continuously by means of an ordinary microscope for 
hours, or even for days, or as long as the patience of the observer persists. 

In addition to the active shearing force there obviously must be some mechanism 
that holds the two active surfaces together and more or less in contact with each 
other. If the active force is transmitted through protein bridges similar to those 
which can be demonstrated in electron micrographs of striated muscle (H. E. Huxley 
and Hanson, 1955 ; A. F. Huxley, 1957), then the same bridges serve both purposes, 
and the two mechanisms are really two aspects of the same. 

It is obvious that the word "streaming" when used in conjunction with the pres- 
ent theory of filament streaming is used in a very special sense, and only for histori- 
cal reasons. It does not mean the streaming of a sol, as in Amoeba or in Physarum, 
or even in Foraminifera as assumed by previous investigators. It means the longi- 
tudinal movement of gel-like filaments, or threads, of protoplasm, carried along by 
active processes on the adjacent surfaces of the threads themselves, that is, on the 
surfaces between the two parallel threads which are "streaming" in opposite 
directions. 

One unanswered question concerning which we have no evidence is whether the 
filaments of gel remain essentially as filaments when the material is all inside of the 
body, or whether they are in the form of undifferentiated protoplasm, which is 
re-formed into filaments when the pseudopodia are re-extended. The tuft-like ap- 
pearance of many simultaneously emerging pseudopods might suggest that the 
structure is not completely destroyed within the body. 

Another unanswered question is how the organism is able to extend or retract 
all pseudopodia simultaneously. Is there a central control of these movements? 
Perhaps, and perhaps not. However, if there is a central control, the present theory 
offers a very simple explanation. The outgoing thread of gel must be formed from, 
or at least released from, the body of the animal, and the ingoing thread must be 
incorporated into the body, either as a stored filament or as dedifferentiated proto- 
plasm. Extension and retraction could be controlled very simply if there were 
control of either the release or the re-incorporation of the filaments, or of both 
of these processes. 

One characteristic of reticulopodia which perhaps should be emphasized is that 
in at least all of the finer and possibly in all of the pseudopods there is no tube of 
plasmagel as in Amoeba proteus and in Pliysaruni. This general characteristic has 
been noted by most of the earlier investigators (e.g., Doflein, 1916; Schmidt, 1937; 
Jepps, 1942). The fact that a gel tube is absent is of great theoretical importance 
from the viewpoint of explaining the mechanism of movement. The absence of a 
gel tube definitely rules out the pressure differential theory, unless one assumes that 
the cell membrane is mechanically capable of performing the functions ordinarily 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 113 

assigned to the tube. The question of the structure of the membrane and even the 
possibility of its non-existence on the reticulopodial surface is discussed below. 

Leidy stated that streaming was always in two directions in all pseudopodia "ex- 
cept those of greatest delicacy." It is possible that he was not able to detect 
oppositely directed movement because of a scarcity of granules in the opposite 
stream. However, it is also possible that some of the "pseudopodia of greatest 
delicacy" are formed by splitting of an undirectional thread from a pseudopodium 
and that this thread is merely pushed passively into the medium. The present 
authors have seen a few temporary pseudopods which could be interpreted in this 
manner. However, such a thread, at least according to the present theory, could 
not develop into an active pseudopodium without developing a return flow. 

The theory, as outlined above, will explain all of the facts as we know them, 
including the six items listed above (Discussion, section 1) which cannot be ex- 
plained by the theory of differential pressure in a sol. 

3. Application of the theory to other materials 

It seems as if the present theory could apply in slightly modified form to the 
reticulopodia or rhizopodia which have a central core of stereoplasm and also to 
axopodia, which have a well denned axial filament. From one point of view the 
only change necessary is to assume that the active mechanism is capable of moving 
along the surface of the stereoplasm or the axoneme rather than only along the 
active surface of an oppositely directed filament. This is the equivalent of intro- 
ducing Noland's idea of molecules crawling upon a solid surface. Another pos- 
sibility is that the active shearing occurs not between the rheoplasm and the stereo- 
plasm or axoneme, but between the outgoing rheoplasm and ingoing rheoplasm 
where they come in contact with each other, peripherally to the stereoplasm or 
axoneme. The central cores and the axonemes may serve architectural func- 
tions, but since they do not exist in Allogromia it is obvious that they are not neces- 
sary to explain either the stiffness or the streaming that exists in the reticulopodia 
of Allogromia. 

Previous investigators (e.g., Doflein, 1916) have pointed out that as the 
pseudopod is extended, more stereoplasm is added at the distal end of the core and 
that this must come from the rheoplasm. If so, and if the present theory also 
applies to species which have a central core, the core is composed merely of tempo- 
rarily inactivated fibers of rheoplasm, that is, of the hyaline material without the at- 
tached granules. 

Many of the stamen hair cells of plants have two-way streaming in fine threads 
of protoplasm which go across (that is, through) the cell vacuole (e.g., Zebrina, 
Tradescantia ) . In many instances it is obvious that streaming is in both direc- 
tions. However, streaming sometimes appears to be undirectional because the 
cytoplasm from one direction has few granules and is therefore difficult to detect by 
observation. It seems in such cases that the observers (especially students in ele- 
mentary classes) are often confused when a few large granules or even chloroplasts, 
are seen moving apparently upstream. According to the present theory, the 
granules are merely moving along in the colorless stream of a granular cytoplasm. 
The granules apparently take no active part in the streaming process, but purely a 
passive one, as in the Foraminifera. 



114 THEODORE L. JAHN AND ROBERT A. RINALDI 

The idea of an active shearing force is not limited to filament streaming but may 
also be applied to the contraction that occurs in the posterior end of Amoeba 
protcus. In various other ameboid organisms and in leucocytes, it seems quite 
definite that contraction of the protoplasm does occur (Mast, 1926; Lewis, 1931; 
review, De Bruyn, 1947), but the supposed contraction of the protein molecules of 
the gel, as proposed by Goldacre and Lorch (1950) is merely a theory based on the 
old idea that muscle contracts by a folding or a spiralling of linearly arranged 
elongated protein molecules. Allen (1955) sucked protoplasm of A. protcus into 
capillary tubes and then observed two-way streaming, during which each stream 
behaved as a structural unit, which could became subdivided to form narrower 
streams, so small as to contain only a single row of granules. This seems similar 
to the two-way streaming in the reticulopodia of Allogromia. If this type of 
streaming can occur in the normal endoplasm of Amoeba it could be the physical 
basis of the contractile process. The possibility of such an explanation is mentioned 
by Noland (1957, quotation above). 

Another alternative to the folding mechanism proposed by Goldacre and Lorch 
(1950) is the limited folding or sliding-folding mechanism suggested earlier by 
Frey-Wyssling (1948). According to this suggestion the sliding is caused by a 
wave of limited folding which passes along an elongated molecule. It is really an 
explanation, without experimental evidence, of how a sliding or creeping of one 
molecule along another might occur. 

The contraction of the transparent pseudopodia of Arcella might also involve an 
active shearing rather than a molecular shortening, but on this point there is no 
evidence whatsoever. 

The idea of an active shearing force can be applied to cyclosis whenever it occurs, 
e.g., in Nitella, Chara, Elodca, Paramecium, Vorticclla, etc. The only assumptions 
needed are that the moving material has a high viscosity and that the active force 
is exerted tangentially on its outer surface by the inner surface of the fixed cortical 
gel, or that the force is exerted from the moving high viscosity sol or gel on to the 
fixed cortex. Similar assumptions can also explain the rotatory movements of frag- 
ments of cells of Nitella and Chara described by Yatsuyangi (1953a, 1953b). 

This possibility is well demonstrated in the work of Jarosch (1958) who has 
succeeded in isolating filaments from the protoplasm of Toyellopsis (Characeae). 
Jarosch has shown that these fibers are actively motile because they produce parallel 
displacement forces, that they have an affinity for small microsomes (cf., granules 
of Allogromia), that they fuse into thick bundles, that they have the consistency of 
a gel, and that they possess elasticity. In brief, Jarosch has described in the fila- 
ments of Toyellopsis exactly the properties needed to explain streaming in Allo- 
gromia. In an earlier note he also mentioned the possibility of applying a similar 
theory to ciliary movement and to movement in axopodia (Jarosch, 1957). 

In summary, it seems as if we can postulate two major types of protoplasmic 
movement in the Sarcodina, and possibly only these two types for all protoplasmic 
movements. These are : 

1) Flow of a sol caused by differential pressure as a result of contraction of a 
partially surrounding gel, and 

2) Movement of gel, which in Allogromia (and probably in plant hair cells) is 
in the form of paired filaments of gel which move by means of active shearing 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 115 

forces, acting oppositely and longitudinally along the adjacent surfaces between the 
threads. In reticulopodia of most other Foraminifera the active force of the fila- 
ments, instead of acting on the other filament, might act against the stereoplasm, 
or, in axopodia, against the axoneme. In cyclosis the force from one layer of gel 
may act against that of another layer of thick sol or gel, or vice versa. 

Furthermore, we have the possibility that the gel contraction that causes pres- 
sure flow may involve an active shearing mechanism as the contractile process of 
the gel. 

4. Ta.vonomic significance of the tzvo types of protoplasmic streaming 

Certainly, if we consider only the Sarcodina as a group, we can state that we 
have both pressure flow and filament streaming as the two types of protoplasmic 
movement. If we ignore the untested possibility that both of these might be 
fundamentally the same, and consider them to be distinct, we are faced with the 
very interesting question of how they are distributed taxonomically. If this is a 
fundamental difference in the type of movement, perhaps all Sarcodina which have 
only filament streaming should be placed in a separate group from those which ex- 
hibit only pressure streaming, and those which have both should be placed in an 
intermediate group. 

If this were done we would have to consider organisms with filopodia, reticu- 
lopodia, rhizopodia, and axopodia more closely related to each other than to those 
which have lobopodia only. This would necessitate changes in the well known 
separation of the Sarcodina into Rhizopodea and Actinopodea, and a re-assortment 
of the organisms placed in the rhizopodean order Proteomyxida, most of which 
have filament streaming. Such a thorough reorganization does not seem justified 
at present. However, the lines of demarcation between the current orders of the 
Sarcodina are so far from being satisfactory that a reclassification may well be 
contemplated in the future when more information becomes available. 

5. Nature of the reticulopodia} surface 

The nature of the protoplasmic surface of reticulopodia has been the subject 
of comment by various investigators. It is commonly agreed that most but not all 
objects normally brought into contact with the pseudopodia will stick, and further- 
more, those that stick zvill be carried along in or on the protoplasmic stream, 
Arnold (1953) mentioned this fact in regard to the food material used by Al- 
logroinia. In our experiments the insoluble dye and the glass particles stuck and 
were carried in both directions by the protoplasmic streams. Some particles do 
not stick tight enough to be engulfed. For instance, Sandon (1934) cites the fact 
that flagellates often stick to foraminiferan pseudopodia, are carried for a consider- 
able distance, and then break loose and swim away. Sandon interprets this as 
evidence of a tough protective pellicle over the pseudopod. However, it could 
better be interpreted as evidence that the membrane is either thin and delicate 
as well as sticky, or even non-existent. 

If one assumes that the streaming protoplasm is actually a thread of gel, then 
there is no need of assuming any membrane whatever in order to explain the 
mechanics of streaming. In fact, the principal reason for assuming the existence 



116 THEODORE L. JAHN AND ROBERT A. RINALDI 

of a membrane is that to assume the opposite would be physiological heresy. It 
seems as if the assumption that a membrane does not exist is just as radical as the 
assumption of the existence of an active shearing force would have been a few 
years ago. 

If we assume that a reticulopodial membrane does exist and that foreign particles 
which move with the flow are sticking to the membrane, then the membrane itself 
must move with the stream, as assumed by Sandon (1934). If so, then the portion 
of the membrane over the outgoing stream is moving oppositely from that over the 
ingoing stream, and the membrane, if it is to be considered a single membrane, must 
be sheared along two longitudinal lines, one on each side, where the circumferential 
margins of the oppositely moving streams are closest to each other. Therefore, 
along these two lines the membrane is continuously subject to longitudinal shearing 
and must be very highly labile ; for all mechanical purposes such a membrane might 
as well not exist. The assumed membrane, then, as far as structure is concerned, 
becomes the membrane, not of the pseudopodium but of each protoplasmic stream, 
and it could well be merely the surface of the gel which makes up the moving proto- 
plasmic thread. 

On the other hand, if we assume that the foreign particles penetrate but are not 
completely covered by the membrane and stick to the moving gel thread, and that 
the membrane does not move, then the large particles, of a size, let us say, ten times 
the diameter of the pseudopodium, must split the membrane as it moves with the 
stream; and the membrane must be re-formed behind the particle. If we assume 
that these large particles upon contact with the gel are immediately covered by some 
sort of a membrane, then we must assume that the membrane is very rapidly 
formed at the anterior edge of the particle, and destroyed in the posterior edge of the 
particle, and this seems even more unlikly. 

For these reasons it seems best to assume tentatively that the membrane of the 
small reticulopodia may be merely the surface precipitation membrane, of possibly 
merely the surface, of the moving thread of protoplasmic gel that constitutes the 
stream. 

This tentative assumption, if made in addition to the theory of mechanism out- 
lined above, results in perhaps the simplest overall concept of reticulopodia that is 
possible . . . merely two adjacent naked filaments of gel, or more exactly two 
parts of the same filament, pushing against each other longitudinally along their 
adjacent surfaces, with resultant two-way streaming. This concept may be over- 
simplified, but for the present there seems to be no reason for assuming without 
evidence the existence of any of the complicating structural considerations found in 
other material. 

SUMMARY 

1. Protoplasmic streaming in the reticulopodia of Allogroinia laticollaris is de- 
scribed. Streaming is always a two-directional movement of two threads of 
plasmagel which together with attached granules seem to make up the entire 
structure of the reticulopodia. There is no outer tube of gel, no central core of 
optically refractive material, and no space for an outer hyaline layer. This seems 
to be the simplest form of filament streaming known to exist. 



FORAMINIFERAN PROTOPLASMIC MOVEMENT 117 

2. It is proposed that the mechanism of filament streaming in Allogromia con- 
sists of active shearing or parallel displacement forces located between the adjacent 
surfaces of the two gel filaments, acting longitudinally and oppositely from one 
filament to the other so as to produce two-way streaming. 

3. Possible applications of the theory of active shearing forces to protoplasmic 
movement in other materials are discussed. 

4. It is suggested that in Allogromia the gel threads of the reticulopodia may not 
be covered by a typical cell membrane but by a surface precipitation membrane or 
that the membrane may be merely the surface of the gel filament itself. 

5. The possible taxonomic significance of the existence of two major types of 
protoplasmic movements, namely, pressure flow and filament streaming, is discussed. 

LITERATURE CITED 

ALLEN, R. D., 1955. Protoplasmic streaming in amoeba. Biol. Bui!., 109 : 339-340. 

ARNOLD, Z. M., 1948. A new foraminiferan belonging to the genus Allogromia. Trans. Aincr. 

Micr. Soc., 67: 231-235. 
ARNOLD, Z. M., 1953. An introduction to the study of movement and dispersal in Allogromia 

laticollaris Arnold. Contrib. Cush. Found. For am. Res., 4 : 15-21. 
ARNOLD, Z. M., 1954. Culture methods in the study of living Foraminifera. /. Paleo., 28: 

404-416. 
ARNOLD, Z. M., 1955. Life history and cytology of the foraminiferan Allogromia laticollaris. 

Univ. Calif. Publ. in Zool, 61 : 167-252. 
BOVEE, E. C, 1952. A possible explanation of the gel-sol changes in amoeboid movement based 

on the muscle contraction theories of Szent-Gyorgi. Proc. loiva Acad. Sci., 59 : 

428-434. 

DE BRUYN, P. P. H., 1947. Theories of amoeboid movement. Quart. Rev. Biol., 22: 1-24. 
DOFLEIN, F., 1916. Studien zur Naturegeschicte der protozoen. VII. Untersuchungen iiber 

das Protoplasma und die Pseudopodien der Rhizopodien. Zool. Jahrb. (Anat.), 39: 

335-384. 

EXGELMANN, T. W., 1879. Flimmer und Protoplasmabewegung. Hermann Handb. d. Physio- 
logic, 1: 343-408. 
FREY-WYSSLING, A., 1948. Submicroscopic Morphology of Protoplasm and its Derivatives. 

Elsevier Press, New York. 
GOLDACRE, R. J., AND I. J. LORCH, 1950. Folding and unfolding of protein molecules in relation 

to cytoplasmic streaming, amoeboid movement, and osmotic work. Nature, 166 : 

497-500. 

GRELL, K. G., 1956. Protozoologie. Springer-Verlag, Berlin. 
HUXLEY, A. F., 1957. Muscle structure and theories of contraction. Prog, in Biophysics, 7 : 

255-318. 
HUXLEY, H. E., AND J. HANSON, 1955. The structural basis of contraction in striated muscle. 

Soc. Exp. Biol. (Gt. Brit.) Symp., 9: 228-264. 
JAROSCH, R., 1957. Zur Mechanik der protoplasma Fibrillenbewegung. Biochcm. et Biophys. 

Ada, 25 : 204-205. 

JAROSCH, R., 1958. Die Protoplasmafibrillen der Characeen. Protoplasma, 50 : 93-108. 
JEPPS, M. W., 1942. Studies on Polystomella Lamarck (Foraminifera). /. Mar. Biol. Assoc., 

25 : 607-666. 
KAMAIYA, N., AND K. KURODO, 1958. Studies on the velocity distribution of the protoplasmic 

streaming in Myxomycete plasmodium. Protoplasma, 49 : 1-4. 

LEIDY, J., 1879. Fresh-water rhizopods of North America. Kept. U. S. Geol. Surv., 12 : 324 pp. 
LEWIS, W. H., 1931. Locomotion of lymphocytes. Bull. Johns Hopkins Hosp., 49 : 29-36. 
MAST, S. O., 1926. Structure, movement, locomotion, and stimulation in Amoeba. J. Morphol, 

41 : 347-425. 

NOLAND, L. E., 1957. Protoplasmic streaming: a perennial puzzle. /. ProtosooL, 4: 1-6. 
SANDON, H., 1934. Pseudopodial movements of foraminifera. Nature, 133 : 761-762. 



118 THEODORE L. JAHN AND ROBERT A. RINALDI 

SCHMIDT, W. J., 1937. tlber den Feinbau der Filopodien, inbesondere ihre Doppelbrechung bei 

Miliola. Protoplasma, 27: 587-598. 

SEIFRIZ, W., 1942. The Structure of Protoplasm. Iowa State College Press, Ames. 
SEIFRIZ, W., 1954. The rheological properties of protoplasm. Chapter 1, pp. 3-156. In: 

Deformation and Flow in Biological Systems, A. Frey-Wyssling, ed., Interscience 

Publishers Inc., New York. 
YATSUYANGI, Y., 1953a. Recherches sur les phenomenes moteurs dans les fragments de proto- 

plasme isoles. 1. Mouvement rotatoire et le processus de son apparition. Cytologia, 

18: 146-156. 
YATSUYANGI, Y., 1953b. Recherches sur les phenomenes moteurs dans les fragments de proto- 

plasme isoles. 2. Mouvements divers determines par la condition de milieu. Cyto- 
logia, 18 : 202-217. 



OBSERVATIONS ON THE NUTRITION OF THE LAND 

PLANARIAN ORTHODEMUS TERRESTRIS 

(O. F. MULLER) 1 

J. B. JENNINGS 
Department of Zoology, University of Leeds, England 

The Turbellaria as a class are carnivorous and previous investigations have 
shown that the range of prey available to these relatively simple animals has been 
greatly increased through the development of efficient feeding mechanisms in 
the form of progressive elaborations in the structure and use of the pharynx 
(Jennings, 1957). The Tricladida in particular, with the protrusible cylindrical 
type of plicate pharynx, are active and successful predators and the nutrition of 
the aquatic forms has already received much attention (Willier, Hyman and 
Rifenburgh, 1925; Kelley, 1931; Jennings, 1957). Little is known, however, of 
feeding and digestion in those triclads of terrestrial habit and hence a representative 
of this group, Orthodemus tcrrestris (O. F. Muller), has been examined to gain 
some measure of the influence of a land existence upon the typical pattern of 
triclad nutrition. 

MATERIALS AND METHODS 

Orthodemus tcrrestris occurs beneath limestone debris and fallen branches in 
the Fairburn, Malham and Settle districts of Yorkshire. Specimens collected 
throughout the year were presented in the laboratory with representatives of the 
fauna associated with them under natural conditions, and their food preferences 
and methods of capture and ingestion of the selected prey observed. The course of 
digestion was traced by histological examination of series of individuals previously 
starved to clear the gut and then killed at progressive intervals after being fed on 
either the natural food or test foods such as frog blood and boiled starch paste. 
After fixation in Susa at 30 C. sections were cut at 8 /* and stained with haema- 
toxylin and eosin, Feulgen, benzidine, periodic acid-Schiff, Alcian blue and Lugol's 
iodine. Food reserves were studied after fixation in Flemming (for fat) and 90% 
alcohol containing 1% picric acid (for carbohydrates and proteins), sections of 
specimens fixed in the latter reagent being stained by the Best's carmine, P.A.S. 
and modified Millon methods. 

OBSERVATIONS 
The food and feeding methods 

Orthodemus feeds mainly upon slugs (Anon sp.) and small earthworms. It 
will also attack small arthropods such as collembola, wood-lice, insect larvae and 
myriapods if these are injured or incapacitated in any way but normally they are 
too active for capture by the flatworm, which lacks any trapping or snaring 

1 Xe\v combination by Hyman (1954). 

119 



120 



J. B. JENNINGS 



devices. The mucus produced during locomotion quickly dries out and plays no 
part in the capture of food, unlike that of some aquatic triclads which persists 
about the habitat as sticky strands to entangle insect larvae and crustaceans. The 
prey appears to be found by chance and starved individuals show no awareness of 
the proximity of either damaged or intact animals until random movements bring 
them into direct contact. 

When an appropriately sized slug or earthworm is encountered the flatworm 
rapidly extends across the width of the prey until it can grip the substratum on 
each side and so pin the captured animal beneath the arched body. The grip on 
both prey and substratum is helped by copious secretions of mucus from the 
ventral surface and is so effective that prey rarely escape. Movement across the 
prey continues until the mouth, which lies ventrally approximately one-third of 
the body length from the posterior end, can lie brought into contact with it. The 
muscular tubular pharynx is then protruded through the mouth and after moving 
rapidly over the surface of the prey is eventually thrust through the body wall 
(Fig. 1). When this occurs the flatworm changes position slightly to bring the 
mouth directly over the point of penetration to enable the pharynx to extend as 
fully as possible into the prey. The precise means of penetration could not be 







FIGURE 1. Orthodemus attacking a slug (S). The protruded pharynx (P) is penetrating 
the integument of the slug to withdraw the body contents. Magnification X 5. 

FIGURE 2. Photographed 15 minutes after Figure 1. Feeding is complete and the flat- 
worm is retracting the pharynx from the remnants of the slug. Magnification as in Figure 1. 

FIGURE 3. A portion of the gastrodermis in Orthodemus showing a "sphere-cell" (S.C) 
surrounded by columnar cells. Haematoxylin and eosin. Scale : 1 cm. = 20 p.. 

FIGURE 4. Gastrodermis of Orthodemus, 4 hours after a meal of starch paste, showing 
columnar cells loaded with phagocytosed material. Periodic acid-Schiff. Scale as in Figure 3. 



NUTRITION OF ORTHODEMUS TERRESTRIS 121 

ascertained, but it would appear to be purely mechanical, with the pharynx merely 
forcing its way inwards through the epidermis and musculature. Complete penetra- 
tion is achieved within 30-60 seconds of first contact and there is no evidence to 
suggest the process is assisted by either regurgitation of solvent juices or the selec- 
tion of external openings. 

Once within the body cavity the pharynx moves around disorganzing the 
softer tissues to pass them back in a finely divided condition into the gut. The 
disruption of the tissues is rapid, and, like the penetration of the body wall, is 
mechanical with the pharynx acting as a simple suctorial tube extracting tissue 
fragments and body fluids. Withdrawal of the body contents continues until 
either only the collapsed and empty body wall is left, or the flatworm is replete, 
when the pharynx is retracted and the remnants of the prey abandoned (Fig. 2). 
Feeding lasts 10-20 minutes and during this period the fiatworm lies in a very 
characteristic position across the prey, with the head region often raised and 
swinging slowly from side to side. In the early stages the food may be abandoned 
if the flatworm is disturbed or the incident light increased but later, when the 
pharynx is inserted and ingestion proceeding, the feeding individual is less suscep- 
tible to external stimuli and in the laboratory can often be manoeuvered into 
situations more suited to observation. 

The laboratory stock of Orthodemus was sexually mature and produced cocoons 
between April and August. Up to six young, 3-4 mm. long, emerged from each 
cocoon within three weeks of laying and these fed in the same manner as the 
adults upon newly hatched or very young slugs. The latter were common in the 
habitat during the flatworm's breeding season and appear to form the staple diet 
of young Orthodemus, for isolated mucus cells and granules of a black pigment 
similar to that of the slug were often found amongst the gut contents of the young 
individuals fixed immediately after collection. 

The structure of the gut and the course of digestion 

The gut in Orthodemus has the typical triclad arrangement with numerous 
lateral caeca arising from each of the three main branches. The pharynx is of 
the cylindrical plicate type, and can be protruded through the mouth by simple 
muscular elongation. 

The gastrodermis (Figs. 3 and 4) consists of a single layer of non-ciliated cells 
standing on a delicate basement membrane. Two types of cell occur. The larger 
and more numerous are columnar, 40-50 p. in height, with basal eosinophilous in- 
clusions which apparently represent phagocytosed food particles, for they disappear 
rapidly during starvation. The second type is spherical or slightly pear-shaped. 
25-30 /x high, and situated between the bases of the columnar cells in the ratio of 
approximately one to every ten of the latter. They contain eight to twelve protei- 
naceous spheres which stain heavily with haematoxylin and modified Millon 
method and disappear only slowly with starvation. These "sphere-cells" appear, 
therefore, to function as sites of protein storage a conclusion supported by the 
absence of changes in appearance or staining reaction which can be correlated with 
digestive processes. 

The presence of discrete particles within the columnar gut cells indicated that 
digestion in Orthodemus is intracellular and this was confirmed by an examination 



122 J. B. JENNINGS 

of individuals fixed at various intervals after observed feeds on slugs. Im- 
mediately after feeding the gut lumen is filled with a heterogeneous mass of cells, 
nuclei and muscle fragments already of suitable size for phagocytosis through the 
extreme disruption caused by the pharynx during ingestion. Phagocytosis begins 
as the first tissue fragments enter the gut and within fifteen minutes engulfed 
particles are found throughout the gastrodermis. They are contained at first in 
small vacuoles near the free distal border of the columnar cells, and phagocytosed 
muscle fragments and nuclei are clearly recognizable. With time, however, the 
fragments lose their identity and condense to homogeneous spheres which pass 
back deeper into the cells to disappear as digestion and absorption proceed. Four 
hours after feeding the columnar cells are loaded with phagocytosed material and 
they show a considerable increase in volume, with their walls becoming indistinct 
and the whole gastrodermis appearing almost syncytial. Complete digestion of 
a meal takes 12-24 hours, depending upon the amount of food taken, but any 
particles too big for phagocytosis, such as occasional large muscle fragments, are 
unaffected and remain unchanged in the gut lumen. 

Experimental demonstration of the complete absence of intraluminar digestion 
was obtained by feeding frog blood and boiled starch paste. The former was 
readily taken by the flatworms but the starch appeared to be less palatable and 
had to be injected into a boiled portion of earthworm to ensure its ingestion. Fixa- 
tion after blood feeding showed that many erythrocytes were ruptured during their 
passage through the pharynx, demonstrating its effective triturating action, and 
the corpuscle fragments, nuclei, and intact erythrocytes were quickly phagocytosed 
by the gut cells. Staining with the Feulgen and benzidine techniques showed 
progressive breakdown and disappearance of phagocytosed nuclei and haemoglobin, 
but in sharp contrast material in the lumen remained unchanged in appearance and 
staining capacity until it was either taken up by the cells or eventually expelled 
from the gut eight to twelve hours after feeding. Similar results were obtained 
after feeding with starch paste ; staining with Lugol and P.A.S. showed digestion 
and absorption of starch within the columnar cells (Fig. 4), whilst that remaining 
in the lumen was quite unaltered. 

It was not possible to determine the pH conditions of intracellular digestion 
owing to the limited number of specimens and the difficulty of administering food 
containing indicators. 

The food reserves 

Fat forms the principal food reserve in Orthodemus and the bulk is stored in 
the mesenchyme as large globules 15-20 /A in diameter, whilst smaller amounts 
are scattered as droplets 3-4 p, in diameter in the columnar cells of the gastrodermis. 

As already stated, protein reserves are found within the so-called "sphere-cells" 
of the gastrodermis (Fig. 3) and in adult Orthodeinus these show a marked 
seasonal variation. Thus in early spring the gastrodermis contains more "sphere- 
cells," each with large, dense and heavily staining spheres, but during the summer 
months when the gonads are mature and the flatworms producing cocoons the 
number of cells decreases and the spheres of those remaining shrink and stain only 
lightly. In the late summer, however, the cells start to increase in number and 
reach a maximum of one to every ten of the columnar cells by October or November. 



NUTRITION OF ORTHODEMUS TERRESTRIS 123 

Thus there is a build-up of reserve protein during the late summer which is rapidly 
depleted in the following breeding season. 

There are no significant amounts of carbohydrate reserves. Staining with Best's 
carmine and P.A.S. reveals only very small amounts of glycogen which occur as 
tiny irregular granules scattered through the mesenchyme and columnar gut cells. 

DISCUSSION 

It is evident from these observations that nutrition in Orthodemus terrestris 
differs very little from that described in the related aquatic triclads (Willier, 
Hyman and Rifenburgh, 1925; Kelley, 1931; Jennings, 1957). The typical 
triclad feeding mechanism, with the pharynx functioning as a suctorial tube which 
penetrates the prey to withdraw the body contents piecemeal, has apparently proved 
adequate to the needs of a terrestrial life and is retained unmodified. It allows the 
flatworm to deal effectively with slugs or earthworms which in the absence of 
devices for trapping more active animals appear to form the bulk of the diet. The 
failure of the mucous locomotory trail to persist and perform the secondary func- 
tion of ensnaring the prey, as it does in aquatic triclads, is due perhaps to the 
terrestrial environment which although damp and humid does not prevent desicca- 
tion of the trail soon after its formation. 

The retention of suctorial feeding, with extreme disruption of the food during 
ingestion, allows phagocytosis by the columnar cells to begin immediately food 
enters the gut. Consequently there has been no stimulus for the development of 
intraluminar digestion and the primitive condition of exclusively intracellular break- 
down persists, exactly as in the aquatic triclads. A further similarity between the 
latter and Orthodemus is seen in the form and location of the food reserves, and 
particularly of protein stored in both cases in special "sphere-cells" in the gastro- 
dermis. 

It would appear, therefore, that the adoption of the terrestrial habit by Ortho- 
demus has not necessitated any fundamental modification of the basic triclad methods 
of feeding and digestion. This is probably true of most other terrestrial triclads, 
for of the few existing accounts which mention nutrition, almost all describe or 
infer suctorial feeding upon earthworms, slugs and occasionally other invertebrates 
(Percival, 1925; Eastham, 1933; Johri, 1952; Froehlich, 1955; Pfitzner, 1958), 
and since this has such a profound effect upon the particle size of food entering the 
gut it is likely that it permits retention of purely intracellular digestion, as in 
Orthodemus. A few South American species, however, are reported to swallow 
their food whole (Froehlich, 1955) so that in these cases, unless preliminary break- 
up within the gut is achieved mechanically as in some rhabdocoels (Jennings, 
1957), at least some degree of intraluminar digestion must occur. 

I wish to thank Professor E. A. Spaul for his advice and encouragement 
during the course of this work. 

SUMMARY 

1. The land planarian Orthodemus terrestris feeds principally upon small slugs 
and earthworms which are captured after chance encounter. 

2. The typical triclad method of feeding, with the protruded cylindrical plicate 



124 J. B. JENNINGS 

pharynx inserted into the prey to disrupt and withdraw the body contents, is used 
without modification. 

3. Disintegration of the food during ingestion is so effective that the resultant 
particles are available for immediate phagocytosis by the gut cells and intraluminar 
digestion is absent. 

4. The food reserves consist of fat stored in the mesenchyme and columnar 
gut cells, and protein stored in gastrodermal "sphere-cells." Protein reserves are 
depleted during the breeding season and replenished in the late summer and 
autumn. 

5. It would appear that the basic triclad methods of feeding and digestive 
processes are quite adequate to the needs of terrestrial life and Orthodemus shows 
no particular adaptation to this so far as nutrition is concerned. 

LITERATURE CITED 

EASTHAM, L. E. S., 1933. Morphological notes on the terrestrial triclad Rhynchodemus brit- 
tanicus Percival. Proc. Zool. Soc. Land., 1933 : 889-895. 

FROEHLICH, C. G., 1955. On the biology of land planarians. Bol. Fac. Filos., Cicnc. Letr., 
Univ. Sao Paulo, Zoologia, 20 : 263-272. 

HYMAN, L. H., 1954. Some land planarians of the United States and Europe, with remarks 
on nomenclature. Amer. Mus. Novitates, no. 1667, 21 pp. 

JENNINGS, J. B., 1957. Studies on feeding, digestion and food storage in free-living flatworms 
(Platyhelminthes: Turbellaria). Biol. Bull., 112: 63-80. 

JOHRI, L. N., 1952. A report on a Turbellarian Placoccphalus kcwense, from Delhi State, and 
its feeding behaviour on the live earthworm Pheretima posthwna. Sci-Cult., 18 : No. 
6, 291. 

KELLEY, E. G., 1931. The intra-cellular digestion of thymus nucleo-protein in triclad flat- 
worms. Physiol. Zool., 4: 515-541. 

PERCIVAL, E., 1925. Rhynchodemus brittanicus, n. sp. A new British terrestrial triclad, with 
a note on the excretion of calcium carbonate. Quart. J. Micro. Sci., 69 : 344-355. 

PFITZNER, L, 1958. Die Bedingungen der Fortbewegung bei den deutschen Landplanarien. 
Zool. Beitr., Berl, 3 : 235-310. 

WILLIER, B. H., L. H. HYMAN AND S. A. RIFENBURGH, 1925. A histochemical study of intra- 
cellular digestion in triclad flatworms. /. Morph., 40: 299-340. 






THE CYTOCHROME SYSTEM IN MARINE 
LAMELLIBRANCH TISSUES * 

KIYOZO KAWAI 
Department of Zoology, University of Kyoto, Kyoto, Japan 

The occurrence of the cytochromes and cytochrome oxidase, very similar to 
the oxidase of mammals, has been proved in certain marine molluscs (Ball and 
Meyerhof, 1940; Humphrey, 1947; Ghiretti-Magaldi, Giuditta and Ghiretti, 1957). 
However, it is still obscure whether the cytochrome system acts as a terminal 
oxidation system in their intact tissues. The mere presence of the cytochromes or 
cytochrome oxidase in a cell does not indicate to what extent the normal respiration 
is mediated actually through the cytochrome system. Recently it was suggested 
that the cytochrome system may not play a major role in the respiratory system of 
the oyster mantle, although the tissue contains cytochrome oxidase (Jodrev and 
Wilbur, 1955). 

The present investigation was undertaken to throw- some light on the con- 
nection of the cytochromes with the respiration of intact tissues of marine lamel- 
libranchs. A portion of this work has been preliminarily reported (Kawai. 1958). 

MATERIALS AND METHODS 

The respiratory studies were made at the Seto Marine Biological Laboratory 
during fall and early winter, and certain enzyme assays were carried out at the 
laboratory in Kyoto. Three species of marine lamellibranchs, the oyster, Cras- 
sostrea gigas, the pearl oyster, Pinctada martensii and the mussel, Mytilus cras- 
sitesta, were used as experimental materials. Specimens of the former two, each 
about two years old and about 11 and 6 cm. in shell height, respectively, were 
obtained from culture-farms in the vicinity of the Marine Laboratory, while 
Mytilus, about 8 cm. in shell height, was collected at a shore reef near the Lab- 
oratory. 

Oxygen uptakes of intact tissues and extracts were measured at 25 C. in 
Warburg manometers, with vessels of about 9 ml. capacity. Respiratory measure- 
ments were carried out with several thin tissue pieces, 50 to 100 ing. in fresh 
w r eight, suspended in 1.5 ml. of sea water buffered at pH 8 with 0.03 M glycine 
(Robbie, 1946) or glycylglycine (Tyler and Horowitz, 1937). To absorb CO,, 
0.2 ml. of 0.5 M or 10% KOH and filter paper were placed in the center-well. 
In the cyanide experiments, NaCN was added to the main compartment and 
0.2 ml. of KCN-KOH mixture (Robbie, 1946) was included in the center-well. 
Both glycine and glycylglycine of the concentration used had no effect on the 
respiration of the lamellibranch tissues. In the experiments of photo-reversibility 
of carbon monoxide inhibition, a 500- watt projector lamp was switched on at some 

1 Contributions from the Seto Marine Biological Laboratory, No. 334. 

125 



126 



KIYOZO KAWAI 



distance away from the water-bath. A slight absorption by the KOH solution 
in the center-well was subtracted from each manometric reading. 

Absorption bands of cytochromes were observed w r ith a low dispersion hand 
spectroscope and more accurate sites of the bands were measured with a Hilger 
wave-length spectrometer. The spectrophotometric studies of cytochrome oxidase 
were made with a Hitachi EPU-II spectrophotometer. 

Carbon monoxide was prepared by decomposing formic acid with warm con- 
centrated sulfuric acid. Cytochrome c was prepared from beef heart according 
to Keilin and Hartree (1945). 

RESULTS 
1. Cytochrome spectra 

The absorption spectra of the reduced cytochromes were examined on intact thin 
tissues or breis, packed about 2 mm. thick, adding a small amount of solid sodium 
hydrosulphite. The characteristic absorption bands corresponding to cytochromes 
a + o,, b and c, could be clearly observed in the lamellibranch heart at room tem- 
perature. Cooling the heart with liquid-air by the method of Keilin and Hartree 
(1949), the bands were very intensified, being slightly shifted towards the violet. 
The sites of these cytochrome bands were estimated with the Hilger spectrometer 
at room temperature as follows: aa + a z a: 603; ba: 562; c a : 550; b/3 + c(3: 520- 
530 in/A. 

In other tissues, such as gill, mantle, adductor muscle, etc., only the band of 
cytochrome b could be detected at room temperature. However, a feeble band of 
cytochrome a + a z and a lesser band of r appeared at liquid-air temperature. Table I 



TABLE I 

Observations on the a-bands of reduced cytochromes in marine 
lamellibranch tissues at liquid-air temperature 



Animal 



Tissue 



Relative intensity of the absorption* 



Cyt. a +03 



Cyt. b 



Cyt. c 



Crassostrea gigas 



Pinctada martensii 



Mytilus crassitesta 



Heart 

Gill 

Mantle 

Adductor muscle 

Digestive diverticula 

Heart 

Gill 

Mantle 

Adductor muscle 

Gonad 

Digestive diverticula 

Gill 

Mantle 



_i __ 



* Very strong absorption is shown by the sign + + + or + + + +, while very feeble band is 
indicated by -| . The sign shows the absence of cytochrome band. 



CYTOCHROMES OF MARINE BIVALVES 



127 



o 0.5 

LO 



0.4 



0.2 



Q_ 
O 



0.1 




30 60 90 120 

TIME (SECONDS) 



150 



180 



FIGURE 1. The oxidation of reduced cytochrome c by cytochrome oxidase from oyster gill. 
Reduced cytochrome c was prepared by reduction with a pinch of hydrosulphite and the excess 
hydrosulphite oxidized by shaking. Total volume 3 ml. Each cuvette contained final concen- 
tration of 2 X 10- 5 M cytochrome c, 0.1 M phosphate buffer at pH 7.0 and 0.15 ml. of the 
extract. I contained 10~ 3 M cyanide; II, no cyanide. Reaction was followed at 25 C. The 
change of optical density was plotted on the logarithmic scale. 

represents the results of these observations. The cytochromes are most abundant 
in the heart, where cytochrome c is predominant or equal to b. In other tissues, 
however, cytochrome b is apparently more dominant than a + a z or c. This fact 
forms a sharp contrast to the situation of the heart. Absence of the bands of 
a + a, or c in certain tissues may be due to their very low concentration. When a 
few drops of pyridine were added to the reduced tissues, a very intense pyridine 
hemochromogen band extending from about 550 to 560 m/i, with a mid-point at 
about 557 m/A, and a w r eak band lying about from 580 to 590 m^ were readily 
produced in all tissues examined. They are considered to be the absorption bands 
of pyridine derivatives of cytochromes b and a group, respectively. 

2. Cytochrome oxidase 

The enzyme activity was determined at 25 C. by two methods, i.e., mano- 
metrically and spectrophotometrically. The extract for the enzyme study was 
prepared by homogenizing the excised tissue with a glass homogenizer in five 
parts of cold 1.24 M sucrose (isotonic with sea water) and squeezing through a 
thin cloth. The addition of an aliquot of the extract to a solution of reduced 
cytochrome c results in a rapid decrease in optical density at 550 m^t, (Fig. 1). 
The activity of molluscan cytochrome oxidase is inhibited by about 94% in the 
presence of 10~ 3 M cyanide. The enzyme activity is also inhibited by carbon 
monoxide in the dark. Under the condition, 90% nitrogen + 10% oxygen in the 
control flask and 90% carbon monoxide + 10% oxygen in the experimental, the 
activity is inhibited by about 62% and the inhibition is completely eliminated by 
the illumination (Fig. 2). 



128 



KIYOZO KAWAI 



60 - 



50 - 



40 



X 

CD 




10 



20 



30 40 50 

TIME (MINUTES) 



60 



70 



80 



FIGURE 2. Effect of carbon monoxide on cytochrome oxidase from pearl oyster gill ; 0.5 ml. 
extract in each Warburg flask. Final concentration : phosphate buffer at pH 7.0, 0.05 M ; 
ascorbate, 0.01 M; cytochrome c, 2.2 X lO" 5 M. Final volume 2.0 ml. I, in 90% N 2 + 10% 
O; II, in 90% CO + 10% Ck Temperature 25 C. The black and white blocks under the 
base line show the periods of dark and light. 

3. Effect of carbon monoxide on the respiration of lamellibranch tissues 

Inhibition experiments were performed using the same gas mixture (CO/O 2 
ratio of 9/1) as in the case of cytochrome oxidase. Controls with a gas mixture 

TABLE II 
Respiration of lamellibranch tissues in a gas mixture of 90% CO and 10% Oz 



Animal, tissue 


No. of deter- 
minations 


02 uptake in 

control (jjl./hr./ 
100 mg. fresh 
wt.) 


CO inhibition 
in darkness* 
(per cent) 


Relative 
affinity 
constant** 


Crassostrea gigas 










Heart 


2 


39 


50 


9.0 


Gill 


3 


53 


51 


8.7 


Mantle 


2 


29 


53 


8.0 


Pinctada martensii 










Gill 


4 


48 


47 


10.1 


Mantle 


2 


15 


54 


7.7 


Digestive diverticula 


1 


27 


46 


10.6 


Mytihis crassitesta 










Gill 


2 


26 


52 


8.3 



* The complete elimination of CO inhibition by the illumination was observed in all tissues 
examined. 

* Relative affinity constant of the tissue respiration for CO and O2 was calculated by the 
Warburg equation. See text. 



CYTOCHROMES OF MARINE BIVALVES 



129 



of nitrogen and oxygen (9/1) were also run, but there was no significant difference 
between oxygen uptakes in this control and in air. 

Figure 3 shows the results of a typical experiment, obtained with the oyster 
mantle, indicating the presence of photo-reversibility of carbon monoxide inhibi- 
tion. Nearly identical results, the inhibition of oxygen uptake in the CO gas 
mixture being about 5Q c /c in darkness and completely eliminated by light, were 
obtained in all tissues examined. These results are summarized in Table II. 
The relative affinity constants of the tissue respiration for carbon monoxide and 
oxygen were also calculated according to the equation of Warburg ( 1949) ; 
K = n/l - >i pCO/pO 2 , w r here n is the fraction of oxygen consumption not 
inhibited, and pCO and pQ., are, respectively, carbon monoxide and oxygen pres- 
sure. The values obtained, ranging from 7.7 to 10.6, are in good agreement with 
the average value of 8.2 reported for yeast cells in young cultures (Warburg, 1949). 
The stimulation effect of carbon monoxide on cell respiration, which has been 
found in certain marine eggs (Rothschild, 1949; Minganti, 1957; Rothschild and 
Tyler, 1958), was not observed in these lamellibranch tissues. 




20 



40 60 80 

TIME (MINUTES) 



FIGURE 3. Effect of carbon monoxide on the respiration of oyster mantle. I, in 90% 
N 2 + 10% O 2 ; II, in 90% CO + 10% O 2 . Each flask contained 100 mg. tissue pieces in 1.5 ml. 
of 0.03 M glycylglycine-buffered sea water. Temperature 25 C. The white blocks under 
the base line show the periods of illumination. 



130 



KIYOZO KAWAI 



50 



40 



30 



<c 

h- 
Q_ 



20 



o 



10 




IV 



(b) 




IV 



20 40 60 80 100 20 40 
T 1 M E ( MINUTES; 



60 80 



100 



FIGURE 4. Effect of cyanide and methylene blue on the respiration of oyster tissues, (a) 
gill; (b) mantle. I, control; II, 1(T 3 M cyanide + 6 X 1(T 5 M methylene blue; III, 10' 3 M 
cyanide + 10" 5 M methylene blue; IV, 10~ 3 M cyanide. Each manometric flask contained 50 
mg. of gill pieces or 100 mg. of mantle pieces in 1.5 ml. of sea water buffered with 0.03 M 
glycine. Manometric measurements were started twenty minutes after the tissues were im- 
mersed in each medium containing the reagents. 

4. Effect of cyanide on the respiration of oyster tissues 

The endogenous respiration of the oyster gill was depressed by 0.001 M cyanide 
to 15-20^ of the control and the inhibition was partly restored to about 40% of 
the control in the presence of 6 X 10~ 5 M methylene blue, or its nearly saturated 
solution in sea water ; while a lower concentration of methylene blue, 1 X IQ~ 5 M, 
was slightly effective to reverse the cyanide inhibition at an initial short period, 
but this effect completely disappeared within 40 to 60 minutes after the respiratory 
measurement was started (Fig. 4a). Very similar results, though the effect of 
6 X 10" 5 M methylene blue was somewhat smaller, were also obtained in the 
cyanide inhibition of the oyster mantle respiration (Fig. 4b). 

DISCUSSION 

The marine lamellibranchs, Crassostrea gif/as, Pinctada niartensii and Mytilus 
crassitesta, used in this investigation have no oxygen carrier such as hemoglobin 
or hemocyanin in the blood. However, as reported here, their visceral cells possess a 
normal cytochrome system consisting of cytochromes a, b, c and o- 3 , i.e., cytochrome 
oxidase. The absorption bands of cytochromes in their hearts being nearly equiva- 
lent to those in the cells of baker's yeast, it is inferred that the hearts probably 
contain as much cytochromes as yeast cells. Such high contents of cytochromes in 
the molluscan hearts, resembling mammalian hearts, may be concerned with their 
active movement. Although the contents of cytochromes are considerably low in other 
tissues, such as gill, mantle, adductor muscle, etc., there is apparently more 
cytochrome b than a + a, and r. This is very interesting when compared with the 
situation of the hearts, where cytochrome c is predominant or equal to b. 



CYTOCHROMES OF MARINE BIVALVES 131 

As the molluscan cytochrome oxidase shows photoreversibility in the carbon 
monoxide inhibition, it should contain iron atom, like the oxidase of mammals, in 
the active site. The relative affinity constant of the enzyme for carbon monoxide 
and oxygen, calculated from the equation of Warburg as described previously, is 5.5 
under the condition employed. This value is very similar to that reported for 
cytochrome oxidase of sea urchin eggs (Krahl, Keltch, Neubeck and Clowes, 1941) 
and the spermatozoa of fresh-water mussels (Kawai and Higashi, 1959), but 
somewhat smaller than the value for mammalian cytochrome oxidase (Ball, 
Strittmatter and Cooper, 1951). 

The presence of photoreversible inhibition of carbon monoxide in the respira- 
tion of lamellibranch tissues demonstrates that cytochrome oxidase acts as a 
terminal oxidase in these intact tissues. In a gas mixture of 90% carbon monoxide, 
the activity of molluscan cytochrome oxidase is inhibited by about 62% in the dark 
and the inhibition of the tissue respiration, though somewhat different in each tissue, 
is roughly 50%. Therefore, on the assumption that the oxidase in vivo is in- 
hibited to the same extent in the extract, the respiration of intact tissues mediated 
through the cytochrome oxidase would be about 80% or more of the total respira- 
tion. 

As is well known in many cells, autoxidizable redox dyes, exemplified by meth- 
ylene blue, can restore the depressed respiration by cyanide. Such reversing effects, 
though not remarkable, could also be observed in the cyanide inhibition of oyster 
tissues using methylene blue (6 X 10~~ 5 M} nearly saturated in sea water. Accord- 
ing to Jodrey and Wilbur (1955), methylene blue, 1.4 X 10~ 5 M at the final 
concentration, was without appreciable effect in reversing the cyanide inhibition of 
the mantle respiration of the oyster (Crassostrea rirginica). From this result they 
suggested that the cytochrome system may not play a major role in the oxidative 
metabolism of the oyster mantle. However, the ineffectiveness of methylene blue 
may be attributed to the matter of concentrations employed, because, in the present 
work, a lower concentration of methylene blue (1.0 X 10~ 5 M) was similarly in- 
effective to reverse the cyanide inhibition of the respiration of oyster tissues. 

The author wishes to express his indebtness to Prof. D. Miyadi of Zoological 
Laboratory, Prof. S. Tanaka of Biochemical Laboratory and Dr. Y. Matsui, the 
Director of Nippon Institute for Scientific Research on Pearls, for their encourage- 
ments and numerous facilities given during this investigation. He is also indebted 
to Dr. R. Sato, Institute for Protein Research of Osaka University, for his kind 
advice concerning the spectroscopic examination, and to the Staff of the Seto Marine 
Biological Laboratory for their help and facilities offered. This investigation was 
supported, in part, by a Grant-in-Aid for Developmental Scientific Research from 
the Ministry of Education. 

SUMMARY 

1. The cytochrome system of the lamellibranchs, Crassostrea gigas, Pinctada 
inartcnsii and Mytilus crassitesta, has been studied in relation to the respiration of 
intact tissues. 

2. The visceral cells possess a normal cytochrome system consisting of cyto- 
chromes a, b, c, and a.., i.e., cytochrome oxidase. The oxidase is strongly inhibited 



132 KIYOZO KAWAI 

by cyanide and carbon monoxide. The carbon monoxide inhibition of the enzyme 
is completely eliminated by light. 

3. Cytochromes are most plentiful in the heart, where cytochrome c is pre- 
dominant or equal to b, while b is apparently more predominant than a + a, or c 
in other tissues. 

4. In a gas mixture of 90% CO and 10% O 2 , the respiration of various tissues 
is inhibited by about 50% in the dark and the inhibition is completely eliminated 
by the illumination. Cyanide, 0.001 M, depresses the respiration of the oyster 
tissues to about 15-20% of the control, and the inhibition is partly reversed in the 
presence of methylene blue (6 XlO~ 5 M) nearly saturated in sea water. 

5. It is concluded that about 80% or more of the total respiration of intact 
lamellibranch tissues proceeds through the cytochrome system. 

LITERATURE CITED 

BALL, E. G., and B. J. MEYERHOF, 1940. On the occurrence of iron-porphyrin compounds and 

succinic dehydrogenase in marine organisms possessing the copper blood pigment hemo- 

cyanin. /. Biol. Chem., 134: 483-494. 
BALL, E. G., C. F. STRITTMATTER and O. COOPER, 1951. The reaction of cytochrome oxidase 

with carbon monoxide. /. Biol. Chem., 193 : 635-647. 
GHIRETTI-MAGALDI, A., A. GIUDITTA AND F. GHIRETTI, 1957. A study of the cytochromes of 

Octopus vulgaris Lam. Biochem. J., 66 : 303-307. 
HUMPHREY, G. F., 1947. The succinoxidase system in oyster muscle. /. Exp. Biol., 24 : 

352-360. 
JODREY, L. H., AND K. M. WILBUR, 1955. Studies on shell formation. IV. The respiratory 

metabolism of the oyster mantle. Biol. Bull, 108: 346-358. 
KAWAI, K., 1958. Cytochrome system in oyster tissues. Nature, 181 : 1468. 
KAWAI, K., AND S. HIGASHI, 1959. The cytochrome system in the spermatozoa of fresh-water 

mussels. Seikagaku, 31 : 97-101. 
KEILIN, D., AND E. F. HARTREE, 1945. Purification and properties of cytochrome c. Biochem. 

J., 39 : 289-292. 
KEILIN, D., AND E. F. HARTREE, 1949. Effect of low temperature on the absorption spectra 

of haemoproteins : with observations on the absorption spectrum of oxygen. Nature, 

164: 254-259. 
KRAHL, M. E., A. K. KELTCH, C. E. NEUBECK AND G. H. A. CLOWES, 1941. Studies on cell 

metabolism and cell division. V. Cytochrome oxidase activity in eggs of Arbacia punc- 

tulata. J. Gen. Physiol, 24 : 597-617. 
MINGANTI, A., 1957. Experiments on the respiration of Phallusia eggs and embryos (ascidi- 

ans). Ada Embryo, ct Morphol. Exp., 1: 150-163. 
ROBBIE, W. A., 1946. The quantitative control of cyanide in manometric experimentation. 

/. Cell. Comp. Physiol., 27: 181-209. 
ROTHSCHILD, LORD, 1949. The metabolism of fertilized and unfertilized sea-urchin eggs. The 

action of light and carbon monoxide. /. Exp. Biol., 26: 100-111. 
ROTHSCHILD, LORD, AND A. TYLER, 1958. The oxidative metabolism of eggs of Urechis caupo. 

Biol. Bull,, 115: 136-146. 
TYLER, A., AND N. H. HOROWITZ, 1937. Glycylglycine as a sea water buffer. Science, 86 : 

85-86. 
WARBURG, O., 1949. Heavy Metal Prosthetic Groups and Enzyme Action. Clarendon Press, 

Oxord. 



STUDIES ON THE MECHANISM OF PHOSPHATE 
ACCUMULATION BY SEA URCHIN EMBRYOS l 

J. B, LITCHFIELD2 AND A. H. WHITELEY 

Department of Zoology and the Friday Harbor Laboratories, 
University of Washington, Seattle 5, Washington 

It was shown by Needham and Needham (1930) that the developing larva of 
Dendraster excentricus increases its total phosphate content from fertilization to 
gastrulation. Since this initial discovery, phosphate accumulation by echinoderm 
eggs has been the object of a number of studies. The use of radioactive phosphorus 
in this analysis was introduced by Brooks (1943). 

P 32 uptake by unfertilized eggs is negligible. An exchange that does not result 
in an increase in the internal concentration of phosphate is thought to take place 
between the external and intracellular phosphate (Chambers and White, 1949, 
1954; Brooks and Chambers, 1954). Lindberg (1949, 1950) considered that the 
surface of unfertilized eggs metabolized phosphate but that no intracellular penetra- 
tion occurred. This surface metabolism involved incorporation of P 32 into ATP. 

The fertilized egg presents a radically different picture. Immediately following 
fertilization there is no change in P 32 uptake, but within 6-30 minutes there is a 
noticeable increase. The rate of uptake increases to a maximum value and re- 
mains constant for as much as seven hours after fertilization. These initial events 
have been described for various species by Abelson (1947), Brooks and Chambers 
(1948. 1954) , Whiteley ( 1949) , and Chambers and White ( 1954) . The maximum 
uptake rate by the fertilized eggs is as much as 160 times as great as the uptake by 
unfertilized eggs (Brooks and Chambers, 1948). The rate is not greatly affected 
by the accompanying decrease in P 31 and P 32 concentrations in the suspension 
medium within the limits 0.7 to 13 ^M (Brooks and Chambers, 1954). Evidence 
that this uptake represents a real penetration is the fact that only 2 to 5% of the 
P 32 activity is removed by continuous washing with sea water (Brooks and 
Chambers. 1948, 1954). 

Radioactive phosphate is largely incorporated into the acid-soluble phosphate 
compounds (Abelson, 1947, 1948; Chambers, Whiteley, Chambers and Brooks, 
1948; Chambers and White, 1949, 1954; Bolst and Whiteley, 1957). This is true 
both in the fertilized and unfertilized eggs. Among the acid-soluble components, 
the easily hydrolyzable phosphate compounds have the highest activity (Abelson, 
1948; Lindberg, 1949, 1950; Chambers and White, 1949, 1954; Bolst and Whiteley, 
1957). 

Inhibition of uptake by fertilized eggs can be achieved by the use of low tem- 

1 This investigation was supported in part by the Research Fund 171 of the University of 
Washington, by an Institutional Research Grant from the American Cancer Society to the 
University of Washington, and by a grant from the National Science Foundation. 

2 Present address : Department of Physiological Chemistry, Woman's Medical College of 
Pennsylvania, Philadelphia. 

133 



134 J. B. LITCHFIELD AND A. H. WHITELEY 

peratures (Abelson, 1947; Villee and Villee, 1952). Metabolic inhibitors such 
as 4,5-dinitro-o-cresol (Abelson, 1947) and cyanide (Brooks and Chambers, 1948) 
also diminish uptake. These experiments have lead to the view that the develop- 
ing embryo has an enzymatic mechanism that controls the penetration and conse- 
quent accumulation of phosphate. Lindberg (1949, 1950) has adduced evidence 
that in the fertilized egg the initial enzymatic reaction in the uptake involves in- 
corporation of phosphate into adenosinetriphosphate. However, Chambers and 
Mende (1953) have determined that the primary penetration of phosphate through 
the plasma membrane still could be a matter of simple diffusion down an activity 
gradient inasmuch as they have found an extremely low concentration of free 
inorganic orthophosphate within the fertilized eggs of Strongyloccntrotus droc- 
bachiensis. Chambers and White (1949) have found that the inorganic phosphate 
pool in S. purpuratus decreases quickly in response to fertilization. Agents that 
affect enzymatic activity might alter phosphate penetration by affecting the magni- 
tude of the internal phosphate pool. 

In the present study the nature of penetration of phosphate into the fertilized 
egg of Strongylocentrotns purpuratus has been examined further. The experi- 
ments have largely involved an analysis of the effects of metabolic effectors (2,4- 
dinitrophenol, arsenate, ATP, and temperature) on the rate of uptake of P 3 - in 
fertilized eggs. 

METHODS AND MATERIALS 

Experimental chamber. The basic experimental approach used in most of the 
experiments to be described was to measure continuously with a Geiger counter 
the accumulation of P 32 by sea urchin eggs that were being perfused with sea water 
containing P 32 as orthophosphate. In some experiments the perfusion fluid in- 
cluded various metabolic effectors. The lucite perfusion chamber was an improved 
version of that used by Chambers and Whiteley (Whiteley, 1949), and Chambers, 
White and Zeuthen (Zeuthen, 1951) (Fig. 1). Temperature was maintained con- 
stant by pumping water through the upper chamber. The eggs, which rested on the 
bottom of the lower chamber, were introduced through an opening in the side that 
was then closed by means of a lucite plug. The flow characteristics of the chamber 
were tested by perfusing it with a dye. The dye spread quickly and fairly homogene- 
ously to all parts of the chamber and a flow of three to four ml./min. changed the 
sea water in the compartment to the extent of 90% in 1H minutes as measured with 
a photo-cell. The volume of the chamber is 1.7 ml. 

Radioactivity measurements. The chamber was placed on a lucite platform 
which in turn rested on the top of an end- window Geiger- Mueller tube ; conse- 
quently the geometrical relationship between the tube and the chamber was always 
the same. The sealer was checked before and after each experiment against a cali- 
brated standard. Several Geiger-Mueller end-window tubes were used during the 
course of the experiments. The thickness of their windows ranged from 2.3 to 3.1 
mg./cm. 2 . In the majority of experiments, corrections were made for the dif- 
ference in sensitivity resulting from these differences in window thickness. 

Conduct of experiments. Prior to entry into the egg chamber, the sea water 
was cooled by passage through coiled glass tubing in a constant temperature bath. 
In most experiments the temperature of the bath, and therefore in the chamber, was 



PHOSPHATE UPTAKE BY EMBRYOS 



135 




\ cm. 



I"[(;i/KK 1. Perfusion chamber, c, cooling chamber; e, egg chamber; g, end-window Geiger- 
Mueller tube; i, inlet tube; o, outlet tube; p, platform; s, No. 1 coverslip. 



18.0 C. ; however for several experiments, the temperature was lowered to 8.0 C. 
Before each of these low temperature experiments, it was established with a thermo- 
couple inserted into the chamber that the temperature of the water flowing through 
the egg chamber was 8.0 C. 

At the beginning of each experiment, the eggs were perfused with sea water for 
fifteen minutes, while a background count for the experiments was obtained. Sea 
water containing P 32 was then turned on and the activity in the chamber reached a 
new level with the unfertilized eggs. Fifty to sixty minutes after the start of the 
experiment, the eggs were fertilized. This was accomplished by injecting 0.1 to 0.2 
cc. of a 10% sperm suspension in P 32 -sea water into the chamber inlet tube with a 
hypodermic syringe. Fertilization and development of the eggs were observed with 
a Zeiss Opton stereoscopic microscope placed above the chamber. At the end of 
each experiment the eggs were removed from the chamber, washed, and allowed 
to develop further to check on their normality and on their recovery from the effects 
of any reagent that was being tested. After each experiment the chamber was 
rinsed alternately with concentrated HC1 and NaOH and perfused with tap water 
and sea water in order to remove P 32 adsorbed on the inner surfaces of the bottom 
compartment. Any radioactivity left in the chamber was accounted for by measur- 
ing the activity of the empty chamber just before each experiment. 

In some experiments, eggs were activated parthenogenetically by the double 
treatment of Loeb (Just, 1939). The butyric acid and hypertonic sea water 
solutions were injected by means of a syringe into the egg chamber containing 
the unfertilized eggs. At the end of the treatment the eggs were perfused with sea 
water, followed by P 32 -sea water as in the other experiments. 

Materials. The eggs used were those of the sea urchin Strongylocentrotus 



L36 



I. B. 1 1 IVHFlKl.n AND A. 11. Will FK1 I'V 



(Stimpson). The eggs from a single animal were used in each ex- 
periment. Shedding was induced cither by injection of 4.J5' Y KO (Tyler, 149), 
or by an electric shock of oO volts, applied intermittently for several minutes 
(Harvey. 15_M. I' gg> were shed into filtered sea water and washed until 5' , 
to 100' , fertili.-.ation was obtained. In the majority of experiments the eggs were 
used within one to three hours after shedding'. Sperm were collected in dry 
Syracuse watch glasses, and suspensions were made up just before use. 

A 0.5'\ egg suspension was prepared and I -ml. aliquots of this suspension were 
counted iu a Sedwick-Rafter counting chamber. Based on this count. 15.000 
eggs were transferred to the experimental chamber. During transfer and distribu- 
tion of the eggs to the chamber 15 to JO 1 , of the eggs were lost, so that in each 
experiment approximately 1J.OOO to K\000 eggs were used. This number of egg< 
covered the bottom of the chamber in a single layer. Control eggs were cultured 
at 18.0 C. during the course of the experiment for comparison with those in the 
chamber. 

Sc'utums. All perfusion solutions were made up in filtered sea water. P 32 
was obtained from the Abbott Laboratories as sodium phosphate in 0.' < XaCl at 
pH 0.5. r :; -'-sea water solutions were made to have an activity of 0.005 tie ml. 
In all but two experiments the amount of carrier phosphate added was considerably 
less than the ^1 to oJ /( g 1. normally found in sea water iu this area. The addi- 
tional phosphate in these two experiments had no adverse effect on the eggs. One 
solution of r ; -'-sea water was prepared for each experiment, and from this 1 V: ' J - 
solutions containing various test substances were made. All solutions were ad- 
justed to pi I 8. 

RESULTS 



'/ P s - by nnjcrtilixcJ. fertilized ami parthenogenetically aetirateii eggs 

Experiments with unfertilized eggs confirm previous observations that there is 
practically no P s ' accumulation at this time. A few minutes after the T'-'-sea 
water first enters the egg chamber, the actixity of the chamber reaches a level that 



LU 

h- 

t ? I 500 



O 



or 



co 

Q h- 



a 
o 



000 
500 



i i i i I 



i r 




60 120 180 

TIME MINUTES 



Kic.i'KK J. Uptake of P 83 by unfertilised cji'S^ of St>\*r t; :y!sccntrsti<s furpiiratus (Stimpson "I 
and adsorption of P'^ by tlic perfusion chamber. The initial low level of activity represents the 
background count, a. unfertilized eggs : b. empty chamber. 



PHOSPHATE UPTAKE BY EMBRYOS 



137 



remains relatively constant for hours. During the course of several hours the 
activity may rise slightly, showing a small accumulation over the level in the sea 
water. The curves obtained with unfertilized eggs and with an empty chamber 
(Fig. 2) are very similar, from which it is concluded that the small rise is due to 
adsorption of P 3 - on the surfaces of the chamber and that no accumulation of P 32 
occurs in the unfertilized eggs. This conclusion is strengthened by the results of 
several experiments in which it was found that the rate of increase in activity 
in the chamber is not affected by varying the number of unfertilized eggs from 5,000 
to 20,000. 

In contrast to the unfertilized eggs, fertilized eggs accumulate P 32 so that the 
activity in the chamber soon greatly exceeds that of the P 32 -sea water. An ex- 
periment that typifies the course of this uptake is given in Figure 3. Two other 



7000 




-6O 



60 I 20 I 80 

TIME MINUTES 



FIGURE 3. Uptake of P 32 by sperm activated and artificially activated eggs. The initial 
low level of activity represents the background count. Activation was at zero time, a, sperm 
activated eggs ; b and c, artificially activated eggs. 



138 J. B. LITCHFIELD AND A. H. WHITELEY 

experiments are similar in all essential respects. This accumulation commences 
after a short lag period that is quite variable, ranging in 24 experiments, 
from 7 to 30 minutes with an average of 18. The maximum rate of uptake 
is not established until approximately 40 minutes after fertilization. This again is 
variable, ranging from 22 to 60 minutes in 14 experiments. While an explanation 
of these variabilities is not at hand, there seems to be no correlation between the 
length of the lag period and the length of time the eggs have been out of the ovary, 
which varied from 1 to 5.75 hours in 24 comparable experiments. It is probable 
that the cause resides in inherent differences in the gametes of different animals. 
Once the maximum rate of uptake is established, it remains constant. This constant 
rate of uptake was observed in experiments that continued for five hours after 
fertilization. 

The characteristic uptake pattern of the fertilized eggs could be dependent to 
some degree on the penetration of the sperm, or it could be inherent solely in the 
potentialities of the eggs. To answer this question, eggs were activated partheno- 
genetically and the uptake of P 32 followed. As is shown in Figure 3, such eggs 
exhibit a P 32 accumulation comparable to that of fertilized eggs. The onset of 
phosphate accumulation is also preceded by a lag period. Uptake was initiated by 
both a single treatment with butyric acid and a double treatment of butyric acid and 
hypertonic sea water. The rate of P 32 uptake in these experiments was approxi- 
mately 75% of the fertilized egg rate. This difference was probably due to the 
activation of only 60-70% of the eggs in the chamber, as measured by membrane 
elevation. Moreover, none of the activated eggs cleaved normally. It seems 
probable that under optimum conditions, with activation approaching 100%, the 
uptake would approach very closely that of the fertilized eggs. 

The time of onset and the magnitude of phosphate uptake appear to be poten- 
tialities of the egg, then, and are independent of the sperm. The increase in uptake 
does not begin until well after the visible cortical events of membrane elevation 
have occurred. It is of interest also that the well-known increase in respiration 
associated with fertilization begins earlier than the phosphate uptake and is not 
clearly associated with it. 

Effect of lozv temperature on P 32 uptake 

Two experiments were carried out in which the temperature was initially 8.0 
C. followed by an increase to 18.0 C. in the middle of the experiment. At 8.0 
C. there was a comparatively low rate of P 32 uptake, which was readily stimulated 
when the temperature was raised to 18.0 C. (Fig. 4). From these experiments a 
Q 10 of 2 and 2.3 was calculated comparable to the value of 2 obtained by Villee and 
Villee (1952) with Arbacia punctulata. 

Effect of 2, 4-dimtro phenol on P zz uptake 

While the establishment of the high uptake rate does not coincide with the 
establishment of the high respiratory rate, and the pattern of uptake during cleavage 
does not resemble the exponentially increasing respiratory rate, the effect of tem- 
perature still suggests that phosphate uptake may be related to energy metabolism. 

2,4-dinitrophenol (DNP) is a metabolic inhibitor known to interfere especially 



PHOSPHATE UPTAKE BY EMBRYOS 



139 



6000 



UJ 




60 120 160 240 

TIME MINUTES 



300 



360 



FIGURE 4. Effect of temperature on P 32 uptake. The initial low level of activity repre- 
sents the background count. Insemination was at zero time. Arrows indicate a change in the 
temperature of the perfusion solution. 



with aerobic phosphorus metabolism with the result that it uncouples phosphoryla- 
tions from oxidations (Loomis and Lipmann, 1948) and so in turn interferes with 
energy-requiring processes (Simon, 1953). Inhibition of phosphate uptake has 
been reported by Abelson (1947) for dinitrocresol, another substituted phenol. 
However, in some unpublished experiments, Whiteley had observed that when 
DNP was added some time after fertilization, there was no inhibition of phosphate 
uptake. A detailed investigation of this point has shown that the time when DNP 
is applied has a direct bearing on its effect on P 32 uptake. In these experiments 
DNP in a concentration of 10~ 4 M in sea water was introduced into the perfusion 
chamber at various times before and after fertilization, and the effect on the rate of 
P 32 uptake was measured. This concentration will reversibly inhibit cleavage. 
When DNP is applied before fertilization or any time within the first thirty 
minutes following fertilization, there is a marked inhibition of P 32 accumulation, 
as may be seen in Figure 5. After thirty minutes, the effect of DNP decreases 
until, when it is added at sixty minutes after fertilization, it has no effect on P 32 



140 



J. B. LITCHFIELD AND A. H. WHITELEY 



6000 



ID 5000 

Z 



(T 

LJ 
CL 



4000 



o 
o 



3000 



>- 

> 

\- 
o 

o 

Q 

o: 



2000 



1000 



i i 



T I I 




-60 



60 120 ISO 240 

TIME MINUTES 



300 



FIGURE 5. Effect of 1(T 4 M DNP on P 32 uptake. The initial low level of activity repre- 
sents the background count. Insemination was at zero time. Arrows indicate the addition of 
DNP to the perfusion chamber, a, DNP added at 60 min. ; b, DNP added at 50 min. ; c, DNP 
added at 30 min. ; d, DNP added at insemination. 



accumulation. If the rate of P 32 uptake is plotted against the time after fertilization 
when DNP is applied, the time course of the inhibition can be clearly seen (Fig. 
6). The maximum inhibition is associated with the first 30 minutes following 
fertilization. The degree of inhibition at 40 minutes is variable. This variability 
may be correlated with the length of the lag period and the onset of the maximum 
uptake rates : if the latter is not established until after the application of DNP at 
40 minutes, the inhibition seems to be appreciable ; if the maximum rate of uptake 



PHOSPHATE UPTAKE BY EMBRYOS 



141 



LJ 



20 



LJ 



15 



=> 

o 
o 



10 



LJ 
Q_ 
O 



5- 




o 
I 



15 
TIME 



30 

MINUTES 



45 



60 



FIGURE 6. Effect of 10" 4 M DNP on the rate of accumulation of P 32 when the DNP is 
added at various times after insemination. Ordinate average slope from insemination to the 
end of the experiment. Abscissa time after insemination when the DNP was added. Open 
circles: fertilized eggs and DNP; solid circles: fertilized eggs; half-open circles: artificially 
activated eggs ; quarter-open circles : unfertilized eggs. 



has been established by 40 minutes, the inhibition is considerably reduced. Once 
P 32 uptake has been firmly established, as at 60 minutes after fertilization, DNP 
has no effect on the accumulation, even in experiments continued for three hours 
after the application of the DNP, and despite inhibition of cleavage. Fertilized 
eggs are most sensitive to DNP, as far as P 32 uptake is concerned, at a time when 
virtually no P 32 uptake has been established. 

Effect of adenosinetriphosphate on dinitrophenol inhibition 

Kriszat and Runnstrom (1951), Barnett (1953) and Kriszat (1954) have re- 
ported that ATP will partially reverse the cleavage block due to DNP. In the 
light of these results it was thought that ATP might overcome the inhibitory effect 
on P 32 uptake produced by DNP applied during the lag period. The two experi- 
ments of Table I were done to test this possibility. To conserve ATP the perfusion 
chamber was not used in these experiments. In each experiment four equal aliquots 
containing about ten thousand fertilized eggs were placed in four small flasks. At 



142 J. B. LITCHFIELD AND A. H. WHITELEY 

four to six minutes after insemination the flasks received P 32 at a final concentra- 
tion of 0.01 /ic/ml. and either sea water, DNP, ATP, or DNP and ATP. Final 
concentrations of DNP and ATP (Sigma crystalline disodium adenosine 5'-triphos- 
phate) were 10~ 4 M in each instance. In Experiment 1 the final volumes were 
100 ml., and in Experiment 2, 50 ml. The pH of the cultures was 7.8 to 8.0 and 
they were maintained with stirring at 15.5 C. 'Orthophosphate concentrations in 
the cultures varied from 2.1 to 3.8 ,uM. Two hundred and seventy minutes after 
adding DNP and ATP in the first experiment and 90 minutes in the second, the 
embryos were gently centrifuged and washed three times with sea water. The 
control eggs removed less than \2% of the available P 32 from the culture solutions. 
It is clear that while DNP is markedly inhibitory when added so soon after fertiliza- 
tion, 1Q- 4 M ATP does not relieve the DNP inhibition. ATP itself, in these and 
one other experiment, depresses uptake 15 to 28%. Jn both experiments cleavage 
was completely inhibited by the DNP, even in the presence of ATP. In the longer 
experiment only 5 to 10 % of the embryos cleaved w r hen subsequently placed in sea 
water at the end of the experiment. Recovery was 90 to 95% in the shorter 
experiment. 

The negative results could mean that DNP damage is not repaired by direct 
addition of ATP, or that insufficient ATP penetrated to activate the energy-de- 
pendent reactions blocked in the presence of DNP. The permeability of developing 
eggs to ATP was tested directly in several experiments in which the concentration 
of ATP in sea water bathing eggs was measured during the first 8 or 9 hours of 
development. In these experiments recently inseminated eggs were suspended in 
sea water containing 5 X 10' 5 M ATP (Sigma, crystalline Na 2 ATP) at pH 8.0 
and at 15.1 C. The egg concentrations were about 1% by volume or 10,000 
eggs/ml. Aliquots of the suspensions were collected immediately and at intervals 
until the ninth hour of development, and were assayed for the concentrations of 
adenine-containing compounds (absorbance at 260 m/0, inorganic phosphate, and 
acid-labile phosphate (phosphate hydrolyzed in 10 minutes in 1 N HC1 at 100 C.). 
Controls consisted of sea water with ATP, but no eggs, and sea water with eggs 
but no ATP. 

If ATP were absorbed to or penetrated into the eggs, the absorbance of 
ultraviolet light and the acid-labile phosphate would diminish proportionately. If 
ATP were hydrolyzed to ADP or AMP by surface-located enzymes without pen- 
etration of the adenine moiety, the acid-labile phosphate would decrease, but not 
the ultraviolet absorbance. In different experiments the removal of adenine- 
containing components by the eggs varied from 0-2.34 X 10~ 4 pinoles/ 10, 000 
eggs/hr., and the maximum observed change in ATP that could be ascribed to a 

TABLE I 
P 32 4 cpm/aliquot 

Exp. 1 Exp. 2 

Sample (in DNP for 270 min.) (in DNP for 90 min.) 

Control eggs 6583 6642 

Eggs in 10- 4 M1 ATP 4758 5362 

Eggs in 10-* M DNP 17 42 
Eggs in 10-W ATP 

and 10-WATP 14 50 



PHOSPHATE UPTAKE BY EMBRYOS 



143 




60 

TIME 



120 180 

MINUTES 



240 



FIGURE 7. Effect of 10 * M arsenate on P 3!! uptake. The initial low level of activity 
represents the background count. Insemination was at zero time. Arrows indicate a change 
in the perfusion solution. 

surface-located ATPase was 7.9 X 1O 4 /mioles/10,000 eggs/hr. Both these 
figures are at the limits of the techniques, and indicate practically no change in the 
ATP in the sea water around the eggs. Therefore, the failure of ATP to relieve 
the DNP inhibition of P 32 uptake during the lag phase may be due to insufficient 
penetration of the added ATP. 

Effect of arsenate on P 3 - uptake 

Arsenate, because of its structural similarity, is a competitive inhibitor of 
phosphate in a number of enzymatic reactions. 

As shown in Figure 7, a concentration of 10~ 4 M sodium arsenate markedly 
inhibits P 32 uptake. Unlike DNP, this inhibition occurs whenever arsenate is 
applied from fertilization to 3 l / 2 hours after fertilization. The inhibition is com- 
pletely reversible upon removal (Fig. 7, Table II). A decrease in the rate of 
P 32 accumulation sets in within 1 to 5 minutes after the arsenate has first entered 
the chamber, and recovery from this inhibition takes place within a comparable 
period of time upon removal of the inhibitor. When arsenate is applied at fertiliza- 
tion, a limited uptake commences after a lag period that is within the range of 
the normal lag period, for example, 26 minutes in one experiment. Therefore, 



144 



J. B. LITCHFIELD AND A. H. WHITELEY 



Effect of 



TABLE II 

M arsenals on P 32 uptake 



Exp. No. 


Interval after fertilization 


Perfusion solution 


Rate of P 32 
uptake, counts/ 
min. /min. 


1 


0-88 min. 
89-143 min. 
144-200 min. 


AsO 4 , P 32 -sea water 
P 32 -sea water 
AsO 4 , P 32 -sea water 


6.0 

25.0 
7.0 


2 


0-54 min. 
55-204 min. 
205-260 min. 


P 32 -sea water 
AsO 4 , P 32 -sea water 
P 32 -sea water 


20.3 
6.9 

28.0 



arsenate does not seem to increase the length of the lag period, but markedly and 
reversibly suppresses penetration during the accumulation phase. 

It has been tentatively concluded by Yeas (1950) that arsenate does not penetrate 
the eggs of the echinoid Lytechinns pictus. This conclusion was based on experi- 
ments that showed that arsenate had no effect on respiration or on cleavage. It 
was assumed that, if it had penetrated, there would have been an interference with 
oxidative phosphorylation, and consequently respiration and cleavage would have 
been affected. An experiment was set up to confirm these observations and extend 
them to S. pnrpnratus. Small numbers of fertilized eggs were placed in finger 
bowls of sea water containing sodium arsenate at various concentrations five 
minutes after insemination, and ninety-five minutes after insemination. The time 
required for these embryos to attain the early cleavages, and the normality of sub- 
sequent development were then determined (Table III). A concentration of 
arsenate of 10"* M has an almost imperceptible effect on early cleavages and shows 
a clear retardation of development only at early blastula stages. More serious 
retardations set in after 8 hours in 10~ 4 M arsenate, leading to death in an early 
blastula condition at 28 hours, when controls are late blastulae. One hundred- 
fold stronger concentrations delay the first three cleavages by only about 20%, 
although development beyond 8 hours involves increasing abnormalities, with 
death at 22 hours. In experiments not included in Table III, 10~ 3 M arsenate 
showed retardations only a little greater than 10~* M. Only small quantitative 
differences were caused by adding the arsenate at 5 minutes as compared with 
95 minutes after insemination. It is doubtful that arsenate penetrates the eggs at 
an appreciable rate during cleavage. Therefore, the effects of arsenate on phosphate 
uptake are interpreted as evidence for a surface location of the penetration mecha- 
nism. 

DISCUSSION 

The evidence, taken as a whole, suggests the view that the rapid penetration of 
phosphate into sea urchin embryos is an enzymatically controlled transport. The 
reaction has a temperature coefficient of 2 to 2.3, which is compatible with this 
possibility though not, by itself, conclusive (Danielli, 1952). It is inhibited by 
arsenate, a competitive analogue of phosphate. Furthermore, other investigators 



PHOSPHATE UPTAKE BY EMBRYOS 



145 



TABLE III 

Effect of arsenate on development of embryos of S. purpuratus. Solutions were at pH 8 .0 and 11.4 C., 

and had an ortho phosphate concentration of about 2 X 10~ 6 M. Arsenate was added at 

5 min. (column A) or 95 min. (column B) after insemination 



Time after 
insemination 


Controls 


10-4 M AsO4 


10-2M AsO4 


A 


B 


A 


B 


1 hr. 50 min. 
1 55 

2 7 


50% 2-cell 


50% 2-cell 


50% 2-cell 


50% 2-cell 


50% 2-cell 


2 55 
3 3 
3 7 
3 25 


50% 4-cell 


50% 4-cell 


50% 4-cell 


50% 4-cell 


50% 4-cell 


4 7 
4 20 
4 55 


50% 8-cell 


50% 8-cell 


50%) 8-cell 


50% 8-cell 


50% 8-cell 


7 20 


32-cell 


50% 32-cell 


16-cell, many abnormal 


11 35 


early blastulae 


very early blastulae 


abnormal late cleavage 


22 35 


rotating blastulae 


early blastulae 


dead very early blastuale 


23 20 


hatching 


early blastulae 





28 27 


late blastulae 


dead 






have found that the uptake is independent of the external phosphate concentration 
over a wide range (Brooks and Chambers, 1954; Chambers and White, 1954). 

That the mechanism of phosphate entry is surface-located seems most probable 
from the results of the arsenate experiments. Arsenate must penetrate only very 
slowly, if at all, into the early cleavage stages. This follows from the observations 
of Yeas (1950) and those reported here, that arsenate, even at the concentration 
of 10" 2 M, shows little effect on cleavage of these eggs, and retardations occur only 
after some hours. The conclusion is strengthened further by Yeas' finding that 
0.05 M arsenate does not inhibit respiration of fertilized and cleaving eggs of 
Lytechinus pictus. In marked contrast the strong inhibition exerted by 10~* M 
arsenate on phosphate uptake is evident in a very few minutes. The arsenate must 
have its effect at the surface by competition with phosphate for a surface-located 
transport mechanism, and the reaction is probably not directly linked to respiratory 
metabolism. 

The specific reaction by which phosphate enters the embryo is not definitely 
elucidated by these experiments, but certain possibilities are suggested by the 
arsenate experiments. Arsenate is known to be a competitive analogue of phos- 
phate, and therefore will substitute for it in enzymatic reactions. The resulting 
arsenate compound is usually unstable and is hydrolyzed instantly. The term 



146 J. B. LITCHFIELD AND A. H. WHITELEY 

arsenolysis has been applied to the action of arsenate in splitting organic compounds 
(Doudoroff, Barker and Hassid, 1947). 

Arsenate has been shown to inhibit the enhancement of P 32 uptake by adenosine 
in mammalian red blood cells (Prankerd and Altman, 1954). In this system 
arsenolysis is presumed to result in a decrease in glyceraldehyde-3-phosphate. The 
conversion of glyceraldehyde-3-phosphate to 1-3-diphosphoglyceric acid near the 
surface of the cell is proposed as the mechanism of phosphate entry into the human 
red blood cell (Prankerd, 1956). The same mechanism could be operative in 
the fertilized eggs, although arsenate would also inhibit other phosphate esterifica- 
tions. 

The time of establishment of the mechanism is within the first 40 to 50 minutes, 
varying with eggs from different animals. Whether its appearance as a functional 
system is during the lag period of 7 to 30 minutes, or whether the subsequent period 
of increasing activity represents the time of establishment is not answered by these 
experiments. The experiments with parthenogenesis show clearly that the estab- 
lishment of the mechanism is not dependent on the sperm, nor on the existence of 
a normal cleavage mechanism. 

There remains to be considered the relation between the egg's metabolism and 
the transport mechanism. The presence of DNP during the first 30 minutes 
after fertilization prevents very markedly the later uptake of phosphate. It is 
assumed that DNP is exerting its characteristic uncoupling action and is con- 
sequently inhibiting the formation of high-energy phosphate by aerobic oxidations 
at this time. However, it appears that there is enough energy for this process at 
60 minutes after fertilization despite the presence of DNP. At this time also, 
DNP is presumably having its effect on oxidative phosphorylation since cleavage 
is reversibly blocked. A correlation between oxidative phosphorylation and cleav- 
age has i)een shown by Clowes, Keltch, Strittmatter and Walters (1950), whose 
experiments demonstrate that the concentration of a substituted phenol that will 
block oxidative phosphorylation in cell-free participate systems of Arbacici pnnctnlata 
will also inhibit cleavage in the intact egg. The conclusion drawn from this 
similarity in effective concentrations is that DNP inhibits cleavage by interfering 
with high-energy phosphate production. 

Two interpretations of the effects of DNP on P 32 uptake present themselves. 
According to one, the initial period may be sensitive to DNP because aerobic 
phosphate bond energy is needed for the synthesis of the enzymes of the transport 
mechanism as is the case with adaptive enzyme synthesis (Monod, 1944), or 
perhaps for the spatial rearrangement of the pre-formed system. The later period 
may be insensitive to DNP because the maintenance and operation of the mechanism 
requires smaller amounts of aerobic phosphate bond energy. 

An alternative idea is based on a suggestion from a paper of Siekevitz and 
Potter (1953). It may be that the energy source for the establishment of a 
phosphate entry mechanism is different from that for its maintenance and opera- 
tion. In experiments with rat liver mitochondria they concluded that the ATP 
generated within the mitochondria diffused out and mingled very slowly with that 
generated by glycolysis outside of the mitochondria. Consequently, there may be 
a separation in the functions of the ATP formed in these two locations. Synthetic 
reactions within the mitochondria would preferentially utilize ATP generated 



PHOSPHATE UPTAKE BY EMBRYOS 147 

locally, while an energy-requiring reaction outside of the mitochondria would be 
served by ATP produced by glycolysis externally. It may be that the establish- 
ment of a phosphate transport mechanism involves enzyme synthesis that is favored 
by ATP formed within the mitochondria. DNP could inhibit such a process by 
its action on the tricarboxylic acid cycle, as the enzymes for this cycle are as- 
sociated with the mitochondria. Once the mechanism is established it might be 
maintained by high-energy phosphate resulting from glycolysis. DNP presumably 
does not inhibit the formation of high-energy phosphate by this means, although this 
has not been fully investigated (Simon, 1953). Glycolysis has been implicated 
in phosphate uptake by yeast (Rothstein, 1954) and by the mammalian red blood 
cell (Prankerd, 1956)' 

The negative results of the experiments testing the efficacy of externally applied 
ATP to overcome the early DNP inhibition are explained by the finding that ATP 
neither penetrates the fertilized eggs of this species, nor is adsorbed to their surface, 
nor is hydrolyzed at their surface in amounts detectable by the rather sensitive 
methods used. This failure of ATP to be efficacious is in contrast to its effect on 
cleavage inhibition by DNP reported by Kriszat and Runnstrom (1951), Barnett 
(1953), and Kriszat (1954). It is doubtful that the tiny amounts of energy that 
could have been available to the eggs, judging from the present data, would be 
sufficient to overcome the inhibitions as found by these investigators. If the eggs 
of their studies were not appreciably more permeable to ATP, one wonders if the 
effects could be ascribed to other substances in their ATP preparations. 

According to the results of Bolst and Whiteley (1957) the rate of penetration 
of phosphate increases rapidly for 35 hours in the embryos of S. pnrpnratus. This 
is in accord with the present findings that the transport system is surface-located 
because, during the development to the gastrula, the number of cells, and therefore 
the surface area of the embryos, increases through cleavage, and it is reasonable 
to suppose that the newly formed surface would possess the transport mechanism. 

SUMMARY 

1. The accumulation of phosphate by the eggs and embryos of the sea urchin, 
Strongylocentrotus purpuratus (Stimpson) was analyzed by determining the 
action of various metabolic effectors on the uptake of P 32 from sea water flowing at 
a constant rate over the eggs in a special perfusion chamber. 

2. The rate of uptake of P 32 by unfertilized eggs is nearly zero. The rate 
for the first 7 to 30 minutes after fertilization (lag phase) is also nearly zero, but 
increases rapidly during the next 20 to 30 minutes (augmentation phase), and be- 
comes maximal 22 to 60 minutes after fertilization (accumulation phase) at a 
level many times that of the unfertilized eggs. 

3. Artificial parthenogenesis, by either the single or double treatment, results 
in the same pattern and magnitude of uptake as does fertilization, even in the 
absence of cleavage. 

4. Phosphate accumulation is markedly inhibited by 10' 4 M 2.4-dinitrophenol 
if this agent is added during the lag phase, moderately inhibited if added during 
the augmentation phase, but is unaffected if added during the accumulation phase. 

5. Addition of 10^* M ATP simultaneously with dinitrophenol early in the lag 
phase does not alleviate the inhibition caused by the latter. 



148 J. B. LITCHFIELD AND A. H. WHITELEY 

6. ATP neither penetrates into cleaving eggs nor is hydrolyzed by an ATPase 
at their surface. 

7. 10~* M arsenate markedly inhibits P 32 uptake at all times after fertilization. 

8. Arsenate does not materially retard early development of these sea urchin 
eggs indicating that it does not penetrate into them. 

9. P 32 uptake has a temperature coefficient of 2 to 2.3 during the accumulation 
phase. 

10. The evidence resulting from the use of these effectors indicates that P 32 
uptake in sea urchin embryos is enzymatically controlled and that the enzymatic 
mechanism is located on the cell surface. The period immediately following 
fertilization is believed to be a time when the uptake mechanism is being established. 
This process appears to be dependent on phosphate bond energy, the production of 
which is DNP-sensitive. During the accumulation phase it is suggested that the 
energy requirements for the operation and maintenance of this mechanism are 
quantitatively much smaller, or are satisfied by phosphate bond energy the produc- 
tion of which is DNP-insensitive. Possible reasons for this difference in sensitivity 
are discussed. 

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PHOSPHATE UPTAKE BY EMBRYOS 149 

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ROTHSTEIN, A., 1954. The enzymology of the cell surface. Protoplasmatologia, 2: E4, 1-86. 
SIEKEVITZ, P., AND V. R. POTTER, 1953. Intramitochondrial regulation of oxidative rate. /. 

Biol Chcm., 201: 1-13. 

SIMON, E. W., 1953. Mechanisms of dinitrophenol toxicity. Biol Rei:, 28: 453-479. 
TYLER, A., 1949. A simple, non-injurious method for inducing repeated spawning of sea urchins 

and sand-dollars. Coll. Net, 19: 19-20. 

VILLEE, C. A., AND D. T. ViLLEE, 1952. Studies on phosphorus metabolism in sea urchin em- 
bryos. /. Cell Com p. Physiol, 40: 57-71. 
WHITELEY, A. H., 1949. The phosphorus compounds of sea urchin eggs and the uptake of 

radio-phosphate upon fertilization. Amer. Nat., 83: 249-267. 
YCAS, M. F., 1950. Studies on the respiratory enzymes of sea urchin eggs. Thesis. Calif. 

Inst. Tech., Pasadena. 
ZEUTHEN, E., 1951. Segmentation, nuclear growth and cytoplasmic storage in eggs of echino- 

derms and amphibia. Pitbbl Stas. Zool Napoli, 23 (suppl.) : 47-69. 



SOME EFFECTS OF TEMPERATURE ON DEVELOPMENT IN 
THE SEA URCHIN ALLOCENTROTUS FRAGILIS 

A. R. AIOORE 

Hopkins Marine Station of Stanford L'nh'crsity, Pacific Grove, California 

The deep water sea urchin Allocentrotus jragilis occurs offshore at Pacific 
Grove at depths of 80 to 100 fathoms. The temperature of this environment is 
fairly constant at 8 C. During the past two years it has been possible, at this 
Station, to study the embryological development of this form under laboratory 
conditions. Normal development of the fertilized eggs proceeded at 7-15 C. 
Lower temperatures were not tried. There was some variation at the upper limit, 
the eggs of some females not developing normally above 14 C. while others yielded 
normal embryos at 16 and 17 C. At ordinary room temperature (20 C.), 
however, cytoplasmic cleavage is not normal if it occurs at all. A study of this 
effect may throw some light on the behavior of cytoplasm and nucleus in cell division. 

In order to determine whether the nucleus might be responsible for the failure 
of cleavage at 20 C., the viability of the nucleus was tested by putting it to develop 
in the hardy cytoplasm of Strongylocentrotus pnrpitratus. The eggs of this purple 
sea urchin develop normally at the higher temperature, and when fertilized with 
the sperm of Allocentrotus jragilis they segment and develop into normal plutei at 
20 C. Similarly the eggs of the sand dollar Dcndrastcr cxccntricns fertilized with 
the sperm of Allocentrotus fragilis segment normally at the tempo of Dendraster. 
Such hybridized eggs develop into plutei, which, in their form, show the cross to be 
a true one. Hence the male nucleus has taken part in development. Thus, while 
the cytoplasm of Allocentrotus is inactivated at a temperature of 20 C., the nucleus 
of this species, if given a proper medium, functions normally. 

Visual proof of the fact that this nucleus also functions in the egg of its own 
species even at 20 C. is shown by putting fertilized eggs of Allocentrotus to 
develop at 20 C. Here the nuclei undergo regular division while the cytoplasm 
remains apparently inert, i.e., undivided (Figs. 1 and 2). Now these eggs had 
formed normal fertilization membranes at the outset before being put at the higher 
temperature. Therefore there is no reason to suppose that here we are dealing with 
a Sugawara ( 1943) effect in which failure of the fertilization membrane to lift results 
in constriction of the egg to its original volume with the results that cleavage of 
the egg is inhibited but nuclear division proceeds. Eventually the eggs with which 
we are dealing, after many nuclear divisions, disintegrate. This experiment gives 
further proof that in the early stages the nucleus is more resistant to higher 
temperatures than its cytoplasm and carries out its divisions independently of the 
cytoplasm. 

In the later stages of development, when the cytoplasm has become comminuted 
into the thousand odd cells of blastula and gastrula, the spatially relative situation 
of nucleus and cytoplasm is quite different. If now the advanced larvae of 

150 



ON DEVELOPMENT IN ALLOCENTROTUS 



151 




3 



ri 



I ./ 



FIGURE 1. Normal 8-16 cell embryos at 15 C. FIGURE 2. Same at 20 C. FIGURE 3. 
Five-day pluteus developed at 15 C. FIGURE 4. Five-day plutei in which the temperature was 
changed to 20 C. after development to gastrulae at 15 C. Magnification 95 X. 

Allocentrotus were transferred from the cold room where they had developed to a 
warm room with a temperature of approximately 20 C., it was found that the 
higher temperature no longer exercised a lethal effect on the cytoplasm, but that 
such larvae reached the pluteus stage (Figs. 3 and 4). These plutei were smaller 
and less elaborate than the normal ones developed at the low temperature. 



DISCUSSION 

Generally speaking the problem of cleavage has been attacked from the point 
of view of colloidal behavior, the relation of cytaster formation to the constitution of 
the cell which is the substance of the egg. Cleavage is a process initiated by the 
entry of the sperm into the egg and the formation of the membrane. In artificial 
parthenogenesis the formation of the membrane is sufficient to initiate the develop- 
ment of cytasters and subsequent cleavage of the egg. A second factor has been 
suggested by Swann (1952) who postulates the active agents in cleavage to be 
catalytic substances released from the chromosomes of the sperm nucleus. These 
he has termed "structural agents." 



152 A. R. MOORE 

In the foregoing experiments we have evidence that the differences in the inter- 
action of nucleus and cytoplasm described depend on the nuclear-plasma relation. 
Thus in the early stages of development the mass of the cytoplasm compared to that 
of the nuclei is relatively enormous and the distance of the nucleus from the 
periphery, it seems reasonable to suppose, may be too great for the cytoplasm to be 
significantly affected by substances diffusing from the nucleus. It should be noted 
that the nucleus in the first division is dominated in the tempo of cleavage by the 
cytoplasm. This has been clearly shown in the case of Dendrastcr which has a 
cleavage time for the first division of 55 minutes at 20 C. If the experiment be 
made of enucleating the egg of Dendrastcr and then fertilizing it with the sperm 
of Strongyloccntrotus, the cleavage time of which is approximately 100 minutes, 
the subsequent division of the nucleus and cytoplasm of the experimental egg takes 
place in the time characteristic of Dendrastcr, i.e., of the cytoplasm and not of the 
nucleus. Thus the normally slow nucleus is forced by the cytoplasm to divide in 
a little more than half the time normal to it (Moore, 1933). 

As to the difference in their reaction to the higher temperature on the part of 
the fertilized eggs of Allocentrotus contrasted with that of blastulae and gastrulae, 
it may be suggested that, in the latter, the nuclei are in such intimate contact with 
the cytoplasm that they confer some of their hardiness on it and are able to do this 
because of the close association of the two phases. Such an effect becomes under- 
standable if we accept Swann's hypothesis as to the part played by the nucleus in 
the cleavage of the egg. Using Chambers' (1951) demonstration in the amoeba 
that the nucleus dynamically affects the edge of the cell next to it, Swann has 
suggested that the origin of the furrow in the first division is caused by a cleavage 
substance which diffuses from the chromosomes of the sperm nucleus to the 
periphery of the egg and initiates cleavage. In the present experiments Swann's 
hypothesis seems not to apply in the early stages of cleavage. In later stages, 
however, this hypothesis may give a reasonable explanation of the division of cells 
in advanced larvae at higher temperatures. Thus, while in the early stages of 
cleavage the cytoplasm dominates the formation of the furrow and the tempo of 
cleavage, the situation is altered later where successive divisions of the cytoplasm 
have brought the chromosomes into intimate relations with the cytoplasmic units. 
This would give play to the diffusion of cleavage substances from the chromosomes 
to the periphery as Swann has postulated. Since the effect of the higher tem- 
perature on the further development of blastulae and gastrulae is to make the 
resulting plutei defective, it seems reasonable to suggest that at the time of change 
only a part of the synthesis of structural elements has been completed and that the 
higher temperature to which they were subsequently exposed has inhibited the 
completion of structural processes essential to the form of the normal pluteus. 

SUMMARY 

1. The eggs of the deep water sea urchin Allocentrotus jragllis develop normally 
to plutei under laboratory conditions at 7-15 C. 

2. At the higher temperature of 20 C. cytoplasmic division fails but the nuclei 
show characteristic mitotic figures. 

3. The sperm nucleus of Allocentrotus jragilis functions normally at higher 



ON DEVELOPMENT IN ALLOCENTROTUS 153 

temperatures in the eggs of Strongyloccntrottts pnrpitratns and Dendraster 
excentricus. 

4. Blastulae and gastrulae of Allocentrotus jragilis brought to a temperature 
of 20 C., which is lethal for the eggs and early division stages, develop into plutei 
of reduced size. 

5. It is suggested that in the advanced larvae hardiness to the higher tem- 
perature is the result of the intimate association of nucleus and cytoplasm in the 
minute cells, and the synthesis of structural elements and processes at the lower 
temperature. 

LITERATURE CITED 

CHAMBERS, R., 1951. Micrurgical studies on the kinetic aspects of cell division. Ann. N. Y. 

Acad. Sci., 51: 1311-1526. 
MOORE, A. R., 1933. Is cleavage rate a function of the cytoplasm or of the nucleus? /. Ex{>. 

Blol, 10: 230-236. 
SUGAWARA, H., 1943. The formation of multinucleated eggs of the sea urchin with proteolytic 

enzymes. /. Fac. Sci. Imp. Unir. Tokyo, 6: 129-139. 
S \VANX, M. M., 1952. The nucleus in fertilization, mitosis and cell division. In: Structural 

Aspects of Cell Physiology, 6: 89-104. 



A REVISION IN THE SPHAEROMID GENUS GNORIMOSPHAEROMA 
MENZIES (CRUSTACEA: ISOPODA) ON THE BASIS OF MOR- 
PHOLOGICAL, PHYSIOLOGICAL AND ECOLOGICAL 
STUDIES ON TWO OF ITS "SUBSPECIES" 

J. A. RIEGEL i 

Department of Zoology, University of California, Davis, California 

During a recent study into the physiology of osmoregulation in sphaeromid 
isopods (Riegel, 1959), an interesting problem was brought to light concerning the 
taxonomy of Gnorimosphaeroma oregonensis (Dana, 1852). Menzies (1954a) 
split the species into two subspecies, In tea and oregonensis. G. o. oregonensis was 
described as a typical intertidal bay form, inhabiting the undersides of rocks in 
waters whose salinity approached that of normal sea water. G. o. Intea was de- 
scribed as a creek or pond dweller confined to waters of low salinities and often 
associated with mud and vegetation. Differences in osmoregulatory ability and 
ecological requirements, as well as morphological differences, lead the writer to 
doubt the validity of Menzies' subspecies. 

Riegel (1959) performed experiments on specimens from fresh-water, estuarine 
and bay populations of Gnorimosphaeroma oregonensis to test their osmoregulatory 
abilities. He found that specimens of the bay form [= G. o. oregonensis (Dana)] 
could neither regulate their body fluid concentration within viable limits nor survive 
for longer than a few days in fresh water. Specimens of the estuarine and fresh- 
water forms of G. oregonensis ( -- G. o. httca Menzies 1954) were able to regulate 
their body fluid concentrations within viable limits and survive for over three weeks 
in salinities ranging from fresh water to 125 per cent sea water. Specimens of 
the fresh-water form, when in 50 per cent sea water or less, could maintain 
significantly higher body fluid concentrations than could specimens of the estuarine 
form. All forms of G. oregonensis were found to regulate hyper-osmotically in 
dilute media and hypo-osmotically in salinities just below r or above normal sea 
water. 

The present paper reports the results of experiments and observations designed 
to clarify the taxonomic position of the "subspecies" of Gnorimosphaeroma 
oregonensis described by Menzies (1954a). The differences which prompted the 
unofficial separation of that species into three habitat groups (i.e., estuarine, fresh- 
water and bay forms) in a previous paper (Riegel, 1959) are no longer under 
primary consideration, so to avoid confusion, Menzies' subspecific names will be 
used throughout the balance of this paper. 

METHODS 

Experiments were conducted to determine the effect of salinity on the mor- 
phology of Gnorimosphaeroma oregonensis littea and G. o. oregonensis. One 

1 Present address : Department of Zoology, State College of Washington, Pullman, Wash- 
ington. 

154 



A REVISION IN GNORIMOSPHAEROMA 155 

hundred young of each "subspecies," newly emerged from the brood pouch, were 
placed in separate ringer bowls containing rocks and normal sea water. The normal 
sea water in which the young G. o. oregonensis were placed was diluted over a 
period of two weeks to ten per cent sea water. Controls consisting of newly- 
emerged young of both "subspecies" were kept in normal habitat water, but other- 
wise under identical conditions. The young isopods were fed slices of frozen 
shrimp occasionally, and the water was changed weekly. 

In another experiment, more than two hundred ovigerous females (plus over a 
hundred males and immature individuals) of Gnorimosphaeroma oregonensis lutea 
were placed in a large plastic tub containing normal sea water and otherwise simula- 
ting the natural habitat as closely as possible. The "brood pouches" of the fe- 
males contained eggs and embryos in all stages of development. The animals were 
fed slices of frozen shrimp occasionally. Samples of twenty young were removed 
from the tub each week (for three months) and examined for possible morphological 
differences created by the high salinity. This experiment was done in order to sub- 
ject specimens of G. o. lutea to high salinity as early in their embryological develop- 
ment as possible, in case the effect of salinity (as measured in the first experiment, 
described above) is no longer exerted on the morphology of the animals after they 
leave the brood pouch. 

To ascertain to what extent (if any) populations of Gnorimosphaeroma orego- 
nensis lutea and G. o. oregonensis intergrade ecologically, the writer made exten- 
sive surveys in San Francisco Bay, Tomales Bay, and coastal inlets from Half Moon 
Bay, San Mateo County, to Stillwater Cove (near Fort Ross), Sonoma County, 
California. 

As the result of handling large numbers of Gnorimosphaeroma oregonensis lutea 
and G. o. oregonensis, the writer observed that the body in the latter form seemed 
relatively broader than in the former form. Therefore, measurements of length 
and width were taken of random samples of the two "subspecies" from various 
localities and salinities. From these measurements, length/width ratios were 
calculated and analyzed statistically. 

RESULTS 

The newly-emerged young of Gnorimosphaeroma oregonensis lutea and G. o. 
oregonensis died slowly over a six- week period. That their deaths were not due to 
salinity alone is indicated by the fact that the controls also died slowly. However, 
during the six- week period, some growth was detected (both by increase in size and 
the presence of cast-off exoskeletons), but no changes in the morphology of the 
animals were seen. 

The young removed from the plastic tub in which were kept the ovigerous fe- 
male Gnorimosphaeroma oregonensis lutea all showed the typical morphology of 
that "subspecies." Of particular significance was the fact that young which under- 
went their entire development in normal sea water showed no change from the 
typical morphological configuration of G. o. lutea. These were young hatched from 
eggs which were in the "brood pouches" of the females when the females were 
originally placed in normal sea water. At the end of the experiment, no ovigerous 
females were found in the plastic tub and many of the young were half grown 
(3-5 mm. long). 



156 J. A. RIEGEL 

The results of surveys designed to determine the degree of ecological intergra- 
dation between Gnorimosphaeroma oregonensis lutea and G. o. oregonensis were 
as follows : G. o. oregonensis was found in only three locations, Stillwater Cove, 
Point San Quentin in San Francisco Bay, and Tomales Bay State Park in Tomales 
Bay. G. o. lutea was found in Pilarcitos Creek draining into Half Moon Bay, 
several creeks draining into Tomales Bay, and in the Napa River and several creeks 
draining into the northern end of San Francisco Bay. In only one location did the 
two "subspecies" occur in the same general area, which was at Tomales Bay State 
Park. At the other locations cited above for G. o. lutea, conditions were probably 
not suitable for G. o. oregonensis, since the substrate was either muddy or sandy 
and lacked the rocks and loose rubble which characterize the habitats from which 
the writer has collected the latter form. At Tomales Bay State Park, where the 
two "subspecies" occurred together, there was a sharp break in their respective 
habitats. G. o. lutea was found in a small creek among the vegetation and under 
rocks. Little more than 50 feet away, but in the intertidal area of the rocky beach, 
G. o. oregonensis was collected from under rocks and among the loose rubble. The 
only known barrier present was the very dilute salinity of the creek water (fresh 
to taste), which would probably prevent G. o. oregonensis from entering the creek. 
Since G. o. lutea is capable of living in salinities approaching normal sea water, 
there appeared to be no salinity barrier to its colonization of the intertidal area. 
However, no specimens referable to G. o. lutea were found in the G. o. oregonensis 
habitat, and no specimens of G. o. oregonensis were found in the G. o. lutea habitat. 
At Stillwater Cove, G. o. oregonensis were collected in the loose rubble and under 
stones intertidally. There was a small stream running into the cove, which ap- 
peared suitable for habitation by G. o. lutea, but that form was not found there. 

From measurements of the body length and width of random samples of ten 
specimens of each of the two "subspecies," the following ratios were found. The 
length/width ratio of the body of Gnorimosphaeroma oregonensis oregonensis 
averages 1.64 0.021, with a range of 1.50 to 1.75. The length/width ratio of the 
body of G. o. lutea averages 1.84 0.018, with a range of 1.70 to 1.96. The 
mean differences were found to be highly significant statistically (t = 7.29). Sub- 
sequent checks of specimens of both "subspecies" from different localities and 
salinities made since the above measurements were taken bear out the decided 
separation in the range of length/width ratios between G. o. oregonensis and G. o. 
lutea. 

DISCUSSION 

Osmoregulatory ability in relation to ecology 

In general, the osmoregulatory abilities of the various experimental groups re- 
ported in a previous paper (Riegel, 1959) and summarized in the present paper, 
agree well with their habitat and distribution according to salinity tolerance. 

The fresh-water form of Gnorimosphaeroma oregonensis lutea is a good osmoreg- 
ulator, which would be expected of a successful invader of fresh water. One 
barrier to complete adaptation to fresh water, namely, the ability to reproduce and 
rear young in that medium, possibly has been circumvented by this form. The 
brood pouches of most isopods are formed by flaps (oostegites) projecting medially 
from the bases of four pairs of thoracic legs. The developing eggs and embryos 



A REVISION IN GNORIMOSPHAEROMA 157 

are held between the overlapping flaps and the sternum. However, in the Sphaero- 
midae, the eggs and embryos are carried in various types of internal "brood 
pouches" (Menzies, 1954b) which in G. oregonensis (sensu lato) are separated from 
the body fluid only by a thin and presumably permeable membrane. . Thus, by regu- 
lating the body fluids osmotically, the sphaeromid probably can regulate the osmotic 
environment of the eggs and embryos. Young G. o. lutea which hatched out in the 
laboratory survived for several weeks in fresh water. Whether reproduction in 
fresh water is dependent upon the internal "brood pouch" of G. o. lutea or due to 
osmoregulatory ability or osmotic resistance of its embryos and eggs is not known. 

Populations of Gnorimosphaeroma oregonensis lutea living in brackish water 
must adjust to salinity variation of at least two types: (1) Daily fluctuations in 
salinity of relatively short duration during the tidal cycles, and (2) seasonal fluctua- 
tions due to rainfall and runoff from melting snow. Measurements of habitat 
salinity and body fluid concentration changes during a portion of a tidal cycle 
(Riegel. 1959) showed the pattern of salinity fluctuations to which an estuarine 
population of G. o. lutea adapted. Between a period from low to high tide, the 
salinity of the habitat varied from fresh water (0.27% sea water) to 65 per cent 
sea water. During the same period, the body fluid concentrations of the resident 
G. o. lutea varied from 50 per cent sea water (in fresh water) to 70 per cent sea 
water (in 65% sea water). Another population of the "subspecies" which lived 
in a pond situation away from tidal salinity influences had to adjust to salinities as 
high as 60 per cent sea water during the late summer and fall, and as low as ten per 
cent sea water during the winter and spring periods of rain and heavy runoff from 
melting snow. 

The internal "brood pouch" of Gnorimosphaeroma oregonensis living in 
brackish water is of possible value as a "buffering" mechanism to prevent damage 
to eggs and embryos by extreme fluctuations in the salinity of the habitat, especially 
in the lower salinity ranges. 

Gnorimosphaeroma oregonensis oregonensis has never been collected from salini- 
ties approaching fresh water. The lowest salinity recorded in its habitat was about 
12 per cent sea \vater, which occurred during a period of particularly heavy rains ; 
presumably, this form is capable of surviving for short periods in such low salini- 
ties. Further, it is improbable that the animals must ever endure prolonged ex- 
posure to low salinity. During low tide, they are always found down in the 
coarse rubble on the beach. During conditions of low salinity in the general habitat, 
it is probable that in their microhabitat the salinities are higher due to water trapped 
by spaces in the rubble, leaching of residual salts from the rubble and evaporation. 

Osmoregulation and other factors in relation to systematics 

As mentioned previously, Menzies (1954a) split Gnorimosphaeroma oregonensis 
into two subspecies, G. o. oregonensis and G. o. lutea. However, because of dif- 
ferences in their osmoregulatory physiology and habitat preference, the validity of 
Menzies' subspecies is questioned by the writer. 

The biological species is defined by Mayr et al. (1953, p. 313) as consisting of 
"groups of actually (or potentially) interbreeding natural populations which are 
reproductively isolated from other such groups." Mayr et al. define a subspecies 
(p. 314) as "a geographically defined aggregate of local populations which differs 



158 J. A. RIEGEL 

taxonomically from other such subdivisions of the species." Let us analyze Gnori- 
tnosphaeroma oregoncnsis in the light of the ahove concepts. 

Gnoritnosphaeroma oregonensis occupies a very extensive range from Alaska to 
central California. From the distribution records (Menzies, 1954a), it appears 
that the species is broken up into fresh-water, estuarine and intertidal bay popula- 
tions throughout its range. Barring the unlikely occurrence of multiple local evolu- 
tions, it is probable that the same mechanism (s) has created the G. o. lutea and 
G. o. oregoncnsis "subspecies" over the entire range. Therefore, at least three 
possibilities concerning the origin of the various forms present themselves. (1) 
The bay and estuarine-fresh-water forms separated long ago (in the sense of geologi- 
cal time) and both are still capable of distributing themselves across open ocean 
barriers. (2) The bay form has given rise to the estuarine-fresh-water form 
recently (or vice versa). (3) The morphological, physiological, and ecological 
differences between the two "subspecies" are salinity-induced and the two forms 
represent ecotypes of the same species. If the first alternative is true, the two 
"subspecies" are probably separate species. If the second alternative is true, they 
may be true subspecies. 

There are only two apparent morphological differences between Gnorimosphac- 
roma oregonensis oregonensis and G. o. lutea a difference in the morphology of the 
pleotelson (Fig. 1) and a difference in body proportions. According to Menzies 
(1954a), the third pleonite in G. o. lutea does not reach the lateral border of the 
pleotelson, but in G. o. oregonensis it does. Examination by the writer of several 
hundred specimens of each form taken from several localities and various salinities 
confirms Menzies' observation and shows also that the difference in pleotelson 
structure is remarkably consistent. Further, there were no intergrades with respect 
to this diagnostic character. The writer observed that the body in G. o. oregonensis 
seemed relatively broader than in G. o. lutea. Measurements of body length and 
width of specimens of the two "subspecies" from various localities and salinities 
bore out this observation and showed that there is a significant separation between 
the two forms in the average length/width ratio of the body. 

The osmoregulatory abilities of the two supposed subspecies show striking dif- 
ferences. Gnorimosphaeroma oregonensis oregonensis was unable to survive in 
fresh water, at least under experimental conditions (see Riegel, 1959). G. o. lutea 
lived for several days in all salinities under the experimental conditions. 

The ecological differences between the two "subspecies" are well-marked and 
possibly associated, at least in part, with the above-mentioned physiological dif- 
ference. Gnorimosphacroma oregonensis oregonensis lives intertidally in bays. 
During periods of low tide, it remains down in the coarse rubble on the beach, 
where it is out of water, but in very moist conditions. During high tide, it leaves its 
hiding places and presumably forages for food. G. o. lutea lives in estuaries and 
fresh-water streams and ponds. It is usually associated with mud and vegetation, 
but occasionally it may be collected from rocky bottoms of small streams. It lives 
among the roots of aquatic plants and in cracks in mud, rock, wood, and in burrows 
made by other animals. Of special interest in regard to the ecological separation 
of the two forms is the fact that G. o. lutea is fully capable of occupying the G. o. 
oregoncnsis habitat (taking into consideration only the former form's salinity 



A REVISION IN GNORIMOSPHAEROMA 



159 



Imm. 



PLEOTELSON 




JPLEONITES 1-3 



TELSON 



A 



PLEOTELSON 




PLEONITES 1-3 



TELSON 



B 



FIGURE 1. A. Diagram of Gnorimosphaeroma oregonensis lutea showing the whole animal 
and the details of its pleotelson morphology. B. Diagram of the pleotelson morphology of 
Gnorimosphaeroma oregonensis oregonensis (A and B after Menzies, 1954a). 



tolerance), but it does not do so. Further, as far as can be ascertained, there are 
no natural hybrids between the two forms. 

The osmoregulatory and ecological characteristics of Gnorimosphaeroma orego- 
nensis oregonensis and G. o. lutea have been considered as adaptive in the sense of 
Allen (quoted by Pantin, 1932), who suggested that adaptation be measured by 
survival. From the teleological vantage point afforded by the experiments and 
observations presented in this paper, it appears that the ability of the two "sub- 
species" to survive within their respective habitats is correlated with their ability to 
osmoregulate and survive in various experimental dilutions and concentrations of 
sea water. Caution must be exercised in making statements concerning the actual 
barrier(s) or "selective factor (s)" which has permitted one form of G. orego- 



160 J. A. RIEGEL 

nensis to invade and exploit a new environment (estuaries and fresh water) to the 
exclusion of the other. From the experimental data, salinity appears to be a major 
factor, but it is possible that such is not the case. Prosser (1957) has pointed out 
that gradual acclimatization to various environmental stresses can often alter an 
organism's response to those stresses. Anderson and Prosser (1953) showed 
that specimens of the blue crab, Callinectes sapidus, collected from dilute salinities 
survived better and maintained higher blood osmo-concentrations in dilute sea 
water than blue crabs collected from higher salinities. However, after acclimatiza- 
tion to 100 per cent sea water for one week, the crabs from dilute sea water ap- 
proached the crabs from higher salinities in their survival ability and tolerance to 
osmotic stress. Anderson and Prosser did not indicate whether gradual ac- 
climatization of specimens of C. sapidus from high salinities to low salinities 
would have altered their osmotic behavior. They stated that the observed dif- 
ferences in osmoregulatory and survival ability between the specimens from dilute 
and more concentrated sea water were due to phenotypic (non-genetic) osmotic 
adaptation. Lockwood and Croghan (1957) found that fresh- water specimens of 
the isopod, Mesidotea entomon, could be acclimatized to 100 per cent sea water, but 
specimens of the species taken from brackish water could not be acclimatized to 
fresh water. Therefore, they felt that the presence of a distinct fresh-water race 
was indicated. A species of sand worm, Nereis diversicolor, has been considered as 
being composed of physiologically distinct races because of differences in ability to 
tolerate dilute salinities between specimens from populations over different parts 
of its wide range. Smith (1955) concluded from studies of chloride ion regula- 
tion of specimens of the species from England, Scotland, and Finland that the experi- 
mental evidence suggested only non-genetic variation between the specimens from 
different populations. In the present work, an attempt has been made to determine 
the effect of salinity on the morphology of the experimental animals when raised 
under abnormal salinity conditions, but nothing has been learned about the effect 
of rearing in foreign salinities on the osmoregulatory response of the animals. 
Adult specimens of Gnorimosphaeroma oregonensis oregonensis were gradually 
(over ten days) acclimatized to low salinities, but they could survive only for 
less than two days in ten per cent sea water or less. 

Referring to the three stated alternatives are Gnorimosphaeroma oregonensis 
oregonensis and G. o. lutea subspecies, ecotypes, or separate species? the writer 
presents the following conclusions. The lack of morphological and ecological inter- 
grades between the two forms and their apparently discontinuous distribution 
rule out, in the writer's opinion, the possibility that they are true subspecies. 
Since there is no evidence, thus far encountered, to indicate that G. o. oregonensis 
and G. o. lutea are ecotypes of the same species, only a remote possibility persists 
that such is the case. The morphological differences between them are not cor- 
related with any known factor in their environment, which is perhaps not to be 
expected, but some adaptive relationship is commonly seen in ecotypes. Further, 
specimens of G. o. lutea hatched and reared in habitat conditions close to those of 
G. o. oregonensis retained (at least for three months) their typical morphological 
configuration. Therefore, the writer is of the opinion that the two "subspecies" 
are actually species. To establish beyond all question their status as true species, 
it would be necessary to extend salinity tolerance tests on early developmental stages 



A REVISION IN GNORIMOSPHAEROMA 161 

and immature individuals and to perform breeding experiments on the two forms 
to establish whether or not actual interbreeding is possible, and if so, whether the 
hybrids are fertile. Until such time as the above-described tests can be accom- 
plished, the best disposition of the case seems to be to propose that G. o. orego- 
nensis and G. o. lutea be considered full species. As stated by Prosser (1957, p. 
363), "most functional variation among animal populations appears to be either 
non-genetic or specific ; relatively little is racial." Thus G. o. oregonensis becomes 
G. oregonensis (Dana, 1852) and G. o. lutea becomes G. lutea Menzies 1954. For 
complete descriptions of the two species, a review of their taxonomy and distribu- 
tion and the disposition of types, the reader is referred to the paper by Menzies 
(1954a). 

The writer wishes to express his gratitude to Professor Milton A. Miller, of 
the University of California, Davis, for his many comments and suggestions in the 
period during which this study was in progress. For their generous comments 
and criticisms during one or another stage in the development of the manuscript, 
the writer conveys sincere thanks to Dr. Ralph I. Smith, of the University of Cali- 
fornia, Berkeley, Professor C. Ladd Prosser, of the University of Illinois, Professor 
Maurice James, of the State College of Washington, and Dr. Robert J. Menzies, 2 of 
the Lament Geological Laboratories, Columbia University. 

SUMMARY 

1. In the writer's opinion, Menzies' determination that Gnorimosphaeroma orego- 
nensis consists of two subspecies is not valid, since there is no apparent morphologi- 
cal and ecological intergradation between the two, and their distribution is dis- 
continuous. 

2. There is no evidence that the two forms are ecotypes. The morphological 
differences between them are not correlated with any known factor in their environ- 
ment, and Gnorimosphaeroma oregonensis lutea hatched and reared for three months 
in habitat conditions close to those of G. o. oregonensis retained their typical mor- 
phological configuration. 

3. It is the opinion of the writer that until such time as extensive rearing and 
breeding tests can be performed, it is best to propose the elevation of the two sub- 
species to full species status. 

LITERATURE CITED 

ANDERSON, J. D., AND C. L. PROSSER, 1953. Osmoregulatory capacity in populations occurring 

in different salinities. Biol. Bull, 105: 369. 
LOCKWOOD, A. P. M., AND P. C. CROGHAN, 1957. The chloride regulation of the brackish and 

fresh-water races of Mesidotea entomon (L.). /. Exp. Biol., 34: 253-258. 
MAYR, E., E. G. LINSLEY AND R. L. USINGER, 1953. Methods and Principles of Systematic 

Zoology. McGraw-Hill Book Co., New York, 328 pp. 
MENZIES, R. J., 1954a. A review of the systematics and ecology of the genus "Exosphaeroma," 

with the description of a new genus, a new species, and a new subspecies (Crustacea; 

Isopoda, Sphaeromidae). Amer. Mus. Nov., 1683: 1-24. 

- It seems only fair to state that Dr. Menzies is not in agreement with the conclusions 
drawn in this paper concerning the elevation of Gnorimosphaeroma oregonensis oregonensis and 
G. o. lutea to species status. 



162 J. A. RIEGEL 

MENZIES, R. J., 1954b. The comparative biology of reproduction in the woodboring isopod 

crustacean, Limnoria. Bull. Mus. Comp. Zool. Harvard, 112: 346-388. 
PANTIN, C. F. A., 1932. Physiological adaptation. /. Linn. Soc. London, 37: 705-711. 
PROSSER, C. L., 1957. The species problem from the viewpoint of a physiologist. Pp. 339-369, 

The Species Problem, E. Mayr, ed., Amer. Assoc. Advanc. Sci., Publ. No. 50. 
RIEGEL, J. A., 1959. Some aspects of osmoregulation in two species of sphaeromid isopod 

Crustacea. Biol. Bull, 116: 272-284. 
SMITH, R. L, 1955. Comparison of the level of chloride regulation by Nereis diversicolor in 

different parts of its geographical range. Biol. Bull., 109: 453-474. 



STUDIES ON THE PHYSIOLOGICAL VARIATION BETWEEN 

TROPICAL AND TEMPERATE ZONE FIDDLER CRABS OF 

THE GENUS UCA. II. OXYGEN CONSUMPTION OF 

WHOLE ORGANISMS x 

F. JOHN VERNBERG 2 

Duke University Marine Laboratory, Beaufort, North Carolina and the Department of Zoology, 

Duke University, Durham, North Carolina 

The year 1936 marked a new era in investigating compensatory adaptation of 
the rate of metabolism in organisms from different latitudes. In this year Sparck, 
Thorson, and Fox independently reported on intra- and interspecific differences in 
rate functions of organisms in Greenland, the North Sea, and the Mediterranean 
Sea. Since then a number of other papers dealing with this subject have appeared 
and recently Prosser (1955) and Bullock (1955) reviewed this area of study. 

Studies of the physiological variation of latitudinally isolated populations of 
fiddler crabs, genus Uca, were undertaken to determine the extent of this variation 
and to correlate these results with their distribution. The first paper in this series 
(Vernberg and Tashian, 1959) dealt with a study of the thermal death limits of trop- 
ical and temperate zone animals as affected by thermal acclimation. The present paper 
reports on their rate of oxygen consumption as influenced by starvation, size, season 
and temperature. Although it may be a question as to what constitutes a valid 
measure of climatic adaptation, it was felt that the rate of oxygen uptake would 
reflect changes in the physiological response of a total organism more nearly than 
studies involving its component parts. Subsequent studies will deal with dif- 
ferences in tissue and enzyme activity in respect to climatic adaptation. 

Rather than attempt to review all the literature relating to this general problem, 
the present paper will be restricted to those papers pertaining to oxygen con- 
sumption. 

Thorson (1936), comparing metabolic rates of lamellibranchs from Greenland 
and the Mediterranean, reported the following general facts (p. 121) : "Hence it 
would seem that species with a northerly distribution have a higher metabolism than 
southerly distributed species of the same genus at the same temperature. . . ." 
He also found a close correlation between oxygen consumption and habits of 
animals, in that epifaunal forms have a comparatively higher metabolic rate than 
digging species and level-bottom species. In addition, he noted that certain arctic 

1 Supported by a grant (G-2509) from the National Science Foundation. Grateful ac- 
knowledgment is made to the University College of the West Indies, Jamaica, The West 
Indies, for supplying laboratory space for a portion of this work. I am especially grateful to 
Dr. R. E. Tashian, University of Michigan, for his valuable assistance in many ways during 
the course of this study. Dr. C. Ladd Prosser has offered his suggestions on the manuscript. 
I wish to acknowledge the technical assistance of John L. Walker, Mary Pinschmidt and J. 
Chipman. 

2 A portion of this work was done while serving as a John Simon Guggenheim Memorial 
Fellow. 

163 



164 F. JOHN VERNBERG 

species are very sensitive to slight increases in temperature. Also working with 
lamellibranchs, Sparck (1936) obtained similar results. 

The work of Fox and subsequent papers in collaboration with Wingfield cite 
further evidence relating to this problem. When comparing metabolic rates of 
pairs of marine organisms from Kristineberg, Sweden and Plymouth, England, 
Fox (1936) found the reverse metabolic-temperature response reported by Thor- 
son and Sparck, i.e., the rates of the warmer-water species were higher than their 
Swedish counterparts when measured at the normal temperature at which the 
species were collected. However, he reported that the rate of respiratory move- 
ments of southern forms at 16.5 C. was the same as northern forms at 5.5. In 
1937 Fox and Wingfield, studying two additional species, one from northern waters 
and the other one from more southern waters, observed that the metabolic-tem- 
perature curves obtained for whole animals and isolated muscle tissue were parallel. 
They concluded that the greater rate of oxygen consumption of warm water 
animals is due to greater non-locomotory metabolism. However, in later papers 
by Fox (1939) and Wingfield (1939), they stressed that when taking into con- 
sideration such factors as body size and season aquatic poikilotherms from the 
north usually will exhibit a higher rate of physiological function at a given tem- 
perature than their more southern relatives. Later Berg (1953) reviewed these 
results of Fox and Wingfield and concluded that most of the exceptions cited by 
them actually showed some degree of acclimation. 

In 1953 Scholander et al. measured the rate of oxygen consumption at various 
temperatures of 38 species of tropical and arctic poikilotherms, including fishes, 
crustaceans, insects and spiders. They concluded that there is considerable, but 
incomplete, metabolic adaptation in aquatic arctic forms relative to aquatic tropical 
species, while terrestrial insects revealed slight adaptation if at all. (They were 
of the opinion that no evidence has been found to show that organisms are adapted 
to temperate fluctuation by being metabolically insensitive to temperature changes?\ 

Reporting on metabolic rates of the two sub-species of Uca pugna.v from ' 
Trinidad, B.W.I., Florida, North Carolina and New York, Tashian (1956) found 
marked differences in the response of species from the tropical and temperate zone. 
Recently Tashian and Vernberg (1958) have elevated these sub-species to the 
specific level. Data on oxygen uptake of latitudinally isolated populations of Uca 
pugilator have been reported by Edwards (1950) and Demeusy (1957). Roberts 
(1957a, 1957b) studied the shore crab, Pachygrapsus crassipes, from different local- 
ities on the west coast of California and Oregon and reported a difference in the 
resting metabolism which could be attributed to compensation for local temperatures. 
Results of the present paper, while further substantiating some of the findings of 
the above workers, also breach the gap between some apparent differences reported 
by various investigators. 

MATERIAL AND METHODS 

Fiddler crabs are an excellent group of animals to study as they are abundant 
over most of the eastern coastline of the Americas and islands of the Caribbean 
(Rathbun, 1918). The various species have either temperate zone or tropical 
zone affinities, and, in addition, there is an area of overlap of some northern and 



OXYGEN CONSUMPTION OF UCA 165 

some southern forms along the northeast coast of Florida (Tashian and Vern- 
berg, 1958). 

Animals used in this study were collected from the following areas : Beaufort, 
North Carolina, latitude 35 ; Alligator Harbor, Florida, latitude 30 ; and Jamaica, 
West Indies, latitude 18. Experimental studies on fiddler crabs from North 
Carolina and Florida were conducted either at the Duke University Marine Lab- 
oratory or at Duke University, and tropical species were studied at the University 
College of the West Indies, Jamaica. The following is a brief description of the 
range and local distribution of the seven species of Uca used in this study. 

Uca minax (Le Conte). Ranges from Massachusetts to Texas (Rathbun, 
1918). In the region of Beaufort, North Carolina this species is typically found 
in the section of the Spartina marsh which is farthest from the banks of the drainage 
ditches and immediately preceding the Salic ornia-Distichlis zone. 

Uca pugilator (Bosc). Ranges from Massachusetts to Texas (Rathbun, 1918). 
Usually this species is associated with the sandy-muddy beaches of either the 
protected areas of the harbor or along the sandy sections of the salt marshes. 

Uca pugnax (Smith). Ranges from Massachusetts to northeast Florida 
(Tashian and Vernberg, 1958). Locally this species is found on mud flats along 
with U. pugilator or in muddy areas of the Spartina marsh. 

Uca rapax (Smith). Ranges from northeast Florida to Brazil (Tashian and 
Vernberg, 1958; de Oliveira, 1939). In Florida this species may be found along- 
side U. pugnax or more generally nearer the high tide level in sandy soil. In 
Jamaica this species was collected in many habitats ranging from sandy soils to 
mangrove swamps. 

Uca mordax (Smith). Ranges from the Bahamas and the Gulf of Mexico 
to Brazil (Rathbun, 1918). In Jamaica this species was frequently collected on 
sandy-clay flats and among mangrove roots. 

Uca thayeri (Rathbun). Ranges from northeast Florida to Brazil. This 
species was abundant in muddy banks of drainage ditches where U. rapax were 
frequently caught. 

Uca leptodactyla (Rathbun). Found from the west coast of Florida and the 
Bahamas to Brazil (Rathbun, 1918). A small-sized species which was found only 
on protected sandy-mud beaches. 

The three species of fiddler crabs studied from North Carolina were Uca minax, 
Uca pugnax and Uca pugilator. In Jamaica, determinations were made on U. 
leptodactyla, U. rapax, U. mordax and U. thayeri. U. rapax and U. mordax 
came from the Port Henderson area, while U. leptodactyla were collected from the 
swamp near the Morant Point Lighthouse, and U. thayeri from Port Morant. Only 
U. rapax were studied from Alligator Harbor, Florida. 

After collecting the animals and bringing them to the laboratory, they were 
rinsed in sea water and placed in aquaria containing about one-half inch of sea 
water. Animals kept as a general supply were exposed to hamburger, fish and 
Pablum once or twice a week for about 12 hours and then the sea water was 
changed. Most of the animals appeared to be feeding and in a good state of 
nutrition as fecal pellets were readily observed and mortality was low. Individuals 
to be used experimentally were isolated in marked glass containers and then sub- 
jected to the condition of the experiment. 



166 F. JOHN VERNBERG 

Oxygen consumption was determined by means of standard manometric tech- 
niques (Umbreit, 1957). A large-sized respirometer flask (volume about 125 cc.) 
was connected to a conventional Warburg manometer for all determinations, except 
in the series of experiments involving the smallest sized species, U. leptodactyla, 
where a flask with a volume of 8 cc. proved to be better. A measured amount of 
filtered sea water was introduced into a flask containing an organism. The salinity 
was not measured each time but sporadic measurements gave values ranging from 
31 to 35 0/00. In all cases a determination of the rate of oxygen consumption 
involved only one organism per flask. Ten per cent KOH was used to absorb 
CO 2 . Flasks were not shaken as this understandably proved to be too excitatory 
to the animals. All results are expressed as p.\. oxygen consumed/minute/gram of 
wet weight. Determinations of oxygen consumption were made over a graded 
temperature series by the use of a thermally controlled water bath. Preliminary 
studies showed that the time interval required for thermal equilibration and for 
the rate of oxygen uptake to reach a somewhat steady level varied inversely with 
temperature. Only oxygen consumption data which were relatively stable over a 
period of time were used. The duration of the experiment also varied with tem- 
perature : at high temperatures rates were determined over a two-hour period, 
while at low temperatures eight hours of observation were used. 

Recent workers have reported on cycles of oxygen consumption in fiddler 
crabs which were correlated with time of day, tide, seasons, and other factors 
(Edwards, 1950, and Brown et a!., 1954). In the present study an attempt was 
made to minimize variation due to rhythmic daily fluctuations by making an equal 
number of determinations in the afternoon and the morning. It is interesting to 
note that a preliminary study comparing determinations made on the same animals 
run in the morning and afternoon did not show any consistent variation. Although 
no attempt was made to correct for possible tidal influence on metabolism, seasonal 
variation was observed in some species and this will be discussed later. 

The recent thermal history of animals from North Carolina and Jamaica was 
the same in that their habitat and laboratory temperatures were alike, although the 
determinations were made at different times of the year. The mean water tem- 
perature at Jamaica varies slightly throughout the year, ranging from 80-82 F. 
while the range during June-August in the Beaufort area was from 77-82 F. 
Work at Beaufort extended from June to September, while studies in Jamaica 
began in October, 1957, and ended in April, 1958. 

In all of these studies only male crabs in the intermolt stage were measured. 
The criteria of Drach (1939) and Guyselman (1953) were used to determine the 
stage of the molting cycle. 

When oxygen consumption data were plotted on logarithmic co-ordinates with 
rate of oxygen uptake (weight-specific) against the weight of the organism, a 
regression equation was obtained of the type 



W 



where Oo is oxygen consumption/unit time, W the body weight (wet weight), and 
a and b are constants, indicating the intercept and the slope of the regression line 
in the log-log plot. Additional statistics calculated were the standard error, S (log 
y. log .i"), and coefficient of correlation (r). 



OXYGEN CONSUMPTION OF UCA 



EXPERIMENTS AND RESULTS 



167 



Influence of starvation on metabolism 

One variable in comparing metabolic rates of animals is the degree of starvation. 
To determine variation due to this factor, 25 specimens of U. pugnax from North 
Carolina were collected, isolated individually, and maintained at room temperature. 
First, their rate oxygen consumption was determined after being exposed to food 
and subsequently determined after the first, third, fifth, seventh, ninth, sixteenth 
and twenty-first day of starvation. Of the original 25 animals, 21 survived for the 
entire period while four animals died after the sixteenth day. Results represented 
in Figure 1 are averages of the 21 animals surviving the entire 21 days of starvation. 




i.o 



DAYS 

FIGURE 1. The influence of starvation on the rate of oxygen consumption of Uca pugnax. 

Determinations made at 28 C. 



A marked drop occurred by day 1 followed by a progressive decline in metabolic 
rate and subsequent insignificant fluctuations. On the basis of these findings, 
animals were starved for one to three days before being used in any experiment 
unless otherwise specified. 

The response pattern to starvation of U. pugnax is similar to results observed 
in Pachygrapsus crassipes (Roberts, 1957a), pulmonate snails (von Brand, Nolan 
and Mann, 1948) and fish (Wells, 1935). 



168 



F. JOHN VERNBERG 



Metabolic rates oj tropical and temperate zone Uca 

Table I represents the rates of oxygen consumption of seven species of fiddler 
crabs from the tropical and temperate zones determined at different temperatures. 
Although an increase in temperature generally resulted in a higher rate of oxygen 
uptake, there appear to be certain thermal ranges within which the metabolic rate 
is little influenced. A specific example can be seen for U. pugna.v in that the rate 
of oxygen consumption at 7 and 12 is similar, but a sharp increase followed when 
determined at 17. Throughout the temperature range of 12 17 (determinations 
made at 12, 15, and 17), U. pugilator and U. minax consumed oxygen at about 
the same rate (Fig. 2). This phenomenon was observed at higher temperature 
ranges as well for these temperate zone animals ; for example, a five-degree in- 
crease from 28-33 only slightly increased the metabolic rate of U. inlna.v. This 
apparent "staircase" effect was noted for tropical animals also, but only at the 
intermediate and higher temperature levels (Fig. 3). 

Changes in the rate of oxygen consumption are expressed as Q 10 , according to 
Van't Hoff's equation, rather than the heat of activation (u) of Arrhenius. Q 10 
values for these seven species found in Table II further help to illustrate this 
"staircase" phenomenon. If determinations had been made only at larger thermal 



4.0, 



3.0. . 



2.0- . 



UJ 

5 



z 
o 

U 

c? 



I) 
u 



1.0 
.9 
.8 
.7 
.6 

.5.. 
.4 . 



.3.. 



.2.. 




-UCA 

O O-UCA 
X X-UCA 



PUGNAX 

MINAX 

PUGILATOR 



I 



I 



I 



10 13 16 19 22 25 28 31 34 
TEMPERATURE CENTIGRADE 



37 40 



FIGURE 2. The influence of temperature on the rate of oxygen consumption of three 

species of Uca from North Carolina. 



OXYGEN CONSUMPTION OF UCA 



169 



TABLE I 

Rate of oxygen consumption of Uca from the tropical and temperate 
zones determined at various temperatures 



Species 


Temperature 
(C.) 


Size of 
sample 


Body weight (gms.) 


Rate of Oz consumption 
(mm.Vmin./gram) 


Mean 


Range 


Mean 


Range 


pugnax 


7 


19 


2.57 


0.94- 4.72 


0.328 


0.156-0.725 




12 


33 


2.70 


1.06- 4.65 


0.357 


0.179-0.730 




17 


31 


3.63 


0.88- 5.95 


0.734 


0.296-1.410 




28 


54 


2.90 


1.15- 5.57 


1.587 


0.485-3.489 




33 


19 


2.95 


0.65- 4.31 


2.140 


1.131-3.705 




39 


18 


3.20 


0.89- 5.57 


3.278 


2.020-4.523 


minax 


7 


17 


6.19 


3.85- 8.56 


0.167 


0.109-0.280 




12 


25 


6.57 


4.54- 9.05 


0.292 


0.135-0.622 




15 


26 


6.48 


4.79- 8.94 


0.281 


0.129-0.567 




17 


26 


6.44 


4.58- 8.61 


0.342 


0.133-0.689 




22 


24 


6.33 


4.41- 8.83 


0.688 


0.350-1.207 




28 


66 


5.77 


1.66- 8.42 


1.185 


0.566-2.554 




33 


23 


6.61 


5.04- 8.28 


1.308 


0.841-1.751 




36 


22 


6.60 


5.01- 8.06 


1.442 


0.915-1.907 


pugilator 


7 


17 


2.06 


0.89- 2.84 


0.311 


0.122-0.627 




12 


13 


2.03 


0.62- 2.95 


0.751 


0.412-1.482 




15 


22 


1.87 


0.68- 2.95 


0.757 


0.379-1.395 




17 


23 


1.93 


0.66- 3.41 


0.723 


0.426-1.264 




28 


63 


2.35 


0.95- 4.87 


1.333 


0.806-2.473 




36 


17 


2.06 


0.93- 3.00 


2.729 


1.962-3.896 


rapax 


7 


24 


2.80 


1.42- 5.12 


0.352 


0.150-0.553 


from Florida 


12 


32 


2.60 


1.25- 5.12 


0.732 


0.280-1.561 




17 


30 


2.51 


1.25- 5.12 


1.017 


0.396-2.048 




27 


97 


1.82 


0.95- 5.12 


2.109 


1.125-3.802 




33 


13 


3.17 


1.66- 5.12 


2.324 


1.558-3.332 




36 


25 


2.00 


1.00- 5.12 


2.838 


1.315-6.042 


rapax 


12 


25 


3.78 


0.57- 9.33 


0.349 


0.167-0.525 


from Jamaica 


15 


40 


4.36 


0.63-11.57 


0.665 


0.272-1.877 




17 


29 


3.51 


0.67- 9.33 


0.958 


0.327-1.931 




22 


24 


3.10 


0.84- 6.56 


1.468 


0.746-2.369 




28 


54 


4.33 


0.58-13.67 


1.663 


0.641-4.139 




33 


26 


3.69 


0.67- 9.54 


1.954 


0.890-3.304 




36 


26 


3.36 


0.58-13.67 


2.400 


0.801-4.597 




39 


23 


4.65 


0.92-13.67 


2.807 


0.683-4.497 


thayeri 


12 


11 


4.38 


1.50- 6.51 


0.240 


0.149-0.333 




15 


12 


5.25 


2.71- 7.96 


0.528 


0.383-0.859 




30 


13 


4.36 


2.71- 6.51 


1.412 


0.675-1.980 




36 


16 


4.60 


1.65- 8.74 


1.838 


1.306-2.471 


mordax 


12 


9 


2.81 


1.53- 3.84 


0.392 


0.216-0.548 




15 


7 


2.41 


0.91- 3.63 


0.582 


0.462-0.713 




22 


8 


2.50 


0.91- 4.26 


1.436 


0.884-2.023 




28 


12 


2.67 


0.87- 4.28 


1.603 


0.942-2.835 




36 


11 


2.49 


0.87- 4.24 


2.885 


1.457-4.460 


leptodactyla 


12 


11 


0.26 


0.15 0.31 


0.294 


0.115-0.545 




15 


13 


0.28 


0.18- 0.35 


0.674 


0.460-1.176 




30 


14 


0.27 


0.18- 0.36 


2.495 


1.748-3.768 




36 


13 


0.28 


0.19- 0.35 


3.842 


2.588-4.973 



170 



F. JOHN VERNBERG 



2 
O 

\ 

z 

2 
\ 
Q 

UJ 

2 

D 

<n 

z 
o 
u 



4.0. 
3-0 

2.0. . 



1.0. 
.9. 
.8. 
.7. 

.6. 
.5.. 

.4.. 



.3.. 



.2.. 




RAPAX 
THAYERI 
MORDAX 
LEPTODACTYLA 



1 



I 



12 15 18 21 24 27 30 33 36 
TEMPERATURE - CENTIGRADE 



39 



FIGURE 3. The influence of temperature on the rate of oxygen consumption of four 
species of Uca from Jamaica, The West Indies. 



intervals, for example every 10 degrees, this type of response would not have been 
very evident. The Q 10 value for U. pugilator for the temperature range of 7-17 
was 2.32 while a value of 5.83 was obtained for the range of 7-12. Tropical 
species afford additional examples ; U. rapa.v had an exceptionally high Q 10 value 
of 8.58 over a three-degree increase from 12-15, whereas a Q 10 value of 4.20 for 
the 10-degree range of 12-22 was observed. 

In general lower Q 10 values were obtained at higher temperatures than at lower 
temperatures for all seven species of Uca examined. A marked difference in 
metabolic behavior between tropical and temperate zone animals was observed at 
the lower temperatures. Between 12 and 15 very high Q 10 values were obtained 
for U. rapa.v, U. leptodactyla and 17. thayeri (range from 8.58-15.9) while a mod- 
erately high value of 3.73 was observed for U. mordax. Although U, minax and 
U, pugilator, both temperate zone forms, showed no increase in metabolic rate 
within this same three-degree range, moderately high Q 10 values were obtained 
between the 7 to 12 range. Determinations made at lower temperatures would 
probably result in still higher Q 10 values for these two forms and also for U. pugnax. 
Thus it would seem that at certain points along a temperature gradient, a slight 



OXYGEN CONSUMPTION OF UCA 



171 



TABLE II 

Qio of oxygen consumption of seven species of Uca from tropical and temperate zones 



Temperature 
range ( C.) 


minax 


pugnax 


pugilator 


rapax 
(Florida) 


rapax 
(Jamaica) 


mordax 


lepto- 
dactyla 


thayeri 


7-12 


3.06 


1.20 


5.83 


4.36 














7-17 


2.04 


2.24 


2.32 


2.89 














7-28* 


2.54 


2.10 


2.00 


2.45 














7-36 


2.10 





2.11 


2.07 














12-15 


1.00 





1.00 





8.58 


3.73 


15.9 


13.7 


12-17 


1.37 


4.23 


1.00 


1.93 














12-22 














4.20 


3.66 








12-28* 


2.40 


2.51 


1.43 


2.02 


2.65 


2.41 


3.28 


2.67 


12-36 


1.95 





1.71 


1.76 


2.23 


2.27 


2.92 


2.33 


15-17 


2.67 





1.00 





6.20 


. 








15-22 





- 








3.10 


3.49 








15-28* 


2.51 





1.55 





2.02 


2.18 


2.39 


1.92 


15-36 


2.18 





1.84 





1.84 


2.12 


2.29 


1.68 


17-22 


4.04 











2.35 











17-28* 


3.09 


1.97 


1.75 


1.86 


1.63 











17-36 


2.45 





2.01 


1.71 


1.62 











22-28* 


2.57 





. 





1.23 


1.32 








22-33 


1.90 











1.29 











28-33* 


1.22 


1.90 





1.42 


1.38 











28-36* 


1.28 





2.46 


1.33 


1.57 


2.03 


2.05 


1.55 


33-36 


2.49 








2.22 


1.98 












* Determinations were made at 27 for rapax (Florida) and 30 for leptodactyla and thayeri. 

increase in temperature results in a marked increase in metabolic rate, whereas at 
other points the rate of oxygen consumption of these animals is relatively tem- 
perature-independent throughout a wider temperature range. Both temperate and 
tropical species exhibit this type of metabolic response but at different points on 
the temperature spectrum : tropical animals were metabolically activated at higher 
temperatures than temperate zone forms. 

The similar Q 10 values for both temperate and tropical zone animals obtained 
at intermediate and higher temperatures are not surprising as the recent thermal 
history of all seven species was very similar and reflected summer or elevated 
temperature conditions. However, at lower temperatures, tropical animals exhibit 
a reduced ability to meet metabolically this environmental stress, while temperate 
zone forms appear to be more labile. 

When comparing the metabolism of tropical and temperate zone fiddler crabs 
over a wide range of temperatures, some rather interesting points can be noted. 
At 12 the range of the average rates of oxygen consumption of the four tropical 
species is from 0.240 to 0.392 while the range for temperate zone forms is 0.292 
to 0.751. These figures would suggest that at this particular low temperature 
northern species tend to have higher metabolic rates. However, as will be dis- 
cussed in more detail, it is necessary to take into consideration the factor of body 
size when comparing species. The following comparisons are of similar sized 
species : pugnax-rapax-mordax and ininax -thayeri. At 12 U. pugnax from North 
Carolina and U. rapax and U. mordax from Jamaica consume oxygen at a similar 



172 F. JOHN VERNBERG 

TABLE III 

Statistical analysis of relation of oxygen consumption to body size of Uca pugnax and Uca 

rapax determined at various temperatures 



Temperature 

(C.) 


No. of determi- 
nations 


6-1 


log a 


Olog |/. log z 


) 



Uca rapax from Jamaica 



12 


25 


-0.377 


-0.301 


0.113 


0.81 


15 


40 


-0.236 


-0.076 


0.143 


0.42 


17 


29 


-0.571 


0.192 


0.139 


0.75 


22 


24 


-0.387 


0.309 


0.075 


0.82 


28 


54 


-0.343 


0.360 


0.146 


0.64 


33 


26 


-0.377 


0.434 


0.136 


0.67 


36 


26 


-0.374 


0.498 


0.135 


0.77 


39 


23 


-0.354 


0.620 


0.143 


0.69 



Uca rapax from Florida 



7 


24 


-0.050 


-0.446 


0.133 


0.00 


12 


32 


-0.474 


-0.010 


0.209 


0.38 


17 


30 


-0.465 


0.150 


0.161 


0.29 


27 


97 


-0.210 


0.356 


0.119 


0.25 


33 


13 


-0.456 


0.574 


0.076 


0.65 


36 


25 


-0.380 


0.557 


0.185 


0.52 















Uca pugnax from North Carolina 



7 


19 


-0.770 


-0.234 


0.094 


0.88 


12 


33 


-0.537 


-0.266 


0.102 


0.63 


17 


31 


-0.309 


-0.003 


0.162 


0.36 


28 


54 


-0.373 


0.321 


0.172 


0.37 


33 


19 


-0.304 


0.423 


0.158 


0.44 


39 


18 


-0.316 


0.650 


0.069 


0.52 



rate, while U. pugilator from North Carolina, with a weight average slightly less 
than these three species, consumed almost twice as much oxygen per unit weight and 
time. U. minax, another northern species, has a higher metabolic rate than U. 
thayeri. Oddly, U. leptodactyla, a very small tropical species, consumed oxygen 
at a rate similar to the larger sized species, while at elevated temperatures (30 
and 36) it had the highest metabolic rate of all seven species. 

Determinations were not made at 7 on tropical species as it has been shown 
by Vernberg and Tashian (1959) that these animals are not able to withstand this 
low temperature for a long enough period : 50% mortality occurred after exposure 
to 7 for 30-40 minutes. 

As noted above the oxygen consumption rate of tropical species was greatly 
increased by a three-degree increase from 12-15 while temperate zone species are 
little affected. 

At 28 U. pugnax, U. mordax and U. rapax again have similar metabolic rates, 
while values for U. thayeri are now higher than U. minax which is the reverse of 



OXYGEN CONSUMPTION OF UCA 



173 



5.0-,- 



4.0-- 




2 468 

BODY WEIGHT -GRAMS 

FIGURE 4. The relation of oxygen consumption to size in Uca pugnax from North Carolina 

when determined at different temperatures. 



174 



F. JOHN VERNBERG 



QC 
O 



I.5-- 



O 



0.4- 




I.O-- 



0.8-- 



0.6-- 



2 46 

GRAMS (WET WEIGHT) 



10 



FIGURE 5. The relation of oxygen consumption to size in Uca rapax from Alligator 
Harbor, Florida when determined at different temperatures. 



the results obtained at 12. Although the temperate zone species U. pugnax 
utilized more oxygen than U. rapax at 33 and 39, the tropical species U. thayeri 
consumed oxygen at a faster rate than U. minax at elevated temperatures. It 
would appear that no uniform metabolic difference between tropical and temperate 
zone species was apparent under the conditions of this study. At any temperature 
point one tropical species may consume oxygen at a faster rate than a similar sized 
northern organism, while at this same temperature a temperate zone species of 
another paired comparison would utilize oxygen faster than its tropical counterpart. 

Influence of size on metabolism 

Numerous workers have stressed the importance of the dependence of metabolism 
on body size when making inter- and intraspecific comparisons of crustaceans 
(Weymouth et al, 1944; Vernberg, 1956; Tashian, 1956; Zeuthen, 1953; Roberts, 



OXYGEN CONSUMPTION OF UCA 



175 



4.0.: 



3.0 



z 

i 
\ 



cr 
o 

N 



D 
O 



H 
Q. 



Z 
O 

o 

z 

u 

o 

> 

X 

o 



I.O-. 



0.8 .: 



O.6.- 



0.4.-- 



39 




12' 



2 46 

BODY WEIGHT -GRAMS 



10 



FIGURE 6. The relation of oxygen consumption to size in Uca rapax from Jamaica, The 
West Indies, when determined at different temperatures. 

1957a; Edwards and Irving, 1943a, 1943b). In general, smaller individuals or 
smaller species consume oxygen at a faster rate per unit weight and unit time than 
larger individuals or larger species. 

Oxygen consumption data determined at various temperatures were obtained 
using different sized animals of U. pugnax and two latitudinally separated popula- 
tions of U. rapax. The comparative data for these two species, using the statistical 
techniques described earlier, are summarized in Table III. Figures 4, 5 and 6 
represent the log-log plotting of the regression curves calculated from these data. 



176 



F. JOHN VERNBERG 



It appears that the slope of the regression curve varies with temperature. For 
U. rapax from Jamaica similar b values were obtained at all temperatures with the 
exception of 15, where the slope was less steep, and 17 where the slope was 
steeper. The b constant for U. pugnax also fluctuated with the highest values, 
being obtained at low temperatures (7 and 12). Oddly at 7, U. rapax from 
Florida showed no correlation of body size and respiration, but at 12 the steepest 
slope was obtained. Although the absolute temperature at which the steepest slope 
was observed was different for these two species, it appeared that a significant break 
occurred at some low temperature. Interestingly this break occurred at a lower 
temperature for the northern species than for the tropical species. In general the. 
slope of the regression curve was less steep for U. pugna.v than U. rapax. 

According to the method of Snedecor (1940, pp. 132-133), the correlation 
coefficients are significant at the 1% level for all points except for U. pugna.v at 17, 
33, and 39 where the level of significance is at the 5% level and U. rapax 
(Florida) at 7 where no significance was observed. 

When the Q 10 is calculated from the linear regression curves for animals weigh- 
ing 1, 3.5 and 9 grams, an apparent difference in response correlated with body 
size is observed (Table IV). One size group may be more sensitive to a given 
temperature change than a different size group, i.e., large specimens of U. rapax 
have a larger Q 10 value than small sized (one gram) individuals at the temperature 
interval of 12-15, while at 15-17 the reverse is observed. 

After a log-log plotting of metabolic data of seven species of fiddler crabs 
obtained at two temperature levels, interspecific differences correlated with body 
size are evident (Fig. 7). At 28 or 30 a regression curve with a slope of -- 0.204 
was obtained which indicates that the small sized species consumes oxygen at a 



TABLE IV 

values of different sized Uca pugnax and Uca rapax based on 
linear regression curves 



Temperature interval in C. 


1 gram 


3.5 grams 


9 grams 



Uca pugnax 



7-12 


1.0 


1.6 


2.4 


12-17 


3.4 


6.0 


9.2 


17-28 


2.0 


2.9 


1.8 


28-33 


1.6 


1.8 


2.1 


33-39 


2.4 


2.3 


2.2 



Uca rapax 



12-15 


5.6 


12.3 


16.1 


15-17 


24.8 


2.2 


1.0 


17-22 


1.9 


2.6 


2.9 


22-28 


1.2 


1.4 


1.0 


28-33 


1.4 


1.3 


1.2 


33-36 


1.6 


1.7 


1.7 


36-39 


2.5 


2.6 


2.9 



OXYGEN CONSUMPTION OF UCA 



177 



2 - 



O 

^ 1.0- - 

z 

.8-- 
\ 6 



.4-- 



5 

:> 

. .2.5+ 
U 

.15-1- 




I 



4- 



H 1- 



0.2 0.4 0.6 0.8 1.0 



GRAMS ( WET WEIGHT ) 



1r 



8 10 



FIGURE 7. The relation of oxygen consumption to size in seven species of Uca measured 
at two temperature levels. Two regression curves were calculated for data at 12 : a did not 
include leptodactyla (1) whereas b did (see text for discussion). 1 is leptodactyla, mi is 
minax, mo is mordax, pg is pugilator, px is pugnax, ra is rapax, and t is thaycri. 



higher rate than a larger sized species. However at 12 the metabolic rate of the 
small sized tropical species is greatly reduced in relation to the other six species and 
the regression curve has a slope of - - 0.038 which is similar to results obtained for 
U. rapax (Florida) at low temperatures. But, if U. leptodactyla data are omitted 
on the basis of this tremendous response to environmental temperature stress, a 
curve with a slope of 0.732 is obtained which seems to correspond to the steep 
slopes at low temperatures for intraspecific data on U. pugnax (a slope of 0.770 
at 7) and U. rapax from Jamaica (a slope of 0.571 at 17). 

Comparisons of metabolism of different populations of Uca rapax 

Data on the oxygen consumption of U. rapax from Florida and Jamaica are 
included in Table I. One of the most obvious differences between the two popula- 
tions is seen in their respective responses at reduced temperatures. The rate of 
oxygen uptake of Florida animals at 7 was very similar to that of Jamaican forms 
at 12. As noted previously data for Jamaican animals at 7 could not be obtained 
as they did not survive exposure to this temperature. Following in this same 
trend, Florida animals consumed oxygen at a slightly higher average rate at 12 
than their more tropical counterpart did at 15. At all subsequent similar tem- 
peratures the subtropical form exhibited the highest metabolic rate. 



178 



F. JOHN VERNBERG 



U. rapax from Florida metabolically behaved like U. pugnax at the low tem- 
perature of 7 while at 12, 17, 27 and 33 rapax had a higher rate. The 
Floridan U. rapax exhibited the highest Q 10 value at 7-12 which is more char- 
acteristically like temperate zone forms than tropical zone species. 



Influence of season on metabolism 

Although results discussed up to this point were made on tropical and temperate 
zone animals which had similar recent thermal histories, experiments were carried 
out at different seasons of the year. Therefore the possibility of seasonal fluctua- 
tions in metabolism had to be investigated. Although metabolic studies involving 
Jamaican species were conducted from October to April, it was assumed that if 
any seasonal variation was to be observed it would be in evidence during this 
period as there is little monthly variation in temperature throughout the year. 

When oxygen consumption determinations on U. rapax made either at 28 or 15 
during October were compared with results of February-March, there was no 
significant difference of means. This indicated that under the conditions of these 
observations no shift in metabolism of U. rapax was observed which could be 
correlated with seasons (Table V). 

Data on U. pugnax collected during the summer months and also during the 
period from November to January show a marked seasonal variation. At 7, 17 
and 28, crabs collected during November and maintained in the laboratory under 
the same thermal conditions as summer animals (22-27) consumed oxygen at a 
significantly faster rate than summer animals run at the same temperatures. How- 
ever, winter and summer animals responded similarly at 33. The Q 10 of 1.61 of 
"winter" animals was lower than that of summer animals (2.24) at low temperatures 
(7-17) but the opposite response was observed at higher temperatures (17-28) 
where the respective values were 2.37 and 1.97. 



TABLE V 

Metabolic rate of Uca pugnax determined at different seasons of the year 
and at various temperatures 









Oxygen 






Month of year 


Temp. 
t r ) 


No. of 
determi- 


consump- 
tion rate 


Standard 
error 


Level of significant difference 
of means 




\ *-"/ 


nations 


(mm. 8 / 


of mean 










gm./min.) 






June to August 


7 


19 


0.328 


0.035 


>.01 highly significant 


November to January 


7 


23 


0.572 


0.039 




June to August 


17 


31 


0.734 


0.047 


>.01 highly significant 


November to January 


17 


40 


0.920 


0.052 




June to August 


28 


54 


1.587 


0.094 


>.01 highly significant 


December to January 


28 


25 


2.379 


0.222 




June to August 


33 


19 


2.140 


0.207 


no significant difference 


December to January 


33 


27 


2.312 


0.113 





OXYGEN CONSUMPTION OF UCA 179 

DISCUSSION 

Results of this investigation demonstrate the existence of differences in one 
physiological response, oxygen consumption rate, between temperate and tropical 
zone fiddler crabs. Other workers, using different measures of climatic adapta- 
tion, have reported the existence of physiological variation between latitudinally 
separated species of poikilotherms (Mayer, 1914, pulsation rate of the bell of 
Aitrclia anrita; Horstadius, 1925, Thorson, 1936, Moore, 1939, 1942, 1949, Dehnel, 
1955, egg development and growth of various invertebrates and frogs; and Rao, 
1953. rate of ciliary pumping of water in a mussel) . 

When making intra- or interspecific comparisons of poikilothermic animals 
from different latitudes, it is generally stated that at any given temperature, within 
limits a northern or cold-adapted form will show a higher metabolic rate than a 
southern or warm-adapted form. This comparison is dependent upon a number of 
other factors, such as body size and season, as pointed out so excellently by Prosser 
(1955), Bullock (1955) and Rao and Bullock (1954). 

However, when comparing similar sized species of fiddler crabs from temperate 
and tropic zones, which have similar thermal histories, no consistent difference in 
metabolism correlated with latitude was observed except at low temperatures. 
But an intraspecific comparison of Uca rapa.v from northern Florida and Jamaica 
shows the classical type of response, especially at low and high temperatures. Us- 
ing the data of Tashian (1956) it can be seen that similar sized fiddler crabs 
(3 gms.) from southern Florida and Trinidad had similar metabolic rates when 
determined at 24, while the New York species was slightly lower. But at 14.1 
14.9 C., animals from New York weighing 3 gms. had a higher metabolic rate 
than the more southern forms. Working at still lower temperatures (1.4 and 
15 C.) Demeusy (1957) reported that Uca pugilator from Woods Hole, Mas- 
sachusetts had a significantly higher rate of metabolism than specimens from 
Florida only at the lower temperature. It is possible that these results might be 
influenced by seasonal temperature changes as this work was started in the fall. 
Whereas Demeusy did not find any difference at 15 between these two popula- 
tions, Edwards (1950) reported the Woods Hole form of U. pugilator to have a 
higher rate of oxygen consumption at 20 than animals from Florida. No men- 
tion was made of either the thermal or seasonal history of the two populations 
studied. However, seasonal studies on U. pugnax indicated that "winter" animals 
from North Carolina had higher metabolic rates in the temperature range of 7 
25 than "summer" animals, while the tropical species, U. rapa.v, did not show any 
seasonal fluctuation. This absence of any seasonal variation in metabolism of U. 
rapax may be correlated with the thermal constancy of their environment through- 
out the year. However, fluctuating yearly temperatures of more northern latitudes 
have resulted in a labile metabolic pattern in U. pugnax which can be correlated 
with thermal acclimation. 

Teal (1959), dealing with the relation of the respiratory metabolism of crabs 
to flow of energy through an ecosystem in Georgia salt marshes, found a marked 
ability of U. pugnax to demonstrate seasonal thermal acclimation. 

The review paper of Bullock (1955) cites numerous examples of seasonal ac- 
climation in many but not all poikilotherms. Recently Roberts (1957b) reported 



180 F. JOHN VERNBERG 

that in the lined shore crab, Pachygrapsus crassipes, seasonal acclimation in metabo- 
lism was not present when determination was made at 16 C. However, he noted 
that respiration rates did bear some relationship to local seasonal temperature 
changes when intertidal sea water temperatures were below the environmental mean 
of 16. It is noteworthy that the annual temperature flutuation experienced by 
Pachygrapsus is much less than that of fiddler crabs from North Carolina and thus 
might explain the difference in degree of response. The high and low mean monthly 
average temperature reported by Roberts was about 12 in January and 22 in 
August. At Beaufort McDougall (1943) and Outsell (1930) reported similar 
average temperature values at 5.5 in February and 28 in July. 

Metabolic rate determinations made at a number of temperature levels give a 
better insight into the influence of temperature on metabolism than generaliza- 
tions from a few widely separated thermal points. The results of the present 
paper show that certain points along a temperature gradient appear to be of a more 

('critical" nature for the organism than others. This type of metabolic response 
when graphed gives a "staircase" appearance. In general tropical and temperate 
fiddler crabs responded similarly at intermediate and elevated temperatures, but 
at low temperatures tropical species were metabolically activated at a higher 
temperature than more northern species. Woodworth (1936) and Vernberg and 
Mariney (1957) observed that terrestrial insects, bees and fruit flies, respectively, 
were relatively temperature-insensitive within a given temperature range. Takatsuki 
(1928) showed a similar response in the heart rate at various temperatures of 
oysters from the tropical and temperate zone seas. When comparing the results 
of this study with those of Teal (1959), a remarkable similarity is observed. To 
cite a few examples : Teal observed U. minax consumed oxygen at the same rate 
at 11.1 as at 15.9; in the present study the temperature range of 12 to 17 
had little effect on their metabolism. U. pugilator was found to be temperature- 
insensitive between 13.2 and 19.4 by Teal and in the present investigation the 
same response was noted from 12 to 17. Noteworthy is the similarity of the 
response of U. pugnax. In both studies, this species behaved differently than the 
other two species of Uca: while pugilator and minax were temperature-insensitive 
in this range of about 11-19, the metabolic rate of pugtiax was greatly increased. 
Additional cases, chiefly terrestrial animals, are cited by Bullock (1955). 

In general this marked influence of a relatively narrow temperature increase 
may be expressed in terms of a high Q 10 (values greater than 3). After reviewing 
and re-evaluating many papers, Rao and Bullock (1954) -concluded that Q 10 was 
dependent on size and temperature of adaptation. Results of the present paper 
present additional data to show that marked differences in Q 10 exist in the semi- 
terrestrial crabs of the genus Uca from the tropical and temperate zones. Scho- 
lander et al. (1953) measured the metabolic rate of U. mordax from Panama 
and reported high Q 10 's at low temperatures. However it is difficult to compare 
results for the following reasons: 1) determinations were made only at 10-degree 
intervals; 2) Q 10 's were estimated by eye-fitted tangents to eye-fitted curves; and 
3) few data were available at 10, as only three out of eight animals survived suf- 
ficiently long to give valid readings. 

Similar results as the present paper were obtained by Thorson (1936), the 



OXYGEN CONSUMPTION OF UCA 181 

most striking example being a Q 10 of 21 over the temperature range of -1 to 1 
for Pec ten groenlandicus. Interestingly this lamellibranch lives in the fjords of 
Greenland where the temperature is constantly below 0. The same type of response 
appears in the results of Sparck (1936) but it is difficult to understand the basis of 
his metabolic-temperature curves as no experimental data are given and no points 
are shown on the curves. Demeusy (1957) and Teal (1959) observed a similar 
pattern of O 1(1 values with their work on Uca. 

Results of the present paper demonstrate the dependence of metabolism on 
body size both when making inter- and intraspecific comparisons. The slope of the 
linear regression (b-1) varies with temperature: the steepest slope is at a higher 
temperature for the tropical species than for its northern counterpart. Although 
there are only a few observations, these results show the same tendency of tem- 
perature to influence metabolism as did O 10 values for the 7 species. Roberts 
(1957a) observed that the slope of the linear regression was the same at 8.5 and 
16 for Pach\grapsns but was significantly less steep at 23.5. In their recent 
review paper, Rao and Bullock (1954) replotted the data of Edwards and Irving 
(1943a) and Edwards (1946) and found that the slope varied with temperature: 
1) with summer animals, the slope was steeper at 12 than at 22; and 2) with 
winter animals the reverse was noted. As a consequence of the change in slope of 
the regression curve, the O 10 values of different sized organisms will be changed. 
Bishop (1950, p. 242) reported that smaller individuals of a species are more tem- 
perature-sensitive than larger ones, while Rao and Bullock (1954) concluded that 
commonly the O 10 increases along with increasing size within normal ranges of 
temperature. Results of the present paper show that the influence of tempera- 
ture on the metabolism of different sized individuals of one species varied with the 
temperature range which was used. Therefore apparent differences reported in 
the literature may have resulted from comparing different temperature ranges of 
animals. 

Interspecific comparisons of fiddler crabs showed the slope of the linear regres- 
sion to be - 0.204 at 28-30 which compares favorably with slopes obtained 
for various groups of organisms (Zeuthen, 1953, -0.20 for crustaceans; Wey- 
mouth ct a!., 1944, -0.174 for various crustaceans; and Vernberg and Hunter, 
1959, -0.21 for cercariae of digenetic trematodes). But, when compared with 
the results at 12 C, marked differences are observed. The metabolic rate of the 
smallest species was depressed greatly by reduced temperature resulting in a regres- 
sion slope of 0.020. Hence, smaller sized fiddler crabs have higher Q 10 's than 
larger sized species with the temperature range of 12 to 28 or 30. 

Results of this paper would suggest that the metabolic response of fiddler 
crabs has real significance to their distribution. In the course of evolution, the 
various populations studied appear to be metabolically adjusted to the temperature 
fluctuations of their habitats. Temperate zone species not only are metabolically 
active at lower temperatures than tropical species but they exhibit a seasonal cycle 
as well. These differences are not only very marked when making interspecific 
comparisons but intraspecific differentiation is observed. The northernmost popu- 
lation of U. rapax behaves more like a temperate zone species at low temperatures 
than its southern relatives. 



182 F. JOHN VERNBERG 

SUMMARY 

1. The rate of oxygen consumption of seven species of Uca from the tropical 
and temperate zones was determined over a graded temperature series. All species 
had a similar recent thermal history. 

2. Starvation resulted in an initial decrease in metabolic rate followed by a 
relatively constant rate in Uca pugna.v. 

3. Generally increased temperature resulted in increased rates of oxygen con- 
sumption. However, in some cases a given temperature range had little or no 
effect on metabolism, while at other temperatures a marked increase resulted. 

4. Q 10 's of temperate and tropical zone species were similar at intermediate and 
higher temperatures but differed at lower thermal levels. Q 10 varied with size 
and temperature levels. Lower O 10 's were obtained at higer temperatures. 

5. Intra- and interspecific comparisons of metabolism-size relationships were 
made on data obtained at various temperatures. The slopes of the regression 
varied with temperature and species. 

6. When comparing the metabolic response of two latitudinally isolated popu- 
lations of Uca rapax with a closely related temperate zone species, the pattern of the 
northernmost population of U. rapax was intermediate between the tropical and 
temperate zone forms. 

7. Although no seasonal variation in metabolism was observed in tropical 
species, fluctuation was observed in a temperate zone species. 

LITERATURE CITED 

BERG, K., 1953. The problem of respiratory acclimation. Hydrobiologia, 5: 331-350. 
BISHOP, D. W., 1950. In: Comparative Animal Physiology, C. L. Prosser, ed. Philadelphia: 

W. B. Saunders Co. 
BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB, 1954. Persistent daily and tidal rhythms 

of Cvconsumption in fiddler crabs. /. Cell. Com p. Physiol., 44: 477-506. 
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AUTHOR'S ERRATA 

In the paper by Giese et a/., which appeared in Volume 116, No. 1, of THE 
BIOLOGICAL BULLETIN, for February. 1958, the following errata occurred : 

Page 53, Table II : The first sign under the column heading "Sex and condition" 

should be "? spent," not "6 spent/' 
Page 53, Table II : The next to last number under the column heading "Gonad 

index" should be 17.7, not 1.77. 
Page 56, footnote 5, line 4: Insert at the beginning of the line the words "the 

variability in." 



Vol. 117, No. 2 October, 1959 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



STUDIES ON THE CARDIAC STOMACH OF A STARFISH, 
PATIRIA MINIATA (BRANDT) 1 

JOHN MAXWELL ANDERSON 
Department of Zoolot/y, Cornell University, Ithaca, N. Y. 

Patina ininiata, the cushion star or sea-bat of the Pacific Coast of North America, 
is perhaps best known to zoologists as a ready source of materials for the study of 
asteroid embryology. Of equal interest, however, are its voracious appetite and its 
unusual feeding habits. Patin'a functions as an omnivorous scavenger of both 
plant and animal materials and to some degree at least as a predator on sessile 
gastropods. Unlike Asterias and Pisaster, it does not open bivalves ; but it has 
developed to a much greater extent than any of the free-armed starfishes the ability 
to evert the cardiac stomach through the mouth and to employ it as a feeding 
organ of great effectiveness. Almost every individual observed undisturbed in a 
tide pool is found with its remarkably voluminous cardiac stomach fully everted and 
widely spread. Specimens maintained in an aquarium spend a good share of the 
time with their everted stomachs closely applied to the glass, perhaps, as suggested 
by MacGinitie and MacGinitie (1949, p. 227). digesting accumulated growths of 
diatoms. Bits of kelp or other seaweed, cracked snails, large pieces of mussel flesh, 
living limpets, or even smaller specimens of Patiria are covered and held against 
the substratum by the body of the starfish, then enveloped by the spreading folds of 
the everted stomach (Fig. 4) until completely digested. Even small pieces of 
soft food, which the animal is capable of ingesting, are first covered and wrapped 
in folds of the stomach before being brought into the cavity of the gut. 

Such observations suggest that the cardiac stomach of Patiria functions in a 
highly specialized manner, being more frequently and more extensively exposed to 
the external environment than the stomachs of other familiar starfishes. It seemed 
likely that the functional peculiarities of this organ might be reflected in significant 
structural specializations, probably involving regional differentiation of the stomach 
wall and a particularly well-developed system of fibrous attachments for holding and 
retracting the everted stomach. Although Patiria is a relatively common intertidal 
species in much of its range along the Pacific coast, no detailed anatomical or 
histological studies of its digestive tract appear to have been made. The present 

1 These studies form a part of a program of investigation begun at the Hopkins Marine 
Station of Stanford University, Pacific Grove, California, during the tenure of a John Simon 
Guggenheim Memorial Fellowship. They were supported by USPHS Grant No. RG-5755 and 
by funds from NSF Grant No. G6007 to Cornell University. 

185 
Copyright 1959, by the Marine Biological Laboratory 



186 JOHN MAXWELL ANDERSON 

study was undertaken to provide a basis for comparison in a contemplated series of 
experiments, and as a contribution to knowledge of comparative and functional 
anatomy and histology in the digestive systems of asteroids. The results of this 
study have brought to light several interesting features of the cardiac stomach of 
Patiria that have apparently not been described previously, either for this species 
or for any of its relatives. 

It is a pleasure to express to the Director and Staff of the Hopkins Marine 
Station my appreciation for their hospitality and helpful cooperation during the 
conduct of these investigations. 

MATERIAL AND METHODS 

Small to moderate-sized specimens of Patiria ininiata were collected in tide 
pools near Pacific Grove, California, and maintained in the laboratory in running 
sea-water. Many observations of normal feeding behavior were made on these 
animals, which were periodically provided with a variety of food such as cracked 
snails, opened mussels, kelp fronds, living limpets, etc. 

For anatomical studies, animals were immersed in isotonic MgCL (8% in tap 
water) until flaccid, then opened by cutting through the body wall all around the 
margin of the body and removing the aboral part, transecting the digestive tract at 
the junction between cardiac and pyloric stomachs. The cardiac stomach and its 
retractor harness were then studied in their normal positions ; or, by cutting all 
the strands binding the stomach to the ambulacral ossicles and transecting the 
esophagus, the stomach could be removed from the body. Portions of such excised 
stomachs, maintained in a relaxed state by continued immersion in MgCl 2 solution, 
were spread and pinned out with fine glass needles in small wax-bottomed dissecting 
trays. After completion of studies of these spread segments in the living condition, 
the salt solution was decanted and the preparation flooded with Kelly's fluid or 
Zenker-acetic. Fixation was continued for 24 hours ; the fixed tissue was then 
removed from the wax, washed for several hours in running water, trimmed, 
dehydrated, and imbedded in paraffin. Carefully oriented serial sections were cut 
at 7/t, in a plane parallel to the mouth. These "horizontal" sections were carried 
through a variety of staining routines, for various purposes. For general histo- 
logical observations, Harris' hematoxylin followed by eosin or light green was used ; 
for delineation of cell boundaries, cytoplasmic granules, and muscular and con- 
nective-tissue fibers, Mallory's phosphotungstic acid hematoxylin (PTA) proved 
excellent. Glycogen and other polysaccharide complexes were demonstrated by 
the use of a periodic acid-Schiff routine (Lillie, 1954, p. 123), controlled by 
salivary digestion. Metachromatic substances were revealed by overnight staining 
in dilute toluidine blue, followed by alcoholic dehydration and differentiation. A 
standard Feulgen technic was used to identify nuclei by selective staining of their 
DNA content. 

Certain special experimental procedures will be described later, in connection 
with the observations they were designed to elucidate. 

OBSERVATIONS 

The retracted cardiac stomach occupies a large part of the central cavity of the 
disk. It lies in the form of a more or less regular series of pouches, extending from 



CARDIAC STOMACH OF PATIRIA 187 

the main lumen both perradially and interradially, above and between the proximal 
ambulacral ossicles of the rays. These pouches are separated by medially and 
upwardly directed folds that extended almost to meet a series of similar folds hang- 
ing from the roof of the pyloric stomach. A relatively slight constriction divides 
the stomach into the conventionally recognized cardiac and pyloric portions, leaving 
a comparatively broad passageway between them ; it is into this passageway that 
the medial folds of the cardiac stomach protrude. The constriction separating the 
upper and lower portions of the stomach is girdled by a glistening band of con- 
nective-tissue fibers related to the retractor harness of the cardiac stomach. 

The retractor mechanism 

What may be termed the extrinsic elements of the retractor harness consist in 
each ray of a pair of fan-shaped bundles extending from broad origins along the 
sides of the ambulacral ridge to rather more restricted attachments on the wall of 
the cardiac stomach (Fig. 2). These bundles bear a superficial resemblance to 
the extrinsic strands described for Asterias forbesi by Anderson (1954) but differ 
markedly in detail. Whereas in Asterias the extrinsic components consist of thick 
marginal bands supporting thinner, mesentery-like sheets, those of Patiria are 
thick and tough throughout, and each of the fan-shaped bands comprises four 
strands. The shortest and most proximal of these originates on the connective- 
tissue coat lateral to the first or proximal ambulacral ossicle and follows a short 
course aborally, skirting between the perradial and interradial pouches, to insert on 
the fibrous girdle described above. The longest of the extrinsic strands forms the 
upper border of the fan-shaped band, originating lateral to the ambulacral ridge in 
the region of the tenth to twelfth ossicle and inserting also on the fibrous girdle of 
the stomach, adjacent to the attachment of the shortest strand. Between the 
shortest and the longest lie two intermediate strands, somewhat thicker, originating 
alongside the ambulacral ridge, passing between the perradial and interradial 
pouches, and disappearing proximally under the perradial pouch. Here these 
strands appear to end rather abruptly but actually break up into branching and 
rebranching intrinsic retractor fibers spreading widely over the surface of the 
cardiac stomach as they approach the oral end of this organ. It will be noted that 
the stomach of Patina lacks the nodules characteristic of the retractor system of 
Asterias, the conspicuous fibrous knots upon which all extrinsic strands insert and 
from which all intrinsic strands branch. Instead, in Patiria the stomach is provided 
w r ith the fibrous girdle which is related only to those elements of the extrinsic 
system that do not contribute to the branching intrinsic fiber systems. 

The intrinsic fiber patterns on the wall of the cardiac stomach in Patiria gen- 
erally resemble those of Asterias. In the portion of the stomach pertaining to 
each of the rays, two major bands pass from the blunt ends of the intermediate 
extrinsic strands, bound together initially but immediately diverging widely to 
course diagonally downward. These soon become closely applied to the outside 
of the stomach and pass underneath the visceral peritoneum to establish contact 
with the muscular and connective-tissue layers of the stomach wall. Each of the 
major strands now branches into two, and below the points of divergence of the 
four strands thus produced a series of four additional dichotomies occurs. Even- 
tually, the final products of all these binary divisions, tapering slowly and growing 
progressively smaller as they approach the lower end of the stomach, disappear 



188 JOHN MAXWELL ANDERSON 

as fine fibers blending into the stomach wall at a line near the upper limit of the 
esophagus. The paired extrinsic bands in each of the rays are thus continued as 
intrinsic strands intimately connected with the fabric of the stomach wall and 
spreading to 64 widely dispersed terminal attachments at its lower end ; in a 
5-rayed specimen, the intrinsic retractor fibers are distributed to a total of 320 
ultimate points of attachment, all arranged along a regular line (cf. Fig. 1). For 
purposes of identification in subsequent discussions and to avoid confusion with 
other components to be described later, the branching strands spreading from the 
ends of the extrinsic bands will be referred to as Class 1 fibers. 

Histological examination shows that these intrinsic strands, like those of 
Asterias, are composed of varying mixtures of connective-tissue and muscle fibers, 
bound together and enclosed by the typical cuboidal epithelium of the visceral 
peritoneum (Fig. 7). As the strands branch, becoming smaller and more in- 
timately connected with the stomach wall, they continually contribute both con- 
nective-tissue and muscle fibers to the corresponding layers in the wall of the 
stomach (Fig. 8). In their lowest levels, near the esophagus, the small terminal 
branches can be recognized only in sections, appearing as characteristically staining, 
somewhat thickened groups of connective-tissue fibers running longitudinally 
beneath the peritoneum. The larger Class 1 fibers are attached to the stomach 
wall only along their sides, often leaving a long, tubular cavity beneath the strand. 
In such spaces, the outer surface of the stomach appears to be composed of its 
muscular and connective-tissue coats, as the retractor strands run inside the 
peritoneum (Figs. 5, 6, 17). 

The Class 1 fibers, in addition to making direct contributions to the muscular 
and connective-tissue layers of the stomach, are also closely related to a secondary 
.system of strands to be designated as Class 2 fibers. These are found in a zone 
lying roughly between the second and fifth forks of the Class 1 system. They con- 
sist of very large numbers of tiny, parallel strands which spring at closely spaced 
luit irregular intervals from the stomach wall, in close proximity to the courses of 
Class 1 fibers (Fig. 6). The Class 2 strands run a horizontal course, leaving the 
stomach wall to branch perhaps once before re-entering the wall and disappearing 
again, in the unsupported pouches between the Class 1 branches (Fig. 3). The 
horizontal fibers are longest high in the stomach, where they arise near the widely 
separated major branches of the Class 1 system; they grow progressively shorter 
in lower levels, where the Class 1 branches approach each other more closely. The 
small and inconspicuous Class 2 fibers are of particular interest in that they con- 
stitute a previously undescribed feature of the retractor mechanism and one that 
does not exist in Asterias. 

Study of the Class 2 fibers in histological sections shows that they originate in 
the muscle layers of the wall of the stomach and hence are formed almost ex- 
clusively of strands that can be identified as muscle fibers (Fig. 6). These join and 
pass out through the more superficial connective-tissue layer to form cylindrical 
bundles of parallel fibers, each enclosed by a layer of peritoneal cells (Fig. 9). 
After a longer or shorter course through the perivisceral coelom near the outer 
surface of the stomach, the strands re-enter the wall, penetrate the connective- 
tissue layer again, and contribute their fibers in a spreading pattern to the muscle 
layers of the stomach (Fig. 10). Class 2 bundles in their free courses over the 
surface of the stomach range approximately from 9 to 14 /A in diameter. 



CARDIAC STOMACH OF PATIRIA 18- 

Still another group of fibers is found outside the stomach of Patiria, lying in 
parallel longitudinal array about its lower end. These, to be referred to as Class 3 
fillers, are the most slender of all the retractor and restraining bands of the stomach 
wall. Each appears to consist of a single, hyaline, homogeneous strand of extra- 
cellular fibrous material enclosed by the vastly extended cytoplasm of the cell that 
produced it ; the nucleus of the cell lies in a thickened area of the cytoplasm some- 
where along the length of the strand (Fig. 13). Class 3 fibers exist in a considerable 
size range, from just under 2 /A to more than 8 ^ in diameter. The coarsest are also 
the longest, originating in the connective-tissue components of small Class 1 fibers, 
passing out through the peritoneum into the coelom, turning downward toward 
the peristome, and finally penetrating the peritoneum again to contribute several 
branching processes to the connective-tissue layer of the esophagus or peristome. 
The origin of such a coarse fiber from a small Class 1 strand is shown in Figure 1 1 ; 
Figure 12 shows the insertion of a similar strand in the peristomial region. The 
most numerous of the Class 3 fibers are smaller and shorter, having both ends 
anchored in the connective-tissue layer of the stomach itself, not related to other 
strands. These run brief courses across folds in the stomach w r all (Fig. 13) or 
across the deep bay formed by the reflection of the esophagus above the horizontal 
peristomial membrane. It should be emphasized that all of the Class 3 fibers, of 
whatever size, consist of single, nucleated cells enclosing fibrous extracellular 
strands ; they contain no muscle fibers, and they are never clothed by a peritoneal 
layer as fibers of Classes 1 and 2 always are. Like the Class 2 fibers, these con- 
stitute a new r feature, previously undescribed. 

In a restricted zone at the lowest end of the stomach, fibers of all three classes 
are present ; that is. Class 1 fibers give off coarse Class 3 fibers in the zone near the 
esophagus in which short Class 2 strands also occur. Here, of course, the hori- 
zontal Class 2 fibers and the longitudinal Class 3 strands run perpendicular to 
each other. By far the majority of the Class 3 fibers, however, are found below 
the level occupied by the lowest of the Class 2 strands. 

The stomach wall 

In Patiria, as in Astcrias, the spreading branches of the intrinsic fibers (Class 1) 
are accompanied by underlying specializations in the structure of the stomach wall. 
These consist of branching gutters, beginning at the lower end of the stomach 
beneath the terminal branches of the Class 1 fibers and running upward, converging 
and joining with each other to form progressively larger channels that ultimately 
widen and fade out at approximately the level where the primary strands of the 
intrinsic retractor system become attached to the stomach. The regular, consistent, 
branching patterns of these gutters are conspicuous in vesicles of the everted cardiac 
stomach ; Figure 1 shows such a pattern in a single everted vesicle, and in Figure 4 
they are clearly visible in several parts of the widely spread stomach. 

Horizontal sections near the oral end of the stomach reveal the intimate relation- 
ship that exists between the gutters and various categories of intrinsic fibers. 
Class 1 fibers are attached longitudinally along the evaginated folds that form the 
gutters, and the horizontal strands of the Class 2 fibers extend from the shoulders 
of these folds to attach again in unspecialized areas between neighboring grooves. 
This arrangement of the Class 2 strands makes it possible for them to maintain 
the gutters by puckering the wall of the stomach and creating the inwardly-directed 



PLATE I 




EXPLANATION OF FIGURES. 

Figures 1 and 2 are photographs of living preparations, and Figure 4 is a photograph of 
a living animal. The remaining figures on this and the following plate are photomicrographs 
of tissues fixed in Kelly's fluid (except Figs. 7 and 20, fixed in Zenker-acetic), sectioned at 7/j., 
and stained as indicated. The magnifications given are approximately correct for the figures 
as they appear here, after enlargement and reduction. 

190 



CARDIAC STOMACH OF PATIRIA 191 

FIGURE 1. Portion of the everted cardiac stomach, showing a part of the system of 
gutters in the stomach wall associated with Class 1 retractor fibers. Note the regular line 
along which the smallest branches begin, in the region termed the esophagus, and the con- 
vergence of the gutters towards the mouth (arrow). 4 X- 

FIGURE 2. Elements of the extrinsic retractor system in one ray, after removal of the 
roof of the animal with all aboral parts of the digestive tract. Note the connective-tissue 
girdle surrounding the stomach (arrow) ; just below this point the shortest and longest retractor 
strands of one side insert on the girdle, close together. Within the angle they form lie the 
two intermediate strands which pass onto the surface of the stomach (to left) and become 
continuous with Class 1 intrinsic fibers. The proximal ambulacral ossicle is indicated at a; 
this is partially covered by a perradial pouch extending from the stomach between the two 
short retractor strands. 4 X- 

FIGURE 3. Horizontal section passing through the level of confluence of two small gutters, 
low in the stomach ; the Class 1 fibers associated with the gutters have not joined at this 
level. The arrow indicates a horizontal Class 2 fiber passing into the plane of the section and 
inserting on the stomach wall ; its origin on the shoulder of the ridge nearby is not shown ; 
cf. Figure 6. Phosphotungstic acid (PTA) hematoxylin ; 70 X- 

FIGURE 4. A large individual feeding on an algal frond ; the vesicles of the broadly-everted 
cardiac stomach have passed through holes in the frond and spread out against the glass wall 
of the aquarium. The arrow indicates a typical vesicle ; note the great size of the stomach, 
and the conspicuous gutter-and fiber patterns in its vesicles. Approximately 0.25 X- (This 
photograph was made by Dr. Allahverdi Farmanfarmaian of the Hopkins Marine Station and 
is printed with his permission.) 

FIGURE 5. Section similar to Figure 3, higher in the stomach, with two small Class 1 
fibers joining. Note the flat floor of the gutter and the general differences in the epithelium as 
between the floor and the sides of the gutter. PTA hematoxylin; 145 X- 

FIGURE 6. Horizontal section, still higher in the stomach. This section demonstrates the 
relationship between the three classes of intrinsic retractor fibers : a large Class 1 fiber is 
shown in cross-section, broadly attached by its margins to the ridge above the gutter. A pair of 
Class 2 fibers proceeds laterally (top and bottom, in the figure) from the shoulders of the 
ridge, and a coarse Class 3 fiber is shown at its origin from one side of the Class 1 strand 
(left arrow). Note the close relationship between the fibers of Class 1 and Class 2. In the 
side-wall area indicated by the right-hand arrow, note the distal deposits of basophilic granules 
in the epithelial cells ; these are always lacking in cells lining the floor of the gutter. The 
dark bodies in the floor are mucous goblets. Harris' hematoxylin, light green ; 145 X 

FIGURE 7. Portion of a cross-section of a major Class 1 fiber, high in the stomach. At 
upper left, the peritoneal covering, followed by a heavy connective-tissue coat; the darkly- 
staining masses are shrunken bundles of muscle fibers, bound together and separated from 
each other by layers of connective tissue. Zenker-acetic fixation, PTA hematoxylin ; 650 X 

FIGURE 8. Floor of a gutter, with attachment of a Class 1 fiber ; the arrow 7 indicates a 
large connective-tissue bundle passing into the connective-tissue layer of the stomach wall from 
the retractor strand (lower left). Above this, the muscular components of the intrinsic fiber 
mingle with the muscular layers of the stomach wall. Lumen of the stomach to right, 
perivisceral coelom to left. Note the thick nerve layer, represented by the light-colored areas 
in arcades between the bases of the epithelial cells. PTA hematoxylin; 650 X. 

FIGURE 9. Longitudinal section of a Class 2 fiber, composed of parallel muscle fibers 
enclosed by peritoneum. Harris' hematoxylin, light green ; 650 X 

FIGURE 10. Tangential section of stomach wall, showing the insertion of a Class 2 fiber 
(arrow) and the wide distribution of its branching strands in the muscle layer of the stomach. 
Note that these muscle fibers are not arranged in circular and longitudinal layers but run in 
all directions. PTA hematoxylin; 320 X- 

FIGURE 11. The origin of a coarse Class 3 fiber from the attachment-point of a Class 1 
fiber to the stomach wall. Note the clear, homogeneous nature of the Class 3 fiber and the 
shriveled coat of cytoplasm surrounding it. This fiber, turning downward towards the mouth, 
passes out of the section at the blurred point, where it runs alongside a second, smaller Class 3 
fiber shown here in cross-section. Harris' hematoxylin, eosin ; 650 X 

FIGURE 12. The insertion of a coarse Class 3 fiber by branching processes penetrating the 
peritoneum and muscle coats to enter the connective-tissue sheet of the stomach wall. The 
very heavy connective-tissue layer with inward extensions is characteristic of the peristomial 
region, where the longest of the Class 3 fibers terminate. PTA hematoxylin ; 650 X - 



192 



JOHN MAXWELL ANDERSON 
PLATE II 




19 



FIGURE 13. A group of short Class 3 fibers lying in the coelom in a fold of the stomach 
wall. Note nuclei in the cytoplasmic masses attached to some. PTA hematoxylin ; 650 X 

FIGURE 14. Nature of the stomach wall at the lip of a gutter, in the lower part of the 
stomach. Note the tall epithelial cells with long, dense nuclei, characteristic of these regions 



CARDIAC STOMACH OF PATIRIA 193 

folds forming their sides. It should he noted that the preparation of this material 
for sectioning involved stretching and spreading segments of the stomach and 
pinning them to flat wax plates ; even such treatment does not obliterate the gutters, 
which must he considered a permanent feature of the wall of the stomach. 

The general histology of the stomach is typical of that repeatedly described, with 
minor variations, for the wall of the asteroid digestive tract (Hamann, 1885; 
Cuenot, 1887; Ludwig and Hamann, 1899; Chadwick, 1923; Hayashi, 1935; 
Anderson, 1954). A flagellated cuboidal epithelium clothes the outer surface. 
Just inside this lie two layers of muscle fibers, which Hamann considers as repre- 
senting an outer longitudinal and an inner circular layer ; in Patina, muscle fibers 
run in all directions, forming an irregular network (Fig. 10), and it is difficult to 
determine which of them make up the principal circular and longitudinal strands. 
Inside the muscle-fiber network a connective-tissue layer is encountered. This 
varies in thickness, being most conspicuous in the neighborhood of Class 1 strands, 
which contribute fibers to it (Fig. 8). The connective-tissue layer serves also as 
a basement membrane, to which are attached the proximal ends of the very tall, 
slender, columnar epithelial cells that line the stomach and constitute the principal 
layer of its wall. In many areas the basal portions of these epithelial cells join 

as of the side walls of the gutters. Lumen of stomach to right, coelom to left ; the random 
fibers lying in the coelom are small Class 3 strands. PTA hematoxylin ; 650 X 

FIGURE 15. Similar epithelium lower in the stomach, near the esophagus, showing a few 
secretory cells packed with coarse spherules. These are interpreted as "mulberry cells." Note 
the distal row of flagellary basal granules and the brush border. PTA hematoxylin ; 650 X 

FIGURE 16. Portion of the wall of a small gutter, at the zone of transition between low 
cells with rounded nuclei in the floor (above) and taller cells with elongate nuclei in the wall 
(right). Lumen of stomach at upper right, coelom to left. The irregular strand passing up- 
ward at the left is part of the Class 1 fiber associated with this gutter ; note that the peritoneal 
layer mounts over the outside of this strand and is absent from the stomach wall beneath it. 
Cf. Figures 5, 6, and 17. PTA hematoxylin; 650 X- 

FIGURE 17. Similar section through the floor of a small gutter; lumen of stomach to 
right, coelom to left, with an attachment point of the associated Class 1 fiber just above left 
center. Note the thick nerve layer in the epithelial arcades, the small spherical nuclei high in 
the epithelium ( amoebocyte nuclei?), and the absence of a peritoneal layer beneath the Class 1 
strand. Several cysts are apparent above the nerve layer in the floor of the gutter. PTA 
hematoxylin ; 450 X 

FIGURE 18. Transition zone in the side of a small gutter. Note the characteristic elongated 
nuclei, distal differentiations of cells, a large mucous goblet ( arrow ) with shrunken nucleus, 
and numerous cysts lying above the basal nerve layer. Lumen of stomach to right, floor of 
gutter at top. PTA hematoxylin ; 650 X 

FIGURE 19. Typical epithelium near the lip of a gutter, showing localizations of PAS- 
positive material after salivary digestion. Note the conspicuous connective-tissue layer (left) 
and granular deposits in the distal ends of the epithelial cells. Lumen of stomach to right. 
Periodic-acid-Schiff, after salivary digestion, counterstained with Weigert's acid iron-chloride 
hematoxylin and light green ; 650 X 

FIGURE 20. Cysts between the bases of tall epithelial cells. These contain clear, re- 
fractile, homogeneous bodies; the nuclei (arrow) are the only Feulgen-positive elements as- 
sociated with the cysts. Zenker-acetic, PTA hematoxylin ; 650 X 

FIGURE 21. Typical rounded-nucleus epithelium spreading from shallow gutters high in 
the stomach. Peritoneum at lower left, lumen of stomach at upper right. Note the long, 
clear mucous goblets, and the numerous cysts ; compare the appearance of the contents of these 
cysts with those in Figures 8, 17, 18, 20. PTA hematoxylin; 650 X- 



194 JOHN MAXWELL ANDERSON 

together in bundles before inserting on the basement membrane, forming arcades 
of various sizes occupied by the fibers of the nerve layer (Figs. 8, 17). Like the 
connective-tissue sheet, the nerve layer is somewhat thicker in the bottoms of the 
gutters, beneath the lines of attachment of Class 1 fibers, but it never forms the 
thickest component of the stomach wall in Patina as it does in some regions of the 
stomach in Asterias forbesi (Anderson, 1954: Fig. 10). 

The epithelium lining the stomach presents no particularly remarkable features. 
Its typical tall columnar cells are covered distally by a brush border, and each cell 
bears a single long flagellum, arising from a conspicuous basal granule somewhat 
eccentrically placed in the distal end of the cell (Figs. 8, 18). Numerous mucous 
goblets are interspersed among the typical epithelial cells, with nuclei usually lying 
in the basal third of the cell and crowded to one side by the accumulated secretion 
(Figs. 6, 18). Other types of secretory cells are encountered very infrequently in 
the cardiac stomach proper ; single cells, long and slender and containing a row or 
two of small secretory spherules, are occasionally found scattered at random among 
the cells of the general epithelium, but these are not at all common. In the region of 
the esophagus there is a considerable representation of secretory cells of a different 
type, tending to lie rather high in the epithelium and to have a somewhat bulbous 
appearance, packed and distended with coarse, deeply-staining spherules (Fig. 15). 
These I have interpreted not as endodermal zymogen cells, such as are abundant in 
the digestive diverticula (pyloric caeca), but as representing the so-called "mulberry 
cells" of the epidermis described by Cuenot (1887). Cells identical in appearance 
and staining behavior are numerous in the adjacent peristomial epithelium and 
gradually disappear in the esophagus ; a very few may be encountered in the lowest 
part of the stomach. 

A considerable degree of regional specialization, or consistent, patterned dis- 
tribution of specific epithelial cell types, occurs in conjunction with the branching 
gutters of the stomach wall, although in Patiria this characteristic is never so marked 
as in Asterias. The flattened floors of the gutters are lined by comparatively low 
columnar cells with clear cytoplasm and small, rounded, granular nuclei (Figs. 17, 
18). In the lower part of the stomach, where the gutters are narrow and deep, such 
cells appear only in their floors ; higher in the stomach, where the gutters become 
broad and shallow, the columnar cells with small rounded nuclei occupy larger 
areas, increase in height, and constitute the most numerous class of epithelial cells 
(Fig. 21). The lateral walls of the deeper gutters consist of tall cells, very 
crowded, in which the nuclei are elongate and slender and stain densely. A narrow 
zone of transition marks the change from one type of epithelium to the other, just 
where the side walls rise from the floor (Figs. 3, 5, 16). The tall cells with dense 
nuclei are most numerous in the walls of the gutters ; they continue over the lips of 
these grooves (Fig. 14) and gradually give way, in the areas between adjacent 
gutters, to equally tall cells with elongate but more granular and lightly-staining 
nuclei. Flagella are never multiple ; each cell bears only one. 

Mucous goblets are numerous in the floors of the gutters as well as among the 
taller cells in their side walls and near their lips. Small cells with spherical, 
granular nuclei, interpreted as amoebocytes, are commonly observed lodged be- 
tween the epithelial cells, most conspicuously at levels above the epithelial-cell nuclei 
(Figs. 17, 18). 



CARDIAC STOMACH OF PATIRIA 195 

Although no detailed histochemical studies have been attempted, special technics 
demonstrate some additional characteristics in various histological components of 
the stomach. Glycogen appears to be very generally distributed throughout, with 
no notable points of concentration, as indicated by results of the periodic acid- 
Schiff routine. After removal, by salivary digestion, of all PAS reactivity at- 
tributable to glycogen, several sites retain strongly positive reactions. These sites 
include, as expected, the connective-tissue basement membrane of the stomach wall 
(Fig. 19) and related components of Class 1 retractor fibers, as well as the contents 
of mucous goblets and free mucus retained on the surface of the epithelium after 
release. In addition, the distal cytoplasm of virtually all the tall epithelial cells 
contains varying amounts of granular, finely-dispersed PAS-positive material 
(Fig. 19), the amount depending on the locations of the cells. Those in the side 
walls of narrow gutters usually contain the largest deposits ; in other locations only 
a small amount of material just under the brush border, surrounding the basal 
granule of the flagellum, reacts positively. In addition to being PAS-positive, 
this material is basophilic (stains with Harris' hematoxylin see Fig. 6) and ex- 
hibits a reddish metachromasia. persisting after alcoholic dehydration and mounting 
in resin, when stained overnight with dilute toluidine blue. These staining reactions 
are consistent characteristics of the distal deposits in tall cells with long nuclei ; it 
is noteworthy that in the epithelial cells with rounded, granular nuclei, localized in 
the floors of deep gutters and more widely distributed in broad, shallow ones, the 
cytoplasm is always clear and never contains even a trace of such material. In 
contrast to the distal metachromatic material in the tall epithelial cells, the contents 
of mucous goblets, while similarly PAS-positive, lose their toluidine-blue meta- 
chromasia during alcoholic dehydration. 

One further unusual feature of the cardiac stomach of Patiria, characteristic of 
all specimens examined, remains to be described, although its significance remains 
in doubt. In all levels of the stomach, the depths of the epithelial layer are oc- 
cupied by numerous cystic growths, of various sizes. They may be small and 
nearly spherical, lying in the arcades between the basal processes of the epithelial 
cells just above the nerve layer (Figs. 16, 17, 18), or they may be much longer 
than their breadth and occur in such masses as to crowd the nuclei and major 
cytoplasmic portions of the epithelial cells into the upper half, or less, of the 
epithelial layer. The cysts are fully as variable in appearance as in size. They are 
always more or less completely filled with inclusions, which are either clear, non- 
staining, refractile spherules less than 2 //, in diameter ; or slightly basophilic spher- 
ules of about the same size, each of which contains a single highly basophilic granule ; 
or somewhat larger masses or groups of very small, deeply staining flocculent or 
granular bodies. The cysts appear to begin, at least, as intracellular bodies ; in 
conjunction with the smaller ones a single large nucleus can usually be demonstrated, 
somewhat distorted and crowded to one side so that it resembles the nucleus of an 
active mucous goblet (Fig. 20). This nucleus is the only body in the cysts that 
gives a positive Feulgen reaction ; although the small, highly basophilic granules 
within the inclusions otherwise stain like chromatin, they are Feulgen-negative. 
It seems reasonable to conclude, at least tentatively, that the cysts are of parasitic 
origin. A detailed study of this material would probably show that the inclusions 
in different cysts, so obviously different in appearance, represent successive stages 



196 JOHN MAXWELL ANDERSON 

in the life-cycle of a sporozoan, widely distributed in Patiria ininiata at Pacific 
Grove. Such a study is beyond the scope of the present investigation. 

DISCUSSION 

Although the cardiac stomach of Patiria is very generally similar to that of 
Asterias, major differences in structural details appear when these organs are 
compared, as indicated briefly at several points in the preceding descriptive section. 
Close resemblance is not necessarily to be expected, as these genera are not closely 
related, belonging to different Orders ; furthermore, their habits and feeding practices 
differ considerably. It is regrettable that comparably detailed studies have ap- 
parently not been made on Asterina gibbosa, a species very close to Patiria that has 
been more generally described by European investigators. It is of interest, how- 
ever, to examine structural differences that exist between Asterias and Patiria, and 
to consider the functional differences to which they may give insight. 

In view of the unusual degree and frequency of eversion of the cardiac stomach 
in Patiria, one might anticipate that provision of mechanisms for anchorage, rein- 
forcement, and restraint of the everted vesicles, and for their rapid retraction, would 
be of the utmost significance. The existence of two supplementary systems of 
fibers here, in addition to the branching intrinsic retractor system (Class 1 fibers) 
found also in Asterias, is undoubtedly related to the functional demands of this 
special situation. Unfortunately, experimental evidence as to the contractility and 
behavior of these fibers in Patiria is not available ; in its absence, deductions as to 
their probable functions may be made from the nature, histological composition, 
and anatomical relationships of the several fiber systems. 

Class 1 strands are stout mixtures of connective-tissue and muscle bundles, 
anchored proximally to the extrinsic retractors (of which they are actually con- 
tinuations), attaching all along their lengths by broad insertions on both the con- 
nective-tissue and muscle layers of the stomach wall, and extending to widespread 
terminal attachments at the extreme lower end of the cardiac stomach. These are 
almost certainly the principal restraining and retractor bands of the stomach ; con- 
tractions in these strands would be very widely transmitted to the everted vesicles 
and would be most effective in compressing them to force the coelomic fluid back 
into the perivisceral cavity, and in drawing the collapsed folds uniformly back 
through the mouth. It is true that the contractility postulated by Anderson (1954) 
for the supposedly homologous intrinsic retractors in Asterias could not, after all, 
be experimentally demonstrated (Burnett and Anderson, 1955). In Patiria, how- 
ever, the Class 1 strands contain a much greater complement of muscle fibers than 
the corresponding structures in Asterias and so may be considered more likely to 
be contractile. 

Muscular contractions in Class 1 fibers may be transmitted even more broadly, in 
areas to which their branches do not extend, through the mediation of the Class 2 
fibers. These originate in the muscle layer on ridges of the stomach wall, and the 
muscle fibers of which they are entirely composed may actually be traceable to the 
contractile components of the adjacent Class 1 strands. At their other ends, they 
distribute their fibers very widely in the muscle network of the stomach wall. In 
addition to maintaining the gutter-patterns in the lower part of the stomach, the 



CARDIAC STOMACH OF PATIRIA 197 

Class 2 fibers are of such composition and anatomical relationships that they could 
serve very importantly in collapsing the everted vesicles of the stomach at retraction, 
or simply in aiding the muscle layers of the stomach wall to resist excessive stretch- 
ing in response to increased internal pressure. 

In contrast, the Class 3 fibers, consisting solely of single strands of fibrous con- 
nective tissue, must function in a purely mechanical fashion. The myriads of short, 
parallel fibers that both begin and end in the connective-tissue layer of the stomach 
wall can only reinforce and restrain the extreme lower end of the stomach where 
it joins the peristome. The coarser, longer strands, originating in smaller numbers 
in the connective-tissue bundles of Class 1 fibers, must function to transmit tension 
from these to the lower stomach wall, in areas not reached by either Class 1 or 
Class 2 branches. 

Little can be said concerning the functions of the extrinsic retractor harness. 
It is of interest, however, to note that the fan-shaped bands, of which there are two 
in each ray, are not of such simple construction as those in Asterias but are com- 
posed of groups of fibers with different relationships. The strands that continue on 
the stomach wall as branching Class 1 fibers are probably contractile, as their contin- 
uations almost certainly are. It is difficult to see how contractility in the strands 
connecting the circumferential girdle of the stomach to the ambulacral ossicles 
could serve any useful purpose ; shortening of these strands would dilate the passage- 
way between cardiac and pyloric stomachs and perhaps depress the floor of the 
pyloric stomach, but the contribution of such an action to the mechanism of eversion 
or retraction of the stomach is difficult to evaluate. At any rate, until experimental 
evidence as to the possible differential behavior of these various strands is available, 
further speculation is fruitless. 

It has been remarked that regional specialization of the tissue components of 
the stomach wall is much less pronounced in Patiria than in Asterias, although the 
gutter-patterns with which this is associated are about equally well developed in 
the two forms. It is particularly noteworthy that the ridges between gutters in 
Patiria are not clothed with tall cells containing huge, densely-staining, cigar-shaped 
nuclei and bearing multiple flagella so characteristic of such regions in Asterias. 
A relatively small number of cells localized here in Patiria do contain long, com- 
paratively dense nuclei, but the difference between these and their counterparts in 
Asterias is striking. If such cells represent sensory receptors, as Smith (1937) 
suggested for Marthasterias gladalis, then Asterias is obviously better supplied with 
gastric sense organs than is Patiria. Yet the stomachs of the two forms are ap- 
parently equally delicate and susceptible to injury and ought to be equally sensitive 
to stimuli from hazardous situations during eversion. The observed differences 
may simply be related to differences in the general characteristics of forms in the 
two Orders to which these species belong; or it may be that different, as-yet- 
tmrecognized cells in the stomach of Patiria are functioning as sensory receptors. 
On the other hand, it may be suggested that the observed differences reflect the 
marked contrast in feeding habits exhibited by Asterias and Patiria. Although 
the stomach of Asterias is less frequently and less broadly everted than that of 
Patiria and hence perhaps not so often exposed to the general vicissitudes of the 
external environment, it is very often insinuated through the minute gape between 
the shells of living bivalves (Burnett, 1955; Lavoie and Holz, 1955; Lavoie, 1956) 



198 JOHN MAXWELL ANDERSON 

and must be provided with patches of specialized sensory epithelium for its guidance 
and protection. Feder (1955) has reported similar behavior in the stomach of 
Pisaster ochraceus attacking clams and oysters ; and in his experiments with 
Evasterias troschelii, Christensen (1957) found that although the tube-feet of 
this species might often be trapped and cut off between the closing valves of an 
imitation clam, folds of the cardiac stomach never were. Patches of specialized 
epithelium closely resembling those found in the stomach of Asterias are very well 
developed also in the cardiac stomachs of Pisaster ochraceus and Pycnopodia 
helianthoides (personal observations, unpublished) ; I have no information on 
Evasterias, but the occurrence of such "sensory" patches in the stomachs of 
Asterias, Marthasterias, Pisaster, and Pycnopodia suggests that this may be a 
common characteristic of members of the Family Asteriidae, related to their habit 
of inserting the everted cardiac stomach into living bivalves. According to my 
observations, the stomach of Patiria operates in no such sophisticated manner ; it 
is merely everted as broadly as possible and wrapped about whatever objects of 
food are available, or perhaps used as a flagellary-mucous feeding organ. 

Experiments using sea-water suspensions of Congo-red-stained yeast cells or 
India ink to trace currents over the surface of the stomach reveal that the gutters 
do not, as might have been expected, serve to conduct food-bearing currents upward 
into the stomach. Such currents as do appear in the vicinity of the grooves are 
directed in such a way as to move material from the depths of the grooves upward 
over their lateral lips and thence away from the surface of the epithelium; there 
appear to be no longitudinal currents, either upward or downward, in the gutters 
themselves. The action of flagella on the ridges between gutters does produce 
currents carrying particles upward into the stomach. These observations are 
essentially in agreement with those reported for Asterias forbesi (Anderson, 1954). 

Digestion of food materials of all kinds surrounded by the everted cardiac 
stomach or drawn into its cavity after retraction is rapid and complete. A small 
specimen of Patiria (radius about 2 cm.), trapped against the glass of an aquarium 
by a large individual (radius about 5 cm.) and enveloped in the folds of its everted 
stomach, was reduced within a day and a half to a small heap of dissociated skeletal 
ossicles ; examples of this kind could be multiplied indefinitely, all testifying to the 
powerful nature of the digestive juices acting in the cardiac stomach. In view of 
this, the extreme paucity of anything resembling zymogen cells in the epithelium 
of the cardiac stomach is surprising and raises the suspicion that perhaps digestive 
enzymes are being produced by cells in the stomach lining that do not resemble 
those customarily identified as the sites of enzyme production in starfishes (Ander- 
son, 1953). An alternative explanation, more in line with classical interpretations 
of asteroid digestive processes, would hold that the stomach itself produces no 
enzymes but serves only as the organ which envelopes the food and mixes it with 
enzymes brought to if from the myriads of typical zymogen cells in the pyloric 
caeca. 

The true state of affairs with regard to these questions was determined by 
simple experiment. All 5 pairs of pyloric caeca were operatively removed from a 
large specimen without interfering with cardiac stomach, pyloric stomach, or other 
parts of the digestive tract ; the specimen was then allowed to recover for a period 
of 8 weeks. Well before the end of this time the integrity of the body wall had 
been restored by healing of the incisions, and with it the ability to evert the cardiac 



CARDIAC STOMACH OF PATIRIA 199 

stomach returned. After 8 weeks, the operated specimen was offered a large, 
distinctively-colored piece of liver and ovary from a snail, which it readily ingested. 
As a control, a similar piece of snail tissue was fed to a normal starfish of the same 
size. After 24 hours, the cardiac stomach of the control animal contained only a 
semifluid, disorganized brei. while the piece of snail tissue in the stomach of the 
operated starfish remained intact and in fact retained its original color. Dissection 
of this specimen revealed that regeneration of the pyloric caeca had begun but had 
advanced only to the extent of producing short, simple, tubular rudiments with none 
of the highly specialized features characteristic of the normal organs, and so pre- 
sumably without any considerable population of zymogen cells. This experiment 
demonstrates that the stomach of Patina, which displays an almost complete lack 
of granular secretory cells, does not secrete digestive enzymes, or at least does not 
produce them in quantities sufficient to bring about normal digestion. The pyloric 
caeca, which contain very large numbers of granule-filled cells interpreted as 
zymogen cells, are obviously necessary for the production of digestive enzymes ; in 
the absence of these organs, digestion does not occur. The enzymatic secretions 
which they normally produce are carried to the cardiac stomach by flagellary 
currents, described for this species by Irving (1924), forming a part of the regular 
circulation that also brings the products of digestion from the stomach into the 
pyloric caeca. 

Many other aspects of structure and function in the digestive system of star- 
fishes await elucidation; detailed anatomical and histological studies, such as those 
reported here for a limited portion of the alimentary system in a single species, 
still remain to be done for a majority of even the commonest asteroids. Such 
studies, continued and broadened, will make possible meaningful comparisons be- 
tween closely and distantly related species, and between species of diverse habits 
and ways of life. Most importantly, detailed and accurate anatomical studies serve 
as a basis for correlation and interpretation of the results of experimental physio- 
logical studies which can. in turn, furnish explanations for anatomical details. 

SUMMARY AND CONCLUSIONS 

The large and voluminous cardiac stomach of Patiria miniata, frequently and 
very extensively everted in the normal feeding behavior of this omnivorous inter- 
tidal starfish, is provided with an unusually elaborate retractor harness. As in 
wisterias, this consists of an extrinsic portion extending from the ambulacral ossicles 
to the stomach, and an intrinsic portion spreading over the stomach in a regular 
pattern of branching strands, here designated Class 1 fibers. A second system 
consists of small, muscular strands extending horizontally in the region of the 
lower Class 1 branches and inserting on the muscle layer of the stomach ; these have 
been termed Class 2 fibers. Still a third set, composed of single strands of con- 
nective tissue, arise either from Class 1 fibers or from the connective-tissue layers 
of the wall of the stomach, take a short, straight, longitudinal course downward, and 
insert again on the connective-tissue sheet in the stomach wall. These Class 3 
strands occur in large numbers, limited to the extreme lower end of the stomach. 
It is assumed that all these accessory components of the retractor harness have 
arisen in connection with special problems presented by the size and mode of 
operation of the cardiac stomach ; the several systems of strands, varying in com- 



200 JOHN MAXWELL ANDERSON 

position and in anatomical relationships, undoubtedly perform specific, different 
functions in reinforcing, restraining, and retracting the spreading vesicles of the 
cardiac stomach. 

The wall of the stomach has essentially the same histological composition as 
that usually found in the asteroid digestive tract, with connective-tissue and nerve 
layers showing localized thickenings in the vicinity of Class 1 fibers. These strands 
are also accompanied by correspondingly branched, specifically distributed pat- 
terns of folds and ridges forming gutters in the stomach wall. Consistent patterns 
of cell localization in the epithelium lining the stomach are related to the gutters ; 
comparatively low cells with rounded nuclei line their floors, while their side walls 
are clothed by taller cells with long, dense nuclei. Mucous goblets are common, 
but there are practically no granular secretory cells. The regional specializations 
in the distribution of epithelial-cell types in Patina are much less marked than in 
Astcrias; in particular, Patiria lacks the conspicuous patches of tall cells with 
huge, cigar-shaped nuclei and multiple flagella (thought to be sensory receptors) 
localized between the gutters in Asterias. This may indicate that the cardiac 
stomach of Patiria is less sensitive to external stimuli than that of Asterias; possible 
explanations for this may involve differences in feeding habits and the behavior of 
the stomach between these two types of starfishes. 

The cardiac stomach of Patiria lacks zymogen cells almost completely ; this is 
surprising in view of the rapidity and versatility of the digestive activities carried 
on by this organ. It has been experimentally demonstrated, however, that individuals 
operatively deprived of their pyloric caeca are unable to digest food ; although they 
ingest it normally and hold it in the cardiac stomach, food remains intact for at 
least 24 hours, a period sufficient for almost complete breakdown of similar food 
in the stomach of a normal animal. It is concluded that the myriads of granular 
secretory cells in the pyloric caeca, usually interpreted as zymogen cells, are the 
source of the digestive enzymes acting to bring about normal digestion of food in 
the cardiac stomach. 

LITERATURE CITED 

ANDERSON, T. M., 1953. Structure and function in the pyloric caeca of Astcrias forbesi. Biol. 

Bull., 105: 47-61. 
ANDERSON, J. M., 1954. Studies on the cardiac stomach of the starfish, Astcrias forbesi. Biol. 

Bull,, 107: 157-173. 
BURNETT, A. L., 1955. A demonstration of the efficacy of muscular force in the opening of 

clams by the starfish, Astcrias forbesi. Biol. Bull., 109 : 355. 
BURNETT, A. L., AND J. M. ANDERSON, 1955. The contractile properties of the retractor 

mechanism of the cardiac stomach in Asterias forbesi. Anat. Rcc., 122: 463-464. 
CHADWICK, H. C., 1923. Asterias. L. M. B. C. Memoir XXV. University Press, Liverpool. 
CHRISTENSEN, A. M., 1957. The feeding behavior of the seastar Evasterias troschclii 

Stimpson. Limnology and Oceanography, 2 : 180-197. 
CUENOT, L., 1887. Contribution a 1'etude anatomique des Asterides. Arch. Zool. exp. et 

gen., Ser. 2, T. 5 bis, Supp., Mem. 2: 1-144. 
FEDER, H. M., 1955. On the methods used by the starfish Pisastcr ochraccns in opening three 

types of bivalve mollusks. Ecology, 36 : 764-767. 
HAMANN, O., 1885. Beitrage zur Histologie der Echinodermen. Heft 2. Die Asteriden 

anatomisch und histologisch untersucht. Fischer, Jena. 
HAYASHI, R., 1935. Studies on the morphology of Japanese sea-stars, I. Anatomy of Henricia 

sanguinolenta var. ohshimai, n. var. /. Fac. Sci., Hokkaido Imp. Univ., Ser. VI, 

Zool, 4 : 1-26. 



CARDIAC STOMACH OF PATIRIA 201 

IRVING, L., 1924. Ciliary currents in starfish. J. Ex p. ZooL, 41 : 115-124. 

LAVOIE, M. E., 1956. How sea stars open bivalves. Biol. Bull., Ill : 114-122. 

LAVOIE, M. E., AND G. G. HOLZ, JR., 1955. How sea stars open bivalves. Biol. Bull., 109:363. 

LILLIE, R. D., 1954. Histopathologic Technic and Practical Histochemistry. Blakiston, 
N. Y. 

LUDWIG, H., AND O. HAMANN, 1899. Echinodermen, II. Buch. Die Seesterne. In: Klassen 
und Ordnungen des Thierreichs, H. G. Bronn, Ed. Bd. 2, Abt. 3. Winter, Leipzig. 

MAcGiNixiE, G., AND N. MAcGiNiTiE, 1949. Natural History of Marine Animals. McGraw- 
Hill, N. Y. 

SMITH, J. E., 1937. On the nervous system of the starfish, Marthasterias gladalis (L). Phil. 
Trans. Roy. Soc., Ser. B, 227: 111-173. 



THE BREEDING SEASON OF THE BRACHIOPOD, 
LINGULA UNGUIS (L.) 

S. H. CHUANG 

University of Malaya in Singapore, Singapore 

Yatsu (1902) believed that the breeding season of Lingiila unguis lasted from 
mid-July to the end of August in Misaki, Japan, since he could not find the larvae 
at any other time of the year. Kume (1956) confirmed Yatsu's observation, and 
also found that the peak of breeding occurred during the first half of August. 
Sewell (1912) found several larvae in December and February in the plankton off 
the south coast of Burma. He attributed this (p. 90) to "either a local peculiarity 
or possibly to the existence of two breeding seasons during the year, one in the 
summer months July and August, and a second from December to February." 
Ashworth (1915) obtained larvae in the southern part of the Red Sea in June 
and October, and in the Indian Ocean in October. He also found a larva in 
Annandale's plankton sample obtained in the Strait of Bab-el-Mandeb in March. 
This led him to believe that in the southern part of the Red Sea a succession of 
spawnings extended at least over the period from the beginning of March to the 
early part of September. 

Yatsu (1902) observed the spawning of Lingiila unguis in captivity on several 
occasions in July and August. He noted that spermatozoa and ova were dis- 
charged forcibly through the median setal tube. Kume (1956) observed that in 
L. unguis spawning in the laboratory occurred twice daily, at sunrise and after 
sunset. 

In the present study plankton samples were collected to determine the occur- 
rence of the larvae of Lingula. The spawning behavior of adult female specimens 
in the laboratory was also studied. 

MATERIALS AND METHODS 

The plankton hauls were made with a fine tow-net at a depth one foot below the 
surface off the north coast of Singapore Island. On each occasion 410 hauls of 
10 minutes each were made, more hauls being made under favorable weather con- 
ditions. The Lingula larvae from the plankton samples were isolated and classified 
according to the number of pairs of cirri. In this study a larva with "n" pairs of 
cirri and a pair of buds, the length of each of which has equalled the diameter of 
the base of the median tentacle, is assigned to the stage of "n +1" pairs of cirri 
(abbreviated to n + 1 p.c. stage). Although the hauls were made 1-2 miles from 
a good bed of Lingula unguis, it was not possible to assign the larvae to this species 
with certainty. 

Specimens of Lingula unguis for the laboratory study of spawning behavior were 
obtained from the north coast of Singapore Island. A plot of muddy sand with 
the greatest concentration of burrows was chosen, and from it all the available 

202 



BREEDING SEASON OF LINGULA UNGUIS 203 

specimens were dug out with a shovel during ebb-tide. The area worked roughly 
amounted to 5-15 square meters per trip. In the laboratory every 25 specimens- 
were laid on their dorsal or ventral side in a rectangular tray, 30 cm. by 45 cm., 
under 4 cm. of natural sea water renewed once a day. These trays were stacked 
on shelves in a darkened room maintained at a temperature of 18-20 C. Every 
morning they were examined with the light of a lamp for spawned ova. Specimens 
that had spawned were transferred into marked petri dishes, 9-20 cm. in diameter 
and with a capacity of 50-400 cc. They were maintained in these dishes with 
daily renewal of water during the rest of the experimental period for easier estima- 
tion of the ova spawned. 

Frequent attempts were also made to find the time of settlement of the larvae 
by searching for the young postlarvae at the Lingula beds along the north and east 
coasts of Singapore Island. 

RESULTS 
/. Occurrence of planktonic larvae 

The larvae of Lingula obtained from plankton hauls during the period July, 
1952-June, 1953 are tabulated according to the number of paired cirri. Dead 
larvae with disintegrating cirri are assigned to the column of unclassifiable specimens 
(Table I). 

Inspection of Table I shows that (1) the larvae appeared in practically every 
month of the year, suggesting a continuous breeding throughout the year. This fits 
in with Orton's rule (1920, p. 353), "that in those parts of the sea where temperature 
conditions are constant or nearly constant, and where biological conditions do not 
vary much, that marine animals will breed continuously." (2) On most occasions 
the free-swimming larvae were at different stages of development. This suggests 
that the larvae of each catch were not the products of any single day's spawning. 
(3) Older larvae of 8 and 9 p.c. stages were rare. (4) Young larvae of 2 and 
3 p.c. stages appeared in July, August, September, October and November of 1952 
and in January, February and June of 1953. (5) The young larvae of 2 and 3 p.c. 
stages were captured two or three times at intervals of 5-7 days in August and 
September. Their occurrences did not bear any relationship to the phases of 
the moon. 

//. Laboratory spawning of the females in Lingula nnf/nis 

Many specimens brought into the laboratory in every month of the year were 
found on dissection to have ripe ova. Observations of laboratory spawning are 
summarized in Table II, which shows that (1) spawning occurred 5-14 days after 
collection among specimens collected on every occasion. (2) Observation on the 
duration of spawning of each batch was completed only for the batch collected on 
July 29, 1952. Observations on other batches were discontinued after various 
periods. (3) The smallest specimen that spawned had a ventral- valve length of 
22.6 mm. and the largest. 46.0 mm. Presumably, the females become sexually 
mature in this locality when they attain the former size approximately, and continue 
to spawn thereafter at intervals. The presence of ripe ova in many specimens 
exceeding 50 mm. in length seems to indicate that the spawning power is retained in 
old age. 



204 



S. H. CHUANG 



TABLE I 

Number of Lingula larvae from plankton samples obtained off the 
north coast of Singapore Island 





Number of larvae with the following pairs of cirri 




Date of collection 


2 


3 


4 


5 


6 


7 


8 


9 


Unclas- 
sifiable 


Total 


14. 7.1952 




28 


2 












1 


31 


22. 7.1952 










1 










1 


13. 8.1952 


10 


5 


3 












2 


20 


19. 8.1952 


20 


31 
















51 


24. 8.1952 




4 


81 


9 


1 


3 






1 


99 


31. 8.1952 























7. 9.1952 























14. 9.1952 






2 














2 


21. 9.1952 


4 


59 


21 


1 












85 


28. 9.1952 


3 


14 














12 


29 


6.10.1952 























26.10.1952 


9 


38 




1 


6 


1 








55 


2.11.1952 























9.11.1952 























16.11.1952 




10 


39 














49 


6.12.1952 























15.12.1952 








1 


1 








3 


5 


3. 1.1953 


80 


15 


2 




4 


6 


3 






110 


2. 2.1953 




4 














6 


10 


26. 3.1953 


















2 


2 


15. 4.1953 























20. 5.1953 






6 


1 












7 


27. 5.1953 








1 


4 


2 




1 




8 


12. 6.1953 




2 




6 


59 


115 


12 




25 


219 


29. 6.1953 












4 


8 


1 




13 


Total 


126 


210 


156 


20 


76 


131 


23 


2 


52 


796 



TABLE II 
Laboratory spawning of Lingula unguis from the north coast of Singapore Island 



Date of collection 


Onset of 
spawning 
(days after 
collection) 


Total spawn- 
ing days, 
when expt. is 
discontinued 


Number of 
males and 
females 
collected 


Number of 
spawning 
females 


Ventral-valve length of 
spawning females (mm.) 


Minimum 


Maximum 


30. 5.1952 








A few 






28. 6.1952 


6 


74 


159 


55 


23.9 


41.0 


14. 7.1952 


8 


95 


134 


6 


30.3 


39.1 


29. 7.1952 


5 


188 


293 


57 


22.6 


46.0 


28. 8.1952 


5 


78 


275 


28 


23.4 


43.2 


13. 9.1952 


14 


52 


93 


11 


27.5 


37.1 


6.10.1952 


12 


82 




11 


27.0 


42.3 


5.11.1952 


6 


80 


59 


9 


27.5 


38.3 


30.11.1952 


11 


59 


65 


9 


26.7 


39.0 


31.12.1952 


10 


29 


316 


10 


32.4 


43.4 


27. 2.1953 








A few 







BREEDING SEASON OF LINGULA UNGUIS 



205 



Further data on the batch collected on July 29, 1952 are summarized in Figure I, 
which shows that ( 1 ) during a period of 6 months or so, there were some specimens 
spawning each week. (2) The most intensive spawning occurred between the 
seventh and thirteenth week of captivity when from 109-135 cases of spawning per 
week were observed. (3) Cases of intensive spawning when several thousand ova 
were extruded by a female per clay occurred during the first half of the observational 
period. 






U. 

o 



Uj 
CO 

3 



I4O 



I2O 



IOO 



80 



60 



40 



2O 



D <ioo ova per day 

IOO- 2OO 

200-1000 



> 1000 




i 



" " 







- m 



3O 



WEEKS OF CAPTIVITY 



FIGURE 1. Liiu/ula unguis. Number of cases of laboratory spawning per week during 
captivity. These cases were compiled from daily records of fifty-seven spawning females 
collected on July 29. 1952 from the north coast of Singapore Island. 

The spawning behavior of 5 females from among the more consistent spawners 
of the July 29, 1952 batch reveals that ( 1 ) all the ova of an individual were not 
extruded at once. (2) Spawning in a female occurred in a series of bursts sepa- 
rated by rest intervals. (3) The first burst was usually the most intensive and 
consisted of 1-23 days of spawning. (4) Other bursts of lower intensity followed 
at irregular intervals over a period of 2-3 months. (5) The laboratory spawning- 
did not have any relationship to the phases of the moon or the tides. 

Spawning occurred both day and night. The ova were forcibly squirted out 
of the central exhalant setal tube, as Yatsu (1902) had previously observed. On 



206 S. H. CHUANG 

a few occasions these came out in the accessory exhalant currents found midway 
along the lateral gape of the animal. In 24 hours a spawning female produced 
from half a dozen to about 3000 ova. When only a few ova were spawned, these 
were found singly in the dish. However, when a few hundred or more ova were 
extruded, they were coated liberally with mucus which enabled them to stick 
together and form big sheets or clumps, as Yatsu (1902) had noted. 

The newly spawned ovum was spherical in shape. The mean of 62 ova was 
95.55 /* 4.60 /*. Each was enclosed in a prominent vitelline membrane 3ju. in 
thickness. The nucleus was visible in the living ovum. After some time the 
unfertilized ovum degenerated. It gradually enlarged, presumably on imbibing 
water, became irregular in shape and finally disintegrated after 1-2 days in sea 
water. 

When a transparent specimen was examined under a binocular microscope 
during its spawning period, the ova were found circulating in the coelomic fluid in 
the visceral cavity. They were also found in the coelomic fluid in the longitudinal 
pallial sinuses. On one occasion the pedicle of a spawning female accidentally 
snapped ; the coelomic fluid that oozed out contained not only the usual coelomic 
corpuscles but also some ova. 

Analysis of further data of the July 29, 1952 batch reveals that ( 1 ) large 
specimens produced more ova than small ones. For instance, 6 females of 41-46 
mm. ventral-valve length averaged 17,250 ova per specimen during the entire 
observation period; 9 females, 30-40 mm. long, averaged 13,000 ova with a maxi- 
mum of 17,800 ova ; 3 others, all below 30 mm., averaged 4000 ova with a maximum 
of 7500 ova. (2) During the entire observation period of 188 days the largest 
specimen, 46.0 mm. long, spawned 28,600 ova in 104 days; the second largest, 43.5 
mm. long, spawned 22,350 ova in 76 days. (3) A specimen, 39.8 mm. long, 
spawned on 125 days out of 188 days. (4) The females dissected at the end of the 
spawning period usually had spent ovaries. 

The production of a large number of ova is presumably necessary to counteract 
the following possible wastages : ( 1 ) wastage of ova through fertilization taking 
place outside the body, when the germ cells are shed into the sea; (2) wastage of 
larvae due to the long pelagic larval period, which, according to Yatsu (1902). 
lasted 1% months. 

Observations on the laboratory spawning of female specimens suggest that the 
long breeding season of Lingnla in the tropics may be due to the following : ( 1 ) 
all the ova were not shed at once but intermittently over a long period of time; (2) 
the staggering of the peaks of individual spawning in a large population; (3) the 
presumable tendency of the postlarvae from the different spawnings to reach spawn- 
ing age at different times of the year. 

The female specimens in L. ungitis continued to spawn in the laboratory in 
complete isolation from the males, contrary to the hypothesis of Yatsu (1902, p. 4) 
that "Until the sperm is discharged the eggs even when well ripened seem to be 
retained within the body." 

The females, even when reared singly in separate petri dishes, continued to 
spawn in the laboratory for several months. Crowding therefore was not necessary 
for the females to continue spawning, contrary to the observation of Kume (1956, 
p. 223) that "the frequency of shedding was greatly reduced in the vessel in which 
only a small number of individuals were cultured." 



BREEDING SEASON OF LINGULA UNGUIS 207 

///. Occurrence of young postlarvac 

Attempts to collect the young postlarvae were unsuccessful in the muddy 
Lingula beds along the north coast of Singapore Island. Along the east coast the 
beds are sandy. Here some young postlarvae with the following minimum ventral- 
valve lengths were obtained on the following dates: April 16, 1952, 6.5 mm.; 
September 24, 1952, 2.7 mm. ; October 22, 1952, 3.7 mm. ; November 3, 1952, 5.8 
mm.; and July 3. 1953, 1.4 mm. Presumably it was only when the spatfall was 
considerable that the young postlarvae could be found. 

SUMMARY AND CONCLUSIONS 

1. The planktonic larvae of Lingula were found in almost every month of the 
year, suggesting continuous breeding off Singapore Island. 

2. Several heavy spatfalls were observed in 1952, indicating several peaks of 
spawning during the year. 

3. The spawning behavior of the female in Lingula unguis was described and 
its bearing on the continuous breeding in the tropics was suggested. 

4. It was suggested that a female spawned a large quantity of ova to counteract 
the possible wastage of ova accompanying external fertilization and the wastage 
of larvae during the long pelagic larval period. 

LITERATURE CITED 

ASHWORTH, J. H., 1915. On larvae of Linc/ula and Pelagodiscus (Discinisca). Trans. Kn\. 

Soc. Edinb., 51 : 45-69. 
KUME, M., 1956. The spawning of Lingula. Nat. Sci. Reft, of Ochanomisu Univ., Tokyo. 

6: 215-223. 
ORTON, J. H., 1920. Sea-temperature, breeding and distribution in marine animals. /. Mar. 

Biol. Assoc., 12 : 339-366. 
SEWELL, R. B. S., 1912. Note on the development of the larva of Liin/ula. Rec. Indian Mus.. 

7 : 88-90. 
YATSU, N., 1902. On the development of Lingula anatina. J. Coll. Sci.. Tokyo, 17: 1-112. 



NUCLEAR SIZES IN RANA MESONEPHROI x 

MARY GRACE CONNELL 2 
/H<>logy Department, The Catholic University of America, Washington, D. C. 

When Jacobj (1925) devised the now classical caryometric method there was 
opened up for the cytologist a whole new field in population dynamics. Jacobj 
demonstrated that for any given organ the nuclear population showed considerable 
variation in size from one individual nucleus to another. By plotting frequency 
distribution curves of nuclear volumes he obtained evidence to show that the volumes 
increased discontinuously according to a logical pattern. The peaks of the curve 
corresponded to nuclear volumes which when arranged in series gave the geometric 
progression 1 : 2 : 4 : 8. Nuclear class series have since been described for a wide 
range of both invertebrate and vertebrate tissues. 

Caryometry has been used extensively in investigations of ploidy, of endomitotic 
growth, and of the interphasic growth of nuclei in a dividing tissue. The concept 
that nuclear size is a function of ploidy has proved fruitful in the study of ploidy 
in amphibians (cf. Fankhauser, 1945; Gallien, 1953). This idea was used ad- 
vantageously in the interpretation of polymodal curves obtained from nuclear volume 
data derived from studies of the kidneys of frogs : it was indicated that polysomaty 
may occur in this organ (Dawson, 1948; Schreiber and Melucci, 1949). Poly- 
somaty or endopolyploidy is understood in this paper as that condition existing in 
a normal diploid somatic tissue in which there is a certain percentage of polyploid 
cells and/or polytene chromosomes. Furthermore, the concept that nuclear size is 
a function of chromosomal reduplication has been helpful in the interpretation of 
data having to do with interphasic growth of nuclei in a dividing tissue. Nuclear 
class series indicative of a mitotic cycle have been described in Ambystoma larvae 
(Swift, 1950) and in Rana pipiens embryos (Sze, 1953). Both of these investiga- 
tions were primarily concerned, not with relationships in size, but with the photo- 
metric determination of amounts of desoxyribose nucleic acid (DNA) in interphasic 
nuclei. The introduction into cytology of photometric techniques has renewed 
interest in the caryometric interpretations of nuclear sizes. 

Nuclear size as a reflection of ploidy relates importantly to amphibian develop- 
ment both in regard to gross morphology and in regard to tissue differentiation 
(Fankhauser, 1945; Gallien, 1953). Also it has been shown in certain molds that 
both cell size and nuclear size changes may accompany morphogenesis (Bonner, 
1957). Furthermore, it has been hypothesized that DNA may show a slight de- 
crease with the progressive differentiation of certain R. pipiens tadpole tissues 
(Moore. 1952). Also of importance is the fact that nuclear size may be related 
to the degree of functional activity of the cells in question. For instance, spinal 

1 A paper submitted to the faculty of the Graduate School of Arts and Sciences of the 
Catholic University of America in partial fulfillment of the requirements for the degree of 
Doctor of Philosophy. 

2 Present address : College of New Rochelle, New Rochelle, New York. 

208 



NUCLEAR SIZES IN RANA MESONEPHROI 209 

cord cells of K. tonporaria show a decrease in volume following narcosis (Krantz, 
1947). On the other hand, nuclear size may increase as a result, not of chromo- 
somal increase, but as a result of protein synthesis in the nucleus. This has been 
shown to be true for certain insect and mammalian tissues by Schrader and 
Leuchtenberger (1950) and by Leuchtenberger and Schrader (1951). 

Nuclear size is, then, of considerable importance in the investigations of some 
of the most fundamental biological problems. The concepts and principles discussed 
here have not been extensively applied to nuclear size relationships of specific organs 
during the metamorphic stages of amphibians. Investigators have concentrated 
mainly on embryos before metamorphosis and on adult tissues. This paper is con- 
cerned with these concepts and principles by way of the study of the mesonephroi 
of Rana sylvatica during metamorphosis by the use of the classical methodology 
of caryometry. 

MATERIALS AND METHODS 

Two clutches of Rana sylvatica eggs were collected in the field and were allowed 
to develop in the laboratory throughout an eighty-day period. The metamorphic 
stages selected for study at arbitrary time intervals were identified by means of the 
criteria established by Taylor and Kollros (1946) for R. pipiens. Some of the 
tadpoles were carried through metamorphosis to young adulthood. Table I gives 
both stage and age for all animals used in this investigation. Immediately after the 
staging of the tadpole the body cavity was opened and the animal was immersed in 
Zenker-formol for fixation. After fixation the mesonephroi were removed, em- 
bedded in paraffin and sectioned either transversely or longitudinally at 6 micra. 
All sections were stained by the Feulgen technique after Stowell (1945). 

Only the center sections of a kidney from each of the 63 animals were examined. 
From these sections an average of 470 camera lucida outline drawings of nuclei 
from the convoluted tubules were made at a magnification of 1350 X diameters. 
The selection of nuclei to be drawn was random. However, it was necessary to 
set up certain criteria as a basis for selection. The first criterion was established 
in the following manner. In order not to cause marked distortion in the data the 
outline drawing of a nucleus was made at the focus showing the greatest nuclear 
diameter. If the nucleus is sectioned it is impossible to determine whether or not 
the greatest diameter lies in the section of tissue studied or in the next succeeding 
section. Therefore, nuclei lying in the two cut surfaces had to be eliminated on 
this basis. If it was possible to focus on the tissue at a level above and at a level 
below the nucleus selected, then it was certain that the entire nucleus was in the 
section. In establishing the second criterion, the long and short diameters were 
determined in mm. for each drawing. From these data the nuclear volumes were 
calculated according to the formula for the volume of an ellipsoid. It is a fact 
that this volume is not often used in similar studies of biologic material because of 
the difficulty of determining a diameter in the third dimension. This is practically 
impossible in sectioned material. However, in the kidneys of R. sylvatica used in 
this study the nuclei appear more or less spherical both in cross-section and in 
longitudinal section. On the basis of observation, therefore, it was concluded that 
most of the nuclei were not markedly flattened. Any nucleus that was definitely 
elliptical from surface view was eliminated. Also, any nucleus that appeared ob- 
viously thin on focusing through it was also eliminated. This device is, of course, 
arbitrary and subjective and does not remove the difficulty of the third diameter. 



210 



MARY GRACE CONNKLL 



TABLE I 

The table shows the modal volumes in mm. 3 for the peak classes, and the ratios X x/766 mm* 

where x is any one of the modal volumes and where 766 mm. 3 is the 

lowest volume in the table 



Stage 


Class I nuclei 


Intermediate class 


Class II nuclei 


Age in days 


3 


949(1.2) 


1288(1.7) 




18 


4 




1310(1.7) 


1762.5(2.3) 


24 






1290(1.7) 






5 




1069(1.4) 




22 






1226.5(1.6) 










1075(1.4) 






6 


766(1.0) 


1105(1.4) 


1378(1.8) 


32 


7 




1225(1.6) 


1364.5(1.8) 


39 






1114(1.4) 






8 




1231(1.6) 


1400(1.8) 


32 








1363(1.8) 




9 




1066(1.5) 


1369(1.8) 


39 








1358.5(1.8) 




10 




1270(1.7) 


1500(1.9) 


37 






1229(1.6) 






11-12 




1130(1.5) 


1511.5(2.0) 


36 






1028.75(1.3) 






13-15 


952(1.2) 


1143(1.5) 




36 






1061(1.4) 






16-17 




1248(1.6) 


1513(2.0) 


39 






1138(1.5) 






18 




1152(1.5) 




43 






1294(1.7) 










1109.5(1.4) 






19 






1375(1.8) 


45 








1369(1.8) 










1355(1.8) 




20 




1246(1.6) 


1376.5(1.8) 


47 








1355.5(1.8) 




21 




1114(1.4) 


1443(1.8) 


47 








1381.25(1.8) 




22 






1512(2.0) 


50 








1926(2.5) 










1811.5(2.3) 




23 




1054(1.4) 




57 






1334(1.7) 










1300(1.7) 






24 




1320(1.7) 


1384(1.8) 


57 






1160(1.5) 






*25 


775.5(1.0) 


1087(1.4) 


1519(2.0) 


80 




846(1.1) 


1097.5(1.4) 


1522(2.0) 






958(1.2) 




1417.75(1.8) 










1349(1.8) 










1433.5(1.9) 





* Young adults. 

In addition, there are difficulties other than those due to diametric differences. 
For instance, there are a number of sources of error, not the least of which are 
those inherent in the investigator. The eye in seeing and the hand in drawing are 
not always coordinated to the same degree. There is the problem of focusing on 



NUCLEAR SIZES IN RANA MESONEPHROI 



211 



the maximum nuclear surface, the possibility of inaccuracy in measurement, the 
effect of the procedures on the nuclei themselves, and according to Merriam and 
Ris (1954) the probable sources of error in the camera lucida projection method. 
Further, there is the fact that the error due to method has been raised to a higher 
power in the formula for determining volumes. However, a search for absolute 
values and statistical certainty is not the purpose of this paper. Only a relative 



180 



o 

z 

ID 

:D 
o 
u 
o: 



60 




\ 

\ 



\ 



\ 



3.0 



LOG NUCLEAR VOLUME 



3.5 



FIGURE 1. Histograms of three samples, one from each of three stages : a) stages 6, b) 

stage 18, c) stage 22. 

value or an approximate value is sought. No attempt has been made here at 
statistical analysis of the data. 

After determination of the nuclear volumes, these values were grouped into 
frequency classes and frequency curves were drawn for each one of the 63 kidneys. 
The modal volume for the peak class was determined for each case and they are 
recorded in Table I. This table shows that the lowest modal volume (766 mm. 3 ) 
occurs in one of the individuals at stage 6. Using this volume for comparative 



212 



MARY GRACE CONNELL 



purposes a ratio can be obtained for each modal volume listed. The ratios form a 
progression, 1 : 1.5: 2, with nearly all intermediates between these values. 

In the histograms the frequency of nuclear volumes is plotted against the class 
interval. Also, one third of the data were plotted by two other methods. In the 
one case, the long diameters of the nuclei were substituted for nuclear volumes ; in 
the other, the logs of the nuclear volumes were plotted against frequency. The 
long diameters or the surface areas are frequently used as a substitute for volume 



100 



o 

UJ 

ID 
O 
UJ 

<r 
u. 



50 




3.0 



3.5 



LOG NUCLEAR VOLUME 

FIGURE 2. Histogram of one of the individuals at stage 24. 

when the nuclei are ellipsoidal. Both values have the advantage over volume 
calculations since it is not necessary to multiply the error by raising the diameters 
to a higher power. In this study one third of the samples were selected and fre- 
quency curves were drawn using long diameters rather than volumes. In all cases 
the curves correspond to those obtained in histograms in which numbers were 
plotted against volumes. It appears, therefore, that for the data presented here 
there was no great advantage of one method over the other, at least in terms of the 
revelation of three classes of nuclei. If, however, a curve of greater symmetry is 



NUCLEAR SIZES IN RANA MESONEPHROI 



213 



desired then the use of logarithms of nuclear volumes has a distinct advantage over 
both volume and the long diameter. The logs of nuclear volumes give a more 
symmetrical curve than the nuclear volume itself (Figs. 1 and 2). Bucher (1954) 
claimed that the logarithmic system of classification is better adapted to a mathemat- 
ical analysis of the curve and that it is the only valid system from a biological and 
statistical viewpoint. On the other hand, it has been stated (Bonner and Eden, 
1956) that a mathematical analysis of the curve is not especially helpful for com- 
parative purposes except in those restricted cases where there is additional informa- 
tion about cell or nuclear growth. 

Finally, the geometric mean of the nuclear volumes was calculated for each 
kidney for purposes of plotting the logarithmic growth curve (Fig. 3). The geo- 



3.3 



< 
UJ 



o 

tr 

H 
UJ 
2 
O 
UJ 



Lt_ 
O 



o 



3.0 







35 



75 



TIME IN DAYS 



FIGURE 3. Logarithmic growth curve. Each point plotted represents from one to four 
individuals. Thus, at 80 days the 10 young adults are represented in the four points. 

metric mean is subject to all those errors inherent in the method and is. like the 
nuclear volume, an approximation. 

OBSERVATIONS 

The frequency distribution curves of all but one of the kidneys studied show a 
single peak and are therefore unimodal. There is one bimodal curve (Fig. 2) in 
which the nuclear volumes give two peak classes. In all cases, when the modal 
volumes (Table I) calculated for these peaks are arranged in a series they form a 
progression in the ratio 1:1.5:2. Therefore, the histograms reveal two major 
classes and one intermediate class (Fig. 1). 

In Class I the nuclei are believed to have the diploid value characteristic of post- 



214 MARY GRACE CONNELL 

mitotic nuclei. Following division there is, possibly, a period of interphasic growth 
of the chromosomes during which the nuclear volumes increase to the size of the 
intermediate class. This may be followed by a second period of interphasic growth 
during which the nuclear volume becomes twice that of the diploid nucleus. Nuclei 
of Class II may, therefore, be tetraploid and ready for division. All intermediate 
values shown in Table I are interpreted hypothetically as intermediate classes which 
would be expected to appear during the two interphasic growth periods. The three 
peak classes are shown in Figure 1 where three of the 63 histograms were selected 
for illustrative purposes. It is to be noted that in both Figures 1 and 2 the log 
nuclear volume has been substituted for nuclear volume. 

Examination of Table I, with the assumptions noted above in mind, will reveal 
that modal volumes of Class I are rare, and that the intermediate class occurs most 
frequently. It is perhaps significant that whereas 30% of the young adults have 
modal volumes of Class I, only about 5% of the tadpoles do. The number of 
kidneys in the tetraploid class (Class II) are most numerous through stages 7-10, 
19-22 and in the young adults. In stage 7 through 10 and in the young adults 
50% of the kidneys sampled give peak classes in the tetraploid range, while in stages 
19-22 approximately 83% of the samples are in this range. It appears, therefore, 
that the greatest periods of mitotic activity occur within the stages indicated. 

The logarithmic growth curve (Fig. 3) also serves to point out some of these 
relationships. The growth curve is a straight line drawn through points which 
represent the intermediate class described above. The scatter above and below the 
line is due to the occurrence of samples which give peak classes in the Class I and 
in the Class II range. Obviously, however, there are no marked increases or de- 
creases in growth that can be correlated with age. All nuclear classes may be 
found in the kidneys of tadpoles which are of the same age, just as all nuclear classes 
may be found in the kidneys of tadpoles in the same stage (Table I). Nevertheless, 
a rough correlation can be made for stages 19-22. The animals in these stages are 
45-50 days old. At 45-50 days the scatter is all above the line of growth indicating, 
perhaps, increased mitotic activity at this time. 

A single bimodal curve was revealed in one of the individuals at stage 24 
(Fig. 2). The two peaks correspond to the intermediate class and to Class II 
nuclei. Other instances of bimodality were suggested in the histograms. But in 
all cases except the one they were not revealed by either of the other graphing 
techniques, and are considered insignificant. With the one exception all histograms 
regardless of the technique were unimodal. However, when nuclear volumes are 
used in the histograms the curves show a marked asymmetry and are skewed to 
the right. This marked asymmetry disappears when the logs of nuclear volumes are 
used in place of the nuclear volume itself. 

DISCUSSION 

The commonly accepted explanation for the appearance of the different nuclear 
classes in the frequency distribution curve is that for some nuclei growth in size is 
arrested at one or another of the growth stages, resulting in an accumulation of 
nuclei in the stage at which growth ceased. The tissue, as a result, carries ac- 
cumulations of nuclei of differing sizes which are responsible for the peaks in the 
curve. Nuclei with volumes in the Class II range and greater, represent tetraploid 



NUCLEAR SIZES IN RANA MESONEPHROI 215 

nuclei and the higher degrees of polyploidy, while those with volumes Class I-Class 
II represent interphasic sizes between successive mitoses. Hertwig (1939) in 
measuring nuclei from the early cleavage stages of mouse ova demonstrated that 
at each mitosis the nuclear volume was halved and he postulated that these volume 
changes could be correlated with similar changes in the genome. Alfert (1950), 
who studied cleavage stages in the mouse by photometric analysis of DNA content, 
reported doubling of DNA amounts in the interphase preceding mitosis. The 
significance of this observation derives from the fact that there is considerable evi- 
dence, obtained principally by the use of quantitative techniques, which suggests 
that DNA is either identical with the genie material or is at least closely associated 
with it. The same quantitative distributional patterns hypothesized by cyto- 
geneticists for genie material can be directly applied to distributional patterns 
hypothesized for DNA. If this generalization is applied here, then a relationship 
must exist between nuclear volume and DNA content. Swift (1950) and Truong 
and Dornfeld (1955), using the photometric method for DNA determination in 
combination with caryometry on different animal tissues, showed that there is a 
definite relationship between DNA amount and nuclear size. Both volumes and 
DNA amounts fall into the ratio 1:2:4. In stable adult tissues (Swift, 1950, 1953 ; 
Pollister, Swift and Alfert, 1951) the DNA classes are clearly demarcated with 
little or no overlapping, while in actively dividing tissues there may be considerable 
overlapping because of the appearance of intermediate classes. The intermediate 
classes are due to gradual DNA synthesis during interphase. When the DNA reaches 
tetraploid level the nucleus enters prophase. After quantitative division of DNA at 
anaphase the diploid (Class I) amount of DNA is restored in the telophase nuclei. The 
cycle of DNA reduplication then repeats itself. Similarly, when labile tissues are 
studied by means of nuclear volume determinations, intermediate classes are re- 
vealed (Schreiber and Angeletti, 1940; Schreiber, 1949). In these cases the 
volume changes are interpreted as resulting from reduplicating phenomena in the 
genome. 

In the kidneys of R. syk'atica used in this study there were no DNA determina- 
tions. Interpreting the results, however, on the basis of the above discussion it 
is possible to conclude that the data reveal the typical nuclear classes of a mitotic 
cycle, and a whole series of intermediate classes between Class I and Class II 
nuclei. The histograms (Figs. 1 and 2) show considerable overlapping. The 
overlapping is so marked that when the individuals are grouped and a combined 
histogram of all 63 kidneys is drawn, only one nuclear class is revealed. The modal 
value of this class falls approximately half way between Class I and Class II. It is 
generally believed that the increase in nuclear volume is rhythmic and that periods 
of rapid growth alternate with periods of relative inactivity. It is not possible from 
the results of this investigation to determine conclusively whether rhythmic growth 
as opposed to continuous growth is present here or not. For one thing, the degree 
of overlapping precludes any affirmative assumption concerning the presence of 
rhythmic growth. Not only are there no sharp demarcations between peaks sug- 
gesting discontinuity but there are also few positive correlations between the peak 
class and the stage and/or age of the animal. Secondly, the mere fact of the 
existence of a series of nuclear classes in a tissue does not imply that the details of 
individual nuclear growth are known, for normal frequency distributions may be 



216 MARY GRACE CONNELL 

obtained not only from actively dividing tissues but from stable tissues as well 
(Bonner and Eden, 1956). However, the low incidence of Class I nuclei in the 
mesonephros of R. sylvatica may be due to a period of very rapid growth im- 
mediately following mitosis, so that Class I nuclei occur only transitionally. There 
is the possibility that the large percentage of nuclei in the intermediate class ac- 
cumulate in this class due to a decrease in nuclear activity. Nuclei from this 
reservoir may be released gradually into the second growth period, at the end of 
which there is an accumulation of Class II nuclei. Schreiber and Angeletti (1940) 
found that in the mitotic cycle of hepatic cells of the carp there is a rhythmic in- 
crease and decrease in nuclear volume which can be correlated with the stage of 
development. In the kidneys of Rana sylvatica there is no such clearcut correlation. 
All nuclear classes may be present irrespective of the stage of development or of the 
age of the animal (Fig. 3). The developmental pattern in the kidney does not, 
therefore, reflect the details of nuclear growth. However, the percentage of 
Class II nuclei is greatest at stages 7-10, 19-22 and in the young adults. This 
suggests not only the possibility of three waves of mitotic activity but also a 
rhythmical growth pattern. 

The nuclear size increase recorded here may not be associated with the duplicat- 
ing process of the genome or with an increase in DNA. It has been discovered that 
in a given tissue, while DNA remains constant per nucleus, the protein content and 
the nuclear size show corresponding increases (Alfert, 1950; Biesele, 1944; Schra- 
der and Leuchtenberger, 1950; Leuchtenberger and Schrader, 1951). Further, 
the chromosomal volume may increase by protein synthesis without an accompany- 
ing morphological change in nuclear size (Biesele, 1944). Also the nuclear volume 
may even be reduced as a result of water loss rather than by a change in the 
genome (Krantz, 1947). Furthermore, differences in nuclear size may have to 
do with nutritional differences that do not affect chromosomal size (Montgomery, 
1910). Even in a normally dividing tissue there may be a size difference due to 
some factor other than that which stimulates cell activity. For instance, the age 
of the animal may affect cell or nuclear size quite independently of a tendency 
toward compensatory hypertrophy (Buchner and Glinos, 1950). 

In view of these considerations a number of possibilities come to mind in regard 
to the increase in nuclear size observed here. This increase has been attributed to 
mitotic activity, or to an increase in chromosomal content. It is conceivable that 
some of this increase is due to imbibition or to an increase in the osmotic con- 
centration consequent upon the synthetic activity of the chromosomes. Or synthetic 
activity in the interphase nucleus may alter the nuclear membrane, causing osmotic 
changes. This may effect an increase in nuclear volume which is not exactly 
paralleled by an increase in the volume of the chromosome. As a result nuclear 
volumes may vary to such an extent that there is considerable overlapping of volume 
classes in frequency curves. The increase in size of a nucleus may be partially due 
to mechanical pressure (Teir, 1949). In addition to the factors postulated above it 
is known that a morphological increase in size may be due to compensatory hyper- 
trophy following such operations as unilateral nephrectomy (Sulkin, 1949) or 
partial hepatectomy (Sulkin, 1943), to hormonal agents like oestrone (Salvatore. 
1950; Alfert and Bern, 1951; Schreiber, 1954), or to agents which stimulate 
chromosomal activity such as thiouracil (Roels, 1954), alloxan (Diermeier et al., 
1951) and colchicine (Bucher, 1951; Fankhauser, 1952). The administration of 



NUCLEAR SIZES IN KANA MESONEPHROI 217 

any one of these agents may be followed by morphological changes in the nucleus, 
but need not necessarily be the direct cause of that change. Undoubtedly the size 
increase involves and is indicative of phenomena which are very complex due to 
the complexity of factors forming the cytoplasmic and nuclear ecology. 

Some of the agents mentioned may not only be causative factors in increasing- 
nuclear size but they may also induce polyploidy. Fankhauser (1952) by the use 
of colchicine induced endopolyploidy in embryos of the axolotl. Some of these 
embryos were originally diploid. It is generally known that polyploidy does occur 
in normal diploid animals. This condition, known as polysomaty or endopolyploidy, 
has been reported in the renal tubules of Leptodactylus, a South American frog, 
by Schreiber and Melucci (1949) and in the renal tubules of Cy dor ana, the Austral- 
ian desert frog by Dawson (1948). Polysomaty does not occur here in the kidneys 
of R. sylvatica. Twenty-six of the kidneys give modal volumes belonging to Class 
II. Conceivably, some of the nuclei in this peak class might be true polyploids with 
twice the diploid number of chromosomes rather than interphasic cells approaching 
a proliferative stage. There is no way of determining this, however, by the method 
used here. The histograms gave no peak classes at higher than the hypothetical 
tetraploid level. Possibly, some of the very large nuclei are octoploids. If so they 
are so rare that there is no great accumulation at this level and therefore the higher 
peaks are not obtained. In tissues showing a relatively high incidence of polyploicl 
cells the frequency increases with age (Swartz, 1956). Polyploid cells may be 
absent then in the tadpole kidneys and in young adults and may appear only in 
older animals. However, it is now a well known fact (cf. Fankhauser, 1945) that 
polyploidy does occur in tadpole tissues. It can only be concluded, therefore, that 
there are few if any polyploid cells in the mesonephroi of these R. sylvatica. This is 
consistent with the general opinion that the incidence of polyploidy in kidney tissues 
is relatively rare, even though it is known to occur in those instances cited above. 

The concept of endopolyploidy, or any concept of variation in chromosome 
number, renders untenable the hypothesis that for a given species the chromosome 
number is the same for all of the somatic cells. The "constancy hypothesis" has, 
therefore, been revised on the assumption that DNA is somehow related to the 
genie material. The constancy of DNA per chromosome set, first proposed by 
Boivin, Vendrely, and Vendrely (1948), and by Ris and Mirsky (1949), is sup- 
ported by the work of Pollister and his school (cf. Pollister, Swift and Alfert, 1951 ) . 
As pointed out above, increased metabolic activity in the chromosomes as shown by 
DNA synthesis is accompanied by an increase in nuclear size. If this increase is 
always proportionate to DNA increase then a constant relationship should exist 
between DNA content per chromosome set and nuclear size. Obviously, such a 
constant relationship does not exist. The phenomenon of variation in nuclear size 
may very well be a secondary event which may or may not be associated with DNA 
content. In terms of "constancy," assumptions concerning chromosome number and 
probable DNA content cannot always be made from nuclear volume data. There 
is also a certain amount of evidence which calls into question the constancy of DNA 
per chromosome set. Roels (1954) working with rat thyroid and Diermeier ct al. 
(1951) with the liver of alloxan diabetic rats have presented evidence to indicate that 
the DNA content of a cell may vary with its degree of functional activity. Pasteels 
and Lison (1950) concluded that the DNA content of certain rat tissues is lower than 
the diploid value because of chromatin diminution. Though this particular evi- 



218 MARY GRACE CONNELL 

dence has been questioned (Alfert and Swift, 1953), chromatin diminution is known 
to occur in Ascaris (Boveri. 1904) and in some other forms (see the brief review of 
Tyler, 1955.) Also, Marshak and Marshak (1955) discuss negative DNA re- 
actions in the Arbacia egg. Their evidence, however, has also been questioned (cf. 
Burgos, 1955; Marshak and Marshak, 1956). The exceptions to DNA constancy 
still raise a question in regard to the chemical nature of the gene, and they must be 
kept in mind when interpreting data having to do with nuclear volumes. 

The occurrence of mitotic activity in the mesonephroi herein investigated in- 
dicates that during metamorphosis the kidney has some cells that are undifferenti- 
ated. At least physiologic inactivity in terms of renal function can be postulated 
for the proliferating cells. Some of the non-dividing cells, however, must be already 
manifesting renal activity. If so then the nuclear sizes may be indicative of a 
differentiating process. The fact that nuclear size and characteristic cell structure 
are intimately related is most dramatically demonstrated in insects. For example, 
in certain honeybee tissues (Merriam and Ris. 1954) the nuclear volume not only 
increases with age but there is a direct correlation between the nuclear size and 
secretory activity. Of equal importance is the fact that in certain mammalian tissues 
such as rat liver the increase in numbers of large nuclei closely parallels the histo- 
logical and functional development of the organ (Sulkin, 1943; McKellar, 1949). 
A somewhat similar relationship has been demonstrated for a species of the slime 
mold, Dictyostelium (Bonner, 1957). In this mold, the stage of development and 
the early stages of differentiation are reflected in both the cell size and in the 
nuclear size which is characteristic of the particular stage. Further, the most 
active cells of this slime mold are located at the anterior end of a migrating sausage- 
shaped mass. The active cells are also the larger and are, according to Bonner, 
responsible for important morphogenetic effects which end in the formation of the 
fruiting body. Another parallel between cell size and differentiating activity may 
be seen in the developing sea urchin egg. It is well known that the micromeres 
formed during the cleavage stages of the sea urchin egg play an important role in 
morphogenesis in that they induce vegetal differentiation. McMaster (1955), 
working with Lytechinus, has shown by photometric determinations of DNA 
amounts in the cleavage cells that the lowest DNA amounts are in the micromeres. 
It is possible that the differentiating effects of these cells may be due to the lower 
DNA values. Once again, therefore, the point to be made here is that cell size is 
associated with a differentiating process. Progressive differentiation may also be 
associated with quantitative differences in DNA amounts in amphibians. Moore 
(1952), in working with haploid and diploid tissues of Rana pipiens embryos, 
found that the range of DNA values in the forebrain was greater in the 7-day 
embryos than in the 11 -day embryos. She found no correlation between mitotic 
activity and the amount of DNA. In explaining this she discusses a possible cor- 
respondence between DNA amount and differentiation. DNA may decrease with 
age and with the maturation of the tissue until it reaches some more or less constant 
value. If this is so then nuclear volumes may also be smaller in adults than in em- 
bryos. Perhaps increased differential activity accounts for the larger percentage of 
Class I nuclei recorded (Table I) here for the young adults of Rana sylvatica. 

At histological maturation differentiation may be influenced by polysomaty. 
Polyploid cells at the time of differentiation may return to a lower value by some 
process such as reduction mitosis (Hnskins. 1948). The genie segregation which 



NUCLEAR SIZES IN RANA MESONEPHROI 219 

accompanies the reductional division may produce cells whose differentiating poten- 
tial is quite different. This interpretation puts stress on ploidy as a causative 
factor in differentiation. In considering polyploid organisms it is known that 
generally they differentiate normally and effects of ploidy, such as larger nuclear or 
cell size, are considered secondary. Mather (1948) has insisted that duplication 
of the chromosome number is not the cause of differentiation. The fate of the cell, 
he concluded, is dependent not so much on the nucleus which in most cases is diploid 
in sexually reproducing animals or plants, but on a cytoplasm which is inherited 
from the past. The action of the nucleus is effective only because the cytoplasm 
has changed at each step along the way. At each division the nuclei are quantita- 
tively and qualitatively alike but each one inherits a portion of cytoplasm, one 
portion of which cannot be equated with the other. If polyploidy occurs it is 
probably in response to the cytoplasm. 

In conclusion, the difficulties of interpreting nuclear size relationships are many 
and varied. Not only are the techniques used for such studies possessed of their 
own difficulties but nuclear size itself may be altered by a number of conditions. 
Size changes may be physiological, mitotic, or due to different degrees of hetero- 
ploidy. If genie changes are involved then these changes may be correlated with 
alterations in DNA amounts. The results strongly suggest that DNA is the genie 
material. But the fact of the matter remains that the gene is a biological concept, 
an abstraction, known only in its effects. The exact relationship between the gene 
and DNA is still unknown. A nuclear size change, itself, may be a phenomenon 
occurring coincidentally with differentiation, as if there were two progressions, 
one of which functions as the cause or the effect of the other. Or instead, all the 
events involved may converge and commingle as an expression of a single phenom- 
enon reaching peak expression in the differentiated living cell. Increases or de- 
creases in nuclear size are morphological and physiological events which take part 
in this convergence. 

SUMMARY 

1. The mesonephroi from 63 Rana sylvatica tadpoles and young adults were 
studied in sectioned material by means of a caryometric method for determination of 
nuclear volumes. Frequency histograms drawn from the data reveal three peak 
classes which, when arranged in series, give the progression, 1:1.5:2. These 
results are interpreted as being due to an interphasic growth preceding mitosis. 

2. The results are discussed in terms of morphological increase and decrease in 
nuclear size, increase and decrease of DNA values, polyploidy, the "constancy" 
hypothesis, physiological activity and histological differentiation. 

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THE ROLE OF ADSORPTION AND MOLECULAR MORPHOLOGY 

IN OLFACTION: THE CALCULATION OF 

OLFACTORY THRESHOLDS 

J. T. DAVIES AND F. H. TAYLOR 1 

Dept. of Chemical Engineering, Pembroke Street, Cambridge, England, and Dept. of Chemistry, 
Imperial College, South Kensington, London, S. W. 7, England 

The surface of the olfactory nerve cell is folded into a number of delicate, 
hair-like protoplasmic filaments (Engstrom and Bloom, 1953; Bloom and Eng- 
strom, 1952) which have long been considered to be the ultimate sensory processes 
of the receptor cells. Admirable photographs showing these hairs, a discussion 
of their possible functions and a description of the anatomy of the entire olfactory 
epithelium of various vertebrates are given by Le Gros Clark (1957) and by 
Allison (1953). 

The filaments serve to give the olfactory receptors a large surface area. That 
this enlarged surface readily adsorbs odorants was shown by Moncrieff (1954, 
1955) who made direct measurements, using a sheep's head, and found that 
odorants are adsorbed strongly and rapidly on the olfactory epithelium and that 
the process is reversible. 

Neither the structure nor the exact composition of the human olfactory cell 
membrane is known. Hopkins (1926) examined the olfactory filaments of the 
frog which he found to be extensions of the olfactory cell membrane ; he noted 
that the hairs reduce osmic acid and are disrupted by organic lipoid solvents, the 
proximal parts only remaining intact. The human olfactory membrane will 
probably resemble that of the frog and, like other nerve cells, will consist of a 
few layers of oriented lipid and protein molecules. This membrane is continually 
bathed by mucus (effectively saline) at a pH of about 7.2. 

Davies and Taylor (1954) used the erythrocyte membrane as a model for that 
of the olfactory nerve cell, and showed that a large number of odorous substances 
act as accelerators of haemolysis by saponin. Moreover, for these substances, 
the logarithms of the olfactory thresholds (for man) are directly proportional to 
the logarithms of the haemolytic accelerating powers. This is shown in Figure L 
Some of these compounds which act as haemolytic accelerators have also been 
shown to cause a leakage of potassium ions across the red cell membrane (Davson 
and Danielli, 1938). This correlation lends support to theories of olfaction such 
as those of Ehrensvard (1942) and Davies (1953a, 1953b) which postulate that 
odorant molecules must first adsorb onto the plasma membrane of the olfactory 
cell. Ehrensvard's theory that the potential changes due to adsorption at this 
interface initiate the nerve impulses ascribes more importance to the specific 
polar groups of the odorant molecules than does the present work. Further 
experiments on potentials on the lines of those of Ehrensvard and Cheesman 
(1941) would be of interest in this connection. 

1 Beit Memorial Fellow for Medical Research. 

222 



QUANTITATIVE ASPECTS OF OLKACTION 



223 



4:0 




16 



13 



IZ 



10 



e 



Loq Olfactory Threshold 

JlO J 

FIGURE 1. Plot of the logarithm of the haemolytic accelerating power against the logarithm 
of the olfactory threshold (in molecules/cc.). The equation of the line which is drawn in can be 
deduced from equation (4) if certain approximations are made (Davies and Taylor, 1957). 

The present work, following the theory of Davies, assumes that the odorant 
molecules can simply dislocate the olfactory cell membrane, permitting an ex- 
change of sodium and potassium ions across it. This exchange of ions initiates 
the nervous impulse as in the general theory of Hodgkin and Katz (1949). 

Recently, Rideal and Taylor (1958) have shown that the logarithm of the 
haemolytic accelerating power of a substance is directly proportional to its free 
energy of adsorption at the oil-water interface ; this means that the effectiveness 
of compounds as haemolytic accelerators depends only on the number of mole- 



224 J. T. DAVIES AND F. H. TAYLOR 

cules adsorbed on the red cell surface and not, in addition, upon such factors as 
molecular shape or size. In olfaction, however, additional factors must be 
important since the slope of the line in Figure 1 is not unity and also because 
there is some scatter of the points about the line. This scatter may, in part, be 
clue to the roughness with which olfactory measurements can be made, but it is 
also likely that the sizes, shapes and flexibilities of the penetrating molecules are 
important (see Davies, 1953a, 1953b). Indeed, Timmermans (1954) and Mullins 
(1955) have recently emphasised this point. Further, among isomers, the one 
with the bulkiest molecule (i.e., with the most branched chains) has the lowest 
olfactory threshold even though it may be less strongly adsorbed at a fatty 
surface than the corresponding linear molecule. This applies to w-amyl alcohol 
(threshold 6.8 X 10 12 molecules/cc.) and to isoamyl alcohol (threshold 6.8 X 10 11 
molecules/cc.) (von Skramlik, 1948) and also to the isomeric decanols; it is true 
for Phormia as well as for humans. 

In this paper, the relative importance of adsorption and of molecular mor- 
phology in olfaction is assessed quantitatively, and an equation derived from 
which the olfactory threshold of any particular compound may be calculated. 

THEORETICAL SECTION 

For mathematical convenience, we assume that an olfactory cell surface is 
divided into n small, non-specific areas or sites, each of area a sq. cm. In order 
to stimulate a cell, a critical number of odorant molecules must be concentrated 
on one site in the cell surface. For the strongest odorant, which is assumed to 
be ft ionone, perhaps only one molecule need occupy one of these areas. Weaker 
odorants, however, such as acetic acid or methanol have a less "dislocating" 
effect on the cell membrane and so p molecules must be concentrated simul- 
taneously on one of the sites to cause a response. 

The quantity 1/p, then, is a measure of the "puncturing" ability of the 
odorant, i.e., of the effectiveness of the molecule in causing the necessary ionic 
leakage across the cell membrane. For the strongest odorants 1/p = 1 whilst for 
weaker odorants 1/p < 1, and for water which is continually bathing the cell 
without causing any stimulus, 1/p = 0. 

To adsorb on the sites, the molecules must pass from the air through the 
aqueous (mucous) phase. This process is reversible (MoncriefT, 1955) and at 
equilibrium the distribution of molecules is given by (1) which is a simplified 
Langmuir isotherm. 

x 

d - - K LIA . 

Here x is the average membrane concentration of odorant molecules/sq. cm., c is 
the average concentration of molecules/cc. in the air, and d is the surface thick- 
ness (about 10 A). KL/A is the adsorption constant for molecules passing from 
air to the lipid-water interface. The number of sites, N, per nerve cell containing 
p molecules where p is greater than the average number ax is given by Poisson's 
equation : 

-** 



p\ 
where n is the total number of sites/cell. 



QUANTITATIVE ASPECTS OF OLFACTION 

At the olfactory threshold, only one site with the p adsorbed molecules will 
be required to stimulate the cell minimally, so that N is unity and c is the olfac- 
tory threshold. By combining equations (1) and (2) we can eliminate x and 
obtain a relation between KL/A, P and the olfactory threshold. The relation in 
its final form is given by (3) and has been derived in full elsewhere (Davies and 
Taylor, 1957). 

-log ft log ft! , 

log O.I. + log KL/A ' - -log ad. 

Calculation shows that for the weakest odorants possible, p lies between 15 and 
30 and an arbitrary value of 24 is taken. For these compounds the quantity 
(log O.T. + log KL/A) tends towards a value of 22. The compound with the 
lowest recorded olfactory threshold (ft ionone; as listed by von Skramlik, 1948, 
and confirmed by the measurements of Neuhaus, 1953b) is assumed to have a 
p value of 1. If any more powerful odorant were discovered and assigned a p 
value of unity, the numerical values of the thresholds predicted would change 
slightly, but their relative values would not. 

Application of these boundary conditions enables values of the constant log n 
and log ad to be found ; equation (3) then takes the form : 

4.64 log/?! 

log O.T. + log KLIA =- + +21.19. (4) 

P P 

Using this equation we can calculate the olfactory threshold of any substance, 
knowing its adsorption constant between air and the lipid-water interface and 
its value of p. 

METHODS 

The adsorption constants tor molecules going from water to the oil-water 
interface have been determined from measurements of the lowering of the inter- 
facial tension at the petroleum ether-water interface (Haydon and Taylor, 1959). 
Non-polar compounds (e.g., hydrocarbons) will dissolve in the lipophilic interior 
of the membrane, and so for these compounds, distribution constants for mole- 
cules passing from water to the petroleum ether (bulk) phase have been used. 
Since we are dealing with a fatty membrane, the adsorption at the membrane 
water interface will be approximately equal to that at the oil-water interface. 

KQ/W KL/W- 

In fact KL/W will be slightly less than KO/W by a factor depending on the 
dielectric constant at the membrane surface. The distribution constant for 
molecules going from air to the aqueous phase has been found from the ratio of 
the solubility of the substance in water at 20 C. to its vapour pressure at the 
same temperature, or, in some instances, from the measurements of partial vapour 
pressures of aqueous solutions of the substances recorded in the literature. 

From KL/W (= KO/W) and KW/A, the term KL/A (=' KOI A} required in equa- 
tion (4) may be obtained directly since 

KLIA KL/W KW/A- 
Values of log KOI A are listed in Table I. 



226 



J. T. DAVIES AND F. H. TAYLOR 



TABLE I 



Compound 


logio Ko/w 


logio Kw IA 


logio Koi A 


Cross-sectional areas A 2 


a 


b 


Methanol* 


1.235 


4.245 


5.48 


20.0 


12.4 


Ethanol* 


1.80 


3.99 


5.78 


26.7 


15.4 


Propanol* 


2.41 


3.81 


6.22 


32.6 


18.3 


Butanol 


2.85 


3.69 


6.54 


38.0 


19.2 


Pentanol 


3.71 


3.42 


7.13 


43.0 


20.0 


Hexanol 


4.39 


3.18 


7.57 


47.8 


20.0 


Heptanol 


4.82 


2.83 


7.65 


52.3 


20.0 


Octanol 


5.30 


3.04 


8.34 


56.0 


20.0 


Decanol 


6.94 


2.04 


8.98 


64.6 


20.0 


/3 ionone 


5.79 


2.56 


8.35 


69.7 


57.6 


Piperonal 


3.41 


3.48 


6.89 


47.3 


41.4 


Menthol 


4.505 


2.015 


6.52 


62.8 


44.8 


Skatol 


4.76 


3.84 


8.60 


53.5 


42.5 


Xylol musk 


4.58 


3.96 


8.54 


74.0 


56.2 


Isoamyl alcohol 


3.71 


3.50 


7.21 


43.0 


26.0 


Isobutanol 


2.26 


3.81 


6.07 


38.0 


24.0 


Camphor 


3.335 


2.64 


5.98 


59.7 


43.6 


Phenol 


3.04 


4.66 


7.70 


37.9 


24.1 


Isoamyl acetate 


3.63 


1.82 


5.45 


52.6 


30.0 


Nitrobenzene 


3.85 


8.14 


6.99 


42.2 


24.1 


Coumarin 


2.83 


4.48 


7.31 


48.0 


41.3 


Pyridine* 


3.25 


4.78 


8.03 


36.3 


24.0 


Cycloheptadecyl lactone 


5.33 


3.42 


8.75 


89.0 


50.0 


Glycerol* 


1.93 


4.00 


5.93 


36.5 


36.0 


Cyclohexanol 


3.92 


4.05 


7.97 


45.5 


27.0 


Water 











14.0 


8.1 


Naphthalene** 


4.67 


1.82 


6.49 


48.5 


42.0 


Benzene** 


3.09 


0.75 


3.84 


36.1 


24.0 


Cyclohexane** 


3.56 


-0.35 


3.21 


43.6 


27.0 


Ethane** 


1.23 


-0.66 


0.66 


24.4 


15.4 


w-butane** 


2.40 


-0.60 


1.80 


36.1 


20.0 


w-pentane** 


2.97 


-0.66 


2.31 


41.2 


20.0 


w-heptane** 


4.15 


-0.59 


3.56 


50.6 


20.0 


w-nonane** 


5.33 


-0.76 


4.57 


59.3 


20.0 


-undecane** 


6.52 


-0.74 


5.78 


67.3 


20.0 


w-butyric acid* 


3.19 


6.17 


9.36 


38.7 


20.0 


?z-valeric acid 


3.78 


5.04 


8.82 


43.8 


20.0 


Caproic acid 


4.38 


4.85 


9.23 


84.5 


20.0 


Oenanthic acid 


4.97 


4.55 


9.52 


53.0 


20.0 



Data needed to calculate olfactory thresholds for a number of odorants. 

* KWIA calculated from published data on the partial vapour pressures of aqueous solutions 
of the substances. 

* KOIW for non-polar compounds refers to passage from water to bulk of the oil phase. 

The cross-sectional areas in column a have been calculated from the molecular volumes 
assuming the molecules to be spheres. The molecular volumes have been obtained by assuming 
the additivity of atomic volumes using data given in Partington (1951). 

Areas in column b have been measured from models. 



QUANTITATIVE ASPECTS OF OLFACTION 



227 



RESULTS AND DISCUSSION 

(a) The calculation of olfactory thresholds 

Since \/p is a measure of the ability of an odorant molecule to "puncture" 
or dislocate the membrane temporarily, it is expected to be a function of molecular 
shape and size. These quantities are difficult to define, however, and for present 



P 0-5 




0-1 



o 10 ao 30 40 50 60 70 

Molecular Cross Section Area (A ) 

FIGURE 2. The relationships which are assumed to hold between \/p and the molecular 
cross-sectional areas in the evaluation of !//>, when (A) cross-sectional areas derived from molecu- 
lar volumes are employed (molecules assumed to be spheres). (B) Measurements on models have 
been employed (molecules uncoiled and orientated in the membrane). 



228 



J. T. DAVIES AND F. H. TAYLOR 
TABLE II 



Compound 


Olfactory thresholds in molecules/cc. 


Observed 


a 


b 


Methanol 


1.10 X 10 16 


8.13 X 10 16 


1.15 X 10 16 


Ethanol 


2.44 X 10 15 


6.61 X 10 14 


2.04 X 10 15 


Propanol 
Butanol 


5.00 X 10 13 
8.20 X 10 12 


5.13 X 10 13 
7.94 X 10 12 


3.00 X 10 14 
1.12 X 10 14 


Pentanol 


6.80 X 10 12 


9.78 X 10 11 


2.14 X 10 13 


Hexanol 


6.72 X 10 12 


1.32 X 10 11 


7.76 X 10 12 


Heptanol 
Octanol 
Decanol 
i3 ionone 


9.00 X 10 11 
3.00 X 10 ln 
3.63 X 10" 
1.60 X 10 8 


5.25 X 10 10 
4.27 X 10 9 
1.78 X 10 
1.60 >? 10 8 


6.46 X 10 12 
1.32 X 10 12 
3.02 X 10 11 
1.60 X 10 8 


Piperonal 
Menthol 


2.00 X 10 11 
2.00 X 10 11 


7.08 X 10 11 
8.32 X 10 11 


3.39 X 10 11 
3.31 X 10 11 


Skatol 


1.80 X 10 9 


4.07 X 10 9 


4.47 X 10 9 


Xylol musk 
Isoamyl alcohol 
Isobutanol 


2.10 X 10 9 
6.80 X 10 11 
8.20 X 10 12 


4.47 X 10 7 
8.13 x'lO 11 
2.34 X 10 12 


1.58 X 10 8 
3.63 X 10 12 
8.13 X 10 13 


Camphor 
Phenol 


5.00 X 10 12 
7.90 X 10 12 


4.90 X 10 11 
5.37 X 10 11 


1.45 X 10 12 
1.82 X 10 12 


Isoamyl acetate 
Nitrobenzene 


1.82 X 10 14 
2.00 X 10 11 


6.92 X 10 12 
1.32 X 10 12 


8.51 X 10 13 
9.55 X 10 12 


Coumarin 


2.10 X 10 11 


2.40 X 10 11 


1.32 X 10 11 


Pyridine 
Cycloheptadecyl lactone 
Naphthalene 
Benzene 


3.10 X 10 11 
1.75 X 10' 
2.60 X 10 12 
4.00 X 10 13 


3.63 X 10 11 
2.76 X 10 6 
1.45 X 10 12 
5.75 X 10 15 


8.91 X 10 11 
5.50 X 10 8 
3.09 X 10 13 
1.38 X 10 16 


Cyclohexane 
Ethane 


1.82 X 10 15 
1.30 X 10 19 


6.46 X 10 15 
1.70 X 10 20 


2.88 X 10 16 
6.31 X 10 19 


w-butane 


7.11 X 10 16 


6.31 X 10 17 


4.57 X 10 18 


-pentane 
-heptane 
w-nonane 


2.73 X 10 16 
1.32 X 10 16 
4.87 X 10 15 


7.59 X 10 16 
8.51 X 10 14 
1.32 X 10 13 


1.41 X 10 18 
7.94 X 10 16 
7.76 X 10 16 


w-undecane 


1.35 X 10 15 


1.15 X 10 11 


4.79 X 10 14 


w-butyric acid 
w-valeric acid 


1.4 X 10 11 
1.2 X 10 11 


1.05 X 10 10 
1.55 X 10' 


1.26 X 10 11 
4.37 X 10 11 


Caproic acid 
Oenanthic acid 


1.2 X 10 12 
1.35 X 10 13 


2.63 X 10 9 

5.13 X 10 s 


1.70 X 10 11 
8.71 X 10 10 



Observed and calculated olfactory thresholds. The observed values have been taken from 
those listed in the publications of von Skramlik (1948), Backman (1917), Morimura (1934) and 
Mullins (1955). The calculated values listed in column a have been obtained assuming that the 
molecules are spherical. Those in column b assume that the odorant molecules are uncoiled and 
orientated. 

purposes the cross-sectional areas of the molecules are employed. These have 
been calculated in two ways: those obtained from the molecular volumes are 
derived assuming that the molecules are spherical ; for straight chain organic 
molecules this means that the chains must be coiled up. This is likely to be so 
when the molecules are in water but is unlikely in a fatty membrane. In meas- 
urements taken from models, therefore, it is assumed that the molecules adsorbed 
in the membrane are orientated with the hydrocarbon chains completely uncoiled ; 



QUANTITATIVE ASPECTS OF OLFACTION 



229 



in a homologous series the cross-sectional area is thus constant for the higher 
members. 

If it is assumed that the dislocating power \/p varies linearly with the molecu- 
lar cross-sectional area, then values of \/p can be calculated, since for [3 ionone 
\/p = 1 and for water \/p = (Fig. 2). Values of the areas used for this pur- 
pose are listed in Table I. 

From \/p and log KO/A olfactory thresholds can be calculated using (4) ; 
values thus obtained are included in Table II. In Figures 3 and 4 the observed 
and calculated values are compared, assuming respectively that the molecules 
are spherical and that they are unfolded and orientated. It is seen that there is 
good agreement between calculated and observed values. This encourages us to 
believe that the "puncturing theory" of olfaction is essentially correct and that, 
to cause a stimulus, more small molecules must be packed into a given area of 
membrane surface than large ones. 

In general, and for the aliphatic hydrocarbons and alcohols in particular, the 
observed thresholds tally much better with those calculated assuming that the 




ZO 19 16 17 16 15 



13 



LoQ O.TT (calculated) 

J (O 



FIGURE 3. A comparison of observed olfactory thresholds and those calculated from equa- 
tion (4) assuming that the molecules are spherical. O Values for normal alcohols; A values 
for normal paraffins; other compounds. 



230 



J. T. DAVIES AND F. H. TAYLOR 



_Q 
O 



7 

a 



v IO 

i. 



II 



h " 

Uf 

2 15 
cr> 

J 

17 

18 
zo 




20 l<1 18 17 16 IS flf 13 12 I' 10 q ft 7 

Loo O.T (calculated) 
tJio 

FIGURE 4. A comparison of observed olfactory thresholds and those calculated from equa- 
tion (4) assuming that the odorant molecules are uncoiled and orientated in the olfactory cell 
membrane. O Values for normal alcohols; A values for normal hydrocarbons; * other com- 
pounds. 



molecules are unfolded (Fig. 4) than with those obtained from the molecular 
volumes. The constancy of \/p for the higher members of a homologous ali- 
phatic series, as can be seen from equation (4), means that the thresholds for 
these compounds will decrease less rapidly than if l/p increased continuously 
with chain length. This "tailing off" for the higher members of a series is ob- 
served for the thresholds of aliphatic alcohols, acids, hydrocarbons and with the 
chloroparaffins. The better agreement shown in Figure 4 (compared with Fig. 3) 
confirms the idea that the olfactory membrane is essentially fatty in nature. 

(b) Conditions at the olfactory cell surface 

In obtaining equation (4) from equation (3) by the use of experimental 
boundary conditions, values for log n (where n is the number of "sites" per cell) 
and a the area of one "site" are obtained. Log n is 4.64 which means that there 



QUANTITATIVE ASPECTS OF OLFACTION 231 

are 4.5 X 10 4 "sites" per cell and a. has the value of 64 A 2 if the depth of the 
surface phase is taken to be 10 A. 

The active adsorbing surface area of each cell then comes to na or 3 X 10~ 10 
sq. cm. This is much smaller than that which can be calculated from the results 
of Bloom and Engstrom (1952) (3 X 10~ 6 sq. cm.) or those of Le Gros Clark 
and Warwick (1946) (ca. 10~ 8 sq. cm.). This could either indicate that the 
active "sites" do not occupy the entire cell surface or that one of the boundary 
conditions used in deriving (4) is wrong. If, for instance, there is a stronger 
odorant than (3 ionone (such that log O.T. < 8.2), then the surface area pre- 
dicted would be larger. 

(c) Prediction of olfactory thresholds 

If the values of KO/W and KWJA are determined experimentally for any sub- 
stance, and if its molecular dimensions are known approximately, then it is 
possible by use of equation (4) to predict its olfactory threshold. Thus for 
cyclohexanol log K /w is 3.92, log KW/A is 4.05 and from its molecular cross- 
sectional area, l/p is 0.37 (using Figure 2). From equation (4) we now predict 
a threshold of 5 X 10 11 molecules/cc. 

Glycerol will have a value of about 0.32 for l/p and the values of log KO/W 
and log KW/A are 1.93 and 4.0, respectively. The predicted threshold is then 
about 10 14 molecules/cc. However, the saturation concentration in the air is 
much less than this (ca. 10 13 molecules/cc.) at room temperature so that it cannot 
be detected. 

(d) The effect of temperature on the olfactory threshold 

The molecular dimensions of an odorant and therefore, presumably, l/p will 
not alter as the temperature is raised and so the right hand side of (4) will be 
constant for any odorant. However, KLIW(KO/W) decreases for organic com- 
pounds as the temperature is raised ; the value of KW/A also decreases numerically 
(thus at 20 C. KW/A for w-hexanol is 1.54 X 10 3 whilst at 40 C. it is 2.29 X 10 2 ). 

For most compounds, therefore, KO/A decreases as the temperature is in- 
creased. This means that as the temperature is raised the olfactory threshold 
should attain higher values. This is in accord with the findings of Morimura 
(1934) who reported that small increases in temperature raise the threshold 
appreciably. 

(e) O 1 faction and chemoreception in insects 

The receptors of olfaction in Phormia (the blowfly) are situated on the an- 
tennae and labellae whilst those of the contact chemical sense are on the tarsi. 
Dethier and his co-workers have been able to measure rejection concentrations 
for one sense by removal of the receptors of the other: thus by using antennec- 
tomized and labellectomized insects Dethier and Chadwick (1948, 1950) were 
able to measure rejection concentrations for tarsal chemoreception uncomplicated 
by olfaction. These concentrations are plotted in Figure 5 against log KO/W 
and there is seen to be a linear relationship between the two quantities, the 
equation of the line being 

log M = 1.17 log Ko/w + 2.83. 



232 



J. T. DAVIES AND F. H. TAYLOR 



Here M is the threshold expressed as the molar concentration of test substance 
in water rejected by 50% of the flies. The slope of the line is very near to that- 
expected for adsorption from solution on to a pure lipid membrane (1.0). 

The olfactory rejection concentrations, however, (given by the molar concen- 
trations in the air rejected by 50% of the insects) when plotted against log KO/A, 



-3-o 



-2.-O 



E 



o 

cr> 
o 



-l-o 



o 



I O 



Log 



10 



K 



o/w 



FIGURE 5. Plot of the logarithm of the molar concentration in water rejected by 50% 
of a number of blowflies against logic KO/W. The concentrations are taken from the publications 
of Dethier and Chadwick (1948, 1950). 



QUANTITATIVE ASPECTS OF OLFACTION 



233 



show a levelling off for the higher members of a homologous series (Fig. 6). This 
confirms that a shape factor in addition to an adsorption factor is significant in 
olfaction. Unfortunately, results are available only for aliphatic alcohols and 
aldehydes for Phormia (Dethier and Yost, 1952; Dethier, 1954). 

Examination of the literature shows that there are many examples of the 
interaction of small molecules with biological membranes where adsorption is all- 
important (as with the chemoreception above or in haemolytic acceleration) and 
others where the effect is dependent on molecular shape or polarity in addition 
to the membrane concentration. Thus in the action of compounds on B. typhosus 



-7-O 



-60 



(fl 
6) 



- 

r- 



-5O 



cro 
o 



-40 



-3O 



-2-0 




7 



8 



IO 



L< 



0/A 



FIGURE 6. Plot of the logarithm of the olfactory rejection concentration for blowflies against 

logu, KOIW. The concentrations are taken from Dethier and Yost (1952). ., line 

with slope expected if \/p were to increase regularly with chain length for alcohols. 

the effect is proportional to log KOIW (Ferguson, 1939) but the action of similar 
substances on chloroplast lecithinase systems (Kates, 1957) involves a shape or 
polarity factor in addition. 

(/) Olfaction in dogs 

Several workers have recently determined olfactory thresholds for dogs 
(Krushinski, Chuvaev and Vollkind, 1946; Uchida, 1951; Neuhaus, 1953a, 
1953b; Ashton, Eayrs and Molton, 1957). The carefully conducted experiments 
of Ashton, Eayrs and Molton, using solutions of fatty acids, yield thresholds in 



234 



J. T. DAVIES AND F. H. TAYLOR 



terms of gm. mols. /liters of aqueous solution. From the values of K W /A given 
in Table I, these can be converted into molecules/cc. in air, assuming that equi- 
librium conditions prevailed during the experiments. The results for four acids 
are given below. 

Thresholds 



Acid 

Butyric 
Valeric 
Caproic 
Oenanthic 



Log molar 

cone, in 

water 

-4.38 
-3.29 
-3.56 
-3.87 



Molecules/cc. in air 

From Neuhaus 
Calculated (1953a, 1953b) 



1.73 X 10 10 
2.82 X 10 12 
2.34 X 10 12 
2.29 X 10 12 



9 X 10 3 
3.5 X 10* 
4.0 X 10 4 



The thresholds are 10 7 times higher than those obtained more directly by Neuhaus 
and by Buytendijk (1921) and are of the same order as those observed for man. 
This means either that the experiments of Ashton, Eayrs and Molton were not 
carried out under equilibrium conditions or that dogs are less sensitive to odours 
than is commonly supposed. The levelling-off of the thresholds with the higher 
acids suggests, once more, that adsorption and molecular shape and size are 
important in olfaction. 

(g) Qualitative aspects of odour and the intensity factor 

The quantitative success of our theory supports, we believe, the idea that 
adsorbed odorant molecules initiate the nervous impulse by causing localized 
permeability changes in the cell membrane. It is clear that to be a strong 
odorant a substance must possess a large value of KL/A and a low value of p. 
Few molecules exhibit both characteristics since, if p is decreased by the intro- 
duction of branched chains, KL/A decreases markedly. Artificial musk (trinitro 
tertiary-butyl xylene) possesses good olfactory characteristics because the mole- 
cule is strongly adsorbed whilst \/p is relatively high. Mullins (1955) has also 
concluded that rigid molecules are more effective stimulants than flexible 
molecules. 

Mullins (1955) has recently shown that carefully purified w-paraffins defi- 
nitely possess an odour and has measured thresholds for these compounds. The 
present theory, unlike those postulating interaction between polar groups, pre- 
dicts this and the threshold values that we have calculated agree well with those 
observed by Mullins (Figs. 3 and 4). 

It should be possible, by an extension of the theory, to investigate the varia- 
tion in intensity of an odour with its concentration in the air about the threshold 
value. The intensity of an odour, however, is difficult to define or to measure 
for humans, and little work has been done on this subject. Experiments on 
Phormia or other insects seem more suitable for work on this parameter. The 
recent electrophysiological experiments of Beidler (1958) have shown that the 
activity of the olfactory receptors in the rabbit increases with the concentration 
of odorant until a maximum level of activity is reached. 

It is not clear, however, whether excitation of an olfactory cell by an odorant 
is an all-or-nothing phenomenon (so that the intensity of smell depends on the 
number of cells stimulated), or whether there are different degrees of stimulation 



QUANTITATIVE ASPECTS OF OLFACTION 235 

of one cell (when the intensity would depend on the number of impulses per 
second, i.e., on the number of small areas per cell occupied by p odorant molecules). 

Repeated exposure of the receptors to concentrations of odorant well above 
the threshold causes a temporary loss of sensitivity. On the theory of olfaction 
proposed here, this represents a simple depolarization phenomenon, the cell being 
unable to build up the "equilibrium" ion concentrations in the periods between 
successive inspirations. This phenomenon of fatigue or adaptation has been 
discussed by Adrian (1950a) with reference to olfaction in the rabbit and in 
general (1950b). Kristensen and Zilstorff-Pedersen (1953) discuss olfactory 
fatigue in man. 

Two problems concerning the qualitative aspect of odour remain. Is there 
a relation between the adsorption and morphology of an odorant molecule and 
the type of odour it possesses? Secondly, is there more than one type of receptor 
in the olfactory epithelium? 

The first point has been considered recently by Amoore (1952) and Timmer- 
mans (1954) ; the general conclusion is that molecular shape is important in deter- 
mining the qualitative characteristics of an odorant. Amoore correlated the type 
of an odour with the shapes and electronic configurations of the molecules whilst 
Timmermans suggested that all molecules smelling like camphor are spherical, 
and conversely, that all spherical molecules have a camphorous smell. Unfor- 
tunately, there are exceptions to this rule which would otherwise be of the greatest 
significance. 

In an exhaustive survey of work on the morphology of the olfactory region 
in vertebrates, Allison (1953) concludes that attempts (of this nature) to discover 
two or more types of olfactory receptor have been unsuccessful. Le Gros Clark 
(1957), however, has noted histological differences between receptor cells in 
different regions of the olfactory epithelium and has considered the possibility 
that this is linked with the problem of differential sensitivity. 

Adrian (1950c) noted from his studies of the electrical activity of the olfactory 
bulb during stimulation that, since the receptors of lower animals are located in 
folds and are not equally accessible to currents of air, discharges set up by different 
smells may differ greatly in their temporal and spatial pattern. This fact alone 
is hardly sufficient to account for olfactory specificity since the olfactory epi- 
thelium in some higher animals, including man, is comparatively flat and exposed 
over its entire surface. His more recent electrophysiological investigations (1951, 
1952, 1954) indicated that there are several types of olfactory receptor but that 
they are specifically sensitive at concentrations only just above the threshold; 
at concentrations much above this they react to more odorants. Beidler and 
Tucker (1955) recorded neutral activity from isolated bundles of primary olfac- 
tory nerves of the opossum. Their results, too, yielded evidence for a certain 
degree of specificity in the receptors. These techniques, however, because of the 
difficulties of placing the electrodes, yield indirect and imprecise information about 
different receptor types. We may conclude (with Allison) that any observed 
differences in receptor response must be attributed to subtle differences in struc- 
ture at the physico-chemical level. Certainly, the specificities of the receptors 
might depend on the ease with which the different membrane layers are distorted : 
slight differences in their structure could render them more readily disoriented 
by molecules of certain shapes than by others. If there are only a few types of 



236 J. T. DAVIES AND F. H. TAYLOR 

olfactory receptor, however, then finer shades of odour must be the result of a 
complex pattern of impulses arriving in the brain as a result of the different 
intensities of stimulation of different receptor types as suggested by Adrian. 
Mullins (1955) and Cheeseman and Mayne (1953) have studied this question 
with regard to olfaction in humans, by examining the interference of one odorant 
with the threshold of another. Mullins suggests that since butanol does not 
disturb the threshold for butane and vice versa, there are at least two types of 
receptor membrane in the olfactory epithelium. 

The authors wish to express their gratitude to Sir Eric Rideal, F.R.S., for 
helpful advice during the course of this investigation. 

SUMMARY 

1. It is proposed that a stimulus is initiated in an olfactory receptor cell only 
when a critical number (p) of odorant molecules is concentrated within one small 
area of the cell membrane. An equation is derived which relates the olfactory 
threshold for humans to this number p and to the adsorption constant for mole- 
cules passing from air to the oil/water interface. Olfactory thresholds are cal- 
culated for a range of odorants on the assumption that \/p is a function of the 
molecular cross-section area of an odorant. The calculated thresholds agree 
with the observed values; that predicted for glycerol exceeds the saturation 
concentration in the air so that this substance is odourless. 

2. The equation suggests that olfactory thresholds should increase as the 
temperature is raised, as has been found experimentally. The results suggest 
that the olfactory cell membrane is lipoid in nature; the calculated "active 
surface area" of each olfactory cell is less than the observed total value. The 
effectiveness of compounds as odorants for Phormia and dogs., as well as for 
humans, depends on the concentration adsorbed on the membrane and upon the 
shape and size of the odorant molecules. Contact chemoreception in Phormia, 
however, is dependent only upon the appropriate adsorption constant and not 
upon molecular morphology. 

LITERATURE CITED 

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encephalog. and Clin. Neurophysiol., 2: 377-388. 

ADRIAN, E. D., 1950b. Olfactory adaptation. J. Physiol,, 112: 38P. 
ADRIAN, E. D., 1950c. Sensory discrimination, with some recent evidence from the olfactory 

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ADRIAN, E. D., 1951. Differential sensitivity of olfactory receptors. J. PhysioL, 115: 42P. 
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1-11. 

ADRIAN, E. D., 1954. The basis of sensation. (Banting Lecture.) Brit. Med. J., 1: 287-290. 
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AMOORE, J. E., 1952. The stereochemical specificities of human olfactory receptors. Perf. and 

Essential Oil Record, 43: 321-323. 
ASHTON. E. H., J. T. EAYRS AND D. G. MOLTON, 1957. Olfactory acuity in the dog. Nature. 

179: 1069-1070. 
BACKMAN, E. L., 1917. The strength of odorous substances and their solubility in water and in 

oil. /. Phys. Path. Gen., 17: 1-4. 



QUANTITATIVE ASPECTS OF OLFACTION 237 

BEIDLF.R, L. M., 1958. The physiological basis of flavor. In: "Flavor Research and Food 

Acceptance." Reinhold, New York. Part 1 : 1-28. 
BF.IDLER, L. M., AND D. TUCKER, 1955. Response of nasal epithelium to odor. Science, 122: 

76-77. 
BLOOM, G., AND H. ENGSTROM, 1952. The structure of the epithelial surface in the olfactory 

region. Exp. Cell Res., 3: 699-701. 

BUYTENDIJK, F. J. J., 1921. L'odorat du chien. Arch. Neerl. de PhysioL, 5: 434-457. 
CHEESEMAN, G. H., AND S. MAYNE, 1953. The influence of adaptation on absolute threshold 

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DAVIES, J. T., 19S3a. Olfactory stimulation; some ideas and possible model systems. Inter. 

Perfumer, 3 : 1 7-22. 
DAVIES, J. T., 1953b. L'odeur et la morphologic des molecules. Industrie de la Parfunieric, 

8: 74. 
DAVIES, }. T., AND F. H. TAYLOR, 1954. A model system for the olfactory membrane. Nature, 

'174: 693-694. 
DAVIES, J. T., AND F. H. TAYLOR, 1957. Molecular shape, size and adsorption in olfaction. 

Second International Conference on surface activity, Volume IV: 329-340. 
DAVSON, H., AND J. F. DANIELLI, 1938. Studies on the permeability of erythrocytes. V. Bio- 

chem. J., 32: 991-1001. 
DETHIER, V. G., 1954. Olfactory responses of blowflies to aliphatic aldehydes. /. Gen. PhvsioL, 

37: 743-751. 
DETHIER, V. G., AND L. E. CHADWICK, 1948. The stimulating effect of glycols and their polymers 

on the tarsal receptors of blowflies. /. Gen. PhysioL, 32: 139-151. 

DETHIER, V. G., AND L. E. CHADWICK, 1950. The relationship between solubility and stimu- 
lating effect in tarsal chemoreception. J. Gen. PhysioL, 33: 589-599. 
DKTHIER, V. G., AND M. T. YOST, 1952. Olfactory stimulation of blowflies by homologous 

alcohols. J. Gen. PhysioL, 35: 823-839. 
EHRENSVARD, G., 1942. The primary processes in the stimulation of chemoreceptors. Ada 

PhysioL Scand., 3 (Supplement No. 9): 1-151. 
EHRENSVARD, G., AND D. F. CHEESMAN, 1941. Specific phenomena at liquid/liquid interfaces. 

Sartyrek ur Svensk Kein. Tid., 53: 126-137. 
ENGSTROM, H., AND G. BLOOM, 1953. The structure of the olfactory region in man. Acta Oto- 

LaryngoL, 43(1): 11-21. 
FERGUSON, J., 1939. The use of chemical potentials as indices of toxicity. Proc. Roy. Soc., 

Ser. B, 127: 387-404. 
HAYDON, D. A., AND F. H. TAYLOR, 1959. On adsorption at the oil-water interface and the 

calculation of electrical potentials in the aqueous surface phase. Proc. Roy. Soc., Ser. A 

(in press). 
HODGKIN, A. L., AND B. KATZ, 1949. The effect of sodium ions on the electrical activity of the 

giant axon of the squid. /. PhysioL, 108: 37-77. 

HOPKINS, A. E., 1926. The olfactory receptors in vertebrates. J. Comp. NeuroL, 41 : 253-289. 
KATES, M., 1957. Effects of solvents and surface active agents on plastid phosphatidase C 

activity. Canad. J. Biochem. PhysioL, 35: 127-142. 
KRISTENSEN, H. K., AND K. ZILSTORFF-PEDERSEN, 1953. Quantitative studies on smell. Acta 

Oto.-LaryngoL, 43(6): 537-544. 
KRUSHINSKI, L. V., A. V. CHUVAEV AND N. I. VOLLKIND, 1946. New data on scent in dogs. 

Zoologicheski ZhurnaL, 25: 373-382. 
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Proc. Roy. Soc., Ser. B, 146: 299-319. 
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J. NeuroL Neurosurg. Psych., 9: 101-107. 

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130: 543-558. 

MORIMURA, S., 1934. t'ntersuchung u'ber den Gerushsinn. Tohokn J. Exp. Med., 22: 417-448. 
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PhvsioL, 35: 527. 



238 J. T. DAVIES AND F. H. TAYLOR 

NEUHAUS, W., 1953b. Die Reichschwellen des Hundes fiir Jonon und Athyl-Mercaptan und 

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RIDEAL, E. K., AND F. H. TAYLOR, 1958. On haemolysis and haemolytic acceleration. Proc- 

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VON SKRAMLIK, E., 1948. The minimum concentration necessary to arouse sensations of taste 

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THE PHYSIOLOGY OF SKELETON FORMATION IN CORALS. 

II. CALCIUM DEPOSITION BY HERMATYPIC CORALS 

UNDER VARIOUS CONDITIONS IN THE REEF 

THOMAS F. GOREAU 1 AND NORA I. GOREAU 

Department of Pliysiology, University College of the West Indies and 
The New York Zoological Society 

In a previous paper (Goreau, 1959a) a method was described for the direct 
determination of calcium deposition in madreporarian corals and other calcareous 
reef building organisms by the use of radioactive calcium-45. In these initial 
experiments calcification rates were measured under a variety of controlled con- 
ditions in the laboratory, but no information was obtained as to whether these were 
comparable with growth rates of corals in the open reef. Preliminary results 
suggested that growth in the natural environment is faster than that which we 
determined under laboratory conditions. In order to study this possibility more 
extensively we modified our method to allow direct assessment of calcium deposition 
in hermatypic corals under conditions approximating those found in the reef. For 
test organisms we selected fifteen of the most important West Indian shallow water 
hermatypic coelenterates, among which were included thirteen Madreporaria and 
two of the Milleporina. 2 These species were all collected from actively growing 
shallow reefs near Lime and Rackham Cays, located respectively two and four miles 
southeast of Port Royal, Jamaica, W. I. A general review of the abundance and 
distribution of these corals together with a description of Jamaican coral reefs has 
been published elsewhere (Goreau, 1959b). 

PROCEDURE 

All the experiments were conducted in the reefs mentioned above and the 
necessary equipment was brought to the site in a power launch which was moored 
in a suitable shallow place in about five or six feet for the duration of the operation. 
A boatman and two divers were reqviired to carry out the various steps. Large 
twelve-liter Pyrex vacuum desiccator jars containing plastic- wrapped lead weights 
were used as the experimental vessels. These were sunk on the reef and selected 
coral colonies were gently detached by one of the divers who then transferred 
them into the jars in such a way that the living surface of the coenosarc was not 

1 Mailing address : Department of Physiology, University College of the West Indies, 
Mona, St. Andrew, Jamaica, B. W. I. 

2 These species were Acropora palmata (Lamarck), A. cervicornis (Lamarck), Porites 
porites (Pallas), P. furcata (Lamarck), P. astreoides (Lamarck), Sidcrastrea siderea (Ellis 
and Solander), S. radians (Pallas), Diploria clivosa (Ellis and Solander), D. strigosa (Dana), 
D. la'iyrinthiforinis (Linnaeus), Colpophyllia natans (Muller), Manicina areolata (Linnaeus), 
Montastrea annularis (Ellis and Solander), Millepora alcicornis (Linnaeus), M. complanata 
(Lamarck). The classification of the Madreporaria is according to Wells (1956), that of 
the Milleporina is according to Boschma (1948). 

239 



240 



THOMAS F. GOREAU AND NORA I. GOREAU 




FIGURE 1. Procedure for loading and setting out the experimental jars on the reef. The 
cross-hatched square object on the bottom of the vessel is a plastic-wrapped lead brick vised 
to weigh down the jar and stabilize it against currents and wave surge. 



touched. The entire procedure was carried out under water and the coral colonies 
did not come in contact with air at any stage of the experiment. For replicate 
runs on the same species, colonies of similar size and shape were used. After load- 
ing, the jars were briefly raised above the surface, the excess water decanted, and 
ten milliliters of sea water containing approximately 0.2 me. Ca* s C1 2 adjusted to 
pH 8 were added. The lids were then put on, the jars gently swirled to mix the 
contents and a water sample taken for counting. The joint was sealed with silicone 
stopcock grease and the lids were secured by a bow clamp. The entire loading 
procedure is shown in Figure 1. Some of the jars were painted black to exclude 
light and one of these was run in each series. The full jars were placed by the 
clivers on the reef at or near the sites from which the corals within had originally 
been taken, and situated so that there was no shading by neighbouring coral colonies. 
The standard depth was five feet, but several runs were also made at depths ranging 
from one to twelve feet. During our experiments the water temperatures were 
between 26 C. and 28 C. with fluctuations of not more than one degree centigrade 
during individual runs. Relative light intensities were estimated with a water- 
proof light meter. Although this instrument was not suitable for measurement of 
incident light, it could be used to give comparative light values by pointing it 
horizontally at a matte white surface about one foot distant. 

The experiments were allowed to run for four to eight hours through the middle 
of the day from about 9:00 A.M. onward. They were terminated by picking up the 
jars from the reef, taking a water aliquot for counting, and transferring the corals to 
large volumes of fresh non-radioactive sea water with a pair of long tongs. The 
time of removal from the radioactive sea water was taken as the end point of the 



CORAL GROWTH IN REEFS 



241 



experiment. The dark jars were allowed to run for only about four hours, and 
opened first to minimize pH changes due to anaerobiosis. 

The living colonies were rinsed for two hours in running fresh sea water to 
remove adhering radioactivity from the coenosarc. Ramose corals were sampled 
by the method described in our previous paper (Goreau, 1959a). Massive non- 
branching colonies were first split into smaller pieces with a cold chisel, cutting the 
samples from the surface in such a way as to include the whole thickness of the 
polypary layer. 

A hollow steel core punch was used to obtain samples having a uniform cross- 
sectional area of 2 cm 1 '. With a hammer the punch was driven in several centi- 
meters so that the entire thickness of the coenosarc was included. Care was taken 
to exclude from the sample any boring sponges, worms, clams, Crustacea and en- 
crusting organisms. A shield and gloves were used to prevent contamination by 
radioactive coral fragments. Different parts of colonies were sampled to measure 
any growth gradients. The resultant cores were extruded by a piston, the method 
being illustrated in Figure 2. Cores from the deeper layers, just beneath the coeno- 
sarc, were taken to measure the diffusion of Ca-45 into the non-living parts of the 
skeleton. The coring method was not used on such corals as ACT op or a cervicornis 
and Porites furcata because of the relatively small size of their branches. These 
were therefore sampled by cutting or hack-sawing off pieces with accurately known 
dimensions from which the approximate surface areas could be calculated with 
suitable formulae. Measurements and sketches were made of all colonies treated in 
this way to locate the areas from which the samples had been taken. As these pieces 
were often large, some of them reaching several grams in weight, they were 
individually dissolved in numbered conical flasks containing 20 ml. dilute HC1 

(1:1). 

After solution, all samples were heated, homogenized with a Potter tissue 



Plunger a> 



Brass 
Striker 



- - 

1.55 cm o.d. 



Punch 



l.6cm i.d. 



{- 0.5 cm w.t. 



Hammer 




Plunger 



Striker 



Punch 



Extruded 
core 




'.$ 







FIGURE 2. The core punch and the procedure for taking coral samples of known 

cross-sectional area. 



242 



THOMAS F. GOREAU AND NORA I. GOREAU 

TABLE I 

Calcium deposition by hermatypic corals 



Family and species 


Branch- 
ing or 
massive 


Calcium uptake in pg. Ca/mg. N/hr. 
Sample numbers in parentheses 


Maximum 
light: dark 
ratio 


Sunshine 


Cloudy 


Darkness 


Acroporidae 












A. palmata 


B* 


43.6 9.81 (8) 


26.3 7.5 (10) 


3.2 0.64 (7) 


13.5 






50.9 8.40 (9) 




4.0 0.51 (8) 


12.8 


A. cervicornis* 


B* 


53.7 7.43 (9) 


39.4 8.65 (7) 


8.4 0.66 (6) 


6.4 






71.5 12.92 (5) 


33.2 6.80 (6) 


4.1 0.35 (8) 


8.5 






61.0 8.78 (6) 












73.7 18.82 (6) 








Poritidae 












P. porites* 


B* 


25.1 3.90 (9) 




7.9 4.60 (9) 


3.2 






18.6 db 3.31 (13) 












23.2 3.54 (8) 












20.7 1.73 (8) 












29.3 db 4.91 (8) 












27.8 5.58 (15) 








P. furcata 


B* 


15.4 3.70 (8) 




1.9 0.20 (6) 


8.1 






17.8 4.25 (9) 








P. astreoides 


M 


6.7 d= 3.65 (8) 




1.5 0.36 (5) 


4.5 






6.8 1.35 (5) 












13.7 db 4.36 (8) 












10.1 2.20 (7) 












5.5 1.55 (7) 












14.1 3.00 (9) 








Siderastreidae 












S. siderea 


M 


13.7 3.02 (11) 












9.2 2.50 (12) 








S. radians 


M 


7.1 1.34 (6) 








Faviidae 












D. divosa 


M 


6.7 1.27 (8) 












6.5 1.00 (8) 








D. strigosa 


M 


6.5 1.38(11) 












5.2 1.11 (12) 








D. labyrinthiformis 


M 


20.9 5.80 (15) 












14.8 2.72 (14) 








C. natans 


M 


14.9 3.80(11) 












10.4 1.85 (8) 




2.5 1.56 (8) 


4.3 


M. areolata 


M 










zooxanthellae 




13.4 7.54(10) 




1.8 0.42 (9) 


7.5 


no zooxanthellae 




0.7 0.31 (15) 









CORAL GROWTH IN REEFS 



243 



TABLE I Continued 



Family and species 


Branch- 
ing or 
massive 


Calcium uptake in /Kg. Ca/mg. N/hr. 
Sample numbers in parentheses 


Maximum 
light: dark 
ratio 


Sunshine 


Cloudy 


Darkness 


M. annularis 


M 


7.3 1.71 (10) 

8.9 2.00 (10) 
11.7 2.00 (12) 




0.3 0.05 (9) 


22.9 


Milleporidae 
.17". complanata 


B* 


35.7 3.89 (9) 
47.1 13.80 (12) 
36.2 4.76 (6) 
36.5 3.92 (11) 


24.6 5.52 (9) 
23.2 4.43 (10) 


4.7 0.36 (10) 


7.5 


M. alcicornis 


B* 




23.6 db 3.52 (8) 







* Apical polyps only. 

grinder, cooled, and diluted to twenty-five milliliters with distilled water. Replicate 
aliquots were taken for counting and nitrogen analysis. The radioactivity was 
determined by the method described in our previous paper and the total amount of 
calcium taken up was calculated from the specific activity of calcium-45 in the 
sea water samples. The final calcification rate was expressed either in /ug. Ca de- 
posited per hour per mg. N, or jug. Ca deposited per cm 2 , per hour. The final results 
were not corrected for isotopic exchange since the calcium-45 incorporated in this 
way amounted to a maximum of 0.5 per cent of the total calcium deposited. 

RESULTS AND OBSERVATIONS 

The highest calcification rates were invariably found in the terminal regions of 
the branching, or ramose corals, whereas lower rates were characteristic for the 
massive non-branching corals. Our results, in terms of calcium deposited per 
hour per milligram of organic nitrogen, are brought together in Table .1. In 
decreasing order, the species we tested ranked approximately as follows: A. cervi- 
cornis, A. palmata, M. complanata, M. alcicornis, P. porites, P. jurcata, D. labyrin- 
thiformis, C. natans, M. arcolata, P. astreoides, A. siderea, M. annularis, S. radians, 
D. clivosa, D. strigosa. The first six species are branching, the others are all 
massive non-branching. 

As the ambient light intensity was previously shown to have an effect on the 
calcification rates of reef corals (cf. Goreau, 1959a), we tested the influence of 
different light conditions on coral calcium uptake in the reef by running some of 
our experiments on cloudy days, and some in complete darkness. Because exact 
measurements of the underwater light intensities could not be made due to lack of 
a suitable meter, we have listed our results in Table I under arbitrary headings, 
depending on whether the light conditions during the experiments were clear and 
sunny, cloudy or no light at all. In all species tested, the calcification rate was 
highest during sunny weather, lower during cloudy weather, and very low in 
darkness. The maximum light : dark ratios varied over a broad range, between 
approximately 3.2 to 22.9. 



244 



THOMAS F. GOREAU AND NORA I. GOREAU 



Previously we have also presented evidence that the calcification rate of corals 
is in part dependent on the presence of zooxanthellae, showing a marked fall in 
their absence (Goreau, 1959a). An opportunity came to repeat this experiment 
under natural conditions when several bleached and zooxanthella-less colonies of 
M. areolata were found alive and in good condition, growing unattached in semi- 
darkness under a large hollow coral head. The polyps of these were expanded, and 
their tissues were colourless and translucent. Controls were run at the same time 
with normal yellowish-brown colonies collected nearby. The results, which are 
included in Table I, show that the normal coral with zooxanthellae calcified about 
nineteen times faster than those which had lost their algae. This is in substantial 
agreement with our previous experiments. The light : dark ratio for normal colonies 
of M. areolata was about 7.5, which means that colonies with zooxanthellae could 
still calcify considerably faster in darkness than could zooxanthella-less colonies in 
the light. An hypothesis concerning a mechanism whereby the calcification process 

TABLE II 

Calcification rates in different parts of branches of A. cervicornis at various light intensities 



Light conditions 
on the reef 


Calcium uptake in /ug. Ca/mg. N/hr. 
Sample numbers in parentheses 


Apical polyps 


2 cm. behind apex 


3 cm. behind apex 


Darkness 


8.4 0.66 (6) 


2. 2 0.72 (6) 




Cloudy weather 


39.4 8.65 (7) 
33.2 6.80 (6) 


15.1 6.20 (8)* 
9.8 3.91 (5)* 




Bright sunshine 


53.3 7.43 (9) 
71.5 12.92 (5) 
61.0 8.78 (6) 
73.7 18.82 <6i 


33.9 3.47 (TO) 
48.8 8.39 (4) 
42. 5 7.15 (7) 
36.8 6.57 (5) 


27.5 4.45 (7) 
28.9 1.00 (5) 
31.6 5.39 (7) 
25.7 3.61 (6) 



* Samples taken 3 cm. behind apical polyp. 

in Madreporaria may be stimulated by photosynthesizing zooxanthellae has been 
published in the paper cited above. 

Considerable variance in the calcification rates is evident in our results, both 
within sample groups taken from the same colony, as shown by the standard devia- 
tion of the means, and between different colonies of the same species. To determine 
the probability that these two forms of variance were due to dissimilar causes, the 
data from four species 3 were analysed by the F test (cf. Snedecor, 1946; pp. 232- 
233). This shows that the F values varied from less than one to less than five 
per cent, thus indicating that differences in calcium uptake rate between uniformly 
sized colonies of the same species growing under similar conditions were significant. 
Similar variances almost certainly occur in most of the other species tested. 

Preliminary tests to determine the effect of depth were carried out on three 
coral species simultaneously at 1, 3, 6, 9, and 12 feet. The results were incon- 
clusive, showing no correlation of the calcification rate with depth over the range 

3 A. cervicornis, P. porites, P. astrcoidcs, M. complanata. 



CORAL GROWTH IN REEFS 



245 



73 7 18.8 (6) 




vt 

a. 
u 

UJ 

K 
Z 
U 

u 



CALCIFICATION 

RATE IN 
)JG CA/HR/MG N 



FIGURE 3. Apical region of a branch of Acropora cervicornis showing the existence of a 
calcification gradient. The large terminal polyp at the top has the highest rate of calcium 
incorporation. 

tested probably because the light intensity was more or less uniform clue to back- 
scatter by sandy patches in that part of the reef where these experiments were 
made. Further work correlated with more precise measurements of the incident 
light intensity over a greater depth range is now in progress. 

Calcification rates in the ramose corals tended to decrease systematically from 
a maximum in the apical polyps to much lower rates in the lateral and basal branch 
corallites. A characteristic gradient is shown in Table II and Figure 3 for the 
staghorn coral, A. cervicornis, \vhere the calcification was measured at one-centi- 
meter intervals away from the branch apex. Systematic variations in the calcifica- 
tion rates were also observed in other branching species, e.g., A. palmata, M. 
coinplanata and P. jnrcata as shown in Table III. In the massive corals tested, 
gradients were absent and such variations as were observed appeared to be random 
over the entire colony. 



246 



THOMAS F. GOREAU AND NORA I. GOREAU 



Some preliminary experiments were also carried out to determine the calcifica- 
tion rate in terms of surface area. This was done by measuring the calcium uptake in 
core samples of known diameter and cross-sectional area. Our results are almost 
certainly too high, due to an uncompensated systematic error introduced by the 
difficulty of measuring the total area of the complicated skeletal surface of corals. 
Pending development of an accurate method for doing this, a problem now under 
investigation in our laboratory, the area of our samples has been calculated from 
their geometry based on smoothed out dimensions, and our results can therefore 
only be interpreted in very approximate terms on the assumption that all errors in 
the method are predominantly in the same direction. In column 1 of Table III 
are shown the approximate calcium uptakes per cm. 2 in four ramose and two massive 
corals. It is interesting to note that the calcification rate per unit area of the two 
non-branching corals seems to be equal to or greater than that of the branching 
species. The reverse is true when calcium uptake is estimated in terms of the 
nitrogen content as shown in column 3 of Table III. In the second column of this 
table are listed the approximate tissue biomasses contained in the sample in terms 
of mg. N/cm 2 . This shows that the two massive faviid corals had a higher content 
of organic matter per unit area than the branching acroporid, poritid, and hydro- 
coralline species that were tested. This confirms what is readily seen by naked 
eye, that the acroporid and hydrocoralline corals have a very thin translucent 

TABLE III 

The relationship of approximate geometric area to protein nitrogen content and calcium 
uptake in corals. Sample number in parentheses 



Species and location of samples 


Branching 
or 
massive 
form 


Approximate 

Mg. Ca/lir./cm. 2 


Approximate 

mg. N/cm. 2 


jtg. Ca/mg. N/hr. 


A. palmata 
Outer edge of frond 
7 cm. behind edge, upper 
7 cm. behind edge, under 


B 


69 9.5 (3) 

52 2.0 (3) 
32 4.0 (3) 


2.4 0.4 (3) 
3.0 0.3 (3) 
1.8 0.7 (3) 


29.5 0.78 (3) 
17.8 2.59 (3) 
17.5 0.05 (3) 


A. cervicornis 
Apical polyps 
3 cm. behind apex 
5-7 cm. behind apex 


B 


20 1.7 (9) 
12 1.8 (6) 
10 1.6 (3) 


0.5 0.05 (9) 
0.6 0.01 (6) 
0.8 0.4 (3) 


41.7 11.7 (9) 
17.4 2.95 (6) 
13.8 0.84 (3) 


J/. complanata 
Superior edge of frond 
10 cm. behind edge 


B 


38 5.7 (9) 
12 1.8 (4) 


1.1 0.1 (9) 
2.0 0.3 (4) 


35.7 3.89 (9) 
5.6 0.22 (4) 


P. furcata 
Apical 1 cm. of branch 
2 cm. behind apex 
5 cm. behind apex 


B 


36 6.7 (8) 
6 1.4 (3) 

4 2.9 (2) 


2.4 0.1 (8) 
3.3 0.9 (3) 
3.1 0.6 (2) 


15.4 3.70 (8) 
3.34 0.94 (3) 
3.07 0.55 (2) 


(.". natans 
Random over whole colony 


M 


80 1.2 (8) 


7.9 1.2 (8) 


10.4 1.85 (8) 


M. annularis 


M 


54 11.9 (3) 


7.3 0.6 (5) 


7.32 1.71 (5) 



CORAL GROWTH IN REEFS 247 

coenosarc ; the poritids a somewhat thicker, more opaque one ; and that the faviids 
on the whole are rather fleshy, especially the species analysed here. The possibility 
thus arises that the calcification powers of the massive corals, which are less marked 
than those of the branching species on the basis of tissue mass as expressed by the 
nitrogen content, may be as great or greater per unit surface area of the basal 
calicoblastic epidermis which is the organ of skeletogenesis in corals. The problem 
is now under more detailed investigation. 

DISCUSSION 

The long-term field observations of Wood-Jones (1910), Mayor (1924), Ed- 
mondson (1929) and the Stephensons (1933) directed attention to the fact that 
corals do not necessarily grow at even rates. The experiments of the Stephensons 
were particularly valuable as they established that individual variations among 
healthy and normal corals of the same species growing for the same length of time 
in the same environment gave very irregular results, leading to the conclusion that 
the use of averages was preferable to individual figures. Our data seem to cor- 
roborate this view to the extent of showing that there are significant variations in 
calcification rates among similar colonies of the same species under identical 
environmental conditions over the comparatively short periods of time during which 
our measurements were made. It is, however, uncertain whether the variances we 
observed in experiments lasting only a few r hours are similar to those described in 
the studies of other investigators, all of which were made under non-uniform con- 
ditions over periods of months to years. It should be understood that because of 
the dissimilarity in aims, methods and expression of results, the calcification rates 
cited in this paper cannot be compared with the coral growth data obtained by 
previous observers who used completely different procedures. We have endeav- 
oured to determine calcium uptake of corals and to use this as an index of physio- 
logical function, not as a direct measure of absolute growth rate. We hope never- 
theless that the hiatus between these two objectives will disappear with further 
work. 

The present results also support our earlier observations (Goreau, 1959a) that 
the calcification rates of individual corallites fluctuate widely, and probably at 
random, even in symmetrical colonies. Statistically, this type of variance appears 
to be separate and distinct from the clonal growth differences that occur among 
individual colonies in a population of a given coral species. 

The local fluctuations in the calcium uptake of individual corallites may be 
related to the manner in which the coral skeleton is laid down and organised. The 
sclerenchyme, or skeleton, consists of a succession of discontinuous layers held 
together by walls and partitions : the dissepiments, thecae and sclerosepta, respec- 
tively. The dissepiments are separated from each other by more or less regular 
horizontal spacings which suggests that the process of deposition is itself discon- 
tinuous, possibly involving local and temporary detachment of the calicoblast from 
the skeletal basis just before the secretion of a new layer, a millimeter or so above 
the old. Circumstantial evidence that the attachment of the polyp to its skeleton 
may be under some degree of facultative control is provided by the observation 
that polyps of starving corals are able to detach themselves completely from the 
corallum. In this phase they can stay alive for some weeks without showing any 



248 



THOMAS F. GOREAU AND NORA I. GOREAU 



evidence of renewed skeletogenesis although they are able to ingest food normally 
(Goreau, unpublished observations). We do not suggest that the coenosarc ever 
becomes entirely detached under normal circumstances, but the fact that it is able 
to do so at all indicates that the polyps are not anchored down quite as securely as 
is at present believed. On the basis of mineralogical evidence, Bryan and Hill 
(1941) have also suggested that madreporarian calcification is a discontinuous 
process involving possible temporary detachment of parts of the polyp. 

TABLE IV 

Comparison of calcification rates of some hermatypic corals with their relative abundance 

on a large Jamaican coral reef 



Family and species 



Acroporidae 

A. cervicornis 

A. palmata 
Poritidae 

P. porites 

P. furcata 

P. astreoides 
Siderastreidae 

S. siderea 

S. radians* 
Faviidae 

D. clivosa 

D. strigosa 

D. labyrinthiformis 

C. natans 

M. areolata* 

M. annularis 
Milleporidae 

M. complanata 

M. alcicornis 



Relative abundance in all 
zones of reef 



4- 



Relative calcification rate 



oooooo 
ooooo 

oooo 

oo 

o 

oo 
o 

o 
o 

ooo 
oo 

00 

oo 

ooooo 
ooo 



Legend : + not common 

+ + common 

+ + + abundant 
+ + + + very abundant and 

dominant in some 

zones 



o 0-9 jug. Ca/mg. N/hr. 

oo 10-19 /xg. Ca/mg. N/hr. 

ooo 20-29 /ug- Ca/mg. N/hr. 

oooo 30-39 /xg. Ca/mg. N/hr. 

ooooo 40-49 /xg. Ca/mg. N/hr. 

oooooo 50+ /xg. Ca/mg. N/hr. 



* Colonies numerous but of small size with low aggregate biomass. 



We have so far found no consistent relationship between calcification rates of 
corals in terms of organic matter, and their prevalence on a coral reef. In Table IV 
the relative calcification rates of the species tested, are compared with the relative 
abundance of the same corals on a large Jamaican reef. For the Acroporidae and 
Milleporidae, which are the predominant hermatypes of the shallowest reef zones, 
there may be some correlation of abundance with calcium uptake. In the Sider- 
astreidae, the much larger and more common 6". siderea appears to calcify faster than 



CORAL GROWTH IN REEFS 249 

the smaller but hardier >S\ radians. However, in the Poritidae, P. astrcoidcs is the 
more frequent and important species in Jamaican reefs, although it calcifies much 
less rapidly than its branching congeners P. ponies and P. fur cat a. In the Faviidae, 
the apparent discrepancy between the occurrence on the reef and calcification rate 
is still more pronounced since the faster calcifying species such as C. natans, D. 
labyrinthiformis and M. arcolata are less prevalent on the reef than the slower 
growing D. strigosa, D. clivosa and M. annularis. 

Although the preeminence of the massive corals may in part be due to the 
greater susceptibility of branching corals to damage by heavy seas, this fails to 
explain why branching corals are in general typical of the turbulent shallow waters 
whereas the massive corals are more characteristic of the deeper calmer regions of 
the reef. Despite the fact that the growth rate of the massive coral M. annularis 
(cj. also Vaughan, 1915, and Vaughan and Wells, 1943, p. 64) is relatively low, in 
Jamaica this species is nevertheless one of the most important hermatypes in regard 
to the total quantity of calcareous material contributed to the framework of the 
reef. It is usually the dominant coral in the deeper regions of the fore-reef, reaching 
a peak of development in the buttress zone where the gigantic size of its colonies is 
evidence of extremely vigorous proliferation (Goreau, 1958a, 1959b). This zone 
is named for the great coral promontories which grow upward and forward into 
deeper water, forming immense dentate projections that are oriented with their 
long axis into the prevailing seas so that they present a maximal area for coral 
growth with a minimal frontal area exposed to the surge of the waves. In the 
buttress zone the growth habit of such massive corals as M. annularis and P. 
astreoides becomes curiously flattened so that the colonies often resemble platters, 
roof shingles or great flow sheets, whereas in other shallower zones in which these 
corals are frequently much more exposed to surf and currents their colonial habit 
is mushroom shaped. If it is assumed on the basis of our preliminary data that the 
growing power of reef corals is proportional to the area of the calicoblast, then the 
formation of buttresses and a flattened growth habit may be interpreted as represent- 
ing adaptive modifications which enable massive corals to compete more successfully 
on an area basis with the faster growing ramose corals. 

This work was in part supported by grant G-4017 from the National Science 
Foundation to the New York Zoological Society, and by institutional funds from 
the University College of the West Indies. We also wish to thank Professor 
D. M. Steven for making available facilities of the University College Marine 
Station at Port Royal, Dr. R. C. Read for statistical advice, Mr. E. I. Finzi for 
constructing the core punch, and members of the Jamaica Branch of the British 
Sub-Aqua Club for their assistance during diving operations. 

SUMMARY AND CONCLUSIONS 

1. The calcium uptakes of 13 hermatypic corals and 2 hydrocorallines were 
measured by a modified calcium-45 method under conditions approximating those 
of the natural environment of the reef in experiments lasting four to eight hours. 

2. When measured on the basis of nitrogen content, the growth rates of the 
branching corals were higher than those of the massive corals. On the basis of 
area, however, the latter appear to grow as fast or faster than the former. 



250 THOMAS F. GOREAU AND NORA I. GOREAU 

3. Light intensity had a profound influence on the growth rate under the con- 
ditions of our experiments. All corals tested deposited calcium fastest in" sunlight, 
less during cloudy weather and least in darkness. Bleached zooxanthella-less 
colonies deposited calcium at lower rates in the light than normal colonies with 
zooxanthellae did in darkness. 

4. Systematic calcification gradients were observed in branching corals but not 
in massive species. 

5. Although there was a considerable variance in the growth rate from place 
to place within individual colonies, we also observed large and significant differences 
in the growth rate between individual colonies of the same species, size and shape 
under similar conditions. 

6. It is suggested that the organisation of the skeleton, which is really composed 
of many separate lamellae with spaces in between, indicates that the calcification 
process itself may be discontinuous, and that this may in turn be responsible for the 
growth fluctuations that were observed within the coral polyp populations of 
individual colonies. 

7. No hard and fast correlation was observed between the calcification rates 
per unit of nitrogen and the relative abundance of the species on the reef. Although 
some of the commonest shallow water corals are very fast calcifiers as well, the 
most important West Indian reef builders are the slower growing massive corals. 

8. An explanation is put forward to the effect that growth of massive corals in 
the reef is enhanced by the formation of buttresses which serve to increase the 
available surface area for calcification. 

LITERATURE CITED 

BOSCHMA, H., 1948. The species problem in Millepora. Zool. Verhandl. Rijksmus. Nat. Hist. 

Leiden, no. 1, pp. 1-114. 
BRYAN, W. H., AND D. HILL, 1941. Spherulitic crystallisation as a mechanism of skeletal 

growth in hexacorals. Proc. Roy. Soc. Queensland, 52 : 78-91. 
EDMONDSON, C. H., 1929. Growth of Hawaiian Corals. Bull. Bishop Mus. Honolulu, no. 58, 

38 pp. 
GOREAU, T. F., 1958. Calcification and growth in reef-forming corals. Proc. of the XVth 

Int. Congr. Zool., Section III, Paper 42. 
GOREAU, T. F., 1959a. The physiology of skeleton formation in corals. I. A method for 

measuring the rate of calcium deposition by corals under different conditions. Biol. 

Bull, 166: 59-75. 
GOREAU, T. F., 1959b. The coral reefs of Jamaica. I. Species composition and zonation. 

Ecology, 40 : 67-90. 
MAYOR, A- G., 1924. Growth rate of Samoan Corals. Pap. Dept. Marine Biol. Carnegie Inst. 

Wash., 19: 51-72. 
SNEDECOR, G. W., 1946. Statistical Methods. Fourth edition. Iowa State College Press. 

485 pp. 
STEPHENSON, T. A., AND A. STEPHENSON, 1933. Growth and asexual reproduction in corals. 

Gt. Barrier Reef Exped. Sci. Rep., 3: 167-217. 
VAUGHAN, T. W., 1915. The geologic significance of the Bahamian and Floridian shoal water 

corals. /. Wash. Acad. Sci., 1915 (5) : 591-600. 
VAUGHAN, T. W., AND J. W. WELLS, 1943. Revision of the suborders, families and genera of 

the Scleractinia. Geol. Soc. Amer. Spec. Pap. 44 : 298 pp. 
WELLS, J. W., 1956. Scleractinia. Chapter in: Treatise on Invertebrate Palaeontology. Ed. 

by R. C. Moore. Part F. pp. 328-443. 
WOOD-JONES, F., 1910. Coral and Atolls. London, Reeve. 392 pp. 



DISTRIBUTION OF A RADIOMERCURY-LABELLED DIURETIC 
(CHLORMERODRIN) IN TISSUES OF MARINE FISH 1 

ROGER L. GREIF AND MARILYN DU VIGNEAUD 

Department of Physiology, Cornell University Medical College, New York City, and the 
Marine Biological Laboratory, Woods Hole, Mass. 

The ability of mercurial compounds to increase urine flow, recognized at least 
since the time of Paracelsus (1493-1541), has been widely applied in clinical 
medicine following the introduction of the organic mercurial diuretics (Saxl and 
Heilig, 1920). The association of suppression of urine flow with poisoning due 
to inorganic mercury compounds (Woodman and Tidy, 1877) also suggests that 
renal tissue is particularly susceptible to the action of such substances. Ludwig 
and Zillner (1890) were among the early investigators who demonstrated by 
chemical analysis that the kidneys of dogs poisoned with mercuric chloride contain 
far higher concentrations of mercury than other tissues. Many subsequent studies, 
with the use of both biochemical and radioisotopic methods, have confirmed this 
finding. 

Hg 203 -labelled chlormerodrin has recently been used to determine more precisely 
the location of mercury deposition in mammalian kidney. In dogs and in rats the 
concentration is highest in the outer renal cortex, decreases in the medulla, and is 
lowest in renal papillary tissue (Greif ct al., 1956). In this connection, Walker and 
Oliver (1941) have pointed out that the rat outer renal cortex is composed largely 
of proximal segment of the tubule. The purpose of the present study was to 
determine, with the use of a radiomercury-labeled organic mercurial diuretic, 
whether mercury is selectively concentrated in the kidneys of marine fish with a 
variety of nephron types. 

MATERIALS AND METHODS 

Specimens of flounder (Paralycthys dentatus), toadfish (Opsanus tan} and 
dogfish (Mustelus canis) were furnished by the Collecting Department of the 
Marine Biological Laboratory, and were stored briefly in live cars before use. 
They were weighed and injected with alkaline solutions of chlormerodrin 2 in 
concentrations indicated in the tables. Most injections were made intramuscularly 
in the fleshy part of the tail, although a few intramuscular injections were placed 
immediately dorsal and posterior to the gills. The fish were placed in a running sea 
water aquarium until sacrificed. At the time of killing, the fish were stunned and 
bled from either the tail vein or the heart with a needle attached to a heparinized 
syringe. Tissue was then rapidly removed from the fish, weighed on a torsion 
balance, and transferred to a test tube for counting in a well-type scintillation 
counter with sealer. Blocks of tissue were removed from the anterior, medial, and 

1 This investigation was supported by Grant A-786 of the National Institute of Arthritis 
and Metabolic Diseases, USPHS, Bethesda, Md. 
- Generously provided by Dr. R. F. Pitts. 

251 



252 



ROGER L. GREIF AND MARILYN DU VIGNEAUD 



posterior portions of the kidney for counting. Results are expressed as milligrams 
chlormerodrin mercury per gram wet weight of tissue. Unless otherwise indicated, 
the middle portion of the kidney was selected for comparison with other excised 
tissues. Urine was obtained either by inserting a pipette into the urogenital 
papilla or aspirating the exposed bladder with a syringe and needle. Phenol red 
accumulation in the excised flounder tubule was studied by the method of Forster 
and Taggart (1950). 

RESULTS 

a) Flounder. Under the present experimental conditions a high tissue mercury 
concentration is always found in the kidneys. This is especially striking at the 
dosage level of one milligram chlormerodrin mercury per kilogram body weight 
(Table I), an amount which will produce diuresis in mammals (Borghgraef ct al., 
1956). The larger dose, 10 milligrams mercury per kilogram (Table II), which 
is toxic to mammals, also proved fatal to some of the fish. Kidney tissue excised 
from flounder surviving the larger dose was unusually friable. On microscopic 
examination, the freshly teased tubules appeared cloudy and granular and failed to 
concentrate phenol red. When such tissue is fixed in Susa fluid and stained with 
hematoxylin and eosin it cannot be readily distinguished from kidney of fish given 

TABLE I 

Mean tissue concentration of radiomercury administered as one milligram labelled chlormerodrin 

mercury per kilo body -weight. All values expressed as micrograms 

mercury /gram wet weight of tissue 



Species 


No. of 
Animals 


Plasma 


Kidney 


Liver 


Bile 


Gills 


Heart 


Stomach 


Gonads 


Spleen 



12 Hours After Intramuscular Injection 



Flounder 


2 


0.7 


52.2 


3.2 


9.7 


0.2 


0.5 


0.4 


0.2 


0.7 


Toadfish 


1 


0.8 


13.2 


0.3 


0.0 


0.5 


0.0 


0.5 


0.0 


0.2 


Dogfish 


1 


0.9 


42.2 


4.4 


21.9 


0.0 


0.0 


0.3 


0.8 


0.5 



24 Hours After Intramuscular Injection 



Flounder 


3 


0.3 


80.1 


3.9 


1.7 


0.2 


0.4 


0.2 


0.2 


0.5 


Toadfish 


2 


0.7 


12.0 


0.3 


0.2 


0.5 


0.5 


0.4 


0.1 


1.0 


Dogfish 


3 


0.6 


14.5 


4.0 


33.6 


0.4 


0.2 


2.0 


0.9 


0.4 



48 Hours After Intramuscular Injection 



Flounder 


2 


3.5 


35.6 


6.7 


2.8 


1.9 


0.2 


0.4 


0.3 


1.0 


Toadfish 


2 


0.3 


9.0 


0.4 


0.2 


0.3 


0.3 


0.2 


1.2 


0.4 


Dogfish 


1 


0.2 


6.4 


2.7 


21.0 


0.9 


0.3 


2.9 


0.7 


0.4 



72 Hours After Intramuscular Injection 



Flounder 
Dogfish 


2 
1 


0.1 
0.1 


24.6 

4.4 


5.7 
7.3 


2.7 
114.0 


0.2 
1.0 


0.2 
0.2 


0.0 
0.4 


0.2 
0.7 


0.7 
0.4 



MERCURIAL DIURETIC IN MARINE FISH 

TABLE II 

Mean tissue concentration of radiomercury after administration of 10 milligrams labelled 

chlormerodrin mercury per kilo body weight. All values expressed as 

micrograiiis mercury /gram wet weight of tissue 



253 



Species 


No. of 
Animals 


Plasma 


Kidney 


Liver 


Bile 


Gills 


Heart 


Stomach 


Gonads 


Spleen 



12 Hours After Intramuscular Injection 



Flounder 
Toad fish 


4 

2 


13.6 
29.5 


288.0 
197.0 


55.6 
15.0 


12.8 
2.6 


12.5 

12.3 


8.2 
23.5 


8.7 
11.7 


7.3 
6.4 


30.2 
16.5 



24 Hours After Intramuscular Injection 



Flounder 
Toad fish 


4 
3 


11.9 

19.7 


146.5 
128.9 


51.8 
29.2 


53.7 
20.0 


11.4 
10.4 


9.3 
21.2 


6.0 
12.0 


6.4 
8.4 


30.1 
13.6 



48 Hours After Intramuscular Injection 



Flounder 
Toad fish 


3 
3 


12.6 
19.0 


77.1 
105.0 


59.2 
36.2 


121.0 
37.8 


10.3 
11.0 


8.6 
21.4 


8.5 
10.2 


9.8 
5.0 


37.5 
16.6 



72 Hours After Intramuscular Injection 



Flounder 
Toadfish 


2 
3 


10.2 
20.8 


89.4 
106.5 


45.1 
40.9 


57.0 
55.7 


7.1 
11.7 


4.2 
29.2 


7.7 
11.5 


6.7 
18.5 


19.6 

15.8 



the smaller dose of mercurial or from that of control flounder, although these latter 
tissues concentrated phenol red and appeared normal when viewed with the dis- 
secting microscope. On the regimen of 10 milligrams Hg per kilogram, high con- 
centrations of mercury are seen not only in the kidney but also in the liver and 
bile of the surviving animals. 

b) Toadfish. In Table I and Table II may be found the distribution of chlor- 
merodrin mercury in the tissues of this species. As in the flounder the mercury 
concentration in the kidney is high, but at 10 milligrams mercury per kilogram the 
concentration in the bile in toadfish does not exceed the concentration in the kidney. 
It was possible to obtain blood and urine samples in 11 toadfish after chlormerodrin 
injection. Despite great individual variation, in all but one animal the urine-to- 
plasma ratio was at least 4, and the mean value for this ratio was 30.7 9.6 
(S. E.). Phenol red, injected with the chlormerodrin, also appeared in the urine. 

c) Dogfish. Tissue mercury distribution in a small number of animals is in- 
cluded in Table I. The most striking difference to be seen in this instance is the 
high mercury concentration in the bile on the one milligram mercury per kilogram 
body weight regimen. 

General. In all animals at the one milligram/kilogram dose the kidney mercury 
concentration is high, and only in the dogfish is the concentration in the liver and 
in the bile impressive. It is notable that in none of the animals on any regimen is 
the mercury concentration in either the gills or the stomach higher than in the 



254 



ROGER L. GREIF AND MARILYN DU VIGNEAUD 















1 




FIGURE 1. Photomicrograph of area of flounder kidney showing transition between an- 
terior portion (right) containing few renal elements, and middle portion in which numerous 
glomeruli and tubules can be seen. 

plasma. The regional distribution of mercury in the kidney is of interest. Both 
flounder and toadfish kidneys contain much lymphoid tissue in which the renal 
elements are imbedded, whereas the dogfish kidney on microscopic section shows 
tubular and glomerular structures surrounded by supporting tissue containing few 
or no lymphoid elements. Figure 1 shows a photomicrograph of the transition zone 



TABLE III 

Regional distribution of radiomercury in fish kidney. Combined data from fish injected with one 
milligram and 10 milligrams chlormerodrin mercury per kilogram and studied at time inter- 
vals up to 72 hours after injection. Results expressed as per cent of mean mercury 
concentration of tissue from the three regions of the kidney sampled 



Species 


No. of 
animals 


Per cent of total mean mercury concentration in the 
kidney contributed by: 


Anterior sample 


Middle sample 


Posterior sample 


Flounder 
Toadfish 
Dogfish 


16 
15 
6 


9.0 8.5* 
26.6 3.6 
24.9 7.6 


48.5 db 5.9 
33.9 5.1 
35.4 3.7 


42.5 5.6 
39.6 6.5 
39.9 8.2 



* Standard deviation. 



MERCURIAL DIURETIC IN MARINE FISH 255 

between the anterior and middle section of the flounder kidney. A few renal 
elements can be seen imbedded in the lymphoid cells in the anterior portion, and 
these elements can be seen in larger numbers in the more posterior portion of the 
section. The mercury concentration of the anterior block from which this particular 
section was made is one fifth that found in the more posterior section. 

Table III represents a summary of the regional distribution of radiomercury in 
the kidneys of the species studied. The mean mercury concentration in anterior, 
medial, and posterior samples is assigned the value of 100%, and the proportion of 
the total contributed by the three regions is also expressed as per cent. It can be 
seen that in each species the lowest mercury concentration is found in the anterior 
portion, but that in the case of the flounder the difference is particularly striking. 

DISCUSSION 

The species of marine vertebrates chosen for this study have nephrons which 
can be classified roughly into three types. The flounder can be considered to have 
kidneys with glomerular and proximal segmental function (Forster and Hong, 
1958) ; the dogfish has a complex nephron, with glomerulus, proximal, and distal 
segment (Kempton, 1943). The toadfish, since the original description by Mar- 
shall (1929), has been considered to be both functionally and anatomically an 
aglomerular species with a nephron consisting of a proximal segment only. 

The present experiments show clearly that the presence of a proximal segment is 
sufficient for a nephron to be capable of concentrating mercury. The high U/P 
ratio for mercury observed in the toadfish agrees with the findings in dogs of 
Borghgraef ct al. (1956) that chlormerodrin mercury is excreted by means of a 
secretory mechanism. Subsequent "stop-flow" experiments have further localized 
this mechanism in the dog proximal tubular segment (Kessler et al.. 1958). 
Giebisch and Dorman (1958) have noted the selective accumulation of radio- 
mercury administered as labeled chlormerodrin in the kidneys of carp, chicken, bull- 
frogs, and turtles, and have shown that in Necturus inaculosus, a species in which 
the proximal and distal tubular segments are anatomically distinct, the proximal 
segment always contains a higher mercury concentration than does the distal. The 
observation of Bieter (1933) that mercuric chloride induces a diuresis in the 
anaesthetized toadfish also suggests that mercurial compounds act proximally. Un- 
fortunately no analyses of toadfish urine for sodium and chloride are given with 
Bieter's experiments. The excretion of phenol red by the toadfish in the present 
experiments was also observed by Shannon (1938) and others. 

The regional distribution of radiomercury noted in Table III deserves some 
comment. The injections of labelled chlormerodrin were made, for the most 
part, into the muscular portion of the tail, and since the kidneys of fishes are supplied 
largely by the renal portal circulation (Smith, 1953). it might be expected that the 
portion of the kidney closest to the injection site would contain the highest con- 
centration of radiomercury. Such an explanation would agree with the results in 
both toadfish and dogfish, as seen in Table III. but in the case of the flounder the 
highest concentration of radioactivity is found in the medial portion of the kidney. 
It seems probable, therefore, that the low concentration of radiomercury in the 
anterior portion of the kidney in this species is a reflection of the small number of 
renal elements imbedded in the lymphoid tissue in this region (Fig. 1) and that 



256 ROGER L. GREIF AND MARILYN DU VIGNEAUD 

this represents an example of the strikingly higher accumulation of mercury by 
renal elements than by lymphoid tissue. 

Mercurial diuresis in mammals is associated with an increased excretion of 
sodium and chloride ions. In the case of the marine fish, excretion of the sodium 
and chloride ions taken in via the mouth appears to be accomplished by the gills 
(Black, 1957). It is therefore interesting to note that these structures do not 
accumulate radiomercury under the present experimental conditions. Certain com- 
pounds of mercury are concentrated by the mammalian kidney to the same degree as 
is chlormerodrin mercury without affecting the excretion of water, sodium, or 
chloride (Kessler ct al., 1957). The present experiments with marine fish furnish 
evidence that the accumulation of mercury against a concentration gradient is a 
process which may occur in tissues not usually involved in sodium and chloride 
excretion. Studies in progress with the nephridia of Phascolosonia gouldi (Greif, 
1957) are in agreement with this evidence. 

SUMMARY 

1. Chlormerodrin labelled with radiomercury has been injected into marine fish 
with different types of nephrons. The kidneys of flounder (Paralycthys dcntatus), 
with glomerulus and proximal tubular segment, of dog fish (Mustdits canis), with 
proximal, distal segment, and glomerulus, and toadfish (Opsanus tail}, with prox- 
imal segment only, all concentrate mercury to a high degree. 

2. High mercury U/P ratios in the toadfish indicate mercury excretion by a 
secretory mechanism. Excised flounder tubules will not concentrate phenol red 
if the animal has previously been injected with a toxic amount of chlormerodrin. 
Accumulation of mercury is also noted under certain conditions in the bile, especially 
in the dogfish, but is not seen in either stomach or gills. 

3. Evidence is reviewed that in many animal species mercury accumulation is 
greatest in the proximal tubular segment. 

LITERATURE CITED 

BIETER, R. N., 1933. Further studies concerning the action of diuretics upon the aglomerular 
kidney. /. Pharmacol. Exper. Therap., 49 : 250-256. 

BLACK, V. S., 1957. In: The Physiology of Fishes. Editor M. E. Brown, Academic Press, 
New York, Volume I, p. 188. 

BORGHGRAEF, R. R. M., R. H. KESSLER AND R. F. PITTS, 1956. Plasma regression, distribution 
and excretion of radiomercury in relation to diuresis following the intravenous ad- 
ministration of Hg 203 - labelled chlormerodrin to the dog. /. Clin. Invest., 35: 1055- 
1066. 

FORSTER, R. P., AND S. K. HONG, 1958. In vitro transport of dyes by isolated renal tubules of 
the flounder as disclosed by direct visualization. Intracellular accumulation and trans- 
cellular movement. /. Cell. Comp. Physiol., 51 : 259-272. 

FORSTER, R. P., AND J. V. TAGGART, 1950. Use of isolated renal tubules for the examination 
of metabolic processes associated with active cellular transport. /. Cell. Comp. Physiol., 
36: 251-270. 

GIEBISCH, G., AND P. J. DORMAN, 1958. Comparative study of uptake and distribution of 
Hg 203 given as labelled chlormerodrin. Proc. Soc. Exper. Biol. Med., 98: 50-52. 

GREIF, R. L., 1957. Uptake of a radiomercury labeled diuretic (Chlormerodrin) by the 
nephridia of Phascolosoma gouldi. Biol. Bull., 113: 327. 

GREIF, R. L., W. J. SULLIVAN, G. S. JACOBS AND R. F. PITTS, 1956. Distribution of radio- 
mercury administered as labelled chlormerodrin in the kidneys of rats and dogs. f. 
Clin. Invest., 35 : 38-43. 



MERCURIAL DIURETIC IN MARINE FISH 257 

KEMPTON, R. T., 1943. Studies on elasmobranch kidney. I. The structure of the renal tubule 

of the spiny dogfish Squalus acantliias. J. Morphol., 73 : 247-263. 
KESSLER, R. H., R. LOZANO AND R. F. PITTS, 1957. Studies on the structure diuretic activity 

relationships of organic compounds of mercury. /. Clin. Invest., 36 : 656-668. 
KESSLER, R. H., K. HIERHOLZER, R. S. GURD AND R. F. PITTS, 1958. Localization of diuretic 

action of chlormerodrin in the nephron of the dog. Amer. J. Physiol., 194 : 540-546. 
LUDWIG, E., AND E. ZILLNER, 1890. tber die Localisation des Quecksilbers im thierischen 

Organismus nach Vergiftungen mit Aetzsublimat. IVien. Klin. Wchnschr., 3: 534. 
MARSHALL, E. K., 1929. The aglomerular kidney of the toadfish Opsanus tau. Bull. Johns 

Hopkins Hasp., 45 : 95-100. 
SAXL, P., AND R. HEILIG, 1920. Uber die diuretische Wirkung von Novasurol und anderen 

Quecksilber-injectionen. Wien. Klin. Wchnschr., 33: 943-944. 
SHANNON, J. A., 1938. The renal excretion of phenol red by the aglomerular fishes Opsanus 

tau and Lophius piscatorius. J. Cell. Comp. Physiol., 11 : 315-323. 

SMITH, H. W., 1953. From Fish to Philosopher. Little, Brown and Co., Boston, p. 36. 
WALKER, A. M., AND J. OLIVER, 1941. Methods for the collection of fluid from single glomeruli 

and tubules of the mammalian kidney. Amer. J. Physiol., 134: 562-595. 
WOODMAN, W. B., AND C. M. TIDY, 1877. Forensic Medicine and Toxicology. Lindsay and 

Blakiston, Philadelphia, Pa., p. 210. 



FURTHER EVIDENCE OF THE DESTRUCTION OF 
BIVALVE LARVAE BY BACTERIA 1 

ROBERT R. L. GUILLARD 2 
The Oyster Institute of North America. Inc. 

Loosanoff (1954) has discussed the significance of large-scale culture of larval 
oysters and hard clams (Venus mercenaria) for experimental purposes and potential 
commercial use. It has been found that various organisms the fungus Sirolpidium, 
for example can destroy larvae in such cultures. However, evidence that bacteria 
are injurious has been largely circumstantial. Davis and Chanley (1956) showed 
that larval mortality was decreased at times by the use of various antibiotics. Walne 
(1956, 1958) found that antibiotics brought about increased spatfall of European 
oysters and parallel decrease of the bacterial population in his culture vessels. 

Davis (1950, 1953), as part of feeding experiments, fed fourteen species of 
bacteria (including a mixture of B. coll and a bacteriophage) to oyster larvae. 
There was no evidence that any bacteria were of value as food. The four species 
used in the first experiments (Vibrio marinofulvus, Micrococcus niaripuniceus, 
Bacillus imomarinus, and a red sulfur bacterium) were harmful at (unspecified) 
high concentrations, but not at low ones. However, three phytoflagellates studied 
produced the same results. In the second experiments, larvae fed bacteria (at 
unspecified concentrations) died within eleven days, while unfed controls grew 
slightly. Apparently mortality was not catastrophic. In this connection, ob- 
servations by ZoBell and Feltham (1938) are significant. They found that adult 
mussels survived and grew when fed 10 s to 10 9 washed bacterial/ml, once a day for 
nine months. Thirty-one clones were used. However, when a non-toxic peptone 
was added to water containing mussels, the animals died when the concentration of 
bacteria was only of the order of 10 r '/ml. They suggested that metabolites in 
actively growing cultures were responsible, but it is possible that a different flora was 
selected by the addition of fresh nutrient solution. Brisou (1955) points out that 
Pseudomonas-like organisms are common in living bivalves. Takeuchi ct <?/., 
(1957) reported a high mortality of adult Ostrca gigas caused by or associated 
with a bacterium of the genus Achromobacter. 

This report presents evidence that two clones of bacteria isolated from an in- 
fected hard clam (Venus niercenaria) larva destroyed healthy larvae, while other 
clones did not under similar experimental conditions. In the final experiments 
larvae were reared under aseptic conditions up to the time of exposure to known 
bacteria, thus excluding the possibility that contaminating microorganisms were 
the direct cause of death, while the bacteria were secondary invaders. 

1 This work was carried out at the Biological Laboratory, U. S. Fish and Wildlife Service. 
Milford, Connecticut. 

2 Present address : Woods Hole Oceanographic Institution, Woods Hole, Massachusetts. 

258 



BACTERIA AND VENUS LARVAE 259 

I am grateful for the cooperation of Harry C. Davis, Paul Chanley, and Spofford 
Woodruff, of the U. S. Fish and Wildlife Service, Milford, Connecticut. 

MATERIALS AND METHODS 

Isolation and growth of bacteria 

Bacteria were seen "swarming" about moribund clam larvae in a laboratory 
culture having heavy mortality. One larva was transferred to a tube of sea water 
broth (essentially medium STP of Provasoli et al., 1957), and the resulting mixed 
flora was subcultured daily. Pour and streak plates were made on the second day. 
Although most colonies appeared to be of two types, twelve obviously different 
clones were isolated. Ten other clones were isolated from contaminated algal 
cultures (Monochrysis lit t her i or Isochrysis galbana) or from niters through which 
laboratory sea water was passed. Cultures of mixed bacteria were obtained by 
adding raw sea water to sterile broth. 

Bacteria were grown at 28.5 C. for about 24 hours, with resulting concentra- 
tions of most clones of the order of 10 9 /ml. STP broth was used in some of the 
first studies, but the medium was later standardized to % strength (Difco) nutrient 
broth made with sea water. In Experiment 2(2) bacteria were also grown in a 
clam broth made by autoclaving a minced adult hard clam in its own volume of 
sea water and decanting the supernatant. Suspensions of bacteria were diluted 
and samples killed and stained with L-KI and counted in a Petroff-Hausser 
chamber. 

Filtrates of bacterial cultures (Experiment 2(3)) were prepared by drawing a 
few milliliters through sterile ultra-fine fritted glass filters. Filtrate was proved 
sterile by plating and inoculation into broth. 

Bacteria were killed (Experiment 2(4)) by heating to 85 C. for five minutes. 
Cultures so heated did not grow upon subculture or streaking . 

The concentration of bacteria to be used in the final experiments was estimated 
from measurement of concentrations in preliminary studies, which were of the order 
of 10 6 /ml, and from observations made at various times during 1957-1958 of con- 
centrations in apparently healthy larval clam and oyster cultures. In plate counts 
from 24-48-hour larval cultures (made on STP agar, ZoBell's No. 2216 agar 
(ZoBell, 1941), or % strength nutrient agar) bacterial concentrations were 10 5 -10 6 
per ml. However, counts of some of these cultures with a Petroff-Hausser chamber 
and dark field or phase contrast illumination yielded numbers of motile or clearly 
recognizable bacteria about an order of magnitude higher. (Concentrations in 
freshly changed cultures were of the order of 10 3 -10 4 per ml., determined by plat- 
ing.) Walne (1958) measured 10 4 -10 5 /ml. in 24-hour laboratory cultures of 
European oysters by plating on ZoBell's No. 2216 agar counted at 48 hours. It 
may be assumed that the actual concentrations were an order of magnitude higher. 

Bacterial concentrations used in the final experiments were 10 to 10 7 per ml., 
provided from dense liquid cultures. Dilution made carryover of nutrients in- 
significant. (Because larvae had been observed to survive exposure to bacterial 
concentrations of the order of 10 s per ml., this also was tried in Experiment 1. It 
was anticipated that larvae might be resistant to the bacteria because of pretreatment 
with antibiotics.) 



260 ROBERT R. L. GUILLARD 

Preliminary assays with non-aseptically-r eared larvae 

Bacterial cultures were assayed by adding 1- to 4-ml. aliquots yielding 10 6 to 
10 7 bacteria per ml. to liter cultures of healthy larvae in freshly changed filtered sea 
water. (Methods of handling larvae are cited in a later section.) Larvae were 
maintained at a concentration of ca. 10/ml. at 24 C. and fed bacteria-free Isochrysis 
galbana, ca. 5 X 10 4 /ml. 

After 18-30 hours larvae were concentrated by screening and swirling and 
examined in a Sedgwick-Rafter cell with a compound microscope (X 150). Be- 
cause the basic purpose of these assays was to screen bacterial cultures for obviously 
virulent strains, quantitative counts of mortality were not generally made. Rough 
quantitative counts were easily made by counting fewer than ten fields. 

It was shown that the sterile nutrient broths were non-injurious to larvae at 
the concentrations used. This was done by adding sterile broth plus the antibiotic 
mixtures described below. 

In each experiment there were two sets of control larvae ; one received only food, 
the other received also an aliquot of sterile broth. In a few experiments bacteria 
developing in this latter beaker during the experimental period destroyed the larvae. 
These experiments were discarded even though the same flora might not have 
developed in the assay beakers. 

Assay with aseptically-r eared larvae 

Straight-hinge clam larvae were obtained free of bacteria by allowing fertilized 
eggs to develop in solutions of antibiotics shown to be harmless to the animals. In 
the first experiment 50 mg./l. each of penicillin G (1625 units/mg.) and strep- 
tomycin sulfate were used. These concentrations were doubled in the second 
experiment and 50 mg./l. of chloramphenicol added. Oppenheimer (1955) showed 
that similar mixtures reduced viable bacteria in sea water to the order of a few 
per ml. in 24 hours. In our experiments, because adult clams were spawned in 
sterile sea water and larvae isolated with a micropipette after exposure to antibiotics, 
chances of contamination were negligible. No bacteria were detected by isolating 
larvae into sea water nutrient broth. Antibiotics carried over in the isolation 
technique were insufficient to prevent growth of bacteria. 

Procedure 

Sea water was autoclaved. Spawning dishes and screens were sterilized with 
ethanol and rinsed. Adult clams of known sex were washed in warm tap water, 
rinsed, and spawned by methods described or referred to by Davis (1953). Water 
was changed if clams did not spawn within a few hours. Fertilized eggs were passed 
through a 100-mesh screen to free them of feces and pseudofeces, then washed on a 
325-mesh screen to free them of excess sperm and to concentrate them. After re- 
suspension and counting, about 2000 eggs were added to 100 ml. of sterile filtered 
antibiotic mixture in 250-ml. Ehrlenmeyer flasks and kept at 24 C. Twenty-four or 
36 hours later the fluid was poured into sterile Petri dishes and apparently healthy 
larvae caught with a pipette under a dissecting microscope. 



BACTERIA AND VENUS LARVAE 261 

Thirty to forty larvae were put into each of a series of 20 X 125-mm. screw- 
capped culture tubes containing 5 ml. of sterile sea water, and fed bacteria-free 
Isochrysis galbana at a concentration of 10 : '/ml. (Control larvae in unchanged 
water in these tubes went to metamorphosis, but more slowly than those kept in 
containers as described by Davis and Guillard, 1958.) Aliquots of counted bac- 
terial suspensions were added by pipette. 

Motility of larvae could be observed through the test tube walls. At the end of 
the experiments, clams were again poured into a Petri dish and re-isolated onto a 
slide, where each w r as examined with a compound microscope using dark field and 
phase contrast illumination as necessary. 

EXPERIMENTAL 
Assay with non-aseptically-reared larvae 

Mortality of larvae exposed for 18-30 hours to the mixed culture derived from 
a moribund clam and to the five succeeding subcultures from it was greater than 
90%. "Swarming" bacteria were numerous in moribund animals and were seen 
swimming freely. No comparable mortality was observed in five trials involving 
the ten clones derived from contaminated algal cultures or the mixed bacteria re- 
sulting from the enrichment of sea water. (Not all clones were included in each 
of the trials.) Larvae were able to survive in concentrations as high as 10 8 /ml. of 
some of these clones, though they developed abnormally in the higher concentrations. 
Mortality was negligible in both sets of controls in all these trials. 

, Two assays were made of the twelve clones isolated from the moribund clam. 
Ten of these caused no significant mortality, but the other two produced mortality 
comparable to that of the mixture from which they were derived. These clones, 
designated 6-1 and 13-1, were the two colony types predominating on agar plates 
of the mixed culture. Both are gram-negative, non-sporogenous, polar monotri- 
chous rods about 0.75 X 1-2.5 microns in size. Both are halophilic to some extent 
and have not become adapted to growth on media without NaCl or sea water. The 
temperature optimum of 6-1 is between 35 and 40 C., while that of 13-1 lies 
between 25 and 32.5 C. Dr. Einar Liefson of Loyola University has undertaken 
further study of both strains. He has assigned 6-1 to the genus Vibrio and 13-1 
to Pscudonwnas. Some criteria are given in Hugh and Leifson (1953). 

Experiment showed that neither 6-1, 13-1, nor the mixture from which they 
came could injure larvae in the presence of antibiotics. Duplicate larval cultures 
were inoculated with ca. 10 7 bacteria per ml. ; to one set was added also 50 mg./l. 
each of penicillin G and streptomycin sulfate. Animals exposed to bacteria alone 
were destroyed in 24 hours, while those treated with antibiotics also were indis- 
tinguishable from controls exposed to neither or to antibiotics alone. This was 
done twice with each bacterial culture. 

Experiments zvitJi ascptically-reared larvae 

Experiment 1. This was undertaken primarily to test the method and to 
determine if bacteria at the concentrations used would kill larvae previouslv ex- 
posed to antibiotics. The experiment proper consisted of eight tubes, as follows : 



262 



ROBERT R. L. GUILLARD 



1. controls, sea water 

2. controls, nutrient broth 

3. clone 13-1, 10 6 /rnl. 

4. clone 13-1, 10 7 /ml. 

5. clone 13-1, 10 8 /ml. 

6. clone 6-1. 10 6 /ml. 

7. clone 6-1, 10 7 /ml. 

8. clone 6-1, 10 8 /ml. 

There were also five tubes in which larvae were grown to metamorphosis. 

Examination through the tube walls showed that most larvae were killed in a 
clay in the highest concentration of each clone. The other tubes were examined on 
the fourth day. Results are summarized in Table T. Larvae exposed to 10 7 /ml. 

TABLE I 

Mortality of clam larvae caused by three concentrations of clones 6-1 and 13-1 
(larvae initially 115-120 microns in size) 



Clone 


Bacteria 


Time 


No of 
larvae 


No. of 
larvae 


%of 
larvae 


Notes 




per ml. 




moribund 


recovered 


moribund 




13-1 


10 8 


1 day 


26 


27 of 30 


96 


vela disintegrating 


13-1 


10 6 


4 days 


3 


29 of 30 


10 


none over 135 /x 


13-1 


10 7 


4 days 


38 


40 of 40 


95 


none over 135 n 


6-1 


10 8 


1 day 


27 


32 of 35 


84 


none over 125 /x 


6-1 


10" 


4 days 


22 


29 of 30 


76 


largest were 160 /u; dead 














larvae were 120-145 M 


6-1 


10 7 


4 days 


11 


11 of ? 


100 - 


vela swollen ; none over 














135 M 


Control 


none 


4 days 





30 





135-160 yu* 



* Another group of larvae from the same spawning was kept in polyethylene containers, fed 
every day and changed every other day. The largest of these larvae were 190 /j. 

of either clone were 95%-100% destroyed by the fourth day. However, 10 6 /ml. 
of 13-1 caused only 10% mortality, while a like number of 6-1 killed 76%. No 
control animals died. 

Bacteria were not counted during or after the experiment. Judging subjectively, 
there were not as many in the medium at the end as might have expected on the 
basis of the number added. Some of the moribund larvae were surrounded by 
"swarming" bacteria. 

Experiment 2. The objectives were: 

1. To compare the effects of bacteria on larvae kept at three different tem- 
peratures; 20, 25. and 30 C. The observation that clones 6-1 and 13-1 grew 
better at high temperatures than did most other clones isolated suggested this 
portion of the experiment. 

2. To compare the effects on larvae of equal numbers of bacteria grown on two 
different media the usual nutrient broth and clam broth. The possibility that 
bacteria maintained on clam broth might remain more virulent than those maintained 
on nutrient broth prompted this portion of the experiments. 



BACTERIA AND VENUS LARVAE 263 

3. To observe larvae exposed to sterile filtrate of bacterial cultures. 

4. To observe larvae exposed to dead bacteria plus tbeir culture broth. Experi- 
ments 3 and 4 were to confirm that only living bacteria kill larvae, as suggested by 
the antibiotic experiment of Section 1. The experiment was carried out in 20 
tubes, as follows : 

Temperature 
Tube C. 

1. Control, food only 25 

2. 6-1, 10 7 /ml. (broth-grown) 25 

3. 13-1, 10 7 /ml. (broth-grown) 25 

4. Control, food only 20 

5. 6-1, 10 7 /nil. (broth-grown) 20 

6. 13-1, 10 7 /ml. (broth-grown) 20 

7. Control, food only 30 

8. 6-1, 10 7 /rnl. (broth-grown) 30 

9. 13-1, 10 7 /ml. (broth-grown) 30 

10. 6-1, grown in clam broth, 10 6 /ml. 25 

11. 6-1, grown in clam broth, 10 7 /ml. 25 

12. 13-1, grown in clam broth, 10 6 /ml. 25 

13. 13-1, grown in clam broth, 10 7 /ml. 25 

14. 6-1 filtrate. 1 ml. (equivalent to 1 6 X 10 9 bacteria/ml.) 25 

15. 13-1 filtrate, 1 ml. (equivalent to 3.2 X 10 9 bacteria/ml.) 25 

16. Heat-killed 6-1 (8 X 10"/ml.') 25 

17. Heat-killed 13-1 (8 X 10 8 /rnl.) 25 

18. Larvae in broth alone as sterility check 25 

19. Larvae in broth alone as sterility check 25 

20. Larvae in broth alone as sterility check 25 

Larvae were examined on the fifth day. In Table II and Table III, respectively, 
are gathered data pertinent to the temperature portion of the experiment and 
comparison of the effects of bacteria grown on different media. 

Mortality was relatively independent of the temperature at which larvae were 
kept: 73% to 76% of the animals exposed to clone 6-1 were dead, as were 30% 
to 48% of those exposed to clone 131. Maximum mortality in controls was 7%. 
It should be noted that growth in the 30 C. controls was poor and that food 
organisms settled in the tubes at this temperature. (Both 20 and 30 controls 
were bacteria-free at the end of the experiment.) 

From Tables I and II it can be seen that there were no consistent differences in 
mortality caused by bacteria grown on the two different media. Greatly increased 
virulence would have been evidenced by early heavy mortality easily visible through 
the culture tube walls. Small but significant differences would not be detected by 
an experiment such as this. 

The hypothesis that only living bacteria destroy larvae was confirmed by the 
findings in tubes 14 through 17, in which larvae were exposed to filtrate correspond- 
ing to more than 10 y bacteria per ml. or to 8 X 10 8 dead bacteria in their broth. 
Of 120 larvae, only two were dead, one in tube Xo. 15 and one in Xo. 17. How- 



264 



ROBERT R. L. GUILLARD 



TABLE II 

Comparison of mortality of larvae exposed to bacteria and maintained at three different temperatures 
for five days (concentration of bacteria 10 1 /ml. Larvae initially 105-110 microns in size) 









Average size 


Maximum size 


Clone 


Temp. 
C. 


% larvae 
moribund 




















Living 


Moribund 


Living 


Moribund 


6-1 


20 


73 


125 


120 


145 


125 


6-1 


25 


75 


130 


115 


150 


125 


6-1 


30 


76 


120 


120 


130 


120 


13-1 


20 


30 


150 


125 


165 


150 


13-1 


25 


46 


145 


125 


160 


140 


13-1 


30 


48 


135 


120 


185 


135 


None 


20 


7 


145 





165 





\one 


25 





140 





165 





None 


30 


3 


125 





140 






ever, larvae grew scarcely at all. They were initially 105-110 microns in size, and 
increased only to 110-120 microns. (The larger animals seen in control tubes 
were 165 microns in size, see Table III.) Moreover, larvae exposed to filtrate or 
dead bacteria were not feeding and were emaciated. This was probably due to the 
high concentration of metabolites. In the preliminary assays (Section 1) in which 
larvae were exposed to living bacteria together with antibiotics, those animals ex- 
posed to both bacteria and antibiotics were indistinguishable from controls, but the 
amount of inoculum in this case corresponded only to 10"-10 r bacteria/ml, rather 
than to lOyml. 

In both experiments 1 and 2, moribund larvae injured by clone 6-1 differed in 
appearance from those injured by 13-1. The former often had vela so distended 
that they exceeded the bodies of the larvae in length. Some vela appeared to be 
decomposing and occasionally they detached from the rest of the clam. The bodies 
of larvae often had striations visible going from the hinge towards the velum. 

Clams injured by 13-1 usually had vela that were frayed or ragged, with cilia 
largely or entirely missing. The bodies were emaciated and granular in appearance. 
Some seemed to be disintegrating from the hinge side towards the velum. Mori- 

TABLE III 

Comparison of the mortality at 5 days of larvae exposed to bacteria grown in (a) 2/3 strength nutrient 
broth and (b) clam broth (larvae initially 105-110 microns in size) 











Average size 


Maximum size 


Clone 


Medium 


Bacteria 
per ml. 


% larvae 
moribund 






















Alive 


Moribund 


Alive 


Moribund 


6-1 


nutrient 


10 7 


75 


130 


115 


150 


125 


6-1 


clam 


10 6 


40 


135 


125 


150 


135 


6-1 


clam 


10 7 


97 


120 


120 


120 120 


13-1 


nutrient 


10 7 


46 


145 


125 


160 


140 


13-1 


clam 


10 6 


59 


135 


120 


140 


135 


13-1 


clam 


10 7 


48 


135 


120 150 


135 


Controls 




none 


none 


140 





165 






BACTERIA AND VENUS LARVAE 265 

bund larvae in laboratory cultures often fit one of these descriptions, but it is not 
known if the appearance is in fact correlated with an infecting bacterium. 

DISCUSSION 

While the mechanism by which clones 6-1 and 13-1 destroy larvae was not 
studied, evidence available favors the hypothesis that it is by invasion or at least 
contact rather than by an exotoxin liberated into the medium. The fact that larvae 
withstood relatively large amounts of glass-filtered, heat-killed, or antibiotic-treated 
bacterial culture shows that an exotoxin, to be the sole agent, would have also to 
be extraordinarily unstable. Further, an exotoxin would be expected to have a 
relatively uniform influence on animals exposed to it, so that larvae would be more 
or less uniform in size. In fact, however, larvae exposed to bacteria varied con- 
siderably in size (Tables II and III), rather in keeping with the hypothesis that 
they continued to grow until invaded. Finally, there are observations that mortality 
in large cultures often followed the pattern of an epizootic, and that bacteria were 
frequently seen swarming in dying or dead larvae. 

It is not implied that bacterial metabolites are without influence on larval 
growth or development. Indeed, the experiment showed that high concentrations 
stopped growth entirely. It has also been observed that bacterial contamination 
of algal food cultures sometimes caused abrupt decrease in larval growth rate 
without immediate extensive mortality. The addition of cultures of bacteria (other 
than 6-1 or 13-1) often did the same. This depressant effect may well be due to 
exotoxins. 

Strains 131 and 6-1 were far more virulent than other bacteria tested and 
clearly are a hazard to larvae under laboratory conditions. Possibly they were 
favored by conditions in the larval cultures and finally dominated the flora, at which 
point the "disease" became obvious. The observation that both strains grow well 
at temperatures over 30 C., which is relatively uncommon in marine bacteria 
found locally, supports this idea. At present it is not possible to tell if these bac- 
teria destroy larvae in nature, where both bacteria and larvae are usually less 
concentrated than they are in cultures. If the "disease" occurs in nature, one 
would expect to find it under conditions of high temperature and restricted water 
exchange. 

It should be mentioned that the use of antibiotics to control bacteria in larval 
cultures is apparently more effective when the water supply is changed regularly 
and two or more antibiotics are used alternately. Probably this prevents the de- 
velopment of a resistant flora. Animals other than bivalves are also benefited; J. 
Hanks (personal communication) found that larvae of the gastropods Poliniccs 
ditphcata and P. licros grew better when penicillin and streptomycin were used in 
this way. It must be emphasized, however, that the same antibiotics will not 
prevent growth of injurious bacteria in algal cultures used as food. If impure 
algal cultures must be used to raise larvae, the algae should obviously be kept at 
the lowest temperature allowing reasonable growth. 

SUMMARY 

1. Twelve strains of bacteria were isolated from a moribund Venus mercenaria 
larva in a laboratory culture. These, ten other clones, and mixed bacteria from 



266 ROBERT R. L. GUILLARD 

sea water were assayed by adding broth culture yielding 10 t; -10 7 cells/ml, to beaker 
cultures of healthy clam larvae. Only the mixed bacterial culture from the mori- 
bund larva and two of the 12 strains isolated from it caused extensive mortality. 
One of the virulent clones (6-1) is a species of Vibrio, the other (13-1) is a 
Pseudovnonas species. 

2. Larvae exposed to virulent bacteria and simultaneously treated with anti- 
biotics were as healthy as controls, showing that active bacteria were necessary to 
destroy larvae 'and that metabolites in the bacterial inoculum were not harmful to 
larvae. 

3. Larvae were grown free of contaminating micro-organisms by allowing 
washed eggs to develop in antibiotic solutions and then isolating straight-hinge 
larvae by pipette. Either virulent clone ( 10 t; -10 7 /ml. ) destroyed 10-100% of such 
larvae. However, exposing the animals to large amounts of glass-filtered or 
heated broth in which bacteria had been grown (corresponding to ca. 10'' bac- 
teria/ml.) caused no mortality, but retarded growth. 

4. Mortality caused by clones 6-1 and 13-1 in groups of clams kept at 20, 
25, and 30 C. did not vary significantly. However, both virulent clones grow 
well at 30 C. and higher ; thus high temperatures in laboratory larval cultures 
favor these strains. 

LITERATURE CITED 

BRISOU, J., 1955. La Microbiologie du Milieu Marin. Editions Medicales Flammarion. Paris, 

France. 

DAVIS, HARRY C., 1950. On food requirements of Ostrca t'irt/itiica. Anat. Kcc., 108: 620. 
DAVIS, HARRY C., 1953. On food and feeding- of larvae of the American oyster, C. riri/inica. 

Bio!. Bull., 104 : 334-350. 
DAVIS, HARRY C., AND P. E. CHANLEY, 1956. Effects of some dissolved substances on bivalve 

larvae. Proceed. National ShcIIfishcrics Assoc., 46 : 59-74. 
DAVIS, HARRY C., AND R. R. GUILLARD, 1958. Relative value of ten genera of micro-organisms 

as food for oyster and clam larvae. L'. $'. Fish & IVildl. Sen 1 ., Fish. Bull., 58 (136) : 

293-404. 
HUGH, RUDOLPH, AND EINAR LEIFSON, 1953. The taxonomic significance of fermentative 

versus oxidative metabolism of carbohydrates by various gram negative bacteria. 

/. Bact.. 66: 24-26. ; 

LOOSANOFF, V. L., 1954. New advances in the study of bivalve larvae. Autcr. Scicn.. 42: 

607-624. 
OPPENHEIMER, C. H., 1955. The effect of marine bacteria on the development and hatching 

of pelagic fish eggs, and 'the control of such bacteria by antibiotics. Copciu. 1955: 

43-49. 
PROVASOLI, L., J. J. A. MCLAUGHLIN AND M. R. DROOP, 1957. The development of artificial 

media for marine algae. Arch. MikrobioL, 25: 392-428. 
TAKEUCHI, T., T. MATSUBARA, H. HIROKAWA AXD A. TSUIKIYAMA, 1957. Bacteriological 

studies on the unusually high mortality of Ostrca yigas in Hiroshima Bay. III. Bull. 

Japanese Soc. Sci. Fish., 23 (1) : 19-23. 

WALNE, P. R., 1956. Bacteria in experiments on rearing oyster larvae. Nature. 178: 91. 
WALNE, P. R., 1958. The importance of bacteria in laboratory experiments on rearing the 

larvae of Ostrca edulis. J. Mar. Biol. Assoc., 37: 415-426. 
ZoBELL, C. E., 1941. Studies on marine bacteria, I. The cultural requirements of heterotrophic 

aerobes. /. Mar. Res., 4: 42-75. 
ZoBELL, C. E., AND C. B. FELTHAM, 1938. Bacteria as food for certain marine invertebrates. 

/. Mar. Res., 1 : 312-327. 



NEUROSECRETORY CELLS IN GANGLIA OF THE ROACH. 

BLABERUS CRANIIFER 1 

SEMAHAT GELDIAY 

Ih'piirtinciit of Zoolo(/v, ['iiircrsitv of Ankara, Turkc\, and Columbia University 

New York 27, X. Y. 

Neurosecretory cells have been described in many groups of insects ( Scharrer 
and Scharrer, 1954). Most observations have been made upon brain, subesophageal 
and frontal ganglia, and corpora cardiaca. There is relatively little information 
concerning neurosecretion by cells within the thoracic and abdominal ganglia of 
insects. Day (1940) described neurosecretory cells in the abdominal ganglia of 
Lepidoptera. and Kopf (1957) found neurosecretory cells in the thoracoabdominal 
ganglion of Drosoplula. E. Thomsen (1954) found no evidence that neurosecretory 
materials were accumulating on either side of ligatures of abdominal connectives in 
Calliphora and concluded that these connectives did not carry neurosecretory 
products. 

During an investigation of the functional roles of the neurosecretory materials 
accumulated within the corpora cardiaca of the roach, Blabcnis (Ozbas and Hodg- 
son. 1958), the search for appropriate controls, i.e., tissues which would contain 
only non-secretory neurons, prompted an examination of the thoracic and abdominal 
ganglia of this species. It immediately became apparent that neurosecretory cells 
of more than a single type were present in all of these ganglia. Since the existence 
of neurosecretory cells had not been previously reported within the thoracic or 
abdominal ganglia of Blattaria, and since this order of insects is commonly utilized 
in experiments on neurosecretion, it appeared worthwhile to make a detailed study 
of the neurosecretory cells at these hitherto unstudied loci. 

The object of the present report is to describe the types of neurosecretory cells 
present in the thoracic and abdominal ganglia of Blabcnis, and in so far as it is 
possible, to trace the movements of secretions produced by these cells, using evi- 
dence from ligation and histological studies. The relationships of the neurosecretory 
cells within the ganglia to secretory cells located elsewhere within the nervous 
system are also considered. 

MATERIALS AND METHODS 

The last three larval instars and adults of Blabcnis craniifcr were studied. No 
significant differences correlated with sex or developmental stages were found 
among specimens of this group. Only adults were used in ligation experiments 
because their larger size afforded technical advantages for the operations. 

Two fixatives and two stains were used on each type of preparation, so that by 

1 This investigation was aided by U. S. Public Health Service Grant No. E-2271 to 
Dr. Edward S. Hodgson. 

267 



268 



SEMAHAT GELDIAY 



comparing the results of the various methods it was possible to rule out artifacts 
produced by any one fixative or stain. Either Bouin or Helly fixatives were used. 
The stains were the chrome hematoxylin phloxin stain of Gomori (1941), hereafter 
designated CHP, or the aldehyde fuchsin stain (Gomori, 1950) as modified by 
Halmi (1952) and Dawson (1953), hereafter designated AF. All sections were 
cut 5 microns in thickness. 

Ligatures were prepared from Clark's Size A black nylon thread. A single 
strand was teased from this thread and tied around one of the two connectives 
which pass between the ganglia. The operations were performed through incisions 
in the ventral sides of unanesthetized animals, and the ligature sites were varied in 
different animals so that blocks of connectives between each of the thoracic and 
abdominal ganglia were represented in the total series of 44 operated animals. The 
operated roaches were maintained in separate pint jars and fed dogfood, fruit, and 
water, this being the same diet given the standard laboratory colony of Blabcrus. 
The animals were sacrificed 5 to 40 days after the operation, the nerve cords dis- 
sected out, and histological preparations made by the methods outlined above. 







FIGURE 1. Diagrammatic representation of a frontal section through the center of a 
thoracic ganglion, showing arrangement of neurosecretory cells. The dotted lines separate the 
peripheral areas which are occupied by cell bodies from the central neuropile mass. Roman 
numerals designate types of neurosecretory cells see text. The arrow under the diagram 
points anteriorly, toward the animal's head. 



NEUROSECRETION IN BLABERUS 269 

RESULTS 
1. Neurosecretory cells witliin flic yanylia 

The thoracic and abdominal ganglia were found to contain three types of 
neurosecretory cells, as shown diagrammatically in Figure 1, and in the photographs 
A, B, and C of Figure 2. These cells can be differentiated from ordinary neurons 
by their size, staining characteristics, and the presence of characteristic granules or 
droplets within their cell bodies and axons. The cell bodies of all three types of 
neurosecretory cells measure 40 to 60 microns in their longest diameters, 35 to 50 
microns in their shortest diameters, and have ellipsoid nuclei measuring 15 to 18 
microns in their shortest diameters and 18 to 22 microns in their greatest diameters. 
The nucleoli are also conspicuous in many of the stained neurosecretory cells. 
The distinguishing characteristics of the three types of cells are described in the 
following paragraphs. 

Type I (see Fig. 2A). The cell bodies and axon hillocks of these cells contain 
granules which stain deep purple in AF and dark blue in CHP. There are 5 to 10 
such cells in each thoracic and abdominal ganglion, and they are located in the 
outer portion of the ganglion near the origin of the connectives (see Fig. 1 ). 

Type II (see Fig. 2B). These neurosecretory cells have very small granules 
distributed uniformly throughout the cell body. The granules stain green with AF 
and red with CHP. These are the most common neurosecretory cells in thoracic 
and abdominal ganglia, and there are at least several dozen cells of this type 
distributed generally throughout the periphery of each ganglion (see Fig. 1). 

Type III (see Fig. 2C). Only two neurosecretory cells of this type appear to 
be located within any one ganglion. They are seen only after Helly fixation and 
never after use of Bouin solution. The two cells are found in the anterior lateral 
part of the ganglion, one cell on each side of the ganglion (see Fig. 1). They are 
characterized by the presence of large droplets which stain orange with AF. These 
droplets have diameters of 3 to 11 microns. They resemble the "colloid droplets" 
described by E. Scharrer (1941) within the cells of the preoptic nucleus of the fish, 
Fundiilus. There was no indication of cycles of formation or alteration of the 
droplets in the roach sections such as reported in Fundulns. 

2. Observations on normal a.vons and connectives 

Neurosecretory materials from all three types of neurosecretory cells were 
observed within axons extending into connectives between ganglia. Small granules, 
such as found in type I cells, are usually found in no more than one or two axons 
per section of connective (Fig. 2F). These granules fill the axons, where found, 
and they appear to move in definite oriented chains. 

Secretory material from type II cells is best seen in axons after staining with 
CHP. This material is the most abundant in axons, and is seen in practically every 
section of connectives between ganglia. Orange material, presumably from type 
III cells, was seen in axons of only two animals. The rarity of this material in 
axons may reasonably be explained by the rarity of the type III cells. When ob- 
served in axons, the orange material was not in the form of round droplets, but 
had the form of elongated rods. 

Many glia cells were seen between axons in the connectives and also in the 
ganglia. These cells were described by B. Scharrer (1939) in the brain, sub- 



270 



SEMAHAT GELDIAY 




FIGURE 2. Histological preparations. A type I cell from metathoracic ganglion of adult 
male (Helly fixation with AF), 1080 X ; B type II cell from prothoracic ganglion of 
adult male (Bouin fixation with CHP), 1080 X ; C type III cell from mesothoracic ganglion of 
adult female (Helly fixation with AF), 1080 X ; D frontal section through connective between 
subesophageal ganglion and prothoracic ganglion (adult male, ligation on animal's right side). 
Seven days after operation. (Helly fixation with AF), 72 x ; F cross-section anterior to 
ligation in left connective between prothoracic and mesothoracic ganglia, control side on right, 
$ adult 20 days after operation (Helly fixation with AF), 144 x ; F frontal section of left side 
connective ligated and broken between subesophageal and prothoracic ganglia, ? adult, 10 days 
after operation (Helly with AF). N neurosecretory materials. G glia cells. L ligature. 



NEUROSECRETION IN BLABERUS 271 

esophageal ganglion, and connectives of Fcriplancia. The glia cells are spindle- 
shaped, have elongated nuclei, and contain gliosomes staining deep purple with AF 
and deep blue with CHP. In some cases, particularly when the nucleus of the glia 
cell is not seen, the glia cells might be confused with neurosecretory particles inside 
connectives, but the latter are in much longer chains and are larger particles than 
the gliosomes. Roles as supporting cells have been ascribed to the glia cells (B. 
Scharrer. 1939). 

3. Ligation experiments 

Since ligation of nerves containing neurosecretory axons has been shown to 
result in accumulations of neurosecretory materials on those sides of the ligations 
where the neurosecretory materials originate (E. Thomsen, 1954). ligation experi- 
ments were used to determine the directions in which the neurosecretory materials 
were moving. The ligation technique has been described above. In a total of 
44 ligation operations, 4 animals died between 10 hours and 12 days after the 
operation. In 24 cases, the ligated connective was found broken and the nylon 
thread found in the hemolymph in the vicinity of the operation site. In 16 cases, 
the connective was not broken and the thread was found still in place (see Fig. 2D). 

Whether or not the connective was broken, portions of connective both anterior 
and posterior to the break or ligation were swollen, as compared to the correspond- 
ing unoperated connectives (see Figs. 2D, 2E) . The operated connectives, as studied 
in histological sections, differed from connectives on the control sides in several 
other ways as well: (a) more glia cells were present on the experimental side, not 
only in the broken or ligated tips of the connectives, but along their entire lengths (see 
Fig. 2D) ; (b) neurosecretory products of types I and II were accumulated in axons 
on both sides of the breaks, not only localized at the broken or ligated areas, but in 
elongated masses accumulated within axons on both sides of the operation (Figs. 
2D, 2F) ; (c) no accumulation of type III neurosecretion was seen, doubtless be- 
cause of its rarity even in the normal connectives. 

It is therefore possible to conclude from the ligation experiments that neu- 
rosecretory materials from neurosecretory cell types I and II within the thoracic 
and abdominal ganglia pass in both anterior and posterior directions through the 
interganglionic connectives. Secretions from type III cells also pass into the con- 
nectives but are present in such limited quantities that their movements have not 
been intercepted in these ligation experiments. 

DISCUSSION 

With the steadily increasing numbers of neurosecretory cells being described, 
particularly as new histological procedures are adopted, it is obviously desirable 
to prevent unnecessary duplication of terminology. Consequently, a more extended 
comparison of the neurosecretory cells described here with types already described 
seems appropriate. The most exact resemblance is between the cells which are 
here designated type II and those designated type B by Nayar (1955) and Kopf 
(1957). These cells also appear identical to ones staining pink with CHP in the 
pars intercerebralis of Blabcrns (Ozbas and Hodgson, 1958). Cells of similar 
staining characteristics have also been seen in the pars intercerebralis of Diptera 
and Hymenoptera (E. Thomsen. 1954; M. Thomsen, 1954). The uniform dis- 



272 SEMAHAT GELDIAY 

tribution of granules within the cell bodies of all these neurosecretory cells and their 
staining characteristics strongly suggests that they might properly be considered as 
homologous neurosecretory cells in several groups of insects. 

Type I cells are most nearly comparable with, but not identical to, the cells 
designated type A by Nayar (1955) and Kopf (1957), and previously seen by 
other workers in the pars intercerebralis of many insects (Scharrer and Scharrer, 
1954; De Lerma, 1956; Dupont-Raabe, 1956; Ozbas, 1957). The similarities 
between type I and type A cells consist of the shapes of the cells and their staining 
properties with CHP. Nayar describes cytoplasm filled with neurosecretory ma- 
terial in type A cells, but in the preparations of thoracic and abdominal ganglia, 
the neurosecretory material is always near the edge of the cell body. This ar- 
rangement of granules within the cell body resembles that described by Scharrer 
(1955) in the "castration cells" of the subesophageal ganglia of Leucophaea. Nayar 
noted no selective staining of type A cells with AF. Although the AF stain used 
here is a later modification of the AF staining technique used by Nayar, typical A 
cells were seen in the pars intercerebralis of Blaberus. Cells of identical staining 
characteristics, but having cyclic secretory activity, were found in the subesophageal 
ganglion of Blaberus during the present study ; these cells have been described 
earlier by B. Scharrer (1941 ). The distinction between type I and Type A cells 
must, therefore, be a real one and not a dissimilarity caused by differences of 
staining technique. 

The type III cells described in this report have not been previously described 
in any insect. Although the droplets which they contain resemble the "colloid 
droplets" found in certain neurosecretory cells of Fundulus (E. Scharrer, 1941), 
this resemblance cannot be interpreted as implying identity of cell types in such 
widely divergent groups of animals. 

The conclusions from the ligation experiments are contrary to those drawn from 
ligation experiments on abdominal cords of Calliphora by E. Thomsen (1954). 
However, the Calliphora ligations were performed only as controls for other experi- 
ments and the results were not studied histologically. Since few axons in the 
abdominal connectives carry neurosecretory materials, it is not surprising that 
Thomsen did not find accumulations of neurosecretions in the ligated abdominal 
connectives which would be seen by inspection of the whole live connectives. The 
small quantity of neurosecretory material which would be detected in unligated 
abdominal connectives probably also explains why none was detected when observa- 
tions were made upon whole central nerve cords using darkfield illumination (M. 
Thomsen, 1954). 

Attempts to detect effects of neurosecretory products from thoracic and 
abdominal ganglia upon spontaneous electrical activity of Blaberus nerve cords 
in vitro, similar to the effects of neurosecretion from the corpora cardiaca (Ozbas 
and Hodgson, 1958), were unsuccessful. This may be due to the small amounts of 
neurosecretory materials present even in whole thoracic and abdominal ganglia. 
Moreover, the most abundant type of neurosecretory cells in these ganglia, type II, 
is relatively scarce in the pars intercerebralis, and all evidence seems to indicate 
that it is not their secretion which accounts for the effects of corpus cardiacum 
extracts upon spontaneous nerve activity (Ozbas and Hodgson, 1958). 

What may be postulated, then, as a normal function of the neurosecretions 



NEUROSECRETION IN BLABERUS 273 

produced in the ganglia and passing anteriorly and posteriorly through the inter- 
ganglionic connectives? Although the histological sections were always examined 
with such a possibility in mind, no clues were found in the form of any areas where 
neurosecretions from the ganglia were normally being accumulated. It must be 
assumed that the sites of release of the neurosecretory products are widely scattered 
throughout the central nervous system, regardless of where their effects are ulti- 
mately exerted. Concerning this problem of the normal functions of the neu- 
rosecretory cells here described, the present observations can really only be an 
introduction. 

This work was done in the Department of Zoology, Columbia University. The 
author wishes to express her thanks to Professor E. S. Hodgson for his help and 
encouragement during the course of the investigation and for his help in preparing 
the manuscript. 

SUMMARY 

1. Three types of neurosecretory cells are found within the thoracic abdominal 
ganglia of the roach Blabcrus craniifer. These three cell types may be characterized 
as follows : type I contains granules staining deep purple with aldehyde fuchsin, 
the granules being distributed around the periphery of the cell body and in the axon 
hillock ; type II the most common neurosecretory cell in the ganglia, contains very 
small granules staining red with chrome hematoxylin and phloxin, the granules 
being distributed throughout the cell body; type III the rarest of the neu- 
rosecretory cells observed within the ganglia, contains large droplets staining 
orange with aldehyde fuchsin. The possible identities or homologies of these cell 
types with others previously described in insects are discussed. 

2. Secretory products from all three types of neurosecretory cells have been 
found within axons in the connectives between ganglia. 

3. Ligation of the connectives between ganglia reveals that at least neurosecre- 
tory products of cell types I and II move in both anterior and posterior directions 
from the ganglia in which they are produced. Neurosecretion from cells of type 
III is very rarely seen in connectives and, consequently, its direction of movement 
could not be established. 

4. No areas of accumulation for any of the neurosecretions were found within 
the central nerve cords, and the normal functions of these secretions are not known. 

LITERATURE CITED 

DAY, M. F., 1940. Neurosecretory cells in the ganglia of Lepidoptera. Nature, 145 : 264. 
DAWSON, A. B., 1953. Evidence for the termination of neurosecretory fibers within the pars 

intermedia of the hypophysis of the frog, Rana pipicns. Anat. Record, 115: 63-69. 
DE LERMA, B., 1956. Corpora cardiaca et neurosecretion protocerebrale chez le Coleoptere 

Hydrous piccus L. Ann. Sc. Nat. Zool, 18 : 235-250. 
DUPONT-RAABE, M., 1956. Quelques donnees relatives aux phenomenes de neurosecretion chez 

les Phasmides. Ann. Sc. Nat. Zool., 18: 293-303. 
GOMORI, G., 1941. Observations with differential stains on human islets of Langerhans. 

Amer. J. Path., 17: 395-406. 
GOMORI, G., 1950. Aldehyde-Fuchsin : a new stain for elastic tissue. Aincr. J. Clin. Path., 20: 

665-666. 
HALMI, N. S., 1952. Differentiation of two types of basophils in the adenohypophysis of the 

rat and the mouse. Stain Tcchnol., 27 : 61-64. 



274 SEMAHAT GELDIAY 

KOPF, H., 1957. Uber Neurosekretion bei Drosophila. I. Zur Topographic und Morphologic 

neurosekretorischer Zentren bei der Imago von Drosophila. Biol. Zbl., 76: 28-42. 
NAYAR, K. K., 1955. Studies on the neurosecretory system of Iphita liinhata Stal. I. Distribu- 
tion and structure of the neurosecretory cells of the nerve rings. Biol. Bull., 108: 

296-307. 
OZBAS, S., 1957. Two kinds of secretions in corpora cardiaca of Locusta niiyratoria (L.) Ph. 

Solitaria. C oiiiin. Faculte Sci., Uuii'. Ankara, 8: 45-57. 
OZBAS, S., AND E. S. HODGSON, 1958. Action of insect neurosecretion upon central nervous 

system in vitro and upon behavior. Proc. Nat. Acad. Sci., 44: 825-830. 
SC'HARRER, B., 1939. The differentiation between neuroglia and connective tissue sheath in the 

cockroach (Pcriplancta aincricana) . J. Coinf>. N enrol., 70: 77-88. 
SCHARRER, B., 1941. Neurosecretion. II. Neurosecretory cells in the central nervous system 

of cockroaches. /. Comp. Ncnrol.. 74 : 93-108. 
SCHARRER, B., 1955. "Castration cells" in the central nervous system of an insect (Leucophaea 

madcrac, Blattaria). Trans. N. Y. Acad. Sci.. 17: 520-525. 
SCHARRER, E., 1941. Neurosecretion. I. The nucleus preopticus of J'ltiidnlns hctcroclitns L. 

/. Cornf. Ncnrol., 74: 81-92. 
SCHARRER, E., AND B. SCHARRER, 1954. Hormones produced by neurosecretory cells. Recent 

progress in hormone research. (Proc. Laitrcntian Hormone Conf.), 10: 183-240. 
THOMSEN, E., 1954. Studies on the transport of neurosecretory material in Calliphora erythro- 

cephala by means of ligaturing experiments. /. /:.r/>. Biol., 31 : 322-330. 
THOMSEN, M., 1954. Observations on the cytology of neurosecretion in various insects 

(Diptera and Hymenoptera ) . Puhhl. Stas. Zool. Napoli. 24: Suppl., 46-47. 



EXPERIMENTALLY INDUCED RELEASE OF N EURO SECRETORY 
MATERIALS FROM ROACH CORPORA CARDIACA 1 

E. S. HODGSON AND S. GELDIAY 

Jlcpiirtiiicnts of y.oolo<j\. Columbia I'nirersity, A'cu 1 York 27. .V. )'., and 

University of Ankara, Turkey 

Although the pars intercerebralis-corpus cardiacum system of insects has heen 
extensively used for experimental analysis of nevirosecretion (B. Scharrer, 1954; 
Wiggles worth, 1954), there are relatively few data to indicate the conditions under 
which this system normally releases neurosecretory suhstances during the life of the 
animal. Variations in the amount of neurosecretory materials within the corpora 
cardiaca are known to occur according to the age and the physiological conditions 
of insects (B. Scharrer. 1952; Wigglesworth, 1954). Variations in the potencies 
of corpora cardiaca extracts in affecting central nervous activity, as tested by the 
method of Ozbas and Hodgson (1958), have recently suggested that corpora cardiaca 
from roaches which have been extensively handled or subjected to prolonged surgical 
procedures contain significantly less neurosecretory material than normal. An 
analogy with the secretion of adrenalin during the response of mammals to stress 
situations is further suggested by the isolation from corpora cardiaca of a substance 
with adrenalin-like effects upon roach and frog hearts (Cameron, 1953; Unger, 
1957). 

The present experiments were designed to test the hypothesis that neurosecre- 
tory materials are released from the corpora cardiaca of the roach when the animal 
is hyperactive or experiences conditions resembling those which produce symptoms 
of stress in mammals. Another objective of this work has been to determine whether 
experimentally induced "stress" conditions produce histologically detectable changes 
in the neurosecretory cells of the pars intercerebralis or other changes within the 
brain. 

MATERIALS AND METHODS 

The roach Blaberus craniifcr was the experimental animal. Each experimental 
group consisted of adult females which had undergone their last molt on the same 
day. This selection was made because some males lack one of the neurosecretory 
products always observed in corpora cardiaca of adult females of this species (Ozbas 
and Hodgson, 1958), and in order to have the experimental animals as Uniform 
as possible. 

Experimental treatments of the roaches consisted of administering electrical 
shocks to the animals or forcing them to be hyperactive. Ten-volt electrical shocks 
of 5 milliseconds duration each were administered from an electronic square wave 
stimulator at the rate of twenty shocks per second. Steel electrodes (size insect 
pins) were inserted into bilaterally symmetrical positions in the roach's head, 

1 This investigation was aided by a U. S. Public Health Service Grant (No. E-2271 ) to 
Dr. Edward S. Hodgson. 

275 



276 E. S. HODGSON AND S. GELDIAY 

near the medial margins of the compound eyes, or else one electrode was inserted 
into the right lateral portion of the third abdominal segment and the other electrode 
inserted into the left lateral portion of the fifth abdominal segment. The shocks 
thus administered represent approximately the minimum amplitude, duration, and 
frequency of electrical stimulation to which these roaches gave behavioral responses 
consisting of abdominal movements and movements of the appendages. Locomo- 
tion was prevented during the administration of shocks by pinning each roach 
through the edges of its pronotum to a dissecting board. Some control animals, 
hereafter designated Cl, were pinned to the board without electrodes, while others, 
designated C2, were pinned and also had electrodes in the head and abdomen but 
received no shocks. 

Sustained hyperactivity of Blabcnts was produced by placing the roach within 
a glass jar and giving the jar a quick shake so that the roach was turned upside 
down. This posture invariably caused the roach to execute violent leg and wing 
movements until it had turned itself over, whereupon the jar was immediately 
shaken again so as to invert the roach and initiate its struggles again. Each 
animal's activity was sustained by repetition of this treatment throughout the 
desired length of time. 

Control and experimental animals were killed by decapitation at various intervals 
after treatment, and the entire head (including the corpora cardiaca) of each roach 
was immediately fixed with either Bouin's or Kelly's solution, as specified below. 
Five-micron sections were cut and stained with either the chrome hematoxylin 
phloxin stain of Gomori (1941), hereafter designated CHP, or the aldehyde 
fuchsin stain (Gomori, 1950) as modified by Halmi (1952) and Dawson (1953), 
designated AF. Since the numbers of animals in each experimental group varied 
according to the numbers of synchronously molting females available, and their 
treatments varied according to the information sought, each experimental series 
will be described separately. 

RESULTS 

Series I consisted of 6 roaches tested 31 days after molting. Two of these 
were used as controls (Cl and C2), treated in the manner described above. Both 
controls were pinned to the dissecting board for 15 minutes and sacrificed im- 
mediately thereafter. Animal No. 3 had electrodes in its head only, and received 
shocks for one minute ; animal No. 4 also had electrodes in the head only, but 
received shocks for 15 minutes; No. 5 had electrodes in the abdomen and received 
shocks for 15 minutes; No. 6 had one set of electrodes in the head and another set 
of electrodes in the abdomen, and it received shocks through both sets of electrodes 
for one hour. All of the animals in this series were fixed in Bouin's solution 
within one minute after the end of treatment, and the sections were stained with 
CHP. 

The sections revealed marked differences in the amounts of neurosecretory 
materials within the corpora cardiaca of these animals. The corpora cardiaca of 
the controls (both Cl and C2) had large amounts of neurosecretory materials 
staining dark blue and pink. These neurosecretory materials were distributed 
throughout the central parts as well as the peripheral regions of the corpora 
cardiaca. Less of both pink and blue staining materials could be seen in sections 
from animals No. 3, but the most striking results were obtained in the cases of 



RELEASE OF NEUROSECRKTORY MATERIALS 277 

animals 4, 5, and 6. These three roaches had very little of either the pink or blue 
staining materials remaining in the corpora cardiaca. and most of the traces were 
found in the peripheral regions of the glands, especially near the aorta. These 
results indicate a significant loss of both kinds of neurosecretory materials from the 
corpora cardiaca during the experimental shock treatments lasting 15 minutes or 
longer. 

Since there is some variation in the initial amounts of neurosecretory materials 
within the corpora cardiaca of different animals, and even in the amounts observed 
within adjacent 5-micron sections from the same gland, the interpretation of an 
apparent partial decrease in neurosecretory content of glands from one animal 
alone, such as No. 3 from this series, would he questionable. The differences 
between the two controls and the animals receiving shock treatments for 1 5 minutes 
or more are very large, however, not only in the total amounts of neurosecretory 
materials within the glands, but also in the distribution of the materials as described 
above. To this evidence must be added the results from the other experimental 
series also. 

Series II consisted of 6 animals tested 49 days after molting. The tests were 
designed to check the reproducibility of the experimentally induced decrease of 
neurosecretory substances found in Series I, and to determine the rate of restora- 
tion of the depleted substances within the corpora cardiaca. Two control animals 
(Cl and C2) were treated exactly as those in Series I. Each of the other 4 
animals received shocks for 15 minutes through electrodes in the abdomen, thus 
duplicating the treatment given animal No. 5 of Series I. The 4 experimental 
animals in Series II were sacrificed at the following intervals after the end of 
their shock treatments : one minute, one hour, 6 hours, and 24 hours. 

Since total amounts of neurosecretory materials were of concern in this series, 
the AF stain, following Kelly's fixative, was chosen as the most convenient way of 
analyzing the results. Allowing for differences in the histological technique, and 
the fact that Series I animals had undergone their last molt more recently, the 
control animals in Series II did not differ greatly from the controls in Series I 
with respect to the amounts of neurosecretory materials within the corpora cardiaca. 
The controls of Series II had, if anything, slightly more neurosecretory material 
than the Series I controls which were tested closer to their time of molting. The 
distribution of the neurosecretory materials within the corpora cardiaca was also 
similar to that observed in the controls of Series I. 

Animals 3. 4 and 5 of Series II had very little neurosecretory material within 
the corpora cardiaca, the amounts and distribution being approximately the same 
as found in animals 4, 5 and 6 of Series I. (Photographs A and B of Figure 1 
show typical sections through the corpora cardiaca of a control (Cl) and animal 
No. 3 from Series II.) This confirmed the previous conclusion concerning the 
effects of the shock treatments lasting 15 minutes. The results from this series 
were inadequate, however, for determining the possible rate of restoration of the 
neurosecretory substances within the glands. Animal No. 6 had more neurosecre- 
tory material in the corpora cardiaca than could be found in the glands of other 
experimental animals of this series, but the amount was still far less than in the 
controls. Unfortunately, not enough suitable animals were available to permit 
more extensive tests concerning this point, but clearly the time required for refill of 



278 



E. S. HODGSON AND S. GELDIAY 




I 



FIGURE 1. Photomicrographs of typical histological sections; all were fixed with Helly's 
solution and stained with aldehyde fuchsin. A cross-section of corpora cardiaca from control 
animal (Cl of Series II), 144 X ; B cross-section of corpora cardiaca from animal (No. 3 of 
Series II) which had received abdominal shocks for 15 minutes, 144 X ; C cross-section of brain 
of an untreated control (Cl of Series III), 72 X ; D cross-section of brain of an animal (No. 
5 of Series III) which had been hyperactive for one hour, 72 X. G glia cells. H hemocytes. 
N neurosecretory materials. P pars intercerebralis (containing neurosecretory cell bodies). 
R recurrent nerve. 



the glands to a condition resembling that of the controls must be measured in days, 
or possibly in weeks, rather than hours. 

Series III consisted of 5 animals tested 42 days after molting to determine 
whether forced activity of the roaches would produce effects upon the corpora 



RELEASE OF NEUROSECRETORY MATERIALS 279 

carcliaca similar to the effects of electric shocks. Two animals were sacrificed with- 
out any experimental treatment to serve as controls ; when stained with AF, their 
corpora carcliaca did not differ significantly from those of the controls in Series II. 
The experimental animals were sacrificed immediately after various periods of 
forced activity: one minute, 15 minutes, and one hour. After one minute of 
activity, no significant reduction of neurosecretory material within the corpora 
cardiaca was observed. There were, however, marked reductions of neurosecretory 
materials in the corpora cardiaca of the animals active for 15 minutes and one hour ; 
their corpora cardiaca appeared similar histologically to those from animals given 
shocks for 15 minutes or longer. These results were interpreted as evidence that 
"stress" situations other than those caused by electric shock treatments could also 
induce release of neurosecretory materials from the corpora cardiaca. Certain 
other effects of the periods of forced activity are presented later in this section. 

Series IV consisted of 6 animals tested 18 days after molting. Since Ozbas 
and Hodgson (1958) have shown that corpus cardiacum extracts depress spon- 
taneous nerve activity in roach nerve cords, this series was used to determine 
whether corpora cardiaca retain their potency for affecting spontaneous nerve 
activity when the glands are taken from animals which have been hyperactive. 
Corpora cardiaca were removed from two control animals which were not given 
any experimental treatment. These extracts were tested in two lots of two glands 
each, and it was found that they depressed spontaneous nerve activity in roach 
nerve cords in essentially the same manner as has been previously described by 
Ozbas and Hodgson (1958). Extracts of corpora cardiaca from the other three 
animals were similarly tested after the animals had been forced to keep active for 
periods of 15, 60, and 120 minutes each. The glands were removed immediately 
after the periods of forced activity. None of the extracts of corpora cardiaca 
from these three experimental animals caused any significant changes in spontaneous 
nerve activity in the nerve cords, thus showing a clear difference from the controls 
and confirming the effects of the forced activity upon the neurosecretory content 
of the corpora cardiaca, using an entirely different method from the histological 
analysis previously employed. 

Since the neurosecretory substances dealt with in this study come to the corpora 
cardiaca from cell bodies located within the protocerebrum of the roach's brain, 
sections were also cut through the brains of roaches in Series I through III. These 
sections were studied particularly with regard to any histological changes in the 
neurosecretory cells of the pars intercerebralis which might have resulted from the 
various experimental treatments. The amounts of neurosecretory materials within 
the cell bodies of these neurosecretory cells varied so much from cell to cell in 
both control and experimental groups that no significant changes could be attributed 
to the experimental treatments. (See photographs C and D of Figure 1 for the 
location of the neurosecretory cells.) 

In sections of the brains of some experimental animals, there was one difference 
from the controls which was very striking this difference being the presence of 
blood cells distributed throughout the brains of the experimental animals. This 
w r as observed in brain sections from all three experimental animals which had 
undergone periods of hyperactivity in Series III. At least several hundred blood 
cells could be seen within the brain of the roach which had been kept active for one 



280 E. S. HODGSON AND S. GELDIAY 

hour (see Fig. ID), and it was estimated that between one and two hundred blood 
cells were present in the brains of the two animals kept active for shorter periods. 
(Exact counts are difficult to make in these cases because the blood cells are some- 
times tightly clustered and the same cells may be seen in more than one serial 
section.) A similar invasion of the brain by blood was also observed in sections 
of the brain of the one roach in Series II which was sacrificed immediately following 
15 minutes of abdominal shocks. Although only 16 blood cells were counted within 
the brain tissue of this animal, the presence of even a few blood cells within the 
roach brain must be regarded as significant because no blood cells were ever ob- 
served in the brains of any of the control animals similarly stained with AF. No 
blood cells were seen in material from Series I (stained with CHP), and the 
absence of blood cells in the other experimental animals of Series II suggests that 
the invasion of the brain by hemocytes is a temporary one, possibly lasting even less 
than an hour, although this is really a separate problem which cannot be analyzed 
adequately from the present results. Some of the other problems raised by this 
movement of hemocytes will be discussed below. 

DISCUSSION 

The experimental induction of the release of neurosecretory materials reported 
here is analogous to the results obtained in several other cases involving both 
arthropods and mammals. Kleinholz and Little (1949) found that asphyxia, like 
injection of eyestalk extracts, produced hyperglycemia in the crab Libinia. It was 
later proven conclusively that the mediation of the sinus gland within the eyestalk 
was essential in such cases of induced hyperglycemia in various crustaceans, and 
that many experimental treatments, including crowding, handling, and anesthesia 
of the animals, were also effective upon the sinus gland (presumably causing the 
gland to release stored neurosecretory substances), thereby producing hyper- 
glycemia (Kleinholz, Havel and Reichart. 1950). The imposition of stress or 
injury to the animal appears to be a common feature of these experimental treat- 
ments affecting the sinus glands (Carlisle and Knowles, 1959). 

An analogous case involving the rat has been reported by Rothballer (1953). 
Release of neurosecretory materials from the neurohypophysis of the rat was 
brought about by application of painful stimuli to the experimental animals, and 
there were indications that even handling the rats might result in loss of neu- 
rosecretory material from the neurohypophysis. Here, too, the imposition of stress 
would appear to be a common feature of the different treatments affecting the 
neurosecretory center. For similar reasons of convenience, the term "stress" is 
useful to indicate a common feature of the stimuli applied to roaches in the present 
experiments that is, these stimuli would be expected to produce discomfort, pain, 
fatigue, or exhaustion, and to elicit rapid secretion of hormones from the adrenal 
medulla of a mammal. No identity of the mechanisms of response to stress in 
mammals and invertebrates is meant to be implied, however. 

The exact mechanism linking the electrical stimulation or forced hyperactivity 
of the roaches and the release of neurosecretory materials from their corpora 
cardiaca is unknown. In the case of the electrical shocks, particularly in view of 
the magnitude of the shocks and their application directly within the head in some 
of the experimental animals, it might be reasonably argued that the shocks were 



RELEASE OF NEUROSECRETORY MATERIALS 281 

directly stimulating the axons or cell bodies of the neurosecretory cells supplying the 
corpora cardiaca. Potter and Loewenstein (1955) have demonstrated the con- 
duction of action potentials along neurosecretory cell axons following electrical 
stimulation in the fish Lophiits. Knowles, Carlisle and Dupont-Raabe (1955) 
used electrical stimulation to elicit the release of a chromactivating substance from 
a crustacean neurosecretory system in vitro. The assumption that arrival of nerve 
impulses, either normally or experimentally initiated, at the ends of the axons of 
neurosecretory cells would lead to release of neurosecretory substances from such 
cells is compatible with much contemporary thought concerning the release of neu- 
roendocrine substances (Welsh, 1959). 

In the case of hyperactivity involving no electrical stimulation, other mecha- 
nisms must be involved. There is already abundant evidence from studies on other 
neuroendocrine systems that the controlling factors may be quite complex and may 
exert their actions through more than one intermediary mechanism (Scharrer, 
1959), even though transmission of nerve impulses along axons of neurosecretory 
cells may be the ultimate trigger mechanisms for release of neurosecretory sub- 
stances from such accumulation centers as the corpus cardiacum. It is unfortunate 
that the operation of severing the neurosecretory axons between the brain and the 
corpora cardiaca prior to the periods of forced activity, which might otherwise be 
expected to test the importance of the innervation of the corpora cardiaca, is an 
operation which necessitates considerable handling and operative trauma to the 
animal. In itself, this procedure would probably affect the amounts of neurosecre- 
tory materials within the glands, as well as deprive them of their source of supply 
of neurosecretory materials. 

Only very tentative hypotheses can be offered as to the role of these processes 
in the normal life of the animal. The nature of the experimental treatments which 
cause the release of neurosecretory materials from the corpora cardiaca suggests 
that the release of such materials may be part of the normal reactions to stress. It 
is already known that corpus cardiacum extracts increase the frequency of the insect 
heart beat (Unger, 1957) ; they also decrease, sometimes after an initial transient 
increase, the amount of spontaneous activity in nerve cords /;; vitro (Ozbas and 
Hodgson, 1958), and probably other metabolic effects remain to be discovered. 
Although certain adrenalin-like effects of corpus cardiacum extracts (Cameron, 
1953) further suggest an analogy with the secretion of adrenalin during the response 
of mammals to stress situations, it remains to be determined whether the compounds 
being released from the corpora cardiaca during experimental conditions such as 
used in the present study actually bear any chemical relationship with adrenalin. 
An inclusive interpretation of these diverse effects would be particularly aided at 
the present time by studies of the effects of stress on central nervous activity, heart 
function, etc., in the intact animal. The invasion of the brain by blood cells, which 
was unexpectedly found to follow certain experimental treatments, has not been 
previously reported. In staining characteristics, the invading hemocytes resemble 
those within blood sinuses (one of which is shown in the lower left corner of 
Fig. 1 D). The hemocytes do not, however, exactly resemble any of the types 
commonly described, but variations from species to species, and with different 
stains, make such precise identifications difficult even under the best of conditions 
(Munson, 1953). The hemocytes were not localized in any one part of the brain, 
and no visible damage to tissues of the brain appeared to follow the experimental 



E. S. HODGSON AND S. GELDIAY 

treatments; these facts would appear to rule out simple hemorrhage or phagocytic 
action as an explanation for the distribution of the hemocytes. There is some evi- 
dence that hemocytes store, transport, and transform nutritive materials (Munson, 
1953), and such might he their functions during the experimental treatments 
administered in the present cases. Further studies on this problem are planned. 

The authors take pleasure in thanking Dr. Berta Scharrer for her helpful 
discussions of this work and critical reading of the manuscript. The responsibility 
for any errors and for the conclusions, however, is entirely our own. 

SUMMARY 

1 . Neurosecretory materials within the corpora cardiaca of adult female roaches 
(Blabents craniifcr) were studied histologically. using chrome hematoxylin and 
phloxin or aldehyde fuchsin stains. 

2. Both the neurosecretory substances which are stained dark blue with chrome 
hematoxylin and those stained pink by phloxine are markedly depleted in the 
corpora cardiaca following administration of electric shocks for periods of 15 min- 
utes or more to the heads or abdomens of the roaches. 

3. Forced hyperactivity of the roaches, when continued for periods of 15 min- 
utes or longer, also causes a marked decrease in the same neurosecretory materials 
within the corpora cardiaca. 

4. Following periods of forced hyperactivity of the animals, there is also a loss 
of the potency of extracts prepared from their corpora cardiaca, when such extracts 
are assayed for their ability to depress spontaneous activity in roach central nerve 
cords in vitro. 

5. It is suggested that the release of neurosecretory substances from the corpora 
cardiaca may be a part of the roach's response to stress situations. 

6. Hyperactivity of the roaches and, to a lesser extent, electric shock treatments, 
result in the invasion of all parts of the brain by blood cells. The significance of 
this phenomenon is not known. 

LITERATURE CITED 

CAMERON, M. L., 1953. The secretion of an orthodiphenol in the corpus cardiacum of the 
insect. Nature, 172: 349-350. 

CARLISLE, D. B., AND F. KNOWLES, 1959. Endocrine Control in Crustaceans. Cambridge 
University Press, Cambridge. 

DAWSON, A. B., 1953. Evidence for the termination of neurosecretory fibers within the pars 
intermedia of the hypophysis of the frog, Rana pipicns. Anat. Record, 115: 63-69. 

GOMORI, G., 1941. Observations with differential stains on human islets of Langerhans. .liner. 
J. Path., 17: 395-406. 

GOMORI, G., 1950. Aldehyde-fuchsin : a new stain for elastic tissue. Awcr. J. Clin. Patli.. 20: 
665-666. 

HALMI, N. S., 1952. Differentiation of two types of basophils in the adenohypophysis of the rat 
and the mouse. Stain Technol., 27 : 61-64. 

KLEINHOLZ, L. H., AND B. C. LITTLE, 1949. Studies in the regulation of blood-sugar con- 
centration in crustaceans. I. Normal values and experimental hyperglycemia in Lihinia 
emarginata. Biol. Bull, 96: 218-227. 

KLEINHOLZ, L. H., V. J. HAVEL AND R. REICHART, 1950. Studies in the regulation of blood- 
sugar concentration in crustaceans. II. Experimental hyperglycemia and the regulatory 
mechanisms. Biol. Bull., 99: 454-468. 



RELEASF. OF NFUROSF.c RFTORY MATERIALS 283 

KNOWLES, F. G. W., D. B. CARLISLE AND M. DUPONT-RAABE, 1955. Studies on pigment 
activating substances in animals. I. The separation by paper electrophoresis of 
chromactivating substances in arthropods. /. Mar. fiiol. .-Issue., 34: 611-635. 

M rxsoN, S. C., 1953. The hemocytes, pericardial cells, and fat body. Chap. 8 in "Insect 
Physiology," edited by K. D. Roeder. Wiley, New York. 

OZBAS, S., AND E. S. HODGSON, 1958. Action of insect neurosecretion upon central nervous 
system in vitro and upon behavior. Prac. Nat. Acad. Sci., 44: 825-830. 

POTTER, D. D., AND W. R. LOEWENSTEIN, 1955. Electrical activity of neurosecretory cells. 
Amer. J. Pliysiol., 183: 652. 

ROTHBALLER, A. B., 1953. Changes in the rat neurohypophysis induced by painful stimuli with 
particular reference to neurosecretory material. Anat. Rcc., 115: 21-41. 

SCHARRER, B., 1952. Neurosecretion. XL The effects of nerve section on the intercerebralis- 
cardiacum-allatum system of the insect Lciicopluica tnadcrac. Biol. Bull., 102: 261-272. 

SCHARRER, B., 1954. Neurosecretion in invertebrates : a survey. Pubbl. Staz. Zool. Napoli, 
24, Snppl.: 8-10. 

SCHARRER, B., 1959. The role of neurosecretion in neuroendocrine integration. Pp. 134-148 
in "Comparative Endocrinology," edited by A. Gorbman. Wiley, New York. 

UXGER, H., 1957. Untersuchungen zur neurohormonalen Steuerung der Herztatigkeit bei 
Schaben. Biol. ZhL, 76: 204-225. 

WELSH, J. H., 1959. Neuroendocrine substances. Pp. 121-133 in "Comparative Endocrinol- 
ogy," edited by A. Gorbman. Wiley, New York. 

\\~IGGLESWORTH, V. B., 1954. The Physiology of Insect Metamorphosis. Cambridge University 
Press, Cambridge. 



EXAGGERATED ELEVATION OF THE FERTILIZATION MEMBRANE 

OF CHAETOPTERUS EGGS, RESULTING FROM 

COLD-TREATMENT 1 

CATHERINE HENLEY 

Marine Biological Laboratory, Woods Hole, Mass., and Department of Zoology, 
University of North Carolina, Chapel Hill, N. C. 

There are many phenomena associated with elevation of the vitelline memhrane 
after activation of invertebrate eggs ; these have long been of interest to embryol- 
ogists. Recently, Costello (1958a) has summarized much of the earlier work, and 
has contributed new observations concerning the behavior of the vitelline membrane 
and the jelly material which is secreted by the egg of Nereis Ihnbata after fertiliza- 
tion. These studies involved the centrifuging and subsequent treatment of Nereis 
eggs with alkaline sodium chloride (pH 10.5), which had earlier been shown by 
Costello (1945) to be effective in producing exaggerated membrane elevation and 
denuding of the eggs. The method is based upon one first described by Hatt 
(1931) and subsequently modified by Novikoff (1939) for the eggs of Sabcllaria 
I'ulgaris. 

Costello (1958a) interpreted his results to indicate that centrifuging of the 
Nereis egg resulted in a displacement of the cortical jelly-precursor material, so 
that upon subsequent activation such centrifuged eggs had an asymmetrical peri- 
vitelline space which was widest at the centrifugal pole. The jelly-layer, which 
forms external to the membrane, was also asymmetrical and thickest at the same 
area. When centrifuged unfertilized Nereis eggs were treated with alkaline NaCl, 
there was a retention of the jelly beneath the vitelline membrane and formation of 
an asymmetrical perivitelline space. 

Activation of the egg of Chaetopterns pergamentaceus is not accompanied by 
cortical changes of so obvious a nature as those reported for Nereis by Lillie 
(1911), Novikoff (1939) and Costello (1949, 1958a). There are, however, in- 
conspicuous rhythmic contractions of the vitelline membrane of the Chaetopterns 
egg, beginning about 20 minutes after fertilization, which were described briefly by 
Lillie (1906) and in more detail by Pasteels (1950). It was suggested by 
Costello (1958a) that (page 180), "These might be occasioned by rhythmic release 
of small quantities of colloidal material in progressive waves sweeping over the 
egg surface from an origin at one pole or the other." Evidence which offers 
support for this hypothesis has been obtained during the course of experiments 
involving the treatment of Chaetopterns eggs with low temperature. 

Brief preliminary accounts of some of this work have been reported (Costello 
and Henley, 1949; Henley and Costello, 1949; Henley, 1958). 

1 Aided by a grant from the National Institutes of Health, U. S. Public Health Service, 
RG-5328, to Dr. D. P. Costello. I am indebted to Dr. Costello for a critical reading of the 
manuscript, and for his assistance with the photographs. 

284 



COLD-TREATMENT OF CHAETOPTERUS ECKJS 285 

METHODS 

Eggs and sperm of the polychaete annelid Chactoptcnts f>cr(jaincntacens were 
obtained and handled by the methods outlined by Costello ct al. (1957). The gen- 
eral plan of the experiments involved the following procedure : Two to five min- 
utes after insemination, the experimental eggs were, for those series which involved 
temperature shock, transferred to six-inch fingerbowls containing pre-chilled (2- 
3 C.). freshly filtered, aerated sea water ; the fingerbowls rested in a nest of cracked 
ice contained in a large fingerbowl, which in turn was kept in the refrigerator. 
In the series of experiments which did not involve temperature shock, the eggs 
( contained in a six-inch fingerbowl of freshly filtered, aerated sea water at room 
temperature) were gradually chilled by placing the fingerbowl in a larger dish of 
cracked ice which in turn was placed in the refrigerator. Usually a period of 
about 60 minutes was required for the temperature of 2-3 C. to be reached in those 
experiments which did not involve temperature shock. 

Appropriate controls were kept for all series ; these dishes were kept surrounded 
by running sea water. 

At the end of the treatment period, the culture dishes in all series were removed 
from the refrigerator and ice-bath to the sea water table, and allowed to return 
gradually to room temperature, which varied from 20 to 24.5 C. during the various 
periods of the experiments. Egg-samples were removed and studied at frequent 
intervals during and following the warming-up period. No temperature shock was 
involved during the post-treatment period for any of the experiments. 

Counts were made to determine the approximate time of 50% cleavage in 
experimental and control egg-populations, so that a quantitative measure could be 
obtained of the cleavage delay brought about as a consequence of treatment. All 
observations and photographs of living eggs and embryos were made without the 
use of coverslips ; drops of culture fluid were examined in uncovered Columbia 
watchglasses. 

For some series, fixed and stained whole-mount preparations were made of con- 
trol and experimental eggs. These were prepared according to the method des- 
cribed by Henley and Costello (1957), being fixed in Kahle's fluid and stained in 
Harris' acid haematoxylin, or by the Feulgen technique. Most of the results re- 
ported here, however, are based upon observations of living embryos and larvae. 

Photographs of living and fixed eggs were made using a Leica camera with 
Micro-Ibso attachment. 

A total of over 30 experiments was performed, during the course of several 
summers at Woods Hole. 

RESULTS 
Membrane elevation 

The most striking single result of cold-treatment of Chactoptcnis eggs is the 
markedly asymmetrical exaggeration of vitelline membrane elevation, which occurs 
during the period of gradual return of the eggs to room temperature (Figs. 1-10). 
This process begins rather slowly and, for the first 10 minutes or so (after moder- 
ately short periods of treatment), appears to involve no atypical changes in the 
egg or membrane. A relatively localized shallow crinkling of the membrane then 
begins at one sector (Fig. 1. lower egg) ; this is probably, at most, only a slight 



286 



CATHERINE HENLEY 






1 








All photomicrographs are of living, fertilized Chaetoptcrus eggs ; the pictures were made 
with a Leica camera and Micro-Ibso attachment. Magnification of all figures after reproduc- 
tion : approximately 250 X. 

FIGURE 1. Eggs cold-treated for 150 minutes with temperature shock and photographed 
11 minutes after the end of treatment. In the lower egg, the first area of membrane wrinkling 
is visible at approximately one o'clock on the egg periphery; the elevation of the entire mem- 
brane is still at about the normal height. In the upper egg, the second area of membrane 
"blistering" has begun to appear, the original area of crenation still being visible at the lower 
pole of the egg. 

FIGURE 2. Another pair of eggs, cold-treated for 150 minutes with temperature shock 
and photographed 21 minutes after the end of treatment. Note the enlarged "blistered"' areas 
in the membranes of both eggs. 



COLD-TRKATMEXT OF CHAETOPTERUS EGGS 287 

exaggeration of the normal membrane wrinklings which occur at the vegetal pole, 
beginning about 20 minutes after insemination ( Lillie, 1906; Pasteels, 1950). 
Within a period of approximately five minutes, a second, much more pronounced, 
localized area of membrane elevation appears in the treated eggs (Fig. 1, upper 
e t?b. ) This second region is the one from which subsequent exaggerated membrane 
elevation proceeds. It is, from its first appearance, quite distinctive and is charac- 
terized by a more blister-like configuration of the membrane (Fig. 2). It appears 
to bear no constant spatial relationship to the original wrinkled vegetal pole area 
from which, as aforesaid, it is entirely separate. 

During the course of the next few minutes, additional deep folds may appear in the 
membrane (Fig. 3) ; eventually these become contiguous and by 35 minutes after the 
end of treatment (approximately equivalent to 40 minutes after insemination) the 
membrane is smoothly and very exaggeratedly elevated from the egg surface (Fig. 10. 
upper egg). The course of membrane elevation during the period from 28 minutes 
to 33 minutes after the end of treatment is shown in Figures 7-10. These pictures 
show clearly that once the process of exaggerated membrane elevation begins, it 
proceeds rapidly (see also Figures 2 and 3. photographs taken one minute apart). 

The ultimate exaggerated elevation attained may be represented by Figure 10 
(top), shown 33 minutes after the end of treatment. The elevation of the mem- 
brane is quite markedly asymmetrical, the point where it is nearest the egg surface 
representing the sector which was first wrinkled (not that second center from which 
the process of elevation proceeds). Even here, however, at the point of closest 
approach to the egg surface, the elevation of the membrane is much wader than 
normal. The asymmetrical nature of this elevation is a characteristic and consistent 
result of cold-treatment. 

Comparison of Figures 1 (lower egg) and 10 (upper egg) will reveal the 
magnitude of the exaggerated membrane elevation ; although Figure 1 represents 
a treated egg, the elevation of the membrane has not yet proceeded beyond the normal 
stage. 

It is important to note that except for the first area of shallow membrane wrinkl- 
ing, the subsequent stages are probably quite separate and distinct (in degree, at 
least) from the normal membrane shape changes described by Pasteels (1950). 
His description of the initial crenated area in an egg 20 minutes after insemination 

FIGURE 3. The same pair of eggs shown in Figure 2, photographed 22 minutes after the 
end of treatment ( one minute after the photograph of Fig. 2 ) . Note that two additional 
localized areas of exaggerated membrane elevation have now appeared in the lower egg. 

FIGURE 4. Egg cold-treated for 6 hours without temperature shock and photographed 
immediately after the end of treatment. In this case, the process of exaggerated membrane 
elevation has begun considerably sooner after treatment than usual. The localized character 
of the elevation and the double nature of the membrane are apparent. 

FIGURE 5. Another egg from the same treatment group as that shown in Figure 4, but 
photographed 50 minutes after the end of treatment. The double nature of the exaggeratedly 
elevated membrane is visible. The small globule between the egg surface and the membrane 
at the right is apparently a polar body. 

FIGURE 6. A third egg from the same experimental group as those shown in Figures 4 
and 5, photographed 50 minutes after the end of a 6-hour cold-treatment. The membrane has 
remained closely apposed to the egg surface around approximately one-half of the egg periphery 
but is exaggeratedly elevated around the other half. The double nature of the membrane is 
clear. 



288 



CATHERINE HENLEY 






8 









10 




FIGURES 7-10. 



COLD-TREATMENT OF CHAETOPTERUS EGGS 289 

(at an undesignated temperature) as having a "finement plisse" appearance is very 
accurate, and his Figure A shows that the subsequent normal wrinklings and 
shape changes are likewise quite shallow. We have repeatedly confirmed his 
findings in the study of normal, control eggs, and they are also illustrated in the 
photographs accompanying the paper by Harvey (1939). 

There is no way of knowing with certainty whether the changes observed in 
our treated eggs are in any way comparable to the normal wrinklings, since the 
exaggerated membrane elevation is so drastic as to obscure any crenations of the 
type seen in untreated eggs. They are not associated with the presence of a coverslip, 
since all our observations of living eggs were made without the use of a coverslip. 
Careful study of living eggs, of fixed whole-mount preparations, and of photographs 
of living eggs has convinced us that the exaggeratedly elevated membrane is 
double in nature (Figs. 4-6). 

Cleavage delav 

At room temperatures of 20-25 C., the first cleavage of Chaetoptents eggs 
normally occurs from 40 to 50 minutes after insemination (Costello et al., 1957). 
In the cold-treated eggs studied here, first cleavage for 50^ of the experimental 
eggs was delayed from 134 to 704 minutes, the magnitude of the delay being in 
direct relation to the duration of treatment (Table I). As noted below in the 
section on the role of temperature shock, cleavage delay was much greater in those 
eggs subjected to temperature shock than in those which were cooled gradually 
(Table II), in series treated for the same length of time. From cytological study, 
it appears that the cold-treated eggs did not develop very far after the onset of 
chilling, until the treatment was ended and a gradual warming process (to room 
temperature) began. During the five-minute period which intervened between the 
time of insemination and the time when treatment was begun, the sperm penetrated 
the egg and, in some cases at least, the male and female pronuclei approached one 
another. Further development did not occur until the conclusion of the treatment. 
From the data reported in Table II, it appears that a period of from three to ten 
minutes was required for resumption of development in all cases where eggs were 
treated with temperature shock. For those eggs which were not abruptly chilled, 
there was apparently a continuation of some of the early stages of development, 
during the start of the cooling process, so that the magnitude of the cleavage delay 
was sometimes slightly less than the duration of treatment. 

It is of considerable interest that even in those eggs treated for 720 minutes 
(12 hours), development could be resumed after cessation of the cold-treatment. 
As noted in Table I, the trochophore larvae developing from eggs treated for this 
period were very abnormal, however. 

FIGURES 7-10. Successive stages of a single pair of eggs, cold-treated for 150 minutes with 
temperature shock. FIGURE 7 : 28 minutes after the end of treatment. Note the pronounced 
asymmetry of exaggerated elevation in this and the succeeding photographs. FIGURE 8 : 29 
minutes after the end of treatment. The small particle of debris has shifted position in the 
interval between this photograph and that of Figure 7. FIGURE 9 : 30 minutes after the end 
of treatment. FIGURE 10: 33 minutes after the end of treatment. The upper egg (unconfined 
by a coverslip) has rolled over since the photograph of Figure 9 \vas taken. The small piece 
of debris has been obscured. See text for further details. 



290 



CATHERINE HENLEY 



TABLE I 
The effects of cold treatment (5-12 hours) on fertilized Chaetopterus eggs 



Duration of 
treatment 



Effects on membrane elevation 



Cleavage delay for 

50% of exper. eggs 

as compared 

with controls 



Other effects 



300 minutes 
360 minutes 
390 minutes 
420 minutes 
540 minutes 
720 minutes 



Variable; some exaggeration by 15 
minutes after end of treatment 

Ca. 90% with exaggerated mem- 
brane elevation immediately after 
end of treatment 

Exaggerated elevation by 45 min- 
utes after end of treatment ; even- 
tual denuding of many eggs 

94% of eggs with exaggerated eleva- 
tion within 5 minutes after end of 
treatment 

90% of eggs with exaggerated eleva- 
tion within 25 minutes after end 
of treatment 

44% of eggs with exaggerated eleva- 
tion within 23 minutes after end 
of treatment 



301-312 minutes 
320-346 minutes 
429 minutes 



590 minutes 



704 minute^ 



Ciliary defects in trocho- 
phores. First cleavage 
products equal in size? 

Abnormal trorhophores ; 
ciliary defects; surface 
blebs 

First cleavage products 
equal in size? 

Abnormal, amorphous 
trochophores 



Abnormal trochophores ; 
some very small. Cili- 
ary defects 



All treatments were begun within 2-5 minutes after insemination. The sea water medium 
was gradually chilled to a temperature of 2-3 C. (no temperature shock). 

* The cleavages in this group of eggs were very abnormal and chaotic, and could not be timed. 

Other effects of low temperature on development 

The embryos and trochophores developing from the cold-treated eggs exhibited 
a number of characteristic abnormalities. In many of the cases where prolonged 
periods of treatment were used, there appeared to lie a suppression of polar body 
formation in at least some of the eggs. This is in accord with the findings of other 
investigators, for other forms (notably amphibians; see the review by Fankhauser, 

TABLE II 
Treatments of comparable durations, with and without temperature shock 



Duration of 
treatment 


Temp, 
shock ? 


Effects on membrane elevation 


Cleavage delay 


180 minutes 
150 minutes 


No 
Yes 


None, or at most very slight 
Asymmetrical exaggerated membrane elevation by 
15-20 minutes after the end of treatment 


162 minutes 
152-160 minutes 


120 minutes 


Yes 


Asymmetrical exaggerated membrane elevation by 
25 minutes after the end of treatment 


134 minutes 



240 minutes 


No 


Slight exaggeration of membrane elevation in some 


220 minutes 


185 minutes 


Yes 


eggs 
Many eggs with exaggerated membrane elevation 
by 45 minutes after the end of treatment 


189 minutes 



COLD-TREATMENT OF CHAETOPTERUS EGGS 

1945). Less drastic exposures to low temperature apparently had no effect on 
polar body formation, so far as could be determined. 

In a few experiments, the two blastomeres resulting from the first cleavage of 
treated eggs were equal or nearly equal in size. (Normally the AB and CD 
blastomeres resulting from the first cleavage of Chaetopterus and other spirally 
cleaving eggs are noticeably unequal.) Tyler (1930) also observed equal cleavage 
after cold-treatment of Chaetopterus eggs, but his experiments are not entirely 
comparable to those described in the present experiments, since he used somewhat 
higher temperatures, applied later in development and for considerably shorter 
periods of time. Tyler (1930) discusses the production of double embryos by such 
treatments ; no evidence of double embryos, nor of the alteration of cleavage planes 
as described by him, was observed in the present study. 

The normal egg-shape changes ("pear" and polar lobe stages) were often not 
clearly identifiable in the treated eggs. 

The trochophore larvae which developed from cold-treated eggs almost in- 
variably moved very sluggishly, if at all, and there were apparently severe ciliary 
defects. Surface blebs were often present on the trochophores. In general, the 
larvae bore a striking resemblance to those obtained after KCl-treatment by Lillie 
( 1902), and after cold-treatment ( 10-14 C. for 14 hours) by Lillie ( 1906) . How- 
ever, we do not think that the trochophores in our cultures resulted from differentia- 
tion without cleavage, as Lillie (1906) suggested, since cytological study (see 
below) revealed that at least a degree of both karyokinesis and cytokinesis had 
occurred in all observed cases. 

There was a wide variety of size among trochophores developing from eggs 
cold-treated for prolonged periods (12 hours). Some larvae were very small, 
suggesting that they might have developed from egg fragments. Such fragments, 
or detached cells from later cleavage stages, were observed in culture dishes of this 
series. 

In almost all experiments there was a high mortality rate among the later 
embryos and trochophore larvae of the experimental groups. 

Denuding of eggs 

After many of the longer durations of treatment (6 hours or more), the process 
of exaggerated membrane elevation continued until there was a bursting of the 
membrane and denuding of the eggs. This denuding was very similar in course 
and end-results to that observed after alkaline XaCl treatment of Nereis and 
Sabcllaria eggs. It is, however, in contrast to the process of denuding which fol- 
lows alkaline NaCl treatment of Hydroidcs eggs (Costello, 19581) ) ; there appears to 
be an actual dissolution of the Hydroidcs egg membrane in the alkali. (It is sug- 
gestive in this connection that Lillie, 1902, described a similar destruction of the 
Chaetopterus egg vitelline membrane in KC1 and CaCl L) solutions.) Subsequent 
development of the denuded Chaetopterus eggs was very abnormal under the 
conditions of these experiments, but no special efforts were made to cultivate such 
embryos further. Costello (1945) and others have shown that denuded eggs are 
extremely sensitive to contact with bare glass surfaces, and coating of the culture 
dishes with a thin layer of agar in sea water is necessary for the successful mainte- 
nance of such embryos. 



292 CATHERINE HENLEY 

After less drastic treatments, the asymmetrical exaggerated membranes retained 
their configuration through at least the first cleavage and denuding did not occur. 

The role of temperature slwck 

Table II describes the results of experiments involving comparable durations 
of treatment time, with and without temperature shock. Even relatively short 
periods of cold-treatment (120 minutes) involving rapid transfer of the eggs from 
an ambient medium at room temperature to one pre-chilled to 2-3 C. were effective 
in producing a marked delay of the first cleavage, as well as the characteristic 
exaggerated membrane elevation. In contrast, cold-treatments of as long as 240 
minutes without this temperature shock resulted in, at most, very slight effects on 
cleavage time and on membrane elevation. Thus, in one experiment, a treatment 
of 180 minutes without temperature shock resulted in a delay of 162 minutes for 
the first cleavage and produced only slight effects on membrane elevation. A 
similar treatment of 185 minutes with temperature shock resulted in a 189-minute 
delay of the first cleavage time for 50% of the experimental eggs (as compared 
with 50% of the controls) ; by 45 minutes after the end of treatment, there was a 
marked exaggeration of membrane elevation in many of the treated eggs. 

Temperature shock involves the beginning of action of cold considerably sooner 
after insemination (five minutes) than the absence of temperature shock, where at 
least 60 minutes may be required to attain the treatment temperatures of 2-3 C. 
This suggests that the effective action of cold in producing exaggerated membrane 
elevation occurs within the first hour of treatment ; in a gradually cooling medium, 
there may be opportunity for at least a semblance of the normal release of cortical 
material to occur, whereas this release is inhibited almost immediately if the eggs 
are plunged into pre-chilled sea water five minutes after insemination. 

Even treatment without temperature shock, however, results in abnormal 
trochophores and ciliary defects, despite the fact that there may be little or no 
membrane exaggeration. The implication here is that the effects on membrane 
elevation may be very different from those affecting subsequent morphogenesis. 
The low temperature may thus be said to have both a direct and a delayed type 
of action. 

Study of fixed eggs 

In one typical group of eggs, cold-treated for 150 minutes with temperature 
shock, and fixed 15 minutes after the conclusion of the treatment (170 minutes 
after insemination), the normal quota of 9 chromosomes at the metaphase of the 
first maturation division was present and countable in most instances. In some 
eggs, the sperm nucleus was still visible in the interior of the egg, as a separate 
entity ; in others, approach and fusion of the pronuclei had advanced further, and 
the male and female components were no longer separable on the basis of ap- 
pearance. Control eggs fixed at the same time were proceeding from the eight- to 
the sixteen-cell stage in a normal fashion. Experimental eggs of the same series 
fixed 205 minutes after insemination (50 minutes after the end of treatment) were 
at the metaphase of the first cleavage, while control eggs were at the metaphase of 
the fifth cleavage. Several multipolar spindles were observed among the experi- 
mental eggs of this group. 



COLD-TREATMENT OF CHAETOPTERUS EGGS 

The cortical regions of the experimental eggs fixed 50 minutes after the end of 
treatment presented a striking picture. There was a marked asymmetry of cortical 
material, apparent as a more lightly staining peripheral hand with its greatest width 
within one relatively localized sector of the egg circumference. These eggs were 
fixed, whole, on coverslips and there is inherent in the method a certain minor 
degree of distortion of the egg shape. However, we observed no comparable 
asymmetry of the cortical material in control eggs of this or any other series, and 
therefore suggest tentatively that this may represent the area where cortical colloid 
material has been released in the observed asymmetrical fashion. The eggs of this 
group were marked by a high incidence of asymmetrical membrane elevation, which 
was first apparent about 30 minutes after the end of treatment, or 20 minutes 
before the eggs were fixed. The asymmetry of the cortical area is not visible in 
the experimental eggs which were fixed 1 5 minutes after the end of treatment ; 
these ova, in the living condition, had not yet undergone exaggerated membrane 
elevation. 

In addition to the multipolar spindles mentioned above, a number of other types 
of cytological abnormality were noted, especially in those eggs which were fixed 
several hours after the end of treatment. Among the abnormalities were chromo- 
some bridges, lagging or lost chromosomes or chromosome fragments, and unequal 
division of chromatin material to the two poles of the division figure. All these 
anomalies are typical of the kinds produced in other material as a consequence of 
low (and high) temperature, among other agents. 

There was some evidence of the production of polyploidy in larvae from cold- 
treated eggs ; although a few of the body cells of trochophores obviously had more 
than the diploid number (18) of chromosomes present, such duplications of indi- 
vidual chromosomes or sets of chromosomes was apparently not the rule in all 
cells of a given larva. The chromosomes of Chactoptcrus are small and tend to be 
crowded on the spindle, so that counting them is difficult, even in early cleavage 
stages ; to do so is almost impossible in the minute cells of later cleavage stages and 
trochophores. The observed anomalies of chromatin distribution may have arisen 
as a result of the polar body suppression mentioned above. 

Cytological preparations of advanced cleavage stages of the treated eggs indicate 
that there was often some suppression of cytokinesis (although karyokinesis had 
proceeded in a variable fashion). In all cases studied, however, at least some 
degree of cytoplasmic division had occurred, and there was no evidence of differentia- 
tion without cleavage. As would be expected, there was a wide variation in cell 
size, in the treated embryos and larvae. 

DISCUSSION 
The possible mechanism of action of Ion* temperature 

The characteristic asymmetry and exaggeration of membrane elevation in the 
cold-treated Cliaetoptenis eggs suggest that cold-treatment may interfere with the 
gradual and rhythmic release of some substance from the egg surface after insemina- 
tion, so that a sudden localized release of such material occurs at the cessation of 
treatment. There may also be a change in the permeability of the vitelline mem- 
brane, so that the colloidal material is retained within the perivitelline space, bring- 
ing about the observed exaggerated membrane elevation. The asymmetry of this 



294 CATHERINE HENLEY 

exaggerated elevation could be a consequence of the accumulation of colloidal sub- 
stance within one relatively localized sector of the egg, or of changes in the egg 
cortex resulting in release of such a substance in a considerably smaller segment of 
the egg periphery than is normal. The Chaetopterus egg secretes no external jelly 
after activation, and the substance whose abrupt release brings about membrane 
elevation in the treated eggs is therefore postulated to be of some other nature. 
Thus, the situation for the Chaetopterus egg differs, in detail at least, from that 
described for the Nereis egg by Costello (1958a). 

Some additional evidence for this idea is afforded by observations on the action 
of gum arabic solutions ( in sea water) applied to eggs with exaggerated membrane 
elevation. In such cases, the membranes promptly collapsed back against the 
surfaces of the eggs. 

Pasteels (1950) has also suggested that there may be a rhythmic and localized 
release of some sort of material from the cortex of the Chaetopterus egg, during 
the post-fertilization period. His experiments involving treatment of eggs of this 
form with KC1 appear to support such an idea if one assumes that the KC1, like 
cold, blocks the process of release during the course of the treatment. 

Pasteels was able to demonstrate a direct correlation in fixed KCl-treated 
eggs between areas of abnormal membrane wrinkling, and cortical and membrane 
alterations of structure and staining capacity. This correlation appears to be 
comparable to that observed in the present study of fixed and stained cold-treated 
eggs. 

Exaggerated membrane elevation as a consequence of other experimental procedures 

Lillie (1902) mentioned briefly the occurrence of exaggerated membrane eleva- 
tion and dissolution in KC1- and CaCU-treated fertilized and unfertilized Chae- 
topterus eggs. A more thorough study of the same problem was undertaken by 
Pasteels (1950), who treated unfertilized Chaetopterus eggs with KC1 by the 
method of Lillie. Pasteels illustrates a process of asymmetrical membrane elevation 
which, in some respects, is reminiscent of that reported in the present study. His 
Figure B shows that by 40 minutes after the end of KCl-treatment (95% sea water, 
5% 2.5 M KC1, for one hour), there are the beginnings of an asymmetrical mem- 
brane elevation which, at this stage, is similar to that observed after cold-treatment. 
The subsequent course of events after KCl-treatment, however, is quite different; 
by 102 minutes after the end of this treatment, there is a very asymmetrical, deeply 
rugose exaggeration of membrane elevation. Apparently this stage is not succeeded 
by a smooth state of exaggerated membrane elevation. 

Redfield and Bright (1921) reported exaggerated elevation of the Nereis egg 
membrane after various types of irradiation. Following beta or gamma radiation, 
the elevation was symmetrical ; after alpha or ultraviolet treatment, it was asym- 
metrical. Redfield and Bright state that the}- did not obtain an increase in the 
volume of the perivitelline space after the irradiation of eggs such as Cuiningia, 
Astenas, Arbacia and Chaetopterus. which do not normally produce jelly as a part 
of the fertilization reaction. However, Moser (1939), Spikes (1944) and Rustad 
(1959) reported the asymmetrical elevation of the sea urchin egg fertilization 
membrane after ultraviolet irradiation of one side of the eggs. It is of interest 
that x-irradiated unfertilized Chaetopterus eggs (treated with 20,000 and 40,000 r 



COLD-TREATMENT OF CHAETOPTERUS EGGS 295 

and then inseminated) showed no exaggeration of membrane elevation (Henley, 
1958). Furthermore, whole-mount preparations of such x-irradiated eggs, fixed at 
the metaphase of the first cleavage, do not reveal the asymmetry of cortical material 
reported above for cold-treated eggs. Although this observation is by no means 
conclusive evidence, it does suggest that the appearance of such cold-treated ova is 
associated with the postulated abnormal retention and delayed release of cortical 
material. 

As noted in the introduction, alkaline sodium chloride treatment (pH 10.5) 
results in a drastic exaggeration of membrane elevation in the Nereis egg (Costello, 
1945 ; Lovelace, 1949) ; this exaggeration, unlike that reported here, is symmetrical 
in nature, except for the circumscribed area of sperm entrance. Alkaline NaCl- 
treatment of Chaetopterus eggs is followed by an extremely rapid elevation of the 
membrane, which results in denuding of the eggs in less than five minutes. The 
exact process involved will be described in a later communication ; suffice it to say 
here that the membrane elevation appears to be only superficially comparable to 
that produced as a result of cold-treatment. 

Moser C1939) illustrates asymmetrical (although not especially exaggerated) 
membrane elevation in saponin-treated unfertilized Arbacia eggs. 

The chronological relationships of events in cold-treated TS. control eggs 

It is interesting to compare the chronological relationships of events in the cold- 
treated eggs and in the normal, untreated eggs. Pasteels (1950) has described 
the following series of membrane shape changes and wrinklings in fertilized Chae- 
topterus eggs ; he did not specify the room temperature but, from the times noted 
for several "landmark" events, this may be assumed to have been about 17-20 C., 
somew r hat lower than those prevailing during our experiments. 

20 minutes after insemination : There is a vegetal-to-animal pole wave of shallow 
membrane wrinklings. 

23 minutes after insemination : The first polar body is given off ; from now until 
after the second polar body appears, the membrane remains smooth. 

30 minutes after insemination : The second polar body is given off. 

32 minutes after insemination : A second vegetal-to-animal pole wave of mem- 
brane wrinkling occurs. 

38 minutes after insemination: "Pear-shaped" stage; there is now an animal- 
to-vegetal pole wave of wrinkling in the membrane, reversing the first two 
waves. 

42 minutes after insemination : Polar lobe stage ; wrinkling of the membrane 
now occurs in a wave from each pole, more or less simultaneously, leaving 
an equatorial band around the egg free of wrinkles. 

66 minutes after insemination : First cleavage begins ; the wrinklings of the 
membrane are accentuated. 

Our observations showed that for all experiments, both with and without tem- 
perature shock, the greatest incidence of exaggerated membrane elevation occurred 
immediately before the first cleavage (at a stage corresponding to the polar lobe 
although, as noted above, the normal shape changes of the egg cytoplasm were often 
not recognizable in the treated eggs). This is a time, of course, when a number of 



296 CATHERINE HENLEY 

important events are occurring within the egg, in preparation for the first cleavage. 
It does not seem unreasonable to suppose that the double wrinkling (animal-to- 
vegetal and vegetal-to-animal) described by Pasteels as occurring shortly before 
the first cleavage, or some featvire of this phenomenon, may be exaggerated to 
result in the reported events. 

SUMMARY 

1. Fertilized eggs of the polychaete annelid, Chaetopterus pcrgamcntaceus, were 
cold-treated for various periods of time, ranging from 150 to 720 minutes, beginning 
immediately after insemination. Two general methods were employed ; in one, the 
eggs were plunged into pre-chilled, filtered aerated sea water (2-3 C.) ; these 
experiments are referred to as involving temperature shock. In the other type, the 
eggs were gradually chilled to approximately the above temperature. At the end 
of the treatment period all eggs were allowed to return gradually to room 
temperature. 

2. When eggs were cold-treated, with or without temperature shock, there 
was a pronounced asymmetrical exaggerated elevation of the vitelline membrane, 
which reached its greatest incidence about 40 minutes after the end of treatment, or 
shortly before the first cleavage. This exaggerated elevation continued in some 
cases after prolonged cold-treatment, so that the eggs were eventually denuded. 

3. Most of the cold-treatments used were followed by delays in the first cleavage 
time for 50% of the experimental eggs as compared with 50% of the control 
population. 

4. In all cases where cold-treatment was initiated with temperature shock, the 
effects on membrane elevation and cleavage time were much more pronounced than 
when the eggs were chilled gradually. 

5. A number of characteristic morphological and cytological abnormalities were 
noted in embryos developing from the treated eggs ; these included severe ciliary 
defects, fragmentation of the embryos, lagging or lost chromosomes or chromosome 
fragments, duplication of chromosome sets and/or individual chromosomes, sup- 
pression of polar bodies, and multipolar spindles. 

6. It is suggested that the findings reported here afford evidence supporting 
Costello's (1958a) hypothesis that the rhythmic wrinkling and shape changes 
reported for the normal Chaetopterus egg by Pasteels may be due to the gradual 
release of some colloidal material from the surface of the egg. This release is 
considered to be blocked in some manner by the low temperature, and when the 
treatment is terminated, the release proceeds in a drastic, non-rhythmic manner ; it 
then appears to be coupled with a change in the permeability of the vitelline mem- 
brane, so that the colloidal material is retained between the egg surface and the 
membrane. This brings about the exaggerated membrane elevation observed. 

LITERATURE CITED 

COSTELLO, D. P., 1945. Experimental studies of germinal localization in Nereis. I. The de- 
velopment of isolated blastomeres. /. Ex p. Zoo]., 100: 19-66. 

COSTELLO, D. P., 1949. The relations of the plasma membrane, vitelline membrane, and jelly 
in the egg of Nereis limbata. J. Gen. Physiol., 52 : 351-366. 

COSTELLO, D. P., 1958a. The cortical response of the ovum to activation after centrifuging. 
Physiol. ZooL, 31 : 179-188. 



COLD-TRKATMENT OK CHAETOPTERUS EGGS 297 

COSTELLO, D. P., 1958b. Membrane removal from the egg of the annelid, Hydroides. Bio!. 

Bull., 115: 349. 
COSTELLO, D. P., AND C. HENLEY, 1949. Gross morphological effects of low temperature on 

the fertilized eggs of Chaetopterus. Bid. Bull., 97: 256-257. 
COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Methods for 

Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory, 

Woods Hole, Mass. 

FANKHAUSER, G., 1945. The effects of changes in chromosome number on amphibian develop- 
ment. Quart. Rev. Biol., 20: 20-78. 
HARVEY, E. B., 1939. Development of half-eggs of Chaetopterus pcrgamentaceus with special 

reference to parthenogcnetic merogony. Biol. Bull., 76: 384-404. 
HATT, P., 1931. La fusion experimentale d'oeufs de "Sabellaria alvcolata L." et leur de- 

veloppement. Arch, de Biol., 42: 303-323. 
HENLEY, C, 1958. Studies on membrane elevation in the eggs of Chaetopterus and Nereis. 

Biol. Bull., 115: 353-354. 
HENLEY, C., AND D. P. COSTELLO, 1949. Cytological effects of low temperature on the 

fertilized eggs of Chaetopterus. Biol. Bull., 97 : 258. 
HENLEY, C., AND D. P. COSTELLO, 1957. The effects of x-irradiation on the fertilized eggs of 

the annelid, Chaetopterus. Biol. Bull., 112: 184-195. 
LILLIE, F. R., 1902. Differentiation without cleavage in the egg of the annelid Chaetopterus 

pergamcntaccus. Arch. f. Entzv., 14: 477-499. 
LILLIE, F. R., 1906. Observations and experiments concerning the elementary phenomena of 

embryonic development in Chaetopterus. /. Exp. ZooL, 3 : 153-268. 
LILLIE, F. R., 1911. Studies of fertilization in Nereis. I. The cortical changes in the egg: 

II. Partial fertilization. /. MorphoL, 22 : 361-393. 
LOVELACE, R., 1949. The effects of precocious sperm entry on the egg of Nereis limbata. J. 

Exp. ZooL, 112: 79-107. 
MOSER, F., 1939. Studies on the cortical layer response to stimulating agents in the Arbacia 

egg. II. Response to chemical and physical agents. /. Exp. ZooL, 80: 447-471. 
XOVIKOFF, A. B., 1939. Changes at the surface of Nereis limbata eggs after insemination. 

/. Exp. Biol., 16: 403-408. 
PASTEELS, J., 1950. Mouvements localises et rythmiques de la membrane de fecondation chez 

des oeufs fecondes ou actives (Chaetopterus, Mactra, Nereis). Arch, de Biol., 61: 

197-220. 
REDFIELD, A. C., AND E. M. BRIGHT, 1921. The physiological changes produced by radium 

rays and ultra-violet light in the egg of Nereis. /. Physiol., 40 : 61-85. 
RUSTAD, R. C., 1959. Consequences of unilateral ultraviolet irradiation of sea urchin eggs. 

Biol Bull., 116: 294-303. 
SPIKES, J. D., 1944. Membrane formation and cleavage in unilaterally irradiated sea urchin 

eggs. /. Exp. ZooL, 95: 89-103. 
TYLER, A., 1930. Experimental production of double embryos in annelids and mollusks. /. 

Exp. ZooL. 57: 347-407. 



HISTOCHEMICAL STUDIES ON THE NATURE AND FORMATION 

OF EGG CAPSULES OF THE SHARK 

CHILOSCYLLIUM GRISEUM * 

G. KRISHNAX 
Department of Zoology, University nf Madras, India 

Pryor (1940) demonstrated that the ootheca of Blatta is composed of a tanned 
protein similar to that of insect cuticle. Since then a number of authors reported 
the occurrence of tanned proteins in the cysts of nematodes, egg shells of helminths, 
setae of earthworm and byssus of Mytilus cdulis (Ellenby, 1946; Stephenson, 1947; 
Dennell, 1949; Brown, 1952; Smyth, 1954). In this connection it is of interest to 
recall the observation of Brown (1950a) that the egg capsules of selachians also 
give evidence of phenolic tanning although it was believed by the earlier workers 
to be formed of a material similar to keratin. In the light of these observations it 
was thought that a study of the nature and composition of the egg capsules of 
shark would be of interest in itself and for comparison with the egg shells of 
helminths and ootheca of Blatta which have been shown to possess a protein con- 
stitution resembling in essential respects that of tanned cuticles of insects. 

The materials used in the following study comprise nidamental glands and egg 
capsules collected from gravid females of Chiloscyllium griscnm. The staining and 
histochemical reagents used are mentioned in relevant context. For localization 
of the oxidase of the egg capsule, the "catechol" technique (Smyth, 1954) was 
applied. The microchemical and chromatographic procedures employed for the 
study of the protein of the egg capsule are described in the text. 

EGG CAPSULE STRUCTURE 

The egg capsules of sharks and rays have been described by a number of 
workers (Beard. 1890; Widakowich, 1906; Clark, 1922; Hobson, 1930; Xalini, 
1940). The shell material is said to be secreted by the cells of the nidamental gland 
and the egg capsule is formed in the caudal part of the oviduct. The sequence of 
events in the formation of the egg case is not known for certain. There is some 
evidence to suggest that a major part of the egg case is formed before the arrival 
of the fertilized egg. The egg case with the enclosed egg is later ejected into the sea. 

The egg capsules of sharks are more or less rectangular in shape with the 
corners prolonged into anterior and posterior pairs of horns, but considerable 
variation exists in shape and size in the different species. There is little precise 
information regarding the nature of the material composing the egg capsule. The 
earlier workers referred to it as chitinous but the term as used by them carried no 
chemical significance. On the other hand Hussakof and Welker (1908) w r ho re- 

1 This work was carried out in the Department of Zoology, University of Madras. I am 
thankful to Dr. E. R. B. Shanmugasundaram of the Department of Biochemistry for help and 
advice in regard to the chemical analysis reported in this paper. 

298 



HISTOCHEMISTRY OF SHARK EGG CAPSULES 



29<) 



ported on the chemical nature of the egg case of two species of sharks, suggested 
that it may be similar to keratin. However, the previous workers are agreed that 
in all selachians the structure and composition of the egg capsule are identical. 
Widakowich (1906) noted that in Scy Ilium the egg capsule is formed of a large 
number of "Flatten" which adhere to each other loosely at first and later, especially 
after contact with sea water, much more closely so that the entire capsule hardens. 
It has also been observed that the capsule when first formed within the oviduct is 
white and soft and gradually hardens, undergoing a change in color to brown and 
later to deep reddish brown. In Chiloscyllium griseum the egg capsules taken from 
the oviducts show a range of coloration varying from very light brown to deep 
reddish brown. The ridge-like thickening bordering the capsule is more deeply 
colored than the rest of it. 

Frozen sections of the capsule wall, which is lightly colored, show an outermost 
narrow yellowish layer containing dark granular inclusions. Internal to it is a 



outer tarnnedaxjcr 




FIGURE 1. Section through the wall of a light colored egg capsule stained with Mallory. 

broad region more or less uncolored and characterized by horizontal laminations. 
On the inner border of the laminated region is a very narrow strip which is yellow 
colored and apparently homogeneous, similar to the outermost layer (Fig. 1). In 
the laminated region a central part may be distinguished by its darker shade. The 
color disappears after treatment with ethylene chlorhydrin and the entire section is 
marked by a diminution of the dark color. This reaction may suggest the presence 
of melanin-like substances in the wall of the capsule (Lea, 1945). Further sup- 
port to the above suggestion is obtained by the results of tests performed on the 
sections of the capsule wall with hydrogen peroxide and potassium permanganate, 
both of which produce a bleaching effect (Pearse. 1954). 

In paraffin sections stained with Mallory, the outer- and innermost layers, which 
are yellowish in unstained preparations, are colored red with acid fuchsin while the 
laminated region is stained blue. Similar results were obtained with Masson's tri- 
chrome stain; the regions coloring red with Mallory are stained with xylidene 
ponceau and the laminated region is colored green. With Heidenhain's haema- 



300 



G. KRISHNAN 



toxylin the outer and inner layers are dark blue while the rest of the thickness of 
the wall comprising the laminated zone is very lightly or not at all stained. In 
the regions of the capsule which are reddish brown, the staining reactions are dif- 
ferent from those reported above. The central wide laminated region, instead of 
staining uniformly green or blue with Masson's and Mallory stains, shows red 
patches filling up the greater extent of this region. Such a change in staining 
reaction may suggest that the substance originally present has undergone a trans- 
formation so as to resemble that present in the outermost layer. 

TABLE I 

Responses of egg capsule wall of Chiloscyllium griseum to chemical tests 



Test 


Egg capsule wall 


Outer 
layer 


Middle 
laminated 
zone 


Inner 
layer 


Millon's 


light brown 


+ 





+ 




deep reddish brown 


+ + 


+ + 


+ + 


Xanthoproteic 


light brown 


+ 





+ 




deep reddish brown 


+ + 


+ + 


+ + 


Biuret 


light brown 





+ 







deep reddish brown 











Argentaffiu 


light brown 


+ + 


+ 


+ + 




deep reddish brown 


+ + 


+ + 


+ + 


Ferric chloride 


light brown 


+ 


+ + 


+ 




deep reddish brown 


+ 


+ 





Ammonium molybdate 


light brown 


+ 


+ 


+ 




deep reddish brown 


+ 


+ 


+ 


Sudan Black B 


light brown 













deep reddish brown 











Chitosan 


light brown 













deep reddish brown 











Pepsin 


light brown 





+ 







deep reddish brown 











Lead acetate 


light brown 













deep reddish brown 











Sodium nitroprusside 


light brown 













deep reddish brown 











Dilute mineral acids 


light brown 





+ 







deep reddish brown 











Boiling H 2 O 


light brown 





+ 







deep reddish brown 












+ = positive. 

= no apparent effect. 



CHEMICAL COMPOSITION 



To test the validity of the above assumption, the nature of the principal chemical 
components of the capsule walls yielding the staining reaction reported above was 
investigated. It is known from previous work that the major constituent of the 
shell substance is protein. In the following study a qualitative estimate of the 
protein constituents was made by color tests and the results are summarized in 
Table I. It is seen that the entire thickness of the capsule wall is positive to tests 
for protein. But the light colored capsules differ in some respects from those 



HISTOCHEMISTRY OF SHARK K(iG CAPSULES 301 

which are deep reddish brown. It has been pointed out that the capsule when first 
formed is white and later turns to light brown which gradually deepens to dark- 
reddish brown, the changes in coloration representing different phases in the growth 
of the capsule. In the earlier phases of growth when the capsule is very light brown., 
the outer and innermost layers give positive Millon's and xanthoproteic reactions", 
while the central laminated region is negative to these tests, but positive to biuret 
test. In the more fully formed capsule, which is deep reddish brown, the central 
laminated zone also is positive to Millon's and xanthoproteic tests. These changes 
in the reaction to protein color tests coincide with those in staining reactions with 
Mallory. Presumably the fuchsinophil substance staining red with Mallory is 
the same as that giving a Millon-positive reaction. 

A positive reaction to Millon's test has been interpreted as indicative of a protein 
containing the hydroxyl-phenyl group in the molecule, and since tyrosine is the 
only amino acid containing it, it may be inferred that in the egg capsule Millon- 
positive sites may contain a protein rich in tyrosine (Pearse, 1954). The coin- 
cidence of the Millon-positive regions with those giving positive xanthoproteic 
reactions supports the above suggestion since the latter test is said to indicate 
protein containing tyrosine, tryptophane and phenylalanine (Pearse, 1954). On 
the basis of the positive results obtained with the above two tests. Blower (1951) 
suggested that the presumptive exocuticle of the myriopods studied by him con- 
tains a protein, rich in phenolic groups and which are involved in the tanning of 
the exocuticle. Similar results have been reported by Krishnan (1956) in the 
cuticle of Scolopcndra. In the light of the above observations, a reasonable inter- 
pretation of the results of tests on the egg capsule is that when first formed it is 
constituted of a simple protein positive to biuret test and soon after, the outer and 
inner layers are modified by the presence of a protein rich in phenolic groups 
which appear to spread throughout the thickness of the wall. These changes are 
correlated with a deepening of the color of the capsule and also an increased 
chemical resistance. 

EVIDENCE FOR QUINONE-TANNING 

The features noted above recall strongly the characteristic change undergone 
by the insect cuticle during hardening by tanning (Dennell and Malek, 1955) and 
appear significant as indicative of a similar tanning process in the egg capsules. 
Brown (1950b) pointed out that if a structural protein dissolves only in sodium 
hypochlorite solution and is itself secreted by tissues containing polyphenols, there 
is circumstantial evidence for quinone tanning. It will be shown in the sequel 
that both the tests are positive with the egg case material. When small pieces of 
dark brown capsule wall were treated for varying periods with a dilute aqueous 
solution of sodium hypochlorite, the color was readily lost and on continued treat- 
ment the egg capsule wall was dissolved. Further evidence of tanning is indicated 
by the presence of a phenol oxidase which is known to be an essential participant of 
the tanning process in insect and crustacean cuticles (Bagvat and Richter, 1938: 
Dennell, 1947a, 1947b; Krishnan, 1951). In recent studies on the formation of 
helminth egg shell, which involves quinone tanning, the oxidase concerned has been 
demonstrated by the red color produced on incubation with a dilute solution of 
catechol (Smyth, 1954). The mechanism of the above reaction is due to the 



302 G. KRISHNAN 

oxidation of catechol to quinone and its condensation with the protein so as to 
produce a tanning effect. This technique was applied to the egg capsules of shark 
with positive results. Light colored capsule walls when subjected to the catechol 
treatment changed to a dark red color resulting from the tanning of the protein. 
Such a color change is less intense with dark colored capsules. That the change 
in color after catechol treatment is really due to the tanning of the protein may 
be inferred from the observation that the color is lost on addition of a dilute 
solution of sodium hypochlorite. Further, sections of material deeply colored 
by catechol treatment when stained with Mallory show correlated change in 
staining reaction, the central laminated region being fuchsinophil. so as to simulate 
the condition of a more full grown and normally reddish brown capsule. It would 
appear that by treatment with catechol the protein of the central layer is artificially 
tanned. The color change noted above was inhibited by cyanide in a concentration 
of 0.001 M, suggesting the enzymatic nature of the process and the role of an 
oxidase in bringing about the tanning effect. 

The above observations suggest that the egg capsule after its formation under- 
goes a process of hardening by phenolic tanning before being ejected into the sea, 
and this would account for the change of its coloration from light brown to deep 
reddish brown. The principal participants in the process appear to be a protein 
probably rich in tyrosine and a phenol oxidase. The results of histochemical tests 
on the egg capsule material (Table I) indicate an absence of lipids which are usually 
associated with tanning in the cuticles of insects and other arthropods. Further, 
although diphenols are indicated in the capsule walls, as may be inferred from 
positive ferric chloride tests, in the absence of a correlation between their accumula- 
tion and the tanning of the protein, their mere presence may not indicate that they 
are involved in tanning. Their persistence in the outermost layer, where tanning 
is more intense than in the rest of the thickness of the wall, may militate against 
the view that they are the tanning phenols. It is suggested that they may be re- 
lated to the formation of melanin occurring in the capsule walls, for diphenols, 
indicated by ferric chloride, accumulate in the laminated zone early in the growth 
of the capsule and their partial disappearance is followed by the occurrence of 
melanin. This feature, together with the presence of a phenol oxidase in the 
capsule wall, may suggest the oxidation of phenols to melanin. Since the latter 
appears even before the onset of tanning in this region it is suggested that the free 
diphenols may not be directly involved in the tanning process. 

HlSTOCHEMISTRY OF THE NlDAMENTAL GLAND 

With a view to investigate further the nature of the tanning process, a study of 
the mode of formation of the egg shell material was made. It is known from 
previous work that the materials forming the egg case are secreted by the nidamental 
gland. The gland is a dilatation of the oviduct at the junction of the caudal and 
cranial parts comprising a glandular body formed of tubules in a more or less 
parallel series. The histology of the gland in Chiloscyllium shows close agreement 
with that of Scylluiin canicula and Scylliuin catalus (Nalini, 1940). In the anterior 
part of the gland, distinguished as the albumin gland, the secretory tubules are 
formed of both glandular and ciliated cells. The secretions are in the form of 
transparent cytoplasmic granules which appear to be extruded by rupture of the cell 



H1STOCHEMISTRY OF SHARK EGG CAPSULES 303 

Avails into the lumen of the gland tube. The succeeding section of the gland, which 
is distinguished as the shell-secreting zone, is formed of cells whose cytoplasm is 
packed with granules during the period when egg capsules are being formed. The 
shell substance appears to be derived from these secretions and in the light of the 
foregoing observations on the egg capsules, one would expect to find in these cells 
the constituents of the tanning system. Accordingly histochemical tests for phenols 
and proteins were applied. The argentaffin, ammonium molybdate and sodium 
iodate tests for phenols were positive in the cytoplasm of the cells of the shell- 
secreting zone. Identical regions of the cells were also positive to biuret tests for 
protein. Malachite green, which is known to show a specificity for proteins in- 
volved in tanning of egg shells of helminths (Johri and Smyth, 1956), gave positive 
reaction in the cytoplasm, the granules taking a vivid green color. The greert 
coloration is said to be due to the dye becoming bonded to the protein. Since the 
cytoplasmic granules in the cells of this region react positively to both the tests for 
phenols and proteins, it is suggestive that the substance reacting may be a phenolic 
protein, similar to that reported to occur in the vitelline gland cells of helminths 
(Smyth, 1954). Frozen sections of this region of the nidamental gland when 
treated with a dilute solution of catechol develop readily a brown coloration in the 
cytoplasm of the cells. This reaction may suggest evidence of the occurrence 
of a protein undergoing tanning and an oxidase in close proximity to it, responsible 
for the oxidation of phenols involved in tanning. It appears probable that the 
oxidase and the substrate are both located in the cytoplasm of these cells. 

NATURE OF EGG CAPSULE PROTEIN 

The foregoing observations indicate that the principal constituent of the egg- 
capsule is a protein secreted by the cells of the nidamental gland, along with an 
oxidase capable of oxidizing catechol to quinone. In the egg capsules two ap- 
parently distinct protein constituents seem to occur, one forming the basal matrix 
which persists for some time in the central laminated region and the other being a 
tanned protein which is distinguished from the former by the chemical and staining 
reactions. In these respects they present very strong resemblance to the basal 
protein and that impregnating the regions destined to be tanned in the cuticle of 
insects like Penplaneta (Dennell and Malek, 1955). Here the basal protein of the 
procuticle stains blue with Mallory, is negative to Millon and xanthoproteic tests 
and lacks chemical resistance, while that impregnating the presumptive exocuticle 
stains red with Mallory, is positive to Millon and xanthoproteic tests and possesses 
considerable chemical stability. That the above characteristics of the protein of 
the presumptive exocuticle may be due to some sort of aromatic bonding is suggested 
by the observation of Kennaugh (see Dennell, 1958) that the staining properties can 
be reversed by treatment with Diaphanol which is known to break up the aromatic 
bonds by oxidation. The change in staining reaction with Mallory from red to 
blue, reported by the above author, may indicate a restoration of the protein 
component to its original state, as is found in the untanned endocuticle. In the egg 
capsule of the shark it is suggestive that the tanned protein is derived from the basal 
protein such as is found in the laminated region in the earlier stages of capsule 
formation. If it is so. it may be possible to restore the tanned protein to the 
original state by breaking up the aromatic bonds as has been done in the insect 



304 G. KRISHNAN 

cuticle referred to above. This was carried out by adopting the method used by 
Dennell (1958) who following Trim (1941), separated the tanning phenols of the 
puparia of Calliphora using alkaline stannite solution for breaking up the quinone 
bonds. Accordingly, small pieces of egg capsule material were left in a mixture of 
2% sodium hydroxide and stannous chloride at 37 C. for nearly a week, by which 
time the protein was solubilized. The protein fraction was separated and tested. 
Unlike the tanned protein it was negative to Millon's test and was easily digested 
by pepsin-hydrochloric acid and showed a marked swelling in boiling water. 

These observations, in addition to suggesting that the tanned protein of the 
egg capsule may be a derivative of the basal protein, also give some indication of the 
nature of the protein. The reaction to pepsin and swelling in boiling water are 
suggestive, especially in the light of the observation of Astbury (1945) that the 
entire egg capsule of shark yields an x-ray diffraction pattern similar to that of a 
collagenous protein. 

With a view to test further the suggestion made above, a microchemical analysis 
of the capsule protein was made using a modification of the method of Spencer, 
Morgulis and Wilder (1937), who applied the above method for a determination 
of collagen content of the muscles of rabbit. The capsule walls were cut into small 
bits and cleaned by scraping with a blunt scalpel to remove all adhering tissue. A 
sample weighing 0.1 gm. was homogenized with an equal quantity of distilled water 
in a Potter-El vehjm homogenizer, and the material was placed in a water bath at 
100 C. for about 15 minutes along with 10 times its weight of water. This was 
later stored in a refrigerator, and next day, it was autoclaved for 3 hours at 20 
pounds pressure so as to convert collagen, if any, into gelatin. The material was 
then centrifuged at 4,000 rev./min. for one hour and the supernatant fluid drawn off. 
An aliquot of this fluid was treated with 3% tannic acid when a copious precipitate 
was obtained. The above evidence in support of the view that a collagenous type 
of protein occurs in the egg capsule was checked by a chromatographic analysis of 
the precipitate. The material was treated with ten times its weight of 6 N HC1 in 
a sealed tube and hydrolysed at 105 C. for 24 hours. The hydrolysate was dried 
in a vacuum desiccator containing potassium hydroxide and this was used for 
analysis by partition chromatography, following the capillary ascent method of 
Williams and Kirby (1948). The hydrolysate was dissolved in a small quantity 
of distilled water and a 20-^1 sample was used for spotting on the filter paper and 
the chromatogram run with butanol-acetic acid-water as the solvent. Simultane- 
ously a number of chromatograms were run under identical conditions using pure 
amino acids for purposes of comparison. A solution of 0.1% ninhydrin in butanol 
was used for spraying. Qualitative analysis of the chromatogram thus obtained 
shows in general an agreement in amino acid make-up with that of mammalian 
connective tissue (Bowes and Kenten. 1949), suggesting that the protein in question 
may be allied to collagen. Further, the pattern of spots was more or less identical 
Avith that of a sample of pure gelatin hydrolysed and otherwise treated in the same 
way as the test material. Most of the amino acids found in the chromatogram of 
the egg case material correspond to those found in the gelatin. 

However, it is seen that the egg case material differs in the absence of hy- 
droxylysine, leucine and valine as well as in the presence of tryptophane. The 
absence of hydroxylysine may suggest a relationship to elastin, but the occurrence 
of tryptophane is unusual for a collagenous type of protein. It is possible that its 



HISTOCHEMISTRY OF SHARK K(1G CAPSULKS 305 

presence may be due to a contaminant. However, it must be mentioned tbat the 
amino acid composition of collagen derived from different sources may vary 
markedly. The collagen of fish skin is known to differ from mammalian collagen 
in having a low hydroxyproline content while serine, threonine and methionine afe 
in greater amounts (Gustavson, 1956). Such quantitative variations occur not 
only in those amino acids considered to be characteristic of collagen but also in 
some of the non-typical residues like tyrosine. A quantitative amino acid analysis 
is therefore necessary for making a valid comparison. However, the present study 
is essentially from a biological viewpoint and such evidence as has been obtained is> 
enough to indicate the nature of the material composing the egg capsule. The 
presence of non-polar amino acids like glycine and alanine, the prominence of 
proline and hydroxyproline and the comparative rarity of aromatic residues are 
features of the egg capsule protein, which together are suggestive that it may be 
allied to the collagen group (Gustavson, 1956). 

DISCUSSION 

The foregoing observations suggest that the egg capsules of Chiloscyllium 
undergo a tanning process resulting in acquisition of mechanical rigidity and chem- 
ical resistance. The process is comparable to that occurring during the formation 
of ootheca of Blatta (Pryor, 1940) but certain differences are significant. Unlike 
in the insect, here the substrate involved in tanning is a protein without a lipid 
component. No free diphenol appears to participate in the process. The resultant 
tanned product is also different from the tough amber colored sclerotin, being only 
yellowish, and retains a reactivity to stains. The protein constitution of the egg 
capsule appears to be such that it cannot yield sclerotin after tanning, for it has 
been observed in arthropod cuticles that unless the protein precursor of tanning is 
impregnated with a lipid constituent, sclerotin may not be the resultant product. 
Sclerotin itself has been considered as a lipoprotein subsequently tanned. It is clear 
that the tanned protein of the egg case is not sclerotin but recalls in its chemical and 
staining reactions the tyrosine-rich protein precursor of sclerotin, found in the pre- 
sumptive exocuticle of an inset like Periplaneta (Dennell and Malek, 1955) or the 
so-called pro-sclerotin described by Blower (1951) in the myriopods studied by him. 
In the above instances the protein in question shows considerable chemical stability 
even before forming a complex with the lipid participant of tanning and stains red 
with Mallory, unlike the protein confined to those regions which do not undergo 
tanning. The chemical stability of the protein has been attributed to the occurrence 
even at this stage of some kind of aromatic tanning which is distinct from the 
subsequent tanning of the lipoprotein complex by free diphenols resulting in 
sclerotin (Dennell, 1958). Such a tanning has been distinguished by the above 
author as "primary tanning" in contrast to the "secondary tanning" which results 
in sclerotin. In the absence of the participation of free diphenols "primary tanning" 
would be presumably by oxidation of tyrosine side-chains of the protein. The 
tanned protein of the shark egg capsule recalls strongly the product of "primary 
tanning" in its chemical nature, staining characteristics, possession of resistant 
qualities and retention of a "tannable" condition having still free amino groups. It 
seems probable from the observations reported in the present study that the mode of 
tanning of the egg capsule protein may involve a process of auto-quinone tanning 
similar to that suggested to occur in the egg shells of helminths (Smyth, 1954). 



306 G. KKiSHNAX 

SUMMARY 

1 . The egg capsules of Chiloscylliuni griseum, when first formed in the oviducts, 
are soft and white and gradually turn light brown to deep reddish brown before 
being ejected into the sea. 

2. Light brown capsule walls show in section an outer and an inner narrow 
layer apparently homogeneous and yellowish in color while a wide central region is 
laminated and uncolored. This layer stains blue with Mallory, indicates the 
presence of a simple protein positive to biuret test and lacks chemical resistance. 
The outer and inner layers stain red with Mallory and contain a protein which is 
positive to Millon and xanthoproteic tests indicative of phenolic groups. In deeply 
colored walls the central laminated layer shows staining and histochemical re- 
actions similar to those of the outer layer. 

3. Evidence has been presented indicating that the above changes may be due 
to the tanning of a basal protein involving a phenol oxidase resident in the capsule 
wall. 

4. The constituents of the tanning system are derived from the secretions of the 
cells of the nidamental gland. The tanning of the egg capsule protein does not 
appear to involve free diphenols so that some form of auto-quinone tanning seems 
to occur. 

5. The tanned protein of the egg capsule is unlike the sclerotin of the insect 
cuticle, but recalls in its staining and histochemical reactions the protein precursor 
of tanning impregnating the presumptive exocuticle of insects like Periplaneta. 

6. The nature of the egg capsule protein has been investigated using micro- 
chemical and chromatographic methods. From the results obtained it is suggested 
that it is allied to the collagen group of proteins. 

7. The results are discussed. 

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HISTOCHEMISTRY OF SHARK EGG CAPSULES 307 

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THE SIZE AND SHAPE OF METAMORPHOSING LARVAE OF 

VENUS (MERCENARIA) MERCENARIA GROWN AT 

DIFFERENT TEMPERATURES 

VICTOR L. LOOSANOFF 

U. S. f'ish and Wildlife Service, Bureau of Commercial I ; ishcrics, Biological Laboratory, 

Milford, Connecticut 

Among the characters to be employed for identification of lamellibfanch larvae, 
their size at metamorphosis has been suggested by some workers (Lebour, 1938). 
Other students have maintained, however, that since there may be correlations 
between size and environmental factors, the size at metamorphosis cannot be used 
as a criterion for recognition of larvae. Sullivan (1948) thought that this was 
confirmed for mature larvae of Mya arcnaria in Canadian waters, which she ob- 
served to metamorphose in Malpeque Bay upon reaching a length of 250 microns, as 
compared with a length of 415 microns reported by Stafford (1912) for larvae of 
the same clam in the St. Andrews region. 

According to Sullivan the difference in size at setting was probably the result 
of the difference in the summer water temperature at these two places because 
larvae developing at the lower temperature (St. Andrews) needed to reach a larger 
size before metamorphosing. This opinion is, indirectly, in agreement with that 
of Erdmann (1934), who thought that the size of larvae of the European oyster, 
Ostrea cdulis, at the time of swarming was regulated by the temperature at which 
the larvae developed. 

So far, no one has offered a definite explanation as to why lamellibranch larvae 
should reach a larger size if grown in colder water. It seems, however, that this 
opinion was formed because it is widely believed that, "In many instances, and 
perhaps as a general rule, the size that an animal attains is greater when it is reared 
at low temperature" (Coker, 1947, p. 102). Furthermore, since it is known that 
large individuals of the same species have a relatively smaller surface area than do 
small ones, the larger size may be considered as an adaptation to an increase in the 
viscosity of the water which accompanies a decrease in temperature. Examples to 
support this assumption can be found in numerous papers, including those of Mur- 
ray and Hjort (1912), and many students of Radiolaria, copepods and certain other 
forms (Hedgpeth, 1957). Kofoicl (1930), for instance, reported that marine 
protozoa living in cold water grow to much larger sizes than do their relatives 
existing at higher temperatures. 

Aquatic biology also offers many instances in which organisms of the same 
species grown at different temperatures may show a somewhat different shape. 
The phenomenon of cyclomorphosis, reviewed by Brooks (1946), is an example. 
In lamellibranch larvae, of course, no radical changes in structure, as observed in 
Daphnia populations, can be anticipated. Yet, it has been reported (Jp'rgensen, 
1946, p. 296) that at least in some lamellibranchs, such as Venus gallina, "the 
larval outline varies from almost square to circular." According to the same author 

308 



TEMPERATURE AND SIZE OF CLAM LARVAE 309 

the veligers of the common mussel, Mytilus edulis, of Danish waters also show a 
remarkable variability in their shape. The water temperature during development 
can again be suspected as a factor affecting the shape of the larva. 

During the past few years larvae of approximately 20 species of lamellibranchs 
have been successfully cultured from fertilized egg through metamorphosis by mem- 
bers of our laboratory (Loosanoff, 1954). The data collected during these studies 
will soon permit us to offer reliable material for recognizing larvae of the species 
with which we have been working. It will include photomicrographs of the larvae 
and length-width measurements of their shells from early straight hinge stage until 
metamorphosis. However, before offering these criteria, it was deemed necessary 
to ascertain the following possibilities, which could reflect on the reliability of our 
material : 

1) We wanted to know whether, as is suggested by some students, individuals 
of larval populations grown at relatively low temperatures reach a larger size before 
metamorphosing than do larvae grown in warmer water. If this is true, special 
corrections, perhaps as formulae, should be offered to show the relationship between 
average size at setting and water temperature. 

2) Since one of the criteria for recognizing a larva nearing metamorphosis is 
its dimensions, i.e., length and width, it was necessary to determine whether the 
length-width ratio is relatively constant or if it changes in conformance with the 
temperature of the water in which larvae develop. 

The questions posed above could be answered only on the basis of well-controlled 
experiments in which the water temperature was the only factor varied. Obviously, 
because of the time and efforts required, it would have been difficult to conduct such 
experiments with larvae of all 20 species of lamellibranchs with which we were 
working. We decided, therefore, to limit ourselves to observations on one or two 
species only. This paper is devoted chiefly to a description of the observations on 
size and length-width ratio at the beginning of metamorphosis of larvae of the hard 
shell clam, Venus (Mercenaries} rncrccnaria, developing at different temperatures. 

Certain aspects of the studies which provided data for this paper have already 
been described (Loosanoff, Miller and Smith, 1951). In brief, they consisted of 
growing larvae of Venus (Mcrccnaria) mercenaria at constant temperatures ranging 
from 15.0 to 33.0 C. at intervals of 3.0 C. Since fertilized eggs that were placed 
in water of 15.0 or 33.0 C. showed abnormal development and heavy mortality, 
few ever reaching veliger stage, growth of larvae at these temperatures will not be 
discussed here. 

The work was done in winter, the time we find most convenient to control the 
water temperature (Loosanoff, 1949). Altogether, four experiments were con- 
ducted. However, in one experiment one of a pair of cultures grown at 24.0 C. 
was accidentally lost, while in the fourth experiment, which was conducted during 
a comparatively warm spell when low temperature was difficult to maintain, no 
cultures were carried at 18. C. As is our practice, the water in the culture jars 
was changed every second day (Loosanoff and Davis, 1950). The larvae were 
fed a mixture of micro-organisms consisting principally of Chlorella sp. 

Samples for larval measurements were taken 48 hours after fertilization and every 
second day thereafter, until metamorphosis. These samples consisted of 50 larvae 
measured at random from each culture vessel, i.e., 100 larvae from each temoerature 
group. The length represented the greatest distance between the anterior and 



310 



VICTOR L. LOOSANOFF 



posterior shell margins, while the width was the distance measured from the tip of 
the umbo to the middle of the ventral shell margin. 

Because the larvae could not be marked individually, their rate of growth and 
size at setting could not be recorded directly on this basis. For this reason we 
used two substitute criteria. One was the average length of the larvae on the day 
the setting was first observed in each culture, and the second, the maximum size of 
the larvae observed during the life of the culture. 

The number of days required for setting to begin at the different temperatures 
in the four experiments is given in Figure 1. Clearly enough, there were dif- 
ferences between the cultures within the same temperature group and also between 



25 



20 



<n 

v 



15 



10 



Y'=-I.OOX+ 37.91 




V 

r\ ' H 



18 



21 24 



27 



30 



TEMPERATUREC 



FIGURE 1. Number of days necessary for clam larvae to begin setting in cultures grown 
at different temperatures. = mean of individual culture ; o mean of all cultures grown at 
the same temperature ; x = mean for given temperature predicted from regression line. 



the groups carried at different temperatures. The most uniform results were ob- 
tained with the 30.0 C. group, where the beginning of setting in the different 
cultures varied between seven and nine days after fertilization, a difference of only 
two days. However, the difference between the shortest and longest periods needed 
for larvae to begin setting became greater in colder water. For example, in the 
18.0 C. group the earliest beginning of setting was recorded 16 days after fertiliza- 
tion and the latest, after 24 days, a difference of eight days (Fig. 1). 

An analysis of variance was carried out to test the significance of the differences 
among the different temperature groups on the number of days required for setting 
to begin. Since the result was highly significant (beyond the .001 level), separate 



TEMPERATURE AND SIZE OF CLAM LARVAE 



311 



"t" tests were run for all possible pairs of temperature groups. All the "t" tests 
were highly significant (beyond the .01 level), with exception of the comparison 
between the 27.0 and 24.0 C. groups, and between the 21.0 and 18.0 C. groups. 
These results show, therefore, that a very strong relationship exists between tem- 
perature and 'date of setting, i.e., larvae reared at high temperatures set significantly 



210 



200 



190 



i 

H 
O 

UJ 



180 



170 



160 



150 




18 



21 24 



27 



30 



TEMPERATURE 



FIGURE 2. Mean length of clam larvae grown at different temperatures on day of beginning 
of setting. Measurements in microns. = mean of individual culture; o = mean of all cultures 
grown at the same temperature. 



earlier than those raised at lower temperatures. This conclusion was expressed in 
the preliminary paper (Loosanoff, Miller and Smith, 1951). 

Plotting of the dates of beginning of setting in the different cultures against the 
temperatures showed that the mean number of days for setting to begin for the 
various temperatures lies on an almost straight line (Fig. 1). Since the line con- 



312 



VICTOR L. LOOSANOFF 



200 



190 



180 



170 



x 

H 

o 



160 



150 



140 



130 








150 



160 



170 



180 



LENGTH 



190 



200 



210 



FIGURE 3. Mean length and width of clam larvae of individual cultures on day of 
beginning of setting. Measurements in microns. 

necting the means of the different temperatures is obviously rectilinear, a regression 
equation was computed and found to be Y' - l.OOX + 37.91, where Y' is the 
predicted setting date and X is the temperature. 

Because the regression line is an excellent fit to the experimental data, it is 
probable that interpolation within the 18.0 to 30.0 C. limits of the experiment can 
be made with a fair degree of confidence. However, extrapolation beyond these 
limits is not justifiable. This was shown by our other experiments, which demon- 
strated that clam eggs placed in water having a temperature of 15.0 or 33.0 C. 



TEMPERATURE AND SIZE OF CLAM LARVAE 



313 



did not develop normally. Therefore, since the lineal regression does not hold even 
for a slightly higher or lower temperature, it cannot be expected to hold for lower 
or higher temperatures. 

An analysis of variance test showed no significant differences among the five 
temperature groups, with respect to mean length of larvae at date of setting. Thus, 
although larvae grown at different temperatures required different periods to reach 
metamorphosis, in all cases they reach approximately the same mean length before 
setting. This observation indicates, therefore, that there was virtually no relation- 
ship between temperature and mean length at date of setting (Fig. 2). Neverthe- 
less, the same figure shows that there was considerable variation in mean length at 
the beginning of setting among the various cultures within each temperature group. 

In our studies we were also concerned with the shape, at metamorphosis, of 
larvae grown at different temperatures because, as has already been mentioned, the 
literature contains several remarks concerning variability of shape of larvae of the 
same species near setting time. Since the simplest method of describing the shape 
of a larva in mathematical terms for statistical analysis is to indicate its length- 
width ratio, measurements were made on larvae of all cultures, except those con- 
stituting the fourth experiment where no width measurements were taken, and the 
correlation between the mean length and the mean width of the larvae of each 
culture, on the day of the beginning of setting, was determined (Fig. 3). The 



1.20 



1.15 



1. 10 



o 
u 



1.05 



1.00 





18 



21 24 27 



TEMPERATURE C 



30 



FIGURE 4. Ratio of mean length to mean width of clam larvae of different cultures on 
days of beginning of setting, in relation to temperature. = mean of individual culture ; o = 
mean of all cultures grown at the same temperature. 



314 



VICTOR L. LOOSANOFF 



results indicated this correlation to be so high (r = .95) that it seems unlikely that 
any analysis made using width as a variable would add anything new to that already 
made with length. 

Continuing the analysis of data that might help in discovering the differences in 
shape of larvae grown at different temperatures, a study was made of the ratio of 
mean length to mean width at the date of setting. It failed to show the existence of 
any significant change in the ratio at different temperatures (Fig. 4). Thus, this 
matter has been satisfactorily solved to assure investigators working with lamelli- 



230 



220 



o 

z 

UJ 



210 



ID 

* 200 
x 



190 



180 




18 



21 24 27 



TEMPERATURE C 



30 



FIGURE 5. Maximum length of clam larvae of different cultures during the life of a 
culture, in relation to temperature. Measurements in microns. = mean of individual culture ; 
o = mean for all cultures grown at the same temperature. 



branch larvae that individuals of the same species display virtually the same shape 
at metamorphosis, even though they are grown at different temperatures. 

Although our studies showed that even if larvae are grown at different tem- 
peratures, they, in all cases, reach approximately the same mean length before 
setting, the question still unanswered is whether there is an appreciable relation- 
ship between the maximum length of larvae on the date of setting and the tem- 
perature. A statistical analysis demonstrated the lack of an appreciable relationship 



TEMPERATURE AND SIZE OF CLAM LARVAE 



315 



between these two variables, giving a correlation of - .16. The lack of relation- 
ship is clearly indicated in Figure 5, which shows that the means of the cultures 
grown at five different temperatures varied from 201 /x to 208 p, a range of only 
seven microns. Nevertheless, we again noticed a considerable variability among 
the cultures within each temperature group, although it was much less pronounced 
within the 18.0 C. group than in certain others. However, again, no definite trend 
in this respect was observed because the variability of the maximum length of the 



15 



10 



z 
o 



' = -5.80 + .51 X 




18 



21 24 

TEMPERATURE 



27 



30 



FIGURE 6. Average daily length increment of larvae grown at different temperatures. 
Measurements in microns. mean of individual culture; o = mean of all cultures grown at 
the same temperature ; x = mean for given temperature predicted from regression line. 



larvae in the different cultures grown at 27.0 C., the second highest temperature 
group, did not differ greatly from that recorded for the lowest, i.e., the 18.0 C. 
group. 

Using the data collected during these studies, we can calculate the average daily 
growth increment for all cultures at given temperatures. By plotting the average 
daily growth increment for each temperature group against the corresponding 



316 VICTOR L. LOOSANOFF 

temperature, an approximately rectilinear relationship becomes evident (Fig. 6). 
The regression equation computed was found to be Y' = -- 5.80 + .51 X, where Y' 
is the predicted average daily growth increment and X is the temperature. The 
relationship indicates, naturally, that faster growth occurred at higher temperatures 
and, theoretically, from the regression equation it can be assumed that if the tem- 
perature were reduced to about 11.3 C, growth would stop completely. 

In discussing our results it must be remembered that they are representative 
only for this series of experiments and that there are many factors which can 
change the daily growth increment. One of these, capable of lowering or in- 
creasing the rate of growth of larvae, is the quality of the food. In our experiments 
the larvae were fed a mixture of phytoplankton, consisting largely of Chlorella. 
However, recent studies of Davis and Guillard (1958) clearly showed that the 
growth of clam larvae varied greatly depending upon the kind of food they were 
given. Davis found that the larvae grew best when fed a culture of mixed flagel- 
lates, including Isochrysis, Monochrysis, Dunaliclla, and Platymonas, while the 
larvae given Chlorella, two species of which were tried, grew considerably slower. 
In the same series of experiments Davis was able to demonstrate that the rate of 
growth of larvae of the American oyster, Crassostrea virginica, also varied greatly 
according to the kind of food organisms available. 

Quantity of food organisms is another factor to be considered. Our earlier work 
(Loosanoff and Engle, 1947; Loosanoff, Davis and Chanley, 1953a) showed that 
heavy concentrations of food cells, such as Chlorella, usually seriously interfered 
with the feeding of adult oysters and that they also either killed the clam larvae or 
retarded their growth. Furthermore, they indicated that the optimum concentra- 
tion of food organisms depended upon the kind and size of their cells. Since, in 
our experiments described in this article, the number of cells was not accurately 
determined and the food did not consist of pure cultures but of a mixture of many 
organisms, we do not know whether the clam larvae were fed the optimum food 
concentrations. This circumstance, however, does not invalidate our comparisons 
because all cultures were given food of the same quality and in the same quantity. 

Finally, the effect of the concentration of larvae in the experimental cultures 
should be considered. Our studies (Loosanoff, Davis and Chanley, 1953b; Loos- 
anoff, 1954) have shown that larvae in crowded cultures grow somewhat slower. 
However, the difference in the rate of growth of larvae in lightly-populated and 
those in densely overcrowded cultures was not too great. For example, we de- 
termined, at the end of the tenth day, that the mean length of larvae in the cultures 
containing only six individuals per cubic centimeter of water was 162 p., whereas 
the mean length in the overcrowded cultures containing 52 individuals per cubic 
centimeter was 144 //,, or only 18 //, less than that recorded for lightly-populated 
cultures. Since, in the experiments described here, we began with the same number 
of larvae in all containers and because during the experiments no excessive mortality 
was recorded in any of the cultures, our larval populations in all jars were not much 
different from each other and, therefore, could not seriously affect the uniformity 
of the experimental conditions. 

In concluding this article a brief reference to one more aspect of the role of water 
temperature on growth of bivalve larvae may be appropriate. It has frequently been 
reported that species living in warmer water have just as long a pelagic life as their 



TEMPERATURE AND SIZE OF CLAM LARVAK 317 

northern relatives, and that, at a given temperature, the eggs of the southern species 
cleave and develop more slowly than those of the northern species of the same genus 
(Fox, 1936; Thorson, 1950). This suggests that even if the eggs and larvae were 
cultured under identical conditions, development of the eggs and larvae of the 
southern clam, Venus (Mercenaria) campcchiensis, would require a longer period 
than is needed for eggs and larvae of the northern clam, Venus (Merccnaria) 
merccnaria. I had the opportunity to verify this contention by the studies con- 
ducted together with my associate, H. C. Davis. Adult Venus (Mercenaria) 
campcchiensis were imported from the Apalachicola area of the Gulf of Mexico in 
November, 1953. Several weeks later these clams were conditioned for spawning. 
A group of large Venus (Mercenaria} mercenaria, natives of Long Island Sound, 
were ripened under identical conditions simultaneously with the southern species. 
When both groups w r ere ripe, spawning was induced by our usual methods (Loos- 
anoff and Davis, 1950). Fertilized eggs of each species and, later, larvae develop- 
ing from these eggs were cultured under identical conditions, the temperature being 
approximately 21.0 C. Triplicate cultures of each species were grown, and 
random samples of 100 larvae from each culture were measured every second day. 
The curves constructed on the basis of this information showed that the rates of 
growth of the larvae of the two species were practically identical. Moreover, 
setting of larvae of both groups began at the same time. The results of this 
experiment contradict, therefore, the conclusion that when grown at the same 
temperature the eggs and larvae of the southern species develop more slowly than 
those of the northern species of the same genus. 

I wish to express my thanks to Mrs. Barbara Myers for the statistical analysis of 
the data and to my associates, Miss Rita S. Riccio and Harry C. Davis, for their help 
in preparation of this article. 

SUMMARY 

1. The mean setting dates for larvae of Venus (Mercenaria) mercenaria grown 
at constant temperatures of 30.0, 27.0, 24.0, 21.0 and 18.0 C. were found to 
lie on an almost perfectly straight line according to equation Y' = - - 1.00 X + 37.91, 
where Y' is the predicted setting date and X is the temperature. 

2. There were no significant differences among the five temperature groups with 
respect to mean length of larvae at time of setting. 

3. There was no apparent relationship between maximum length of larvae at 
time of setting and temperature. 

4. The correlation between mean width and mean length of larvae at time of 
setting was very high ( r = .95 ). 

5. No apparent relationship was found between shape of larvae (i.e., ratio of 
mean length to mean width) at time of setting and temperature. 

6. The average daily growth increment for all cultures at given temperatures 
under the conditions prevailing during the experiments was determined. 

7. The rate of growth of larvae of the southern clam, Venus (Mercenaria) 
campechiensis, was the same as that of the northern species, Venus (Mercenaria) 
merccnaria, when the temperature and other conditions were identical. Moreover, 
setting of larvae of both species began at the same time. 



318 VICTOR L. LOOSANOFF 

LITERATURE CITED 

BROOKS, J. L., 1946. Cyclomorphosis in Daphnia. I. An analysis of D. retrocurva and D~ 

galeata. Ecol. Monographs, 16: 409-447. 
COKER, R. E., 1947. This Great and Wide Sea. The University of North Carolina Press, 

Chapel Hill, N. C, pp. 1-325. 
DAVIS, H. C., AND R. R. GUILLARD, 1958. Relative value of ten genera of micro-organisms as 

foods for oyster and clam larvae. U. S. Dept. of the Interior, Fish and Wildlife 

Service, Fish. Bull. 136, 58 : 293-304. 
ERDMANN, W., 1934. Untersuchungen uber die Lebensgeschichte der Auster. No. 5 Uber die 

Entwicklung und die Anatomic der "ansatzreifen" Larven von Ostrea edulis mit 

Bemerkungen uber die Lebensgeschichte der Auster. Wiss. Meeresunt. Abt. Helgo- 
land.. 19: 340-341. 
Fox, H. M., 1936. Rates of cleavage of sea urchin eggs in different latitudes. Nature. 178: 

389. 
HEDGPETH, J. W., 1957. Treatise on marine ecology and paleoecology. Waverly Press, 

Baltimore, Md., 1 : 1-1296. 

J0RGENSEN, C. B., 1946. Reproduction and larval development of Danish marine bottom in- 
vertebrates. 9. Lamellibranchia. Medd. Komm. Danmarks Fish. Havundcrs. Ser. 

Plankton, 4: 277-311. 
KOFOID, C. A., 1930. Factors in the evolution of the pelagic Ciliata, the Tinntinnoinea. Western 

Soc. Nat. Contr. Marine Biol., 1-39. 
LEBOUR, M. V., 1938. Notes on the breeding of some Lamellibranchs from Plymouth and their 

larvae. /. Mar. Biol. Assoc., 23: 119-145. 
LOOSANOFF, V. L., 1949. Method for supplying a laboratory with warm sea water in winter. 

Science, 110: 192-193. 
LOOSANOFF, V. L., 1954. New advances in the study of bivalve larvae, .liner. Scientist. 42: 

607-624. 
LOOSANOFF, V. L., AND H. C. DAVIS, 1950. Conditioning J-'. mercenaria for spawning in winter 

and breeding its larvae in the laboratory. Biol. Bull., 98 : 60-65. 
LOOSANOFF, V. L., AND J. B. ENGLE, 1947. Effect of different concentrations of micro-organisms 

on the feeding of oysters (O. virc/inica). Fisher \ Bull. 42 of Fish and Wildlife Service, 

51 : 31-57. 
LOOSANOFF, V. L., H. C. DAVIS AND P. E. CHANLEV, 1953a. Behavior of clam larvae in 

different concentrations of food organisms. Anat. Rec., 117: 586-587. 
LOOSANOFF, V. L., H. C. DAVIS AND P. E. CHANLEY, 1953b. Effect of over-crowding on rate of 

growth of clam larvae. Anat. Rec., 117: 645-646. 
LOOSANOFF, V. L., W. S. MILLER AND P. B. SMITH, 1951. Growth and setting of larvae of 

Venus mercenaria in relation to temperature. /. Mar. Research, 10: 59-81. 
MURRAY, J., AND J. HJORT, 1912. The Depths of the Ocean. London, Macmillan, pp. 1-821. 
STAFFORD, JOSEPH, 1912. On the recognition of bivalve larvae in plankton collections. Contr. 

Canad. Biol. Rep. XIV, 221-242. 
SULLIVAN, C. M., 1948. Bivalve larvae of Malpeque Bay, P. E. I. Bull. Fish. Res. Bd. Canada. 

77: 1-36. 
THORSON, G., 1950. Reproductive and larval ecology of marine bottom invertebrates. Biol. 

Rev., 25 : 1-45. 



ANTIGENIC DIFFERENCES BETWEEN STEM AND 
HYDRANTH IN TUBULARIA * 

JOHN B. MORRILL, JR.^.s 

iic Institute, Florida State University, Tallahassee, 1'Iorida 

The marine hydroid, Tubularia crocea, consists essentially of two layers of tissue 
which are differentiated into a hydranth with hypostome, two groups of tentacles 
and gonophores, and a stem portion surrounded by a chitinous perisarc. Chemical 
differences may underlie these morphological differences between the hydranth and 
stem regions. Since the morphallactic regeneration of a hydranth from the stem 
region involves a remodeling of a certain portion of the stem tissues, further 
chemical changes may accompany visible morphological changes during regenera- 
tion of a hydranth. Because proteins and other complex molecules are the principal 
substances of organic forms, basic changes in morphogenesis probably involve pro- 
duction and transformation of such molecules and their aggregates. 

The key role presumably played by the metabolism of complex molecules and 
especially proteins has interested developmental biologists for some time. A num- 
ber of workers have used immunological methods to study differentiation of such sub- 
stances and to correlate changes in their composition with visible morphological 
differentiation [reviewed by Irwin (1949), Ebert (1955). Tyler (1955, 1957), 
Woerdeman (1955), Schechtman (1955), Nace (1955), and' Brachet (1957)]. 
While there have been many immunochemical studies concerned with embryonic 
development, immunological methods have rarely been used in studying problems 
of regeneration although the usefulness of such methods in studies on regeneration 
has been mentioned by Woerdeman (1953). De Haan (1956) and Laufer (1957) 
have used immunological techniques to study muscle differentiation in the regen- 
erating limb of axolotl larvae. 

In spite of numerous investigations on regeneration in Tubularia there is little 
information on the chemistry of this organism. A rational prerequisite to the 
investigation of chemical changes during hydranth regeneration would seem to 
involve the determination of chemical differences between the two main regions, 
the hydranth and the stem. Accordingly, the present study was undertaken to 
determine the similarities and differences in the antigenic composition of Tubularia 
crocea hydranths and stems (cf. preliminary note by Morrill, 1958). 

MATERIALS AND METHODS 

A species of Tubularia, T. crocea, was collected from the St. John's River 
jetties in Florida, where the organism is abundant from October to June. Only 

1 Part of a dissertation submitted in partial fulfillment of the requirements for the degree 
of Doctor of Philosophy, Florida State University. Aided by National Science Foundation 
grant to Dr. C. B. Metz. Contribution from the Oceanographic Institute, Florida State 
University. 

2 The major portion of this study was performed during tenure of a National Science 
Foundation Predoctoral Fellowship. 

3 Present address : Department of Biology, Wesleyan University, Middletown, Connecticut. 

319 



320 JOHN B. MORRILL, JR. 

colonies whose stems were relatively free of macroscopic epizooites were used. 
After their collection the animals were kept in jars of sea water for not more than 
36 hours. 

Clean whole animals, stems, and hydranths were frozen in a dry-ice bath. 
Hydranths and stems were prepared by first cutting off the hydranths and then 
removing the distal 4 millimeters of the stem plus any basal region of the stem 
that had epizooites. Stems were frozen within 20 minutes after hydranth removal. 
Hydranths were frozen within 4 hours after they had been severed from the stems. 
Separation of hydranths and steins on a sexual basis proved to be impractical. 
The frozen tissues were lyophilized and stored at -- 20 C. 

Preparation of saline extracts for use as antigens 

One-gram quantities of lyophilized tissue were ground in a mortar and extracted 
with 10 ml. of buffered saline (9 g. NaCl/1., 0.1 M phosphate buffer, pH 7.0) for 
8 to 12 hours at 4 to 10 C. At the end of this period the supension was homoge- 
nized in a glass homogenizer in an ice bath. The homogenate was kept at 4 to 10 C. 
for two more hours. Then the homogenate was centrifuged for 30 minutes at a cen- 
trifugal pressure of approximately 4500 g and the cloudy supernatant collected and 
stored at -- 20 C. The nitrogen content of the extracts, determined by a modified 
nesselerization method (Hawk et al., 1954), varied as follows: whole animal, 0.15 
to 0.50 mg. N/ml. ; hydranth, 0.25 to 0.82 mg. N/ml. ; stem, 0.23 to 0.68 mg. N/ml. 

The degree of contamination with insoluble particulate matter may have varied 
in the extracts. Therefore, equilibration on a nitrogen basis is at best an approxi- 
mate equalization of antigen concentration. 

Preparation of antisera 

Nine male rabbits were used in the preparation of antisera. None of the pre- 
injection rabbit sera precipitated the extracts. Several injection routes were 
employed. These included intravenous, intermuscular with oil emulsion adjuvant 
(Freund, 1947), and intraperitoneal injections. The intraperitoneal route yielded 
the most satisfactory antisera. Two rabbits received three series of intraperitoneal 
injections over a two-month period. Each series consisted of 10 to 15 milliliters of 
whole animal extract administered within 6 to 8 days. The extracts injected into 
these rabbits were not adjusted on a nitrogen basis. One of these rabbits (Rabbit 
4) was injected with the supernatant fraction of the homogenate. The other 
(Rabbit 6) was injected with the entire homogenate. 

Blood was collected in sterile 50-milliliter centrifuge tubes 5, 7, and 17 days 
after the last injection of each series and allowed to clot at room temperature for 
4 to 5 hours. The clots were loosened and the tubes stored at 4 to 10 C. for 24 
hours. The serum then was decanted, centrifuged, and stored in vials at -- 20 C. 
until used. 

Serological tests 

In order to test for antigenic differences between hydranth and stem tissues two 
types of precipitin tests were used. The first test employed was the standard 
interfacial ring test. When this test failed to reveal any antigenic differences, even 
when antiserum absorbed with hydranth or stem extract was used, the Ouchterlony 



ANTIGENS IN TUBULARIA 321 

agar gel diffusion method was employed in order to determine the spectrum of 
individual precipitin reactions and what differences might exist between the spectra 
of precipitin lines produced by anti-whole animal serum and stem and hydranth 
extracts. 

Interfacial ring tests were performed by layering 0.05 ml. Tubularia extract over 
0.05 ml. antiserum or over saline or pre-injection serum controls in 3 X 20 mm. 
capillary tubes. The tubes were capped with plastocene clay, incubated at 37 C.. 
and examined after one and two hours for the presence of a precipitating ring at 
the interfaces. The highest antiserum titer (dilution of antiserum) of the sera 
from the two rabbits was 64. The highest antigen titer of the saline extracts was 
16,000. These titers were obtained with the sera collected 5 and 7 days after the 
third injection series. No precipitates formed in control tests where pre-injection 
serum plus saline extracts and antiserum plus saline alone were employed. 

The agar gel diffusion technique of Ouchterlony (Ouchterlony, 1949) was 
employed with modifications by Nace (personal communication). Details of the 
method are as follows. Four per cent agar was prepared and washed in distilled 
water for several days. This was used to prepare an aqueous 2 per cent agar 
containing aqueous merthiolate (0.25 ppth) and methyl orange (40 ing. per 500 ml. 
agar). A basal layer of this melted 2 per cent agar was poured on the bottom of 
petri dishes. After this layer had hardened additional agar was added and a Incite 
well mold placed in position. When the agar had hardened the mold was removed. 

The wells were filled with antigens and antisera 0.30 ml. in the large square 
well and 0.15 ml. in the narrow rectangular wells. The center well of each plate 
was filled with antiserum or pre-injection serum and the four surrounding wells 
with saline extracts or saline. The plates were incubated in high humidity in an 
air-tight container at 37 C. for 7 days. They were then brought to room tem- 
perature for 6 to 24 hours and finally left at 5 C. for an additional 7 days. 

At the end of 14 days the agar plates were fixed in 5 per cent formalin with 
methyl orange added, mounted between thin glass plates, and inserted into a photo- 
graphic enlarger. The focused image of the wells and precipitin lines was so faint, 
particularly in the region of coalescence of lines, that photographing the preparations 
proved to be impractical. Therefore, the enlarged diagram of the lines was re- 
corded by tracing on paper . 

The antisera were absorbed in the following way : antisera and saline extracts or 
saline controls were mixed in various proportions in small sterile tubes, incubated 
at room temperature for two hours, placed in the refrigerator for 36 to 48 hours, 
and centrifuged. The supernatant was then subjected to interfacial ring tests and 
agar gel diffusion tests. 

RESULTS 

In order to test for differences in the antigenic composition of Tubularia hy- 
dranths and stems three tests were employed the precipitin ring test, the Ouchter- 
lony agar gel diffusion test, and the absorption test which was used in conjunction 
with the other two tests. 

Precipitin ring tests 

Precipitin antigen titers were determined in order to test for quantitative and 
qualitative differences between stem and hydranth extracts. Such extracts were 



322 JOHN B. MORRILL, JR. 

equilibrated on a nitrogen basis, serially diluted and layered over anti-whole animal 
antiserum. No differences in the titers were observed. Evidently the two types 
of extracts were similar on a gross quantitative basis. To test the possibility of 
the existence of qualitative differences, absorption experiments were performed. 
Partial absorption of anti-whole animal antiserum by either extract produced anti- 
serum which still reacted equally with both types of extract ; complete absorption by 
either extract produced sera which failed to form a precipitin ring with either ex- 
tract. The precipitin ring tests then failed to reveal any distinct antigenic differences 
between hydranth and stem extracts. 

Because these tests failed to reveal any antigenic differences, it seemed desirable 
to utilize a method where the precipitate of the precipitin ring could be separated 
into a spectrum of one or more precipitin reactions. Accordingly, the antigenic 
composition of the extracts was examined by means of the agar gel diffusion tech- 
nique of Ouchterlony. 

Ouchterlony tests 

Tests with unabsorbed sera. Preliminary Ouchterlony tests of anti-whole 
animal sera revealed multiplicity of precipitating systems. Similarities as well as 
differences existed between the precipitin patterns with stem and hydranth extracts. 
As expected, the number of lines formed varied with the antisera from the different 
rabbits. No precipitin lines appeared with pre-injection sera and saline controls. 
It was found that the pattern of precipitin lines varied in repeated tests with a given 
antiserum, possibly because the saline extracts used as test antigens were prepared 
at different times, even though the extracts were prepared by a standard procedure 
and adjusted on a nitrogen basis. In addition, antisera from a rabbit taken at 
different times following a series of injections exhibited variations in position and 
in number of lines in the precipitate patterns when tested with hydranth and stem 
extracts. This is probably because the maximum concentration of antibodies for 
the several antigens did not occur at the same time (Abramoff and Wolfe, 1956). 
The most complete precipitin line patterns were obtained with antisera from Rabbit 
4 and Rabbit 6 (see under Methods) obtained 5 and 7 days after the third series 
of intraperitoneal injections. 

The best precipitin pattern produced by anti-whole animal sera with hydranth 
and stem extracts is given in Figure 1. This antiserum from Rabbit 4 produced 
a total of seven precipitin lines with hydranth extract and seven precipitin lines with 
stem extract. Six precipitin lines with hydranth extract coalesced with five lines 
produced with stem extract. One additional line \vas restricted to the hydranth 
extract. Two precipitin lines and a spur on a coalescing line were restricted to 
the stem extract. Fewer lines were produced with antiserum of Rabbit 6. In the 
best pattern with antiserum from this rabbit four lines produced by hydranth extract 
coalesced with three lines formed by hydranth extract. In addition two precipitin 
lines were limited to reactions with components of stem extract, and possibly one 
was limited to hydranth extract. 

In the several experiments the coalescing lines formed complex patterns. In 
addition to the fusion of single lines formed by the two extracts, there were instances 
where two hydranth precipitin lines coalesced with one line formed by stem extract 
and vice versa. These results may be interpreted as being due to superimposed 



ANTIGENS IN TUBULARIA 



323 



precipitin lines in the reaction of the antiserum with one extract. It is also possible 
that in one extract an antigenic substance had haptens in common with the haptens 
of two antigenic substances in the other extract (Kaminski and Ouchterlony, 1951). 
In nearly all the experiments one of the stem lines that coalesced with a hydranth 
line had a spur which extended beyond the region of coalescence (Fig. 1). This 
indicates that this stem antigen had two haptens, one in common with a hydranth 
antigen and one not found on any of the hydranth antigens. This interpretation is 
in accord with the explanation for the appearance of spurs given by Kaminski and 
Ouchterlony (1951). 

The large number of lines formed in the Ouchterlony patterns suggested that 
some of the lines might be due to excessive quantities of certain antigens or anti- 
bodies (Kaminski, 1954). It was also possible that one or more of the single lines 
might be due to several superimposed precipitates (Grasset et a!., 1956). Attempts 
to resolve the complexity of the patterns, however, by diluting either the antisera or 




FIGURE 1. Diagram of precipitin pattern of anti-whole animal serum, Rabbit 4, with 

stem and hydranth saline extracts. 

the hydranth and stem extracts resulted in patterns with fewer and more diffuse 
lines. 

The number of lines was also checked by the immunoelectrophoretic method of 
Grabar and Williams (Grabar and Williams, 1953, 1955). In the first experiment 
the antigens of whole animal extract were separated in 2 per cent agar electro- 
phoretically (veronal buffer, pH 8.6, ionic strength 0.1, 10 ma., 24 hrs.). After 
the separation, anti-whole animal serum of Rabbit 4 was placed in a trench parallel 
to the path of migration and allowed to diffuse into the agar. After seven days' 
incubation, nine precipitin lines in the form of distinct arcs were detected. In an- 
other experiment extracts of hydranths and stems were separated electrophoretically 
(veronal buffer, pH 8.6, ionic strength 0.05, 30 ma., 5 hrs.) and tested with anti- 
hydranth serum. This antiserum produced six curving precipitin lines with each 
extract. The electrophoretic mobilities of the several arcs were similar. This 
particular antiserum when previously tested by the Ouchterlony method had pro- 
duced six lines with hydranth extract and five lines with stem extract. 



324 JOHN B. MORRILL, JR. 

The patterns formed by coalescing lines in the Ouchterlony tests demonstrate a 
number of antigens to be common to both hydranth and stem. The immunoelectro- 
phoretic patterns showed that at least six of the hydranth antigens had electro- 
phoretic mobilities comparable to those of six stem antigens. In addition to the 
similar antigens the tests with unabsorbed sera showed one antigen to be limited to 
the hydranth and at least two antigens limited to the stem. 

Tests with absorbed anti-whole animal scrum. In order to resolve further the 
antigenic differences between hydranth and stem extracts, absorbed serum (Rabbit 
6) was used in Ouchterlony tests. Antiserum absorbed with hydranth extract still 
produced two distinct and one faint, diffuse precipitin lines with stem extracts. 
Antiserum absorbed with stem extract produced one precipitin line with hydranth 
extract. The absorbed sera failed to produce bands with the absorbing antigen. 
These results again indicate there are at least two antigenic substances restricted to 
stems and one restricted to hydranths. 

DISCUSSION 

The results demonstrate that precipitating antibodies can be obtained against 
saline extracts of Tubularia. The agar diffusion tests show that a spectrum of 
precipitating antigens is present in stems and hydranths. 

It is possible that some of these antigenic substances are not actually part of 
the tubularian tissues. In spite of the precautions described (see methods section), 
the antigens restricted to the stem extracts may be from organisms associated with 
the perisarc which is limited to the stem region. The antigenic differences may also 
be due to breakdown products of ingested food. The discussion presented here is 
subject to these reservations. 

A number of antigens appear to be common to both hydranths and stems. It 
was previously reported (Morrill, 1958) that there were seven common antigens. 
Re-examination of Ouchterlony patterns has revealed that at least three antigens 
are common to both regions of the animal. In addition, one stem antigen has 
antigenic sites similar to those on two hydranth antigens. Immunoelectrophoretic 
experiments with anti-hydranth serum showed six antigens of hydranths and stems 
to have similar electrophoretic mobilities. This method resulted in distinct non-over- 
lapping lines and should prove useful in future studies on the antigenic composition 
of this organism. Ouchterlony tests with non-absorbed and absorbed anti-whole 
animal sera indicate that at least two antigenic substances are limited to stems and 
one to hydranths. 

With the establishment of antigenic relations between stems and mature hy- 
dranths further investigations need to be conducted to determine the antigenic 
relations between stems, regenerated hydranths, and hydranths at different stages 
of regeneration. The antigens need also to be characterized. Preliminary ex- 
periments show that antisera inhibit hydranth regeneration. Hydranth-specific and 
stem-specific antibodies should now be tested for inhibiting action on the regenera- 
tion of hydranths. 

I wish to express my appreciation to Dr. Charles B. Metz for his guidance, 
encouragement, and interest during the course of this investigation. 



ANTIGENS IN TUBULARIA 325 

SUMMARY 

1. The antigenic composition of hydranths and stems of Tubnlaria crocca has 
heen studied by means of the precipitin ring tests, Ouchterlony agar gel diffusion 
tests, and immunoelectrophoresis. 

2. Precipitin ring tests showed that antiserum against whole animals contained 
precipitating antibodies but failed to reveal antigenic differences between hydranths 
and stems. 

3. Ouchterlony tests of anti-whole animal serum and saline extracts of hy- 
dranth and stem tissues revealed the following : 

a. Four antigens common to both regions of the animal. 

b. One stem antigen with hapten sites similar to those on two hydranth antigens. 

c. Two antigenic substances limited to stems. 

d. One antigenic substance limited to hydranths. 

e. One stem antigen with at least two haptens one in common with a hydranth 
antigen and one which was not related to any precipitating hydranth antigens. 

4. Six stem antigens and six hydranth antigens had comparable electrophoretic 
mobilities. 

LITERATURE CITED 

ABRAMOFF, P., AND H. R. WOLFE, 1956. Precipitin production in chickens. XIII. A quanti- 
tative study of the effect of simultaneous injection of two antigens. /. Immunol., 77 : 
94-101. 

BRACKET, J., 1957. Biochemical Cytology. Academic Press, New York, 516 pp. 

DE HAAN, R. L., 1956. The serological determination of developing muscle protein in the 
regenerating limb of Amblystonw nie.vicanum. J. Exp. Zool., 133: 73-86. 

EBERT, J. D., 1955. Some aspects of protein biosynthesis in development. In: Dorothea 
Rudnick, ed., Aspects of Synthesis and Order in Growth. Princeton Univ. Press, 
Princeton, pp. 69-112. 

FREUND, J., 1947. Some aspects of active immunization. In: C. E. Clifton, ed., Annual Review 
of Microbiology. Vol. 1. Annual Reviews, Inc., Stanford, Calif., pp. 291-308. 

GRABAR, P., AND C. A. WILLIAMS, JR., 1953. Methode permettant 1'etude conjuguee des 
proprietes electrophoretiques et immunochimiques d'un melange de proteines. Applica- 
tion au serum' sanguin. Biochim. et Biophys. Ada, 10: 193-194. 

GRABAR, P., AND C. A. WILLIAMS, JR., 1955. Methode immunoelectrophoretique d'analyse de 
melanges de substances antigeniques. Biochim. ct Biophys. Acta, 17 : 67-74. 

GRASSET, E., E. PUNGRATZ AND T. BRECHBUHLER, 1956. Analyse immunochimique des con- 
stituants des venins de serpents par la methode de precipitation en milieu gelifie. 
hist. Pasteur (Paris} Ann., 91 : 162-186. 

HAWK, P. G., B. L. OSER AND W. H. SUMMERSON, 1954. Practical Physiological Chemistry. 
Ed. 13. Blakiston Co., New York, 1439 pp. 

IRWIN, M. R., 1949. Immunological studies in embryology and genetics. Quart. Rev. Biol., 
24: 109-123. 

KAMINSKI, MARIE, 1954. Quelques observations sur la technique de precipitation specifique 
en milieu gelifie d'Ouchterlony. II. fitude du role des concentrations respectives en 
antigenes et en anticorps et du coefficient de diffusion de 1'antigene dans le cas d'un 
melange de deux systemes precipitants. Soc. dc Chim. Biol. Bull., 36: 289-293. 

KAMINSKI, MARIE, AND O. OUCHTERLONY, 1951. Qualitative and quantitative immunochemical 
studies of the proteins of egg white. Soc. dc Chim. Biol. Bull., 33: 758-770. 

LAUFER, H., 1957. Some immunochemical studies of limb regeneration in the newt. Anat. 
Rec., 128: 580-581. 

MORRILL, J. B., 1958. Antigens in Tubnlaria crocca. Anat. Rcc., 132: 479. 

MACE, G. W., 1955. Development in the presence of antibodies. Ann. N. Y. .lead. Sci., 60: 
1038-1055. 



326 JOHN B. MORRILL, JR. 

OUCHTERLONY, O., 1949. Antigen-antibody reactions in gels. II. Factors determining the 

site of the precipitate. Arkiv for Kem., 1 : 43-48. 
SCHECHTMAN, A. M., 1955. Ontogeny of the blood and related antigens and their significance 

for the theory of differentiation. In: E. G. Butler, ed., Biological Specificity and 

Growth. Princeton Univ. Press, Princeton, pp. 3-31. 
TYLER, A., 1955. Ontogeny of immunological properties. In: B. H. Willier, P. A. Weiss and 

V. Hamburger, eds., Analysis of Development. W. B. Saunders Co., Philadelphia, 

pp. 556-573. 
TYLER, A., 1957. Immunological studies of early development. In: A. Tyler, R. C. von 

Borstel and C. B. Metz, eds., The Beginnings of Embryonic Development. The 

American Association for the Advancement of Science, Washington, D. C., pp. 341-382. 
WOERDEMAN, M. W., 1953. Serological methods in the study of morphogenesis. Arch. 

Nederland de Zool., 10: 144-159. (Supplement 1.) 
WOERDEMAN, M. W., 1955. Immunobiological approach to some problems of induction and 

differentiation. In: E. G. Butler, ed., Biological Specificity and Growth. Princeton 

Univ. Press, Princeton, pp. 33-53. 



ADENOSINETRIPHOSPHATASE OF MYTILUS SPERMATOZOA. 
II. EFFECTS OF SULFHYDRYL REAGENTS, TEMPERA- 
TURE AND INORGANIC PYROPHOSPHATE 1 

LEONARD NELSON - 

Department of Anatomy, University of Chicago, Chicago 37, III. and 
Marine Biological Laboratory, Woods Hole, Mass. 

The enzyme concentrated in the sperm flagellum of the mollusc, Mytilus edulis, 
which splits adenosinetriphosphate (ATP) has been classified as a "true" ad- 
enosinetriphosphatase (ATP-ase) (Nelson, 1955). Mohri (1958) has confirmed 
this in studies on the flagellar ATP-ase of sea urchin sperm. This class of enzyme 
liberates only one phosphate group from each ATP molecule, even on prolonged 
incubation. The Mytilus sperm enzyme exhibits optimum activity at about pH 
8.4 in veronal and glycine buffers, while for sea urchin sperm, pH 8.8 is optimum 
in Tris and veronal buffers. Magnesium has a pronounced activating effect on 
both the molluscan and the echinoderm sperm ATP-ase ; however, calcium exerts 
considerably less activation, and, moreover, antagonizes the potentiation due to 
magnesium. Dilution of filtered-sea-water or isotonic KC1 suspensions of Asterias 
and Mytilus sperm tails with large volumes of glass-distilled water or 10'* M 
MgCU causes slow precipitation of the tails ; this may be accelerated by the addition 
of small amounts of ATP. However, extruded threads produced from these sperm 
tail suspensions do not contract ; this may be attributed to the lack of continuity of 
the components the individual sperm tails (unpublished observation). Salts of 
heavy metals and other sulfhydryl reagents which serve as spermicidal agents act 
to halt sperm motility and also interfere with a regulatory mechanism of sperm 
respiration ; low concentrations of inhibitor permit "uncontrolled" acceleration of 
oxygen consumption, while higher concentrations completely stop O, uptake and 
motility (Barren et al., 1948; MacLeod, 1951). It has also been observed that 
sodium pyrophosphate, apparently by forming firm complexes with the divalent 
cations essential for optimal activity, exerts an inhibitory action on sperm ATP-ase 
(unpublished). The first paper in this series dealt with some of the characteristics 
of the Mytilus sperm tail ATP-ase. The effect of some additional agents (SH- 
inhibitors, temperature, pyrophosphate) on spermatozoan ATP-ase is considered in 
the investigations reported here. 

MATERIALS AND METHODS 

Since it is difficult to induce spawning in the Woods Hole Mytilus either by 
temperature shock or the injection of KC1, it was necessary to obtain the sperm 
from minces of the gonads. The sperm were harvested and prepared as reported 

1 This work has been supported by a Fellowship from the Lalor Foundation, and by the 
Population Council, Inc., New York, New York. 

2 Present address: Department of Physiology, Emory University, Atlanta 22, (in. 

327 



328 



LEONARD NELSON 



previously (Nelson, 1955). "Decapitation" was effected by means of a stainless 
steel, piston-type homogenizer, and the heads and tails separated by repeated gentle 
centrifugation (1000 X g for ten minutes each) in isotonic KC1. The sedimented 
heads were discarded. The dilution-precipitation effect was exploited in the 
further isolation and purification of the tails from the pooled supernatant fractions 
by the addition of at least five volumes of ice-cold 10~ 4 M MgCl 2 . After standing 
in the refrigerator for 6 to 12 hours, the tails had settled out and the clear supernate 
was decanted and discarded. The flocculent precipitate was further concentrated 
by centrifugation at 7000 X g for 10 minutes. The sperm tail concentrate was 
then taken up in two to four volumes of isotonic (M/2) KC1. (All solutions were 



.40 



30 
tfi 
ti 

+- 

3 
C 

6 
o 

.20Q. 




.10 



.0 



275 280 285 290 295 300 305 

Temperature. (Absolute) 



310 



315 



FIGURE 1. Temperature dependence of Mytilus sperm tail ATP-ase. Reaction mixture: 
0.05 M KG; 0.04 M Tris buffer, pH 8.6; 10" 4 M MgCk: 10' 3 M ATP; 0.1 ml. sperm tail 
suspension; total volume: 1.0 ml. Incubation time, 10 minutes. Ordinate, phosphate liberated 
in 10 minutes ; abscissa, temperature, degrees, Absolute. 

made up in de-ionized water.) Since the tail preparation resisted solubilization in 
mild alkaline and detergent solutions, no further effort was made to extract them 
and the experiments were performed on the "intact" tails. The reactants, mixed 
by lateral agitation in 12-ml. Pyrex conical centrifuge tubes, consisted of 0.1 or 
0.2 ml. of sperm tail suspension, 0.8 or 07 ml. Tris buffer (Sigma), pH 8.6 
(0.05 M KC1, 10- 4 M MgCL, 0.04 M Tris), unless otherwise noted. After the 
reactants had equilibrated in a thermostat at 24 to 25 C, 0.1 ml. 1Q- 3 M ATP 
(Sigma disodium salt, neutralized with NaOH to bromthymol blue endpoint) was 
added and the mixture allowed to incubate for ten minutes. Addition of one ml. of 
ice-cold 10% trichloroacetic acid terminated the reaction. The precipitate was 



ATP-ASE OF MVTILUS SPERM. II 



329 



removed by centrifugation and the entire supernate analyzed for orthophosphate 
by the microcolorimetric method of Taussky and Shorr (1953). Optical density 
was measured in a Coleman, Junior spectrophotometer at a wave-length of 660 
millimicrons. Protein content of the sperm tail samples was estimated by a 
modification of the method described by Nielsen (1958). 

RESULTS 

Temperature-dependence of flagellar ATP-asc. Duplicate determinations of the 
enzyme activity at various temperatures over a range from 1 to 41.5 C. show a 
fairly constant increase in rate up to about 20 C., virtually a doubling for each 



1.70 



1.60 



.1.50 



.1.40 




.1.20 



.1.10 



.1.00 



.0.90 



3.20 



3.30 



3.40 
= X10 3 



3.50 



3.60 



FIGURE 2. Temperature dependence of Mytilus sperm tail ATP-ase. Arrhenius plot of 
data in Figure 1. Ordinate, logarithm of rate of phosphate liberated; abscissa, reciprocal of 
temperature, I/degrees, Absolute. 

ten-degree rise (Q 10 -- 2) (Figure 1). Between 20 and 30, the rate of dephos- 
phorylation levels off and then declines fairly uniformly. From the slope of the 
Arrhenius plot (Fig. 2), the activation energy of the reaction was calculated to be 
- 10,450 cal./degree/mole. Since these determinations have been made on crude 
preparations, on "whole" tail suspensions, rather than purified enzyme extracts, 
this finding suggests that the broad temperature range of maximum enzyme action 
may simulate the situation which occurs during natural spawning. This coincidence 
may be of significance in that when associated with other factors (chemotactic, 
antigenic, etc.; cf. review by Rothschild, 1956) which may operate to assure maxi- 
mum fertilization, optimum swimming activity of the spermatozoa may further serve 



330 



LEONARD NELSON 



to increase the number of effective sperm "collisions" with activatable eggs. The 
dependence of spermatozoan motility on utilization of ATP has been established 
(Mann. 1945 ; Rothschild and Mann, 1950; Nelson, 1958a). 

Effect of sodium pyrophosphate on sperm ATP-ase 

Presence of an inorganic pyrophosphatase in flagella. Preliminary observations 
indicated that when the sperm tail incubation medium contained ATP and sodium 
pyrophosphate (NaPP) in the ratio of 1.6:1, the amount of inorganic phosphate 



60 



NaPP 4- 1 xlO 3 M ATP 



_50 



3 

C 



-40 



.30 



o 

L 
Q. 




NaPP alone x 



/ 

- 



ATP-ase Activity 

IxlO" 3 M ATP 

atone 




NaPP cone. (M/l) 



FIGURE 3. ATP-ase and IPP-ase activity of isolated Mytilus sperm tails. Reaction 
mixture: 0.05 M KC1 ; 0.04 Tris buffer, pH 8.6; 5 X 1Q- B M MgCU (dotted line and Curve 1, 
open circles, and Curve 2, closed circles) or 10" 4 M MgCU (Curve 3, open squares and Curve 4, 
closed squares) ; 2 X 1Q- 3 M ATP (Curves 1 and 3 and dotted line) ; varying concentrations 
of NaPP; 0.1 ml. sperm tail suspension. Total volume, 1.0 ml. Incubation: 10 minutes, 
24.5 C. Ordinate, 7 phosphate liberated/mg. protein/10 minutes, abscissa NaPP concentration 
(M/liter). 

liberated was 88% of the control (no NaPP). When the molar ratio of ATP to 
NaPP was 0.8:1, only 57% of the control activity was found. Tentatively, this 
was interpreted as an inhibition caused by the removal of the activating divalent 
cations through their chelation by the pyrophosphate. Verification was deferred 
until the present. 

The sperm tail suspension, incubated for 10 minutes at 24.5 C. in 2 X 10" 3 M 



ATP-ASE OF MYTILUS SPERM. II 



331 



ATP and 5 X 1O 5 M MgCL, splits off about 30 y phosphate per mg. of sperm tail 
protein (dashed line, Fig. 3). When varying amounts of NaPP are added to this 
mixture, the increase in phosphate liberation evidently depends on the relative con- 
centrations of NaPP and MgCU in the medium. Curve 1 (open circles) shows 
that a peak of activity is reached at 10~ 5 M NaPP in the ATP-containing medium; 
while at a somewhat higher MgCU (10~ 4 M) concentration, curves 3 and 4 (open 




FIGURE 4. ATP-ase and IPP-ase activity of isolated Mytilus sperm tails. Reaction 
mixture: 0.05 M KC1; 0.04 M Tris buffer, pH 8.6; 5 X 10' 5 M MgCl 2 ; varying concentration 
of NaPP (Curves B, C, D, open, closed and half circles, respectively) ; varying concentrations 
of ATP (Curve A, open triangles) ; 10~ 3 M ATP (Curve C), 5 X 10" 3 M ATP (Curve D) ; 
0.1 ml. sperm tail suspension (more concentrated than in previous figures). Total volume 
1.0 ml. Incubation: 10 minutes, 24 C. Ordinate, 7 phosphate liberated/mg. protein/10 minutes. 
Abscissa, concentration of phosphate ester (NaPP curves B, C, D ; ATP curve A). 

and closed squares) exhibit peaks of activity at 5 X 10~ 4 M NaPP, both in the 
presence and absence of ATP . 

It is apparent from these results that (i) the sperm tail preparations exhibit an 
active inorganic pyrophosphatase (IPP-ase) ; (ii) that this enzyme activity is ad- 
ditive to that of the ATP-ase; (iii) that above an optimum substrate (NaPP) 
concentration there is a depression of the IPP-ase; (iv) that this optimum may be 
related to the magnesium concentration; and (v) that the presence of ATP may 
even aggravate the depression of IPP-ase activity under certain circumstances. 



332 



LEONARD NELSON 



These observations were confirmed and the situation elucidated with a fresh sperm 
tail preparation. The conditions of the experiment were adjusted by doubling the 
MgCL concentration (from 5 X W~ 5 M to 1O 4 M MgCl 2 ), by making up the ATP 
in the Tris buffer-KQ-MgCl 2 medium to maintain the total [MgCl 2 ] constant while 
varying the [ATP] and by increasing the sperm tail content in the incubation 
mixture (Fig. 4). The ATP-ase activity curve (A) approaches a maximum 
velocity of 70 y phosphate/mg. protein/10 min. with increasing substrate concentra- 
tion. The IPP-ase activity approaches a maximum velocity about half that of the 
ATP-ase (34. 5 y phosphate/mg./protein/lO min., Curve B). When, as shown in 
Curve C, increasing amounts of NaPP are added to the sperm tail incubation 

TABLE I 



1 


2 


3 


Substrate concentration (M/liter) 


Phosphate liberated (y) 


Difference 








Observed 














or extra- 


If ATP 


If NaPP 


7 Phosphate 


ATP 


NaPP 


ATP 
+ NaPP 


polated 














a 


b 


c 


a -b 


a c 


0.001 






56 


56 


32.5 





23.5 




0.000001 




2.5 












0.001001 


56 


56.5 


32.5 


-0.5 | 23.5 




0.00001 




6.5 














0.00101 


60 


57 


32.5 


3 


27.5 




0.0001 




25 














0.0011 


81 


60 


33 


21 


48 




0.001 




32.5 














0.002 


93.5 


62 


33.2 


31.5 


60.3 


0.005 






67.5 


67.5 


34.5* 





33 




0.005001 




71 


67.5 


34.5* 


3.5 


36.5 




0.00501 




75 


67.5 


34.5* 


7.5 


41.5 




0.0051 




90 


68 


34.5* 


22 


55.5 




0.006 




78 


69* 


34.5* 


9 


43.5 



* Vmax calculated from Lineweaver-Burk plot (Fig. 5). 

ATP-ase and IPP-ase activity. Reaction mixture: 0.05 M KC1, 0.04 M Tris, pH 8.6, 5 X 
10~ 5 M MgCU; 0.1 ml. sperm suspension ; and varying concentrations of ATP and NaPP. Total 
volume, 1.0 ml. Incubation conditions: 10 minutes, 24.5 C. Enzyme activity = y phosphate 
split/mg. protein/10 minutes incubation. 

medium containing 10~ 3 M ATP, the activity is increased by 50 to 60 y phosphate 
at all concentrations of NaPP, so that activity curves B and C appear parallel. 
However, when the medium contains 5 X 10~ 3 M ATP plus increments of NaPP 
(Curve D), the initial increase in rate of dephosphorylation approximates that at- 
tributable to the increase in ATP concentrations. Subsequently the rate increases 
slowly, approaching the maximum attained in Curve C, although at about % the 
NaPP concentration. Thereafter the velocity of the enzymic action declines 
rapidly. 

The data summarized in Table I support the conclusion that two distinct 
enzymes are involved in the dephosphorylation of ATP and NaPP. If all the hy- 



ATP-ASE OF MYTILUS SPERM. II 



333 



drolyzable phosphate were present as ATP, in Curve C of Figure 4, the maximum 
amount of phosphate split under the conditions of this experiment (enzyme con- 
centration limiting) could not greatly exceed 62 y instead of 93.5 y (column 2) ; if 
the total source of P were NaPP, then the maximum P liberated probably could be 
only 33.2 y. Similarly, if total hydrolyzable P in Curve D were present in the 



.0.16 



.0.14 



.0.12 



..030 



..020 



.0.1 



V 



-08 



..06 



.04 



.02 



.0 



..010 



ATP - ase 



1000 2000 
1 

[SJ 



5000 



IPP-asc 



10,000 



50,000 
1 

[5] 



100,000 



FIGURE 5. ATP-ase and IPP-asc activity of isolated Mytilus sperm tails. Lineweaver- 
Burk plot. Reaction mixture : 0.05 M KC1 ; 0.04 M Tris buffer, pH 8.6 ; 5 X 10- r> M MgCl,., 
0.1 ml. sperm tail suspension; varying concentrations of substrate; total 1.0 ml. Incubation: 
10 minutes, 24 C. Open circles IPP-ase; inset-open triangles ATP-ase. Ordinate, re- 
ciprocal rate of phosphate liberation/mg. protein/10 minutes ; abscissa, reciprocal of substrate 



concentration. 
= 70. 



KLsc-IPP-ae 4.3 X 10 



?-ase 34.5. KM-A 



= 3.1 X 10- : V 



m ax- ATP-a s 



form of ATP, the maximum liberated P would not exceed 69 y, or if present in the 
form of NaPP, probably would not be in excess of 34.5 y. It is further evident that 
when both sperm tail content and MgCL concentration in the incubation mixture 
are increased, in contrast to the situation in Figure 2, the NaPP does not interfere 
with itself as suggested by the optimum in curve 4, Figure 2, but that addition of 



334 



LEONARD NELSON 



ATP at high enough concentration duplicates this phenomenon' (Curve D, Fig. 3), 
Mohri (1958) and Nelson (1955) have shown the distinctive magnesium activa- 
tion of the sperm tail ATP-ase. Evidence from the literature suggests that, with 
one notable exception cited in Discussion, regardless of enzyme source, inorganic 
pyrophosphatase activity is generally limited by the magnesium content of the 
medium. In this respect, the flagellar IPP-ase is not unique. The mutual de- 
pendence of these enzymes on adequate magnesium ion suggests the validity of the 
original concept that by chelation, NaPP competes with ATP for the magnesium 
ions and thereby could exert an inhibitorv influence on the ATP-ase activitv. The 



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ATP-ase IPP - ase. 10 




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Conc.-SH Inhibitor (M/t) 



FIGURE 6. Effect of sulfhydryl inhibitors on Mytilus sperm tail enzymes, a. Effect of 10 4 
M SH-inhibitor : Ordinate, per cent of control enzyme activity; abscissa, bar 1, control; bar 2, 
monoiodoacetate ; bar 3, N-ethyl maleimide ; bar 4, p-chloromercuribenzoate. b. Curves show- 
ing effect of varying concentrations of N-ethyl maleimide and p-chloromercuribenzoate on 
sperm tail ATP-ase. Ordinate, per cent of control ATP-ase (no inhibitor) ; abscissa, con- 
centration of SH-inhibitor (M/liter). Incubation: 10 minutes, 24.5 C. 

possible significance for sperm motility of this mutual interaction of substrates and 
enzyme activities will be considered later. 

When the Lineweaver-Burk (1934) analysis is applied to these unpurified 
preparations, the Michaelis constants K m , and the maximum velocities V mnx of the 
respective enzymatic reactions may be determined graphically (Fig. 5). For the 
IPP-ase, K ra is 4.3 X 10~ 5 , and V max is 34.5 y P/mg. protein/10 min., while for 
ATP-ase K m is 3.1 X 10' 4 , and V max is 70 y P/mg. protein/10 min. (K m for 
erythrocyte IPP-ase equals 5.4 X 10"* according to Bloch-Frankenthal, 1954; and 
K m for skeletal muscle ATP-ase equals 1 X 10~ 4 to 3 X 10' 4 , according to Watanabe 



ATP-ASK OF MYTILUS SPKRM. 11 335 

ct ul., 1952.) Under these conditions, Q p of the ATP-ase equals 300 as compared 
to 150 for the bull sperm ATP-ase (Nelson, 1954), and would probably be some- 
what higher for purified sperm tail extracts. Further evidence substantiating the 
belief that two separate enzymes are involved derives from comparison of the 
effects of sulfhydryl reagents on the dephosphorylation of the two substrates. 

Sulfhvdryl inhibition of flagcllar ATP-ase mid IPP-ase. The different sulfhy- 
dryl inhibitors caused varying degrees of inhibition of the enzymes. ATP-ase is 
more sensitive than IPP-ase to the action of both X-ethyl maleimide and p- 
chloromercuribenzoate at an inhibitor concentration of 10 4 M, while sodium 
monoiodacetate inhibits both enzymes only very slightly (bar graphs. Fig. 6a). 
(HgCL at the same concentration completely inhibits the ATP-ase.) P-chloro- 
mercuribenzoate, a mercaptide-forming agent, is more effective at higher con- 
centrations, while X-ethyl maleimide, an alkylating agent, is relatively more potent 
at lower concentrations in inhibiting the sperm tail ATP-ase (Fig. 6b). This may 
reflect the fact that while N-ethyl maleimide is a specific sulfhydryl inhibitor, p- 
chloromercuribenzoate at the higher concentrations may be combining with other 
reactive and essential sites on the protein side-chains in addition to sulfhydryls 
(Boyer, 1959). Unfortunately, no attempt was made at the time to reactivate the 
enzymes by treatment with sulfhydryl compounds such as BAL (2. 3 dimercapto- 
propanol), glutathione or cysteine. 

DISCUSSION 

The studies on the temperature dependence and the effects of sulfhydryl inhibitors 
are relatively straightforward. Kielley and Bradley (1956) reported that with 
calcium as activator, when approximately one-half of the sulfhydryl groups of 
myosin are titrated with either p-chloromercuribenzoate or X-ethyl maleimide a 
marked increase in ATP-ase activity occurs. Other distinguishing features of the 
myosin- and actomyosin-ATP-ase and sperm tail ATP-ase have already been con- 
sidered (Xelson, 1955). Under the conditions of the present experiments, the 
flagella again differ from the muscle ATP-ases in exhibiting none of the sulfhydryl 
reagent activation ; whether this is characteristic only of the purified enzyme or of 
Ca-activated ATP-ase, remains to be investigated. 

Of particular interest has been the finding that the sperm tails actively de- 
phosphorylate pyrophosphate. Mohri ( 1958) concludes that Hemicentrotus sperm 
tails which hydrolyze ATP are enzymatically inactive to a number of other phos- 
phate esters among which he includes inorganic pyrophosphate. Heppel and 
Hilmoe (1951 ) describe an inorganic pyrophosphatase in bull seminal plasma, with 
a sharp optimum at pH 8.6. The bull seminal IPP-ase has nearly maximal activity 
in the absence of magnesium, while firefly (McElroy ct a!., 1951. 1953) and yeast 
IPP-ase have an absolute requirement for Mg +H ", as apparently does that of Mytilus 
sperm tail (although this requires further study). The bull seminal IPP-ase, unlike 
that of yeast and the sperm flagellum. shows no inhibition by increased substrate. 
Since metaphosphate, which also forms firm complexes with Mg ++ , but is not acted 
on by the enzyme, inhibits yeast IPP-ase in the same concentration range as pyro- 
phosphate, Heppel and Hilmoe (/or. r/7.) attribute inhibition by high pyrophosphate 
to Mg ++ binding. This interpretation is substantiated by the present studies in- 
volving the combined action of ATP and XaPP. Moreover, inorganic pyro- 



336 LEONARD NELSON 

phosphate inhibits magnesium-activated myofibrillar ATP-ase when the total con- 
centration of ATP and NaPP exceeds that of the MgCL (Perry and Grey, 1956), 
and decreases the light scattering of actomyosin solution in the presence of magne- 
sium (Tonomura ct at., 1952) even though the pyrophosphate is not split. A 
number of the nucleoside triphosphates also possess this property of modifying, or 
interfering with the myosin or actomyosin interaction with ATP, and so the be- 
havior of inorganic pyrophosphate is not unique in this respect (Hasselbach, 1956). 
However, when considered in conjunction with the activity of the enzyme inorganic 
pyrophosphatase, this substance assumes peculiar significance and invites specula- 
tion as to its possible role as a regulator of a specific cellular energetic reaction. 
To cite several instances, in addition to vertebrate muscle, for which inorganic 
pyrophosphate may also serve as an extractant, both the substrate and enzyme may 
be involved in such diverse activities as firefly luminescence and insect flight. Mc- 
Elroy and his co-workers (1953) report that the decrease in light intensity after 
mixing ATP, luciferin, luciferase, O. 2 , and Mg ++ is due to the formation of an 
inactive complex of luciferase which depends on magnesium and a second protein, 
namely, IPP-ase. Addition of pyrophosphate causes a sharp increase in the light, 
but inhibitors of the pyrophosphatase (Mn ++ , Ca ++ , F~) must be added to prevent 
the rapid decay of the high light intensity obtained with the pyrophosphate. (How- 
ever, iodoacetate, even at concentrations of 10" 3 and 10~ 2 M does not inhibit 
this pyrophosphatase.) Gilmour and Calaby (1953) suggest the possibility that 
pyrophosphate hydrolysis may have some importance as a source of energy for 
cellular processes, since locust thoracic muscle pyrophosphatase is three times 
higher than that of femoral muscle, and also refer to the report that the heat 
of hydrolysis of pyrophosphate is approximately 9000 cal./mole (Ohlmeyer and 
Shatas, 1952). It is unlikely that such an enzymatic reaction is without physio- 
logical consequence. An interpretation in harmony with the wide variety of evi- 
dences of pyrophosphate involvement in cellular processes may be deduced from 
evidence that pyrophosphate is one of the naturally occurring "relaxing" or plasti- 
cizing factors. Pyrophosphate duplicates the softening effect of ATP in glycerlnated 
muscle fibers and sperm flagella, so-called ''cell models" (Bishop, 1958a). Magne- 
sium is essential for the production and maintenance of the extensibility and 
plasticity of glycerol-extracted muscle (Bozler, 1954a), by physiological concentra- 
tions both of ^ATP and of NaPP. Bozler (1954b) proposed that relaxation is 
caused by the inactivation of bound calcium and that the relaxed state is due to the 
formation of an enzymatically inactive protein-ATP-Mg complex. He suggests 
(p. 157) that "the effect of ATP depends on a balance between two antagonistic 
actions, contraction, which is caused by the breakdown of ATP. and a softening 
action like that caused by PP. Whether contraction or relaxation occurs then 
depends on which of these effects predominates." 

["Elementary processes in muscle action," Morales ct al. (1955) should be 
consulted for a review of the actions of two other naturally occurring modifying 
factors, myokinase and the system ATP-creatine-transphosphorylase + creatine 
phosphate, as well as EDTA, and the features held in common by these very 
different substances.] Bishop (1958b) believes that one or more of the relaxing 
systems may play roles in sperm model "motility," since glycerinated sperm twitch 
repetitively on addition of ATP, ADP, or ITP, while pyrophosphate increases the 
amplitude of the twitch induced by these substances in rodent sperm. However, 



ATP-ASE OF MYTILUS SPERM. II 337 

these models are capable of very little, if any, progressive movement, the rate oi 
oscillation is usually slower than that of fresh sperm, the wave is not propagated, 
and the movements are occasionally restricted to one or another portion of the tail. 
In this connection, it is worth noting that inorganic pyrophosphatase is a water- 
soluble enzyme, and as such is one of several components extracted upon glycerina- 
tion (Nelson, 1959). Isolated fresh sperm tails, also, may oscillate or twitch 
(Gray, 1958), and so the control or regulation of the undulatory flagellation is 
most likely an autonomous function of the flagellum itself. Initiation of motile 
activity may depend on "extraneous" excitatory factors, e.g., the so-called dilution 
effect, hormones, partial pressure of O,, or CO 2 , etc. (cj. Mann, 1954). But once 
the sperm is activated, propagated contraction waves progress down each of the 
nine outer longitudinal fibers in sequence in such a fashion that while one fiber is 
contracting, the ones opposite are plastic, undergoing relaxation-activation cycles 
which immediately succeed their own contraction waves (Nelson, 1958b). This 
sequence could impart the spiral twist observed in the undulatory wave and perhaps 
as well the spiral thrust which characterizes the progressive movement of the sperm. 
In cytochemical terms this may be visualized as follows : The contractile protein- 
Mg-ATP complex is the condition of the "active" state. Upon contraction, a 
rigor-like state might ensue, except that pyrophosphate then combines with the 
contracted fiber through Mg ++ , inducing the relaxation phase; but since NaPP 
cannot replace the contraction-inducing property of ATP, and the kinetics of 
clephosphorylation suggest that the two substances are mutually inhibitory at physio- 
logical levels, the NaPP "block" must be removed from the spatial proximity of the 
contractile site. This may be effected enzymatically by the Mg ++ -activated IPP-ase. 
Now, locally resynthesized ATP may recombine with the protein in complex with 
Mg + " released from combination with pyrophosphate. This type of contraction- 
relaxation cycle, resembling a spatially compact reciprocating mechanism, obviates 
the necessity for invoking "long-range" migrations of reactants. The highly 
speculative nature of this scheme may eventually be resolved when the mechanism 
and site of resynthesis of the inorganic pyrophosphate are discovered, although this 
should not be an insurmountable objection to the working hypothesis, since both 
pyrophosphate and pyrophosphatase apparently occur in a wide variety of biological 
systems (cj. Bloch-Frankenthal, 1954). 

SUMMARY 

1. Mytilus sperm tail ATP-ase temperature coefficient (Q in )=2; temperature 
optimum occurs in the range between 20 and 30 C. 

2. The sperm tails actively dephosphorylate sodium pyrophosphate (NaPP) as 
well as ATP. Two separate enzymes are involved, which together with their 
substrates apparently compete for the magnesium ions in the medium. 

3. K M _ ATP _ ase ^ 3.1 X 10-*; K M _ IPP _ ase : : 4.3 >: lO' 5 . 

4. In low concentrations of ATP and NaPP. the amount of inorganic phosphate 
liberated is additive, while at higher concentrations, inhibition occurs. 

5. ATP-ase is more sensitive than IPP-ase to sulfhydryl inhibition, although 
iodoacetate has only slight effect on both enzymes. 

6. An hypothesis is proposed that regulation of the undulatory flagellar wave 
primarily resides within the flagellum itself, and is a function of the reciprocal 



338 LEONARD NELSON 

activity of two enzymes, ATP-ase in the contraction phase, and IPP-ase in the 
relaxation phase. 

LITERATURE CITED 

BARRON, E. S. G., L. NELSON AND M. I. ARDAO, 1948. Regulatory mechanisms of cellular 

respiration II. The role of soluble sulfhydryl groups as shown by the effect of 

sulfhydryl reagents on the respiration of sea urchin sperm. ./. Gen. Physiol., 32: 

179-190. 
BISHOP, D. W., 1958a. Mammalian sperm cell models reactivated by ATP. Fed. Proc., 

17: 15. 
BISHOP, D. W., 1958b. Relaxing factors in ATP-induced motility of sperm models. Altai. 

Rec., 132: 414-415. 
BLOCH-FRANKENTHAL, L., 1954. The role of magnesium in the hydrolysis of sodium pyro- 

phosphate by inorganic pyrophosphatase. Biochcm. J., 57 : 87-92. 
BOYER, P. D., 1959. Sulfhydryl and disulfide groups of enzymes. Chap. 11, pp. 511-588 in 

The Enzymes, Vol. I, Ed. 2, P. I). Boyer, H. Lardy and K. Myrback, eds., Academic 

Press, N. Y. 

BOZLER, E., 1954a. Interactions between magnesium, pyrophosphate and the contractile ele- 
ments. /. Gen. Physiol.. 38: 53-58. 

BOZLER, E., 1954b. Relaxation in extracted muscle fibers. /. Gen. Physiol., 38 : 149-160. 
GILMOUR, D., AND J. H. CALABY, 1953. Myokinase and pyrophosphatase of insect muscle. 

Engymologia, 16 : 3440. 

GRAY, J., 1958. The movement of the spermatozoa of the bull. /. l'..\-p. Biol., 35: 96-108. 
HASSELBACH, W., 1956. Interaction of various nucleoside triphosphates with actomyosin. 

Biochim. Biophys. Ada, 20: 355-368. 
HEPPEL, L. A., AND R. J. HILMOK, 1^51. Purification of yeast inorganic pyrophosphatase. 

/. Biol. Chan.. 192: 87-94. 
KIELLEY, W. W., AND L. B. BRADLEY, 1956. The relationship between sulfhydryl groups and 

the activation of myosin adenosinetriphosphatase. /. Biol. Client. , 218: 653-659. 
LINEWEAVER, H., AND D. BURK, 1934. The determination of enzyme dissociation contants. /. 

Amer. Client. Soc., 56 : 658-666. 
MACLEOD, J., 1951. Sulfhydryl groups in relation to the metabolism and motility of human 

spermatozoa. J. Gen. Physiol., 34: 705714. 
MANN, T., 1945. Studies on the metabolism of semen II. Glycolvsis in semen. Biocliein. J., 

39: 458-465. 

MANN, T., 1954. The Biochemistry of Semen. Methuen and Co., Ltd., London. 
MCELROY, W. D., J. COULOMBRE AND R. HAYS, 1951. Properties of firefly pyrophosphatase. 

Arch. Biochcm. Biophys., 32: 207-215. 
MCELROY, W. D., J. W. HASTINGS, J. COULOMBRE AND V. SONNENFELD, 1953. The mechanism 

of action of pyrophosphate in firefly luminescence. Arch. Biochem. Biophys., 46: 

399-416. 
MOHRI, H., 1958. Adenosinetriphosphatases of sea urchin spermatozoa. /. Fac. Sci., Tokyo 

U., 8: 307-315. 
MORALES, M. F., J. BOTTS, J. J. BLUM AND T. L. HILL, 1955. Elementary processes in muscle 

action : an examination of current concepts. Physiol. Re?'., 35 : 475-505. 
NELSON, L., 1954. Enzyme distribution in fragmented bull spermatozoa I. Adenylpyro- 

phosphatase. Biochim. Biophys. Ada, 14: 312-320. 
NELSON, L., 1955. Adenosinetriphosphatase of Mytilus sperm. I. Effects of pH, calcium and 

magnesium, and concentration of enzyme and substrate. Biol. Bull., 109: 295-305. 
NELSON, L., 1958a. ATP an energy source for sperm motility. Biol. Bull., 115: 326-327. 
NELSON, L., 1958b. Cytochemical studies with the electron microscope I. Adenosinetri- 
phosphatase in rat spermatozoa. Biochim. Biophys. Ada, 27 : 634-641. 
NELSON, L., 1959. Inorganic pyrophosphatase distribution in spermatozoa. /. Histochcni. 

Cytochein. (in press). 
NIELSEN, H., 1958. Quantitative micro-determination of proteins and peptides. Ada Chem. 

Scand., 12: 38-43. 
OHLMEYER, P., AND R. SHATAS, 1952. Heat of hydrolysis of some phosphate compounds. 

Arch. Biochem. Biophys., 36: 411-420. 



ATP-ASK OF MYTILUS SPERM. II 

PERRY, S. V., AND T. C. GREY, 1956. Effects of substrate concentration and certain relaxing 

factors in the magnesium-activated tnyofibrillar adenosinetriphosphatase. Biochem. J ., 

64: 184-192. 

ROTHSCHILD, LORD, 1956. Fertilization. J. Wiley and Sons, N. Y. 
ROTHSCHILD, LORD, AND T. MANN, 1950. Carbohydrate and adenosinetriphosphate in sea 

urchin semen. Nature, 166: 781. 
TAUSSKY, H. H., AND E. SHORR, 1953. A microcolorimetric method for the determination of 

inorganic phosphorus. /. Biol. Client. , 202 : 675-685. 
TONOMURA, Y., S. WATANABE AND K. YAGI, 1952. Mechanism of muscular contraction I. 

Interaction between actomyosin and adenosinetriphosphate. /. Biochem. (Japan), 

40: 27-54. 
W ATA x ABE, S., Y. TONOMURA AND H. SHIOKAWA, 1952. Mechanism of muscular contraction 

II. Kinetic studies on muscle adenosinetriphosphatase. /. Biochem. (Japan), 40: 

387-402. 



VASCULAR BUDDING IN BOTRYLLOIDES 

HIDEMITI OKA AND HIROSHI WATANABE 

Zoological Institute, Tokyo Kyoikn {'nit'crsity, Tokyo, 



In our previous paper ( 1957) it has been demonstrated that in Botryllus friini- 
gcnits, in addition to the ordinary palleal (peribranchial) budding, new buds are 
formed also from aggregations of blood-cells at the base of ampullae, and the epithet 
"vascular" has been proposed for that kind of budding. The question at once 
arises whether this vascular budding occurs also in other members of the family 
Botryllidae. Our researches along this line have revealed that Botrylloides i'io- 
laccnin under certain circumstances propagates by vascular budding entirely anal- 
ogous to that of Botryllus. 

In this brief note the process of vascular budding in Botrylloides will be de- 
scribed and then compared with that of Botryllus. 

MATERIALS AND METHODS 

The materials on which the following observations were made were living 
colonies of Botrylloides violaccuin Oka, commonly found in the vicinity of Shimoda 
Marine Biological Station, Shimoda, Japan. As is well known, in Botrylloides the 
zooids are arranged in meandering systems instead of in circular systems as in 
Botryllus. 

To facilitate observation, colonies were fixed on glass slides. The technique 
used for fixing the colonies, setting out the slides in the bay, etc. was essentially the 
same as that described in the paper of Oka and Usui (1944). Observations on 
living materials were supplemented, if necessary, with examination of sections. 

We take this opportunity and express our thanks to the Director and staff of 
the Station for providing us facilities for carrying out this research. Thanks are 
also due to Miss Yoshiko Oshima for her helpful assistance in laboratory works. 

OBSERVATIONS 

Developmental cycle in the colony of Botrylloides 

Developmental cycle in the colony of Botrylloides is exactly the same as in 
Botryllus. In both, the zooids in a colony are perfectly coordinated, so we can 
speak of the phases of a colony as a whole. A colony has four successive phases, 
which constitute a developmental cycle. For particulars see our previous paper 
(1957). 

1 Contributions from the Shimoda Marine Biological Station, No. 109. 

2 The cost of this research has been partly covered by the Scientific Research Expenditure 
of the Department of Education of Japan. 

340 



VASCULAR BUDDING IN BOTRYLLOIDES 



341 




FIGURE 1. Test vessels immediately after isolation. < ca. 60 

Conditions for the appearance of vascular buds 

In the normal growth of the colony of Botrylloides violaccum, buds are formed 
exclusively from the palleal wall, i.e., no sign of vascular budding is seen. When, 
however, a small piece containing ampullae and vessels but no zooids is cut out 
from the marginal part of a colony, vascular buds appear in it after 2 or 3 days. 

In Botrylloides colonies, as in Botryllus colonies, numerous blood vessels traverse 
the test (Fig. 1) and terminate in contractile ampullae at the periphery of the 




FIGURE 2. Test vessels in a one-day-old piece. X ca. 60 



342 



HIIJKMITI OKA AND HIROSHI WATANABK 





FIGURE 3. Vascular Inuls in a 3-day -old piece, x; ca. 60 




FIGURE 4. Vascular buds in a 4-day-old piece. X ca. 60 







FIGURE 5. Section of a bud from a 2-day-old piece. X ca. 2000 



VASCULAR BUDDING IN BOTRYLLOIDES 



343 



colony. In a cut-out piece, the whole vascular system, inclusive of ampullae, 
strongly contracts. At the same time many club-shaped branches are sent out, 
and finally a dense network of anastomosing vessels is formed (Fig. 2). It is 
seen that a flow of blood is maintained in it. On such vessels the vascular buds 
are formed. 

Formation of the vascular bud and its jurtJier development 

It is seen in sections that the formation of the vascular bud is initiated by gather- 
ing of lymphocytes (diameter ca. 3^1 /*) under the epidermis of the blood vessel 
(Fig. 5). The initial number of lymphocytes is about 15-20 as in Botrylliis. The 
development of a new zooid out of this cell mass follows exactly the same course as 
in Botrylliis. At first, through intensive cell division a hollow blastula-like structure 
(diameter ca. 30-40 /x) is formed (Fig. 6) ; at the same time, the local epidermis of 




FIGURE 6. Section of a bud from a 3-day-old piece. >< ca. 2000 

the blood vessel gradually protrudes so as to wrap up the vesicle in itself (Fig. 3). 
The bud is now distinctly visible as such even in living materials. Morphologically, 
the vascular bud of this stage (diameter 50 /i) corresponds to the palleal bud of 
stage 3 except that it has no ova. Further development is the same as in the 
palleal bud. As an example, buds in a 4-day-old piece are shown in Figure 4. 

Time of appearance 

At whatever phase in the developmental cycle of the colony isolation may occur, 
vascular budding always begins two to three days after isolation and lasts about 
24 hours. This means that vascular budding is not related to any definite phase 
of the original colony. 

Vascular budding is strictly restricted to that period, for, as it seems, further 
bud-formation from the vascular wall is inhibited by growing vascular buds, and the 



344 HIDEMITI OKA AND HIROSHI WATANABE 

new zooids, once formed by vascular budding, propagate exclusively by palleal 
budding. 

Site of appearance 

At the time of budding, the vascular system in the isolated piece is represented 
by a network of blood vessels, the diameter of which varies from 45 ^ to 120^,. 
Unlike Botryllus, the vascular buds never appear at the bases of ampullae. Nor do 
they appear on the newly formed club-shaped endings. They are always formed 
along the walls of the old blood vessels. 

Degeneration of the huds 

At the end of the budding period, i.e., 3 to 4 days after isolation, we could often 
count 60-70 newly formed buds, but not all of them continued to develop. As in 
the case of Botryllus, only those which surpass a certain size can develop and form 
perfect zooids, while the remaining ones undergo involution. An example of 
various sizes of buds is shown in Table I. 

TABLE I 

Various sizes of vascular buds in a piece 4 days after isolation 

Diameter Number of 

(in n) buds 

ca. 10 9 

ca. 15 8 

ca. 20 25 

ca. 30 1 7 

ca. 40 6 

ca. 50 1 

ca. 60 1 

ca. 70 1 

ca. 80 1 

ca. 90 1 

Total 70 

Of these 70 buds, those larger than 40 /A continued to develop (11 buds, or 16% ) , 
while those under 30 /A soon began involution and finally disappeared without 
leaving any traces behind (59 buds, or 84%). In another case, of 35 buds once 
formed, only 6 (17%) developed into perfect zooids and formed a colony with two 
systems. Thereupon the colony began to grow by palleal budding. 

DISCUSSION 
Differences between Botryllus and BotryUoides 

The formation of the bud itself is precisely alike in both forms. Yet, as to the 
conditions for and the time and site of the appearance of the vascular buds, there 
are differences between Botryllus priniic/enus and BotryUoides violaceiini as stated 
below : 

1. In Botryllus, vascular budding occurs in active colonies concomitantly with 
palleal budding. In BotryUoides, vascular budding is never seen under normal 



VASCULAR BUDDING IN BOTRYLLOIDES 345 

conditions. Only when a small piece of a colony devoid of zooids is isolated, 
vascular budding is, so to speak, evoked in it. 

2. In Botryllus the appearance of the vascular buds is limited to a certain phase 
(late phase B) in the developmental cycle of the colony. In B otrylloides vascular 
buds can be formed at any phase of the original colony. 

3. In Botryllus the buds are located strictly at the bases of the ampullae, while 
in Botrylloides the buds are scattered across the colony along the walls of the 
vascular system. 

Actually all these differences are the same as existing between Botryllus priini- 
t/cnus and Botrylloides gascoi except on one point. In Botrylloides violaceum we 
could repeatedly observe vascular budding in isolated pieces. In Botrylloides 
gascoi, however, an isolated piece devoid of zooids never regenerated a colony, and 
none of the ampullae in such a piece showed the least tendency towards budding 
(Bancroft, 1903, p. 451). Probably this led Bancroft to suppose that the presum- 
able vascular buds observed by him in an aestivating colony were developed not 
from vessels but from the zooids of the original colony before these had degenerated 
entirely and later severed their connections with the parent zooids. It is to be 
hoped that some future investigator will repeat the experiments with Botrylloides 
gascoi. 

Regulation acting upon the vascular buds 

Botryllids are know r n for their zooids being most- perfectly coordinated. 

In Botryllus, the vascular buds are formed a little later than the corresponding 
palleal buds, but they are soon synchronized with these. Buds formed too late are 
forced to degenerate, thus being eliminated from the colony. All this regulating 
influence is supposed to come from the pre-existing active zooids. 

In Botrylloides, the vascular buds are formed in the absence of any pre-existing 
zooids. Yet, sooner or later, all the newly formed zooids are synchronized, and, as 
in Botryllus, buds formed too late are eliminated from the colony. Possibly with 
the growth of early buds a new coordinating system is established in the piece and 
this regulates the growth of late-coming buds. 

Budding in Botryllidae 

Now that vascular budding has been demonstrated also in Botrylloides, it is to 
be assumed that this kind of budding is rather widely distributed among the family 
Botryllidae. 

It is generally believed that stolonial budding of which vascular budding is 
only a special form is a rather primitive type of budding, while palleal budding is 
phylogenetically a relatively late acquisition. Moreover, palleal budding is so 
unique in nature that it cannot be derived from any other known kind of budding. 
So it has been assumed "that the primitive pleurogonid, undoubtedly derived from 
an enterogonid, had already lost any such capacity for budding, and that within 
the new order the Botryllinae have re-acquired it by a new method" (Berrill, 1950, 
pp. 50-51). That the original capacity for budding has not been completely lost in 
botryllids is clear from our investigations on Botr\llus and Botrylloides. Only, 
with the rise of the new method of palleal budding, it has been more and more 
suppressed. In Botryllus primigenus vascular budding still takes part, though 
concomitantly with palleal budding, in the natural growth of the colony. In 



346 HIDEMITI OKA AND HIROSHI WATANABE 

Botrylloides riohtceuin, and probably Botrylloides yascoi, vascular l)udding is 
totally suppressed in the ordinary life of the colony. Only in the absence of zooids 
the otherwise latent capacity of forming buds from the walls of the blood vessels 
is called forth as a means to save the colony from extinction. 

SUMMARY 

1. Generally Botrylloides violaccitni Oka propagates by palleal budding alone. 
Only when a small piece of a colony devoid of zooids is isolated, new buds are 
formed from the walls of the test vessels, i.e., vascular budding appears. 

2. As in Botryllus, these new buds are formed from aggregations of lymphocytes 
under the wall of the test vessels. 

3. Unlike Botryllus, the buds are not bound to any definite sites, but are dis- 
tributed irregularly along the walls of the vascular system. 

4. The buds generally appear 2-3 days after isolation, at whatever phase of the 
original colony the isolation may occur. 

5. Major difference between Botryllns and Botrylloides is that in the former 
vascular budding coexists with palleal budding, while in the latter vascular budding 
is totally suppressed in the normal life of the colony. 

LITERATURE CITED 

BANCROFT, F. W., 1903. Aestivation of Botrylloides </ascoi Delia Valle. Mark Anniversary 

Volume, 147-166. 
BERRILL, N. J., 1950. The Tunicata with an account of the British species. London, Ray 

Society. 
OKA, H., AND M. Usui, 1944. On the growth and propagation of colonies in Polycitor mutabilis 

(Ascidiae compositae). Sci. Rep. Tokyo Bunrika Daigaku (Section B), 7: 23-53. 
OKA, H., AND H. WATANABE, 1957. Vascular budding, a new type of budding in Botryllus. 

BioL Bull., 112: 225-240. 



AXENIC CULTIVATION OF THE BRINE SHRIMP ARTEMIA SALINA 

LUIGI PROVASOLI AND KAGEHIDE SHIRAISHI 

Haskins Laboratories, Nciv York 17, N. Y., and Dcpt. of Fisheries, I'aculty of 

Tohokn University, Sendai, Japan 



In a previous paper (Provasoli, Shiraishi and Lance, 1959) we have added to 
Gibor's (1956) observations that related species of algal flagellates may be either 
good or bad food for Artcmia. This idiosyncrasy may depend upon nutritional 
deficiencies in the algal food, on toxic metabolic products, or even upon some 
nutrient in excess. One way to attack this ecological problem is to grow Artcmia 
on a non-living medium as a step toward a chemically defined medium and, finally, 
identification of all its nutritional requirements. 

The present paper concerns the first stage, i.e., the growth of Artcinia on a 
IK in-living complex medium. 

MATERIALS AND METHODS 

The amphigonic American race of Artcmia salina was employed. Utah brine 
shrimp eggs (Aquarium Stock Co. Inc., 31 Warren Street, New York 7, New 
York) proved more satisfactory than other samples tried in respect to percentage 
of hatching and speed of development. 

Sterilization of the eggs. Durable eggs of Artcmia obtained commercially 
always contain many dead dried eggs whose chorion is cracked. These eggs are 
lighter and cannot be disinfected as rapidly as the intact viable ones and should 
be eliminated at the onset to avoid infections from the inoculum. The technique 
of disinfection is a modification of the one employed by Gibor (1956). 

The dead eggs, being lighter than the viable ones, are eliminated by the 
flotations in sea water. The eggs are disinfected in screw cap tubes for 10 minutes 
in Merthiolate solution (1 : 1000 in H,O)+ 0.2 ml.^ of a lO^c solution of Aerosol 
OT. to improve wettability. The disinfectant is decanted and the eggs washed in 
three baths of sterile sea water. The egg slurry is distributed into several tubes of 
a sterility-test medium (STP, Table I) and allowed to hatch 2-3 days at 22- 
26 C.). Contamination generally shows in 2-4 days. The new-born nauplii de- 
velop to second metanauplii at the expense of the reserves of yolk within 4-7 days ; 
the third metanauplii. if not fed, die. 

The metanauplii are transferred into a nutrient medium 1-2 days after the first 
nauplii have hatched, to secure a more uniform inoculum in respect to age; 
hatching is spread over several days. 

The growing larvae consume the participate food rapidly and must be transferred 
approximately every 6-8 days, especially after the fourth stage. Transfers are 
made with Pasteur pipettes connected to a mouthpiece by rubber tubing, to allow 
a clear view while fishing the larval forms. The later larval stages and the young 
adults defy suction unless sucked head first, while swimming toward the tip of the 
pipette. To avoid air-borne infection we used a transfer hood (top and back glass. 

347 



348 LUIGI PROVASOLI AND KAGEHIDE SHIRAISHI 

TABLE I 

STP medium 

Sea water 80 nil. 

H,O 15 ml. 

Soil extract 5 ml. 

NaNO 3 20 mg. 

K,HPO 4 1 nig. 

Xa H glutamate 50 mg. 

Glycine 10 mg. 

DL-Alanine 10 mg. 

Vitamin No. 8A* 0.1 ml. 

Trypticase (B.B.L.) 20 mg. 

Yeast autolysate (Albimi) 20 mg. 

Sucrose 0. 1 g. 
pH 7.5 

* See Table 4, p. 408 in Provasoli ct al. (1957). 

open in front) with a "Letheray" germicidal UV lamp (see description and figure 
in Provasoli, Shiraishi and Lance, 1959). 

Preparation of media. The following medium (Table II) allows growth to 
adulthood. 

TABLE II 

Complete media in 

STP(l) 100 ml. 

Cholesterol (2) 200 M g- 

Dehydrated liver infusion No. L 25 (3) 100 mg. 

Trypticase (B.B.L.) 300 mg. 

Alkaline-hydrolysed nucleic acid (4) 40 mg. 

Acid-hydrolysed" DNA (5) 10 mg. 

Sucrose 200 mg. 

Vitamins mix Art. II (6) 1 ml. 

Paramecium factor (7) 5 mg. 

Glutathione (8) 30 mg. 

Ascorbic acid (8) 3 mg. 

Horse serum (aseptic) 5 ml. 

Rice starch (9) 500 mg. 
pH 7.5 

(1) See Table I. 

(2) Dissolved in ethanol. 

(3) Oxoid Ltd., England. 

(4) Yeast RNA brought to pH 9.0 with NaOH and steamed for one hour. 

(5) Herring DNA brought to pH 1.5-2.5 with H 2 SO 4 and steamed for two hours. 

(6) Vitamins mix Art. II 

Thiamine HC1 10 mg. % 

Biotin 0.5 mg. % 

Folic acid 7 mg. % 

Nicotinic acid 50 mg. % 

Choline 500 mg. % 

Ca pantothenate 70 mg. % 

Pyridoxine HC1 8 mg. % 

Carnitine 20 mg. % 

Riboflavin 0.1 mg. % 



AXENIC CULTURE OF ARTEMIA 349 

(7) Paramecium factor was kindly supplied by Dr. D. M. Lilly (1 part dried yeast 
cells + 1 part H 2 O, autolyzed at 58-60 C. for two hours ; the particles are 
centrifuged and the supernatant vacuum-dried ; to obtain a fairly good suspen- 
sion, bring to a boil. 

(S) Fresh solutions of glutathione and ascorbic acid, sterilized by glass-filtration, 
are added before inoculation. 

At first the starch was added to autoclaved media as a slurry. Rice-starch powder 
is mixed with glass beads (200 ^ diameter-Superbrite type 100. Minnesota Mining 
and Manuf. Co.) and sterilized for two hours at 180 C. ; sterile water is added to 
form a slurry. 

We found later that Artcinia ingests cooked starch equally well; this eliminated 
one aseptic addition. To prevent the starch from forming a semi-solid mass during 
sterilization, 500 mg.^o of starch powder is added to the complete medium (minus 
other aseptic additions) before sterilization; the medium is brought to a boil while 
being stirred vigorously with a glass rod or on a heating plate equipped with a 
magnetic stirrer. The starch, on boiling, forms small floccules which become 
larger upon cooling but remain acceptable to Artcinia. The medium is then tubed 
and autoclaved. 

Miscellaneous preparations: 

a ) The cholesterol is generally added as an alcohol solution. It has also been 
employed absorbed on cellulose powder ("Celluflour," Turtox) following the 
techniques of Singh and Brown (1957) (1 mg. of cholesterol dissolved in 10 ml. 
ether is mixed with 200 mg. Celluflour ; let dry, then added to media before 
autoclaving). 

b) Fatty acids absorbed on starch: 980 mg. of the following fatty acid mixture 
(proportions of House and Barlow. 1956) palmitic acid 200 mg. + stearic acid 
100 mg. + oleic acid 480 mg. + linoleic acid 150 mg. + linolenic 50 mg. are dis- 
solved in ether and mixed with 10 g. of starch; after evaporation of the ether, the 
powder is sterilized in ethylene oxide for twelve hours ; the powder is made 
aseptically into a slurry with water, and a solution of XaOH or Ca(OH) 2 added to 
neutralize the fatty acids. 

c) Albumen (bovine) fraction V (X.B.C.) is dissolved in sea water, glass- 
filter-sterilized, and added aseptically. 

Fraction V was employed also as a carrier of cholesterol and the fatty acids 
mixture. Sterile solutions of fraction V and cholesterol (dissolved in ethanol) 
are mixed and added after autoclaving. Two ml. of a sterile 8% solution of Frac- 
tion V were mixed aseptically with 2 nil. of an autoclaved fatty acid mixture (dis- 
solve in 50 rnl. of FLO, 1 ml. of concentrated NaOH, stearic acid 24 mg., palmitic 
acid 54 mg., linoleic acid 36 mg., linolenic acid 12 mg., oleic acid 0.12 ml., adjust to 
pH 8.0 with HC1. autoclave). 

INTERPRETATION OF RESULTS 

To compare the nutritive value of different foods and supplements we needed 
to recognize the various larval forms by characters easily visible by inspection of 
the tubes with a hand lens. "We could not resort to the fine external morphological 



350 LUIGI PROVASOLI AND KAGEHIDE SHIRAISHI 

characters employed by Heath (1924) to divide in 13-15 instars the development 
of Artcmia from egg to adult; these differences can be evaluated only with a micro- 
scope. We decided therefore to employ, slightly modified, the arbitrary nomen- 
clature of Barigozzi (1939) which divides the life-cycle in stages of development 
clearly distinguishable with the hand lens. 

The nauplius is small, roundish, yellow-pink (first instar of Heath). Met- 
anauplius I is small, triangular, yellowish (second instar of Heath). Metanauplius 
II is similar but bigger (third instar of Heath). 

Metanauplius III. "Small" III T-shaped, longer, thin, shows a visible seg- 
mentation in the upper thoracic region (fourth instar of Heath) ; "big" III (1.5 
mm.) ; the first 3-5 thoracic limbs are well developed but do not move well (fifth 
instar of Heath). The first three metanauplii stages are easily distinguished from 
the other stages, even with the naked eye, by their jerky swimming: at these stages 
propulsion depends upon the characteristic backward and forward paddle-like 
movement of the long second antennae. 

Metanauplius IV. "Small" IV (2 mm.). The first thoracic limbs are now 
moving well and the movement of Artcmia is a combination of jerks and swimming 
in circles (sixth instar of Heath) ; "medium" IV (2.5 mm.) swims on its back 
gracefully in circles ; most of the thoracic limbs are fully articulated and move 
rhythmically like rippling waves (seventh instar of Heath) ; "big" IV (3.5 mm.) 
bigger in size, abdomen longer and slender, the second antennae smaller than the 
limbs and lying parallel to the head (eighth instar of Heath). 

"Juveniles" (5-7 mm.) have stalked eyes, the abdomen elongates and becomes 
segmented, at the tip of the abdomen the furca becomes evident ; they have a slender 
appearance and resemble adults but are much shorter (ninth-eleventh instars of 
Heath). 

Adults (7-10 mm.). The males have long claspers (modified overgrown 
second antennae) ; the females have a slender head and a conspicuous egg-pouch 
right below the last pair of limbs (twelfth-fifteenth instars of Heath) . 

We record twice a week the stage reached in each tube. As in many insects, 
some phases of the life-cycle of Artcmia seem more critical than others: the transi- 
tion from the third to the fourth stage and the one between "big IV" to "juveniles." 
In general, a good way to evaluate the effect of the different supplements is to 
compare (a) the days required to reach the "small IV" metanauplius, (b) the days 
needed to reach adulthood, and, if they do not become adults, (c) the stage reached 
at death and days elapsed since birth. 

We generally inoculate 5-8 larvae per tube. Not all develop into adults even 
in the best media although in these media most do. Quite often, especially from 
the III metanauplius onward, "black disease" develops: black spots, consisting of 
fine melanin granules, develop at the lobes of the phyllopodia, especially on the 
dactylopodite. The incidence of black-spotted individuals is sporadic and could 
not be correlated with any particular nutritional deficiency ; it might be simply a 
difficulty in molting, I.e., left-over parts of the previous cuticle may impede normal 
development. We cannot say how much these spots affect normal growth and if 
they are harmful; in complete media (Table II) the adults often had black spots 
from the IV metanauplius on, yet they could become adults. Black disease is 
often common ; until its causes and effect on the health of the larvae are known it 



AXENIC CULTURE OF ARTEMIA 351 

is impossible to use accurately the percentage survival of a mixed population of 
normal and black-spotted larvae as an index of the nutritional status. 

RESULTS 
a) P articulates 

Artcmia is a voracious particle-feeder as we amply observed when rearing them 
on living flagellates. We thought that this behavior could be exploited to increase 
the ratio of ingestion (drinking) of the nutrients added as solutes, because little 
absorption can be expected by an arthropod except from the middle intestine, the 
rest of the body being clad in chitin. An ideal situation would be to have a 
nutritionally inert attractive particle and to supply nutrients as solutes, thus per- 
mitting the application of the usual microbiological techniques for replacing complex 
organic substances with chemically defined components. We tried a variety of 
particles, many of them nutritionally rich, because we did not know whether the 
organisms could withstand a medium rich enough in solutes to support their 
growth. Early experiments had shown that 0.6% Trypticase inhibits Artcmia 
and that Tigriopits is even more sensitive to organic solutes. The following com- 
pounds were ground fine (between 5-20 /A) in a colloidal mill, sterilized by dry 
heat, and added aseptically as water suspensions to the liquid part of the medium. 
The liquid (STP, Table I, + a vitamin mix, and 100 mg.% of "Oxoid" liver in- 
fusion, no. L 25) employed at the time is nutritionally deficient and. in the absence 
of particles, Artemia grow only up to medium-sized III metanauplii. Additions of 
150 mg.^f of participate blood fibrin, yeast cells, corn protein, lactalbumen, Cero- 
phyll, casein, Celluflour, and rice polishings were ineffective, while fish meal or 
gluten permitted reaching the IV in 23-25 days ; rice starch did so in 35-40 days. 
Rice starch was selected because it is, if digested, mainly a carbon source. It is 
almost devoid of impurities of other important nutrilites (as is gluten) and therefore 
offers the possibility of defining requirements for amino acids, proteins, fats, and 
vitamins. 

Larval forms of Artemia are voracious: suspended particles are quickly trans- 
formed into fecal pellets. We therefore raised the participate starch to 0.5% 
and kept it suspended as much as possible by shaking and homogenizing the medium 
twice daily. Later on, when solutions allowed growth beyond the third meta- 
nauplius, we had to transfer the growing metanauplii every 7 days to a fresh medium 
and to increase the volume of the medium from 5 ml. to 10 ml. Five ml. of 
medium in 20 X 125 mm. tubes are better for the growth of young larvae (up to 
the early stages of "small IV") because such larvae swim poorly, feed mostly at 
the bottom, and need a medium well aerated by a large surface exposed to the air. 
Later stages are continuously swimming and stirring the medium. Perhaps the 
later larval stages would grow faster if the media were changed even more often, 
but this requires much patience and increases contamination (see "Methods"). It 
was found later that the starch can be added before sterilization if it is precooked 
while stirring the medium (see "Preparation of Media") ; the resulting floccules 
are still ingested, and remain more easily in suspension. 

When we found a medium allowing growth to adulthood (Table II), we re- 
investigated the necessity of particles. The liquid part of the medium (excepting 
the heat-sensitive components of the medium) was autoclaved, filtered through 



352 LUIGI PROVASOLI AND KAGEHIDE SHIRAISHI 

paper, then glass-filter-sterilized, and dispensed aseptically into tubes. Sterile 
solutions of glutathione, ascorbic acid, and the serum were added last. 

This medium is clear and devoid of visible particles. The nauplii of Artemia 
in this medium reached at best the stage of "big III" metanauplii. In the same 
batch of medium to which was added aseptically a sterile starch slurry, Artemia 
reached adulthood. A similar experiment was done recently but \vith another 
medium allowing growth to adulthood; again the absence of particles prevented 
growth beyond the third metanauplius. 

b~) Solutes 

Trypticase and nucleic acids. In preliminary experiments it was soon dis- 
covered that addition of Trypticase (0.3%) and nucleic acids to the liver extract 
speeded growth greatly; the IV metanauplius stage was reached in 12-19 days but 
growth stopped at medium-size metanauplii. Whole blood (1 ml./lOO) and a 
suspension of red blood cells, as substitutes for the starch particles, did not speed 
growth or allow a more advanced stage ; yeast cells (autoclaved) were inhibitory. 

Vitamins. Since the level of the vitamin mixture initially used (Table I) was 
far below the levels for insects, we suspected that the medium was mainly deficient 
in vitamins. Tentatively, we chose concentrations and ratios similar to those 
employed for insects, but at the lower limits because in earlier experiments we 
found that cholesterol was already inhibitor}- at values which are low for many 
insects. Biotin, pyridoxine. folic acid, nicotinic acid, and choline, added singly 
and in combinations, either affected general vitality (i.e., vigorous swimming), 
speeded the time required for reaching the fourth metanauplius stage, or permitted 
growth up to "very big" IV metanauplii. Therefore we designed richer and more 
complete vitamin mixtures (see latest in Table II). To see whether some vitamins 
were present in suboptimal or toxic concentrations, we removed singly each vitamin 
and added it at different concentrations. Thiamine and folic acid proved limiting, 
indicating that Trypticase, liver extract, and serum at the levels employed in the 
complete medium (Table II) are inadequate sources of these vitamins for Artemia. 
Adulthood was reached in the complete medium and no inhibitions were found up 
to the following maximal concentrations tried (wt./lOO ml. final medium) : thiamine 
200/i,g., biotin 30 /xg., folic acid 300 /^g., nicotinic acid 1 mg., pantothenic acid 3 
mg. Choline did become inhibitory between 3 and 10 mg., pyridoxine between 50 
and 100 p.g.%, and riboflavin between 0.1 and 1.0 mg.%. 

Serum, glutathione, and paramecium factor. Adults were not obtained until 
horse serum, glutathione, and paramecium factor were added to the medium. Serum 
alone reduced the time to reach the IV metanauplius from 29 days (no serum) to 
19 days (2 ml./lOO), and to 13 days (4-10 ml./lOO) ; increasing the serum allowed 
growth up to "big" IV metanauplii. 

In the absence of serum, 20-40 mg. c /c of glutathione enabled the metanauplii 
to reach the IV stage in 16 days and become "small" IV. Paramecium factor 
alone or in combination with glutathione seems ineffective, but when added to the 
combination glutathione plus serum, allows adulthood. Under our conditions, 
only "very big" IV or juveniles were produced by serum + glutathione; this com- 
bination was quite effective in speeding growth ; the IV metanauplius was reached 
in 13 days. For production of adults the serum should reach the level of 3 ml. /1 00 



AXENIC CUI/ITKK <)! ARTKMIA 

or more (up to 10 ml./lOO) ; when the serum was below 3 ml./lOO, (lej)ending on 
the quantities of serum added, only "big." "medium," or "small" IV metanauplii 
were produced. 

Cysteine can substitute for glutathirne in eliciting adulthood: 10 mg.% cysteine 
was as effective as 20-30 mg. c /c glutathione. 

Horse serum can be substituted with a filter-sterilized solution of dried beef 
serum (Difco). 

The active factors present in serum are heat-resistant : both supernatants of the 
autoclaved horse or Difco beef serum, glass-filter-sterilized and added aseptically, 
are as effective as serum. 

Cholesterol and fatty acids. Some attempts were made to replace serum. Five 
milliliters of serum supply, inter alia, much neutral fat, lecithin, cephalin, and 
cholesterol. The medium without serum, except for the concentrations brought 
in possibly by 100 mg. of Oxoid liver, has no fatty acids and only 200 p.g.% 
cholesterol. Some insects require long-chain unsaturated fatty acids (linoleic acid, 
linolenic acid) ; all require cholesterol. Early experiments had shown that choles- 
terol above 500/xg.% becomes inhibitory, but these experiments were done with 
poor media. In comparable media, linoleic acid was indifferent at 1 mg.% and 
inhibitory at 10 mg.%. With better media, cholesterol absorbed on Celluflour 
inhibited at or above 6 mg.^o and linolenic acid was inhibitory above 1 mg.% and 
became rapidly toxic. Neither cholesterol or linolenic acid replaced the serum. 
The fatty-acid mixture absorbed onto starch powder was indifferent at 2-5 mg.% 
and quite toxic above. Thinking that the toxicity might be caused by the acidity 
of the acids, the sterile fatty-acid starch slurry was adjusted with NaOH or 
Ca(OH) 2 ; the Na salts are far more toxic than the Ca salts. 

Cholesterol, or the fatty acid mixed with serum albumen fraction V, also did 
not replace serum. However, 200 mg.% of fraction V alone allowed normal adults 
in two months instead of 25 days ; growth up to the IV metanauplius was as in 
serum. Soya lecithin became rapidly toxic; "Glicldex I" (a refined "lecithin" 
containing 4% lecithin, 29% cephalin, 55% inositol phosphatides, and 4% soybean 
oil) absorbed either on starch or casein, is well tolerated up to 10-15 mg.%. How 
ever neither lecithin, Gliddex I or other "lecithins" replaced serum. 

DISCUSSION 

While Artemia can be grow r n without living food and tolerates concentrations 
of solutes sufficient to permit development to adults, it does not grow wholly on 
solutes. The particles are needed to stimulate filter-feeding, thus allowing ingestion 
of enough nutrient solutes for a normal growth rate. 

The studies of Croghan (1958a, 1958b) on osmotic regulation in Artemia show: 
a) that the cuticle of the branchiae (metepipoclites) of the first 10 pairs of the 
phyllopods is the only part of the external body cuticle that is appreciably permeable 
in the adults ; in the young larvae, where the phyllopods have not yet developed, the 
neck organ is the permeable region; b) the branchiae are the site of active NaCl 
excretion; c) the gut epithelium controls internal water balance by water uptake; 
d) Artemia continues to swallow the medium even when devoid of particles. The 
swallowing behavior of the adults was indicated by the red coloration of the gut 
walls a few hours after Artemia were put into a filtered phenol red solution. 



354 LUIGI PROVASOLI AND KAGEHIDE SHIRAISHI 

Our experiments show that the rate of swallowing of a particle-free medium is 
probably very low in the early metanauplii stages certainly insufficient to provide 
enough nutrient solutes for growth. Particles stimulate swallowing. Since all the 
nutrients in our media are in solution, Trager's (1936) conclusion, based on Aedes 
aegypti, that solutes are utilized for growth, applies to Crustacea. Although other 
invertebrates, like some ciliates, can be grown on solutes, perhaps some phagotrophs, 
including Crustacea, living in oligotrophic waters, may be obligate or partial 
phagotrophs because participate feeding, besides increasing the ingestion of nutrient 
solutes, does not affect their inability to withstand concentrations of organic solutes 
high enough to support growth (Provasoli, 1956). 

The medium allowing growth to adults (Table II) is still quite complex; it 
offers, however, more possibilities of dissection than the only other known axenic 
medium for a crustacean the blood-glucose mixture of Treillard (1924) for 
Daphnia. It was exciting to have started with an inadequate medium because each 
experiment permitted the demonstration of some nutritional needs, some of them 
reflecting obvious requirements, as was the effect of Trypticase and nucleic acids. 
It is remarkable that thiamine and folic acid were required even in the presence of 
Trypticase and liver extract which are ordinarily adequate sources of these vitamins. 
Glutathione is also required and was replaceable by cysteine. So far only another 
arthropod, the mosquito Aedes aegyptii, requires glutathione (Singh and Brown, 
1957), even in the presence of adequate cysteine. Interestingly, the "feeding re- 
action" of Hydra is controlled by glutathione which acts as a specific "feeding 
hormone"- it is not replaceable by cysteine, ascorbic acid or other donors of SH 
groups (Loomis, 1955). 

Serum is required for the full development of adults. The active components 
of serum are heat-stable. We could not replace serum with mixtures of fats and 
cholesterol. These results are only indicative : lack of effect may be due to tox- 
icities of some components of the fatty acid mixture or failure to avoid toxicity by 
presenting them to Artemia on the proper fat carrier. 

However, if the toxicity of fatty acids is the cause, it might explain some of 
the nutritional idiosyncrasies found previously (Provasoli, Shiraishi and Lance, 
1959). Utilization of flagellates as food may depend as much upon their providing 
Artemia with all the nutrilites needed as with their lacking toxic substances and 
vice versa when they are not utilized as food. 

For Artemia we found that especially in the Chlorophyta several species, even 
strains, were inadequate as food while others were not. Chlorophytes are known 
to produce toxic unsaturated fatty acids such as chlorellin (Spoehr et al., 1949). 
Indeed this might well be a characteristic of the Chlorophyta. Proctor (1957) 
found that Haematococcus is particularly sensitive to palmitic, oleic, and linoleic 
acids, and also to substances produced by cultures of Chlamydomonas reinhardii. 
The substances produced by Chlamydomonas are steam-distillable and fat-like, 
quite probably a mixture of unsaturated fatty acids. The accumulation of oil 
droplets is readily observed in different species of Polytoma, the colorless counter- 
part of Chlamydomonas. During the logarithmic phase the cells are full of 
paramylum granules, but as they pass the peak of growth the starch is replaced 
in great part by fat droplets. This might explain the toxicity of aged cultures of 
Chlorclla to Daphnia magna found by Ryther (1954). 



AXENIC CULTURE OF ARTEMIA 355 

This work was supported in part by contract NR 104-202 of the U. S. Office of 
Xaval Research. We are grateful for the help given by Mr. R. Zuzolo in doing 
experiments on the need of particulat.es and the heat stability of the serum factors. 

SUMMARY 

1. Artemia salina can be grown aseptically to adults in a non-living medium. 

2. The components of the medium are : sea water, Trypticase, liver infusion, 
hydrolysed RNA and DNA, serum, sucrose, cholesterol, paramecium factor, gluta- 
thione, a mixture of B vitamins, and starch particles. 

3. Glutathione, thiamine, and folic acid were found essential even in the presence 
of Trypticase and serum. Glutathione can be replaced by cysteine. Horse or beef 
serum (Difco) supply unidentified heat-stable nutrients. Cholesterol and mixtures 
of fatty acids become rapidly toxic, and do not replace serum. 

4. Artemia is a voracious particle feeder and transforms the starch particles 
rapidly into fecal pellets. In the absence of starch particles, the liquid part of the 
medium, though containing all the nutrients, supports growth only to the third-stage 
metanauplii. This indicates that the rate of ingestion (swallowing) of liquids is 
too low to support continuous growth and that the particles are necessary to in- 
crease the swallowing reaction. 

LITERATURE CITED 

BARIGOZZI, C, 1939. La biologia di Artemia salina studiata in aquario. Atti. Soc. Ital. Sci. 
Nat., 78: 137-160. 

CROGHAN, P. C., 1958a. The mechanism of osmotic regulation in Artemia salina: the physi- 
ology of the branchiae. /. Exp. Bio!., 35 : 234-42. 

CROGHAN, P. C., 1958b. The mechanism of osmotic regulation in Artemia salina: the physi- 
ology of the gut. /. Exp. Biol, 35 : 243-49. 

GIBOR, A., 1956. Some ecological relationships between phyto- and zooplankton. Biol. Bull., 
Ill : 230-34. 

HEATH, H., 1924. The external development of certain Phyllopods. /. MorphoL, 38: 453-83. 

HOUSE, H. L., AND J. S. BARLOW, 1956. Nutritional studies with Pseudosarcophaga affinis, a 
dipterous parasite of the spruce bud worm, Choristoncura fumifcrana. V. Effects of 
various concentrations of the amino acid mixture, dextrose, potassium ion, the 
mixture, and lard on growth and development ; and a substitute for lard. Canadian 
J. Zool, 34 : 182-189. 

LOOMIS, W. F., 1955. Glutathione control of the specific feeding reactions of Hydra. Ann. 
N. Y. A cad, Sci., 62 : 209-228. 

PROCTOR, V. W., 1957. Studies of algal antibiosis using Haematococcus and Chlamydomonas. 
Limnology and Oceanography, 2: 125-139. 

PROVASOLI, L., 1956. Alcune considerazioni sui caratteri morfologici e fisiologici delle Alghe. 
Boll. Zool. Agrar. e Bachicoltura, 22 : 143-188. 

PROVASOLI, L., K. SHIRAISHI AND J. R. LANCE, 1959. Nutritional idiosyncrasies of Artemia 
and Tigriopus in monoxenic culture. Ann. N. Y. Acad. Sci., 77: 250-61. 

RYTHER, J. H., 1954. Inhibitory effects of phytoplankton upon the feeding of Daphnia magnet 
with reference to growth, reproduction and survival. Ecol., 35 : 522-33. 

SINGH, K. R. P., AND A. W. A. BROWN, 1957. Nutritional requirements of Aedes aegyptii. J. 
Insect Physiol, 1 : 199-220. 

SPOEHR, H. A., J. SMITH, H. STRAIN, H. MILNER AND G. HARDIN, 1949. Fatty Acids Anti- 
bacterials from Plants. Carnegie Instit. of Wash. Publ. 586, 67 pp. 

TRACER, W., 1936. The utilization of solutes by mosquito larvae. Biol. Bull. 71 : 343-352. 

TREILLARD, M., 1924. Sur 1'elevage en culture pure d'un crustace Cladocere Daphnia magna. 
C. R. Acad. Sci., 190: 1090. 



LARVAL DEVELOPMENT OF THE SAND CRAB EMERITA TALPOIDA 

(SAY) IN THE LABORATORY 1 - 2 

GEORGE H. REES 
Dtikc ( 'iihrrsity Marine f*<ihi>mtry, Beaufort, N. C. 

Present knowledge of the larval development of Emerita talpoida (Say) is 
limited to two early reports, both under the generic name Hippa. Smith (1877) 
described three zoeal stages, which he called second, third, and last zoea, and a 
megalops stage, all from the plankton of Vineyard Sound, Mass. Smith was unable 
to obtain a first zoea, as females brought into the laboratory invariably cast off their 
eggs before they hatched. Faxon (1879a) was able to obtain the first zoea from 
eggs hatched in the laboratory. He was unable to rear any larvae through the 
first molt into the second stage, but ventured the opinion that one or more stages 
remained to be discovered between the first and the earliest described by Smith. 

The larvae of two other species of Emerita have been investigated, Emerita 
asiatica by Menon (1933), and Emerita, analoga by Johnson and Lewis (1942). 
Johnson and Lewis were able to obtain the first zoea from eggs hatched in the 
laboratory, but were unable to maintain the larvae through the first molt. One 
individual did enter the second stage after 34 days, but died soon afterwards. On 
the basis of the first zoea obtained in the laboratory and other stages from the 
plankton, Johnson and Lewis describe five zoeal stages. In addition, they state 
that a number of specimens were collected which appeared to be intermediate 
between Stage III and Stage IV, and which they called, for convenience, "Lower 
Stage IV." Menon (1933) lists five zoeal stages for Emerita asiatica, all of which 
were obtained from the plankton. 

The present paper is a description of the larval development of Emerita talpoida 
(Say) based on observations of larvae reared in the laboratory. 

The author wishes to express his sincere appreciation to Professor C. G. Book- 
bout, under whose guidance and direction this work was done and who read and 
suggested improvements in this manuscript. Thanks are also due to Dr. John D. 
Costlow, Jr., for his many helpful suggestions during the course of the experiment. 

METHODS 

Ovigerous females of Emerita talpoida were collected on the beach at Fort 
Macon, N. C., and held in the laboratory in large fingerbowls until hatching oc- 
curred. If unmolested, the females remained quiet in the fingerbowls until the 
time of hatching. At this time they became active and swam in short spurts 
around the sides of the container. At each spurt of swimming activity a cloud of 

1 Part of a thesis submitted to the graduate faculty of Duke University in partial fulfillment 
of the requirements for the degree of Master of Arts. 

2 Present address : Radiobiological Investigations, U. S. Fish and Wildlife Service, 
Beaufort, North Carolina. 

356 



LARVAL DEVELOPMENT OF EMERITA 357 

larvae was released. The larvae began actively swimming immediately upon hatch- 
ing, with no intervening prezoeal stage. 

Groups of ten newly hatched larvae were placed in four-inch fingerbowls of sea 
water which had been filtered through glass wool and inoculated with 200,000 units 
of penicillin per liter. There were ten such bowls. Other larvae were reared in 
mass cultures in f.ngerbowls containing approximately 200 individuals each. Each 
day the larvae were transferred by means of a pipette to clean bowls of filtered, 
inoculated sea water. To each bowl each day was added a quantity of Nitzschia 
sp. and newly hatched Artemia nauplii. The exception to this was one of the mass 
culture bowls, to which only Nitzsclria sp. was added. The zoea were observed to 
capture and feed upon the Artemia nauplii quite readily. 

The fingerbowls which originally contained ten larvae each were examined 
daily and a record made of the number of individuals surviving and the number in 
each stage of development each day, based upon exuviae found. Larvae in each 
stage were removed from the mass culture dishes and preserved in 70 per cent 
alcohol. 

Throughout the experiment the larvae were maintained at a temperature of 
30.0 C. and under constant illumination from daylight fluorescent lights. The sea 
water in which the larvae were reared varied in salinity from 28.2 parts per 
thousand to 35.1 p.p.t., with a mean of 32.2 p.p.t. 

RESULTS 

In the mass culture dish to which only Nitzschia sp. was added all individuals 
died while still in the first zoeal stage. In the other bowls, to which newly hatched 
Artemia nauplii were added in addition to Nitsschia, some of the individuals 
eventually entered the megalops stage after passing through six or seven zoeal 
stages. The majority of the individuals which became megalops did so after 
passing through six distinct zoeal stages ; a few individuals went through an 
additional molt between the sixth zoeal stage and the megalops. The only mor- 
phological difference apparent between the sixth zoeal stage and the seventh zoeal 
stage was an increase of one or two setae on the exopods of the maxillipeds. The 
appearance of a seventh zoeal stage in a few individuals does indicate, however, that 
the number of molts through which an individual passes during larval development 
is not fixed and/or inflexible. 

The number of individuals which entered each stage and the average duration of 
each stage are given in Table I. Although a few deaths occurred during the inter- 
molt period, most of the individuals which died did so at the time of molting. The 
difficulty in molting was usually the result of the old exuvia adhering to the new 
exuvia, generally on the maxillipeds and near the tip of the rostral spine. This 
failure of the old exuvia to detach from the new may be due to a physiological 
weakness existing in some of the zoea. Whether this weakness also exists in 
nature is a matter for speculation. 

The shortest length of time that it took any individual to become a megalops was 
23 days, the longest was 33 days. The average length of the pelagic larval life in 
the laboratory was 28 days. 

With each zoeal molt the number of setae on the exopods of the first and second 
maxillipeds increased by either one or two. This change in the number of setae 



358 



GEORGE H. REES 



TABLE I 

Number of individuals out of the original 100 -which entered each stage of development 

and the average duration of each stage. 



Stage 


I 


II 


III 


IV 


V 


VI 


VII 


Megalops 


Number of indi- 


















viduals 


100 


94 


75 


71 


63 


45 


5 


15 


Average number 


















of days spent 


















in stage 


3 


4 


3 


4 


5 


9 


3 





on the maxillipeds was found to be an accurate indication of the number of molts 
through which an individual reared in the laboratory had passed. 

DESCRIPTION OF THE LARVAE OF E. TALPOIDA 
FIRST ZOEA (Fie. 1) 

The first zoeal stage is similar to the first stage of Emerita asiatica, as described 
by Menon (1933), and E. analoga, as given by Johnson and Lewis (1942). The 
smoothly rounded carapace is translucent, colorless, and without the lateral spines 
which are characteristic of subsequent stages. The rostrum is short and broad. 
The eyes are stalked. The eyestalks are short and thick and lie close against the 
carapace, directed somewhat posteriorly. The abdomen projects almost straight 
downward from the carapace and is flexed so that the telson is carried beneath and 
nearly parallel to the carapace. The exopods of the maxillipeds bear four plumose 
setae. 

Antennules (Fig. 8). These short, un jointed appendages are thick at the 
base and taper to a blunt point where three setae of about equal length are borne. 
These setae are slightly longer than the body of the appendage. 

Antennae (Fig. 14). The antennae at this stage are rather stubby appendages, 
produced on the outer side into a spine-like process. From the base of the outer 
spine there arises a somewhat slenderer dentiform process of about the same length. 
At the base of this inner process there is a much smaller spine. The form of the 
antennae is relatively unchanged through the first four zoeal stages, the first in- 
dication of a flagellum not appearing until the fifth zoeal stage. 

Mandibles (Fig. 20). The mandibles grow out ventrally and then make a 

TABLE II 

Relative size of larvae in each stage reared in the laboratory. Based on average measurements 
of 10 or more specimens. Dimensions are given in mm. 



Stage 


I 


II 


III 


IV 


V 


VI 


Megalops 


Max. length of carapace 


0.57 


0.68 


0.88 


1.13 


1.60 


1.90 


2.30 


Max. width of carapace 


0.46 


0.56 


0.67 


0.84 


1.00 


1.30 


1.80 


Length of abd. plus telson 


0.80 


0.84 


1.30 


1.50 


1.80 


2.40 


1.90 


Length of rostrum 


0.20 


0.68 


1.27 


1.55 


2.40 


2.90 





Length of lateral spine 





0.30 


0.44 


0.60 


0.80 


1.00 





LARVAL DEVELOPMENT OF EMERITA 



359 




FIGURE 1. First zoea. 
FIGURE 2. Second zoea. 



FIGURE 3. Third zoea. 
FIGURE 4. Fourth zoea. 



360 GEORGE H. REES 

right angle bend towards the median line so that their crowns are opposed. The 
crown is armed on its ventral edge by a stout, rather blunt tooth, followed by two 
shorter, sharp, triangular teeth. Next, there are three or four long slender, setae- 
like teeth and, finally, a sharp, triangular tooth on the dorsal edge. The entire 
crown slopes gradually from the ventral to the dorsal edge. These appendages 
change very little, except for a general increase in size, throughout the zoeal stages. 

First maxillae (Fig. 22). These are fleshy appendages, adapted for handling 
food. The endopod bears three stiff setae at its tip and one short seta about half- 
way down its inner side. The exopod is twice as large as the endopod and flattened 
dorso-ventrally. It is divided at its distal end into two tapering horns. Part way 
down the outer side of the exopod is a small lobe, bearing a single long, curved 
seta. 

Second maxillae (Fig. 24). Each of these appendages is divided into two 
parts. The protopod is a lobe, tapering anteriorly, where it bears a cluster of three 
setae. The scaphognathite is sickle-shaped, broader posteriorly than anteriorly, 
and very thin and foliaceous. Along its anterior-outer margin are nine setae. 
The posterior and inner margins of the scaphognathite are naked. 

First maxillipeds (Fig. 1). These appendages are composed of a short coxopod, 
a long basipod, a two-segmented exopod and a four-segmented endopod. The 
basipod bears a group of three setae on its posterior margin just behind the joint 
with the endopod. One of these setae is shorter and stouter than the other two 
and armed with minute spines. Behind these is a group of two setae, then a short 
distance back, a single seta, and finally a single seta close to the joint with the 
coxopod. The endopod consists of four, cylindrical segments, each bearing setae. 
The first segment bears three setae just below the joint. One of these is shorter 
and stouter than the other two and armed with minute spines. The second segment 
bears two setae just below the joint, one of which is short, stout, and armed with 
spines as above. The third segment has two setae below the joint. The terminal 
segment bears four setae at its tip. The outermost two are the longest, curve 
downward at the ends and bear small spines along their inner margins. The exopod 
consists of a proximal segment as long as the endopod and a very short terminal 
segment which bears four long, plumose setae. 

Second maxillipeds (Fig. 1). These are very similar to the first maxillipeds 
except the endopod is somewhat longer than the exopod. The basipod bears three 
setae along its inner margin; a group of tw r o just behind the joint with the endopod 
and a single seta about halfway between this and the joint with the coxopod. No 
rudiments of other thoracic appendages are visible posterior to the second maxil- 
lipeds at this stage. 

Abdomen. The abdomen is composed of five segments, the first of which is 
not clearly differentiated from the abdomen at this time. The sixth segment is 
consolidated with the telson ; this becomes apparent when the uropods appear. No 
rudiments of abdominal appendages are visible. 

The telson is slightly broader than long, and faintly concave. The lateral 
margins curve smoothly to a stout tooth at each side of the posterior margin. The 
posterior margin of the telson is armed with a complicated series of small spines, 
with minute denticles between them. The eighth spine from each side is the 
longest, and between these two longest spines are either nine or ten spines of 
intermediate length. Thus, in some cases there are twenty-five spines on the 



LARVAL DEVELOPMENT OF EMERITA 



361 



posterior margin and in others there are twenty-six. This arrangement holds 
true for all the zoeal stages, there being sometimes twenty-five and sometimes 
twenty-six spines on the posterior margin of the telson. 

SECOND ZOEA (Fio. 2) 

Two lateral spines are now present on the carapace, projecting posteriorly and 
downward. The rostrum has increased in length tremendously and is now as 
long as the carapace. The eyestalks are longer and the eyes are carried some- 
what farther forward than in the first stage. The exopods of the maxillipeds bear six 
plumose setae. 

Antennules (Fig. 9). Each of these appendages now bears a single stout seta 
instead of the three which were present in the first stage. 

Antennae (Fig. 15). These are the same as in the first zoea. 

Mandibles. As in the first zoea. 

First maxillae. As in the first zoea except that the exopod bears three long 
teeth, the outer one showing no articulation at the base. 

Second maxillae. As in the first zoea. 




FIGURE 5. Fifth zoea. 



THIRD ZOEA (Fie. 3) 

The shape of the carapace has changed somewhat. In the first zoea the 
carapace is practically hemispherical, in the second zoea it is less so, and now in the 
third zoea, its lateral outline is pear-shaped. The rostrum has continued to lengthen 
in comparison to the carapace and now exceeds the length of the carapace. 
Uropods appear on the telson. The exopods of the maxillipeds bear eight plumose 
setae. 



362 GEORGE H. REES 

Antennules (Fig. 10). Each of these bears three setae at its tip, much as in 
the first zoea stage. 

Antennae (Fig. 16). As in the first and second zoea. 

Mandibles. As in preceding stages. 

First maxillae. As in the second zoea. 

Second maxillae. As in the first and second zoea, the number of setae on the 
scaphognathite is nine. 

Maxillipeds. The exopods bear eight plumose setae. 

Uropods. These appear on the anterior ventral surface of the telson and consist 
of a short basal segment with a long, flattened lobe extending from it. This lobe is 
the exopod, as will be seen from later development, and bears two long setae at 
its tip. 

The eyestalks have enlarged and project downward and forward. There is 
no evidence of any additional thoracic or abdominal appendages at this stage. 

FOURTH ZOEA (Fie. 4) 

This stage is characterized by the presence of ten plumose setae on the exopods 
of the maxillipeds and the fact that the uropod bears four setae on its exopod. 

Antennules (Fig. 11). Each of these appendages bears four setae, three at 
the tip and one a short distance down on the inner side. 

Antennae (Fig. 17). These are unchanged except for general growth. 

Mandibles. These are somewhat slenderer than in preceding stages. 

First maxillae. As in preceding stages. 

Second maxillae. There are fourteen setae on the anterior-outer margin of 
the scaphognathite. 

Maxillipeds (Fig. 4). These bear ten plumose setae at the tips of the exopods. 

Uropods. The exopod bears two long and two short setae. No evidence of 
endopod as yet. 

Abdomen. Each of the four free segments of the abdomen bears two small, 
round thickenings on its inner side, the evidence of future pleopods. No additional 
thoracic appendages are visible through the carapace. 

FIFTH ZOEA (Fio. 5) 

The fifth zoea is characterized by the presence of eleven or twelve plumose 
setae on the exopods of the maxillipeds, and the appearance of the rudiment of the 
endopod on the uropods. The rudiments of five future thoracic appendages are now 
visible through the carapace, posterior to the second maxillipeds. 

Antennules (Fig. 12). Each bears six setae; a group of three at the tip, a 
group of two lower down on the inner margin and a single seta below these. 

Antennae (Fig. 18). The rudiment of the flagellum is visible as a conspicuous 
knob, about half as long as the dentiform process, on the inner side of the antenna. 

Mandibles. As in preceding stages. 

First maxillae. As in preceding stages. 

Second maxillae (Fig. 25). The number of setae along the anterior-outer 
margin of the scaphognathite has increased to nineteen. 

Maxillipeds (Fig. 5). There are eleven or twelve plumose setae on the tips of 
the exopods. In the first four stages the number of setae on the exopods was 



LARVAL DEVELOPMENT OF EMERITA 



363 



constant at four, six, eight, and ten, respectively ; now there is some variation. 
Although twelve appears to be the more usual number, about one-third of the 
specimens examined had eleven setae on one or more of the maxillipeds. Indi- 
viduals were found with twelve setae on the first maxilliped of the right side and 
eleven on the first maxilliped of the left side, and vice versa. This was also found 
to be true for the second maxillipeds. No individuals, in this stage of development, 
were found with less than eleven or more than twelve setae on the exopods of the 
maxillipeds. 

Abdomen. As in the fourth zoea. 

Uropods. The rudiment of the endopod now appears as a small bud below the 
exopod. The exopods have increased in length and each now bears five long setae 
at its tip. 




FIGURE 6. Sixth zoea. 



SIXTH ZOEA (Fie. 6) 

There are now uniramous pleopod buds on the four free segments of the 
abdomen. The exopods of the first and second maxillipeds bear thirteen or fourteen 
plumose seta. The rostrum continues to increase in length relative to the carapace 
and is cne and one-half times the length of the carapace. 

Antennules (Fig. 13). These appendages bear eleven setae in four groups: 
four at the tip, a group of four below this, a group of two below that, and finally a 
single seta below these. 

Antennae. The flagellum has increased enormously, dwarfing the dentiform 
processes and extending well beyond the antennule. 



364 



GEORGE H. REES 




FIGURE 7. Megalops. 



LARVAL DEVELOPMENT OF EMERITA 365 

Mandibles (Fig. 21). The crown is armed much as in previous stages, but the 
shaft of the mandible is not so stout in proportion to its length. 

First maxillae (Fig. 23). As in preceding stages. 

Second maxillae. There are now 29 setae which extend all the way around 
the anterior-outer margin of the scaphognathite. 

Abdomen. A pair of uniramous, unsegmented pleopods now appears on the 
second through the fifth segments of the abdomen. 

Uropods. The endopod has increased considerably and is now two-thirds the 
length of the exopod. The exopod bears six setae of unequal length at its tip. 

Ma.riliipeds (Fig. 6). The number of plumose setae on the exopods is now 
thirteen or fourteen occurring with about equal frequency. No individuals were 
found with less than thirteen or more than fourteen. 

Five thoracic limb buds are plainly visible through the carapace. The first of 
these is the largest and extends below the edge of the carapace. This is the 
rudiment of the third maxilliped. The rudiment of the fifth pereiopod is not 
visible at this time. 

Most of the individuals which became megalops did so at the molt following 
this stage. A few, however, went to a seventh zoea before becoming megalops. 
The only difference between this seventh zoea and the sixth was the appearance 
of additional setae on the maxillipeds, making the number fifteen or sixteen. 

MEGALOPS (Fie. 7) 

In general form the megalops resembles the adult, the most obvious difference 
being that the eyes are still relatively large and the abdomen bears four pairs of 
pleopods which are quite unlike those of the adult. The megalops carries the 
abdomen flexed, with the telson between the bases of the pereiopods, though it is 
not as strongly flexed as in the adult. In swimming the megalops sometimes, but 
not always, extends the abdomen, thus utilizing the pleopods as swimming ap- 
pendages. This tendency to swim with the abdomen extended decreased Avith 
time and individuals which had been in the megalops stage as long as one day 
were rarely seen to extend the abdomen. 

Antennules (Fig. 26). These appendages are now as long as the peduncles of 
the antennae and composed of three basal segments and a six-segmented flagellum, 
which is not noticeably delineated from the basal segments. The first segment 
bears one, and the succeeding five segments each bear two plumose setae on the 
outer margin. The segments of the peduncle and the two lower segments of the 
flagellum may bear a single seta on the inner side. The secondary flagellum of 
the adult is not present at this stage. 

Antennae (Fig. 27). These now possess all the important features of the adult 
form. There is a scale-like exopod and an endopod composed of three segments 
and a long flagellum. The flagellum is stout, tapers gradually to a rather blunt 
tip, and is composed of eighteen segments. The segments are short proximally but 
gradually increased in length distally until they are longer than broad near the tip. 
Each segment bears four setae. There are two long, plumose setae which curve 
inward at their tips, and within these are two shorter, straight, unarmed setae. 

Mandibles (Fig. 28). The mandible has undergone a complete change in 
structure and function. It is no longer an organ of mastication but is adapted, as in 



366 



GEORGE H. REES 




30 



LARVAL DEVELOPMENT OF EMERITA 367 

the adult, for the purpose of scraping the antennae and passing food to the mouth. 
The mandible is now composed of two parts, a broad, foliaceous outer lobe bearing 
two short, stout setae on its lateral margin, and an inner palp fringed with setae 
along its anterior and median margins. A very noticeable difference between the 
appendages of the megalops and those of the adult is the relatively sparse setation 
of the megalops appendages. In the adult all the appendages are densely fringed 
with setae. 

First ma. \' iliac (Fig. 29). The first maxillae now possess all the parts of the 
adult appendage. The inner lobe (endopod) is broad, bluntly rounded at the tip 
and armed with stout setae of varying length around the tip and part way down 
the inner margin. The outer lobe (exopod) is longer and much narrower at the 
base but broad and round at the tip. It is armed with short, stout setae at the 
tip and along the inner margin. There is one long plumose seta at the beginning 
of the outer margin. The palpus is a sac-like lateral projection near the base of 
the inner lobe. It bears a single long seta at its tip. 

Second maxillae (Fig. 30). These appendages are like the adult form except 
for the relative proportions of the endites and the sparsity of setae as compared to 
the adult. In the megalops the endites compose three lobes, a small inner lobe bear- 
ing long curved setae down its inner margin, a much larger outer lobe fringed with 
short setae along its anterior-inner margin, and a small papiliform lobe between 
these two, bearing a single long seta. Between the outer endite and the scaphogna- 
thite is a small, triangular lobe representing the endopod. The scaphognathite is 
broad, thin and has much the same form as in the zoeal stages. It now tapers 
more acutely anteriorly and the fringe of setae has extended around the broad 
posterior margin. 

First maxillipcd (Fig. 31). The anterior segment of the protopod is elongated 
into a flat, blade-like process bearing setae around its margins, with a series of much 
longer, plumose setae on the posterior portion of the inner margin. The endopod 
is represented by a soft, slender lobe arising from near the base of the inner side of 
the two-segmented exopod. The exopod consists of a long basal segment bearing 
short setae along its outer margin, and a shorter, broader, paddle-shaped terminal 
segment bearing very long setae at the tip and shorter setae along its other margins. 

Second maxillipeds (Fig. 32). The four-segmented endopod of the second 
maxilliped differs slightly frcm the adult form. The third segment makes a right 
angle bend in the adult, while it is practically straight in the megalops, and the 
terminal segment is proportionally much shorter and stouter in the megalops than in 
the adult. The exopod is two-segmented ; the basal segment does not taper 
anteriorly as acutely as in the adult and the oval terminal segment is proportionally 
smaller in the megalops than in the adult. As in previous cases, the adult append- 
age is very heavily fringed with setae, while in the megalops the setae are shorter 
and much sparser. 

FIGURES 8-13. Antennule. first to sixth zoea. FIGURE 26. Antennule, megalops. 

FIGURES 14-19. Antenna, first to sixth zoea. FIGURE 27. Antenna, megalops. 

FIGURE 20. Mandih'e, first zoea. FIGURE 28. Mandible, megalcps. 

FIGURE 21. Mandible, sixth zoea. FIGURE 29. First maxilla, megalops. 

FIGURE 22. First maxilla, first zoea. FIGURE 30. Second maxilla, megalcps. 

FIGURE 23. First maxilla, sixth zoea. FIGURE 31. First maxilliped, megalcps. 

FIGURE 24. Second maxilla, first zoea. FIGURE 32. Second maxilliped, megalcps. 

FIGURE 25. Second maxilla, fifth zoea. FIGURE 33. Third maxilliped, megalops. 



368 GEORGE H. REES 

Third maxillipeds (Fig. 33). These broad, opercular appendages bear a three- 
segmented palp at their distal end. The rounded prominence at the articulation 
of the palpus is lacking in the megalops. The palpus is stouter and the terminal 
segment much shorter than in the adult. 

Pereiopods. The pereiopods are so much like those of the adult that a detailed 
description of them is unnecessary. The first, second, and third pairs project 
anteriorly; the fourth pair projects posteriorly and, like the first three pairs, is 
especially adapted for burrowing in the sand. The fifth pair of pereiopods are 
very slender, chelate appendages, which are held concealed within the branchial 
cavity. 

Abdomen. The abdomen is, for the first time, composed of six segments, 
similar in form and proportion to those of the adult, and a triangular telson. The 
first segment, as seen dorsally, is a small plate, filling a curved sinus in the posterior 
margin of the carapace. The second segment is the largest of the abdominal segments 
and is about five times as wide as it is long. Its width is due to a broad lamellar 
expansion on each side. The third, fourth, and fifth segments are rounded at the 
outer margins and each is slightly shorter and narrower than the one before. The 
sixth segment is nearly as wide as the fifth and is as long as it is wide, being the 
longest of the abdominal segments. The second, third, fourth, and fifth segments 
of the abdomen each bear a pair of biramous pleopods. The pleopods are made 
up of three portions ; a long basal segment, a paddle-shaped exopod and a knob-like 
endopod. The pleopods of the second and third segments bear ten plumose setae 
each on the exopods and those of the fourth and fifth segments bear eleven plumose 
setae on the exopods. The small endopods increase in length successively from the 
first to the fourth pleopod, and bear at their tips a series of small hooks. These 
hooks can engage those of the endopod opposite it. thus joining the pair of pleopods 
so that they move as one. 

Uropods. The appendages of the sixth abdominal segment are essentially the 
same as those of the adult. There is a two-segmented protopod, the proximal 
segment of which is short and round and the distal segment, much longer, stouter 
and flattened. The exopod and endopod are nearly alike : oval, broadly rounded 
at the tip and fringed with setae which are very long at the tips but shorter along 
the sides. 

DISCUSSION 

The first zoea of Emerita talpoida, obtained from the egg, corresponds with 
the first zoea described by Faxon (1879a). The larvae described by Smith (1877) 
from the plankton as the second, third, and last zoea do not correspond exactly with 
any of the zoeal stages reared in the laboratory. The results of the present experi- 
ments indicate that each zoeal molt results in an increase in the number of setae 
borne on the exopods of the first and second maxillipeds. In the laboratory their 
number never increased by more than two setae at any one molt. Using the 
number of setae on the maxillipeds as an indication of the number of times that an 
individual has molted, Smith's three zoeae correspond to the third, fourth, and 
fifth zoeal stages reared in the laboratory. In each case, however, the zoeae from 
nature possess features (appearance of thoracic limb buds, pleopods, etc.) which 
show them to be farther advanced in development than the corresponding laboratory 
stages. 



LARVAL DEVELOPMENT OF E.MERITA 369 

Smith's last zoeal stage bears twelve setae on the exopods of the first and second 
maxillipeds. A number of individuals in this stage were observed by Smith to 
change to megalops at a single molt. If setation of the maxillipeds is an accurate 
index to the number of zoeal molts, then these individuals from nature completed 
their larval development in five molts, whereas those in the laboratory went through 
six or seven molts before becoming post-larvae. 

Gurney (1942) has questioned the normality of larvae reared in the laboratory 
and believes that abnormal stages may be reared under artificial conditions. How- 
ever, Gurney also states that extra stages, through which each individual need not 
pass in development, occur in nature. Other references to these extra stages have 
been made by Faxon (1879b), Lebour (1940), Gurney and Lebour (1941), and 
Broad (1957a). Johnson and Lewis refer to a "Lower Stage IV" in the larvae of 
E. analoga, which was intermediate between Stage III and Stage IV. They could 
not say whether this was a distinct instar or simply a variable in Stage IV. This 
is apparently another case of a stage in larval development through which not all 
individuals pass. If each larval form found in the development of a decapod is de- 
scribed as a stage, then we may expect to find individuals who skip stages or pass 
through extra stages in the course of normal development, depending upon the 
number of stages previously defined. 

Broad (1957b) working with Palaemonetes larvae, has shown that a direct 
relationship exists between the diet of the larvae and the rate of larval development 
and frequency of molting. His results show that larvae may respond to sub- 
optimal conditions of diet by a prolonged larval life and a greater number of larval 
intermolts. It is probable that a number of other environmental factors also affect 
the tempo of larval development, and that normal development of Etnerita and 
other decapods varies according to the variations in trophic conditions during the 
breeding season. 

SUMMARY 

1. Emcrita talpoida were reared in the laboratory from eggs to the megalops. 
Of individuals hatched in the laboratory 15 per cent survived to the megalops. 

2. The average length of time required to pass through the pelagic larval stages 
in the laboratory was 28 days. A table is given which shows the average duration 
of each of the zoeal stages. 

3. Six zoeal stages and a megalops are figured and described. A seventh zoeal 
stage, which was skipped by most of the larvae, is described. 

4. Larvae reared in the laboratory are compared with three zoea stages described 
from the plankton by Smith. 

5. The larval development of Emerita talpoida should not be regarded as con- 
sisting of a fixed number of stages determined by a fixed number of larval intermolts. 

LITERATURE CITED 

BROAD, A. C., 1957a. Larval development of Palaemonetes pugio Holthuis. Biol. Bull., 112: 

144-161. 
BROAD, A. C., 1957b. The relationship between diet and larval development of Palaemonetes. 

Biol. Bull.. 112: 162-170. 
FAXON, WALTER, 1879a. On some young stages in the development of Hippa, Porcellana, and 

Pinni.ra. Bull. Mus. Comp. Zool. Harvard, 5: 253-268. 



370 GEORGE H. REES 

FAXON, WALTER, 1879b. On the development of Palaemonetes vulyaris. Bull. Mus. Comp. 

Zool. Harvard, 5 : 303-330. 

GURNEY, R., 1942. The larvae of decapod Crustacea. The Ray Society, London. 
GURNEY, R., AND MARIE V. LEBOUR, 1941. On the larvae of certain Crustacea Macrura, 

mainly from Bermuda. /. Linn. Soc., 41 : 89-181. 
JOHNSON, M. W., AND W. M. LEWIS, 1942. Pelagic larval stages of the sand crabs Emerita 

analoga (Stimpson), Blepharipoda occidentalis Randall, and Lepidope myops Stimpson. 

Biol Bull., 83: 67-87. 
LEBOUR, MARIE V., 1940. The larvae of the British species of Spirontocaris and their relation 

to Thor (Crustacea Decapoda). /. Mar. Biol. Assoc., 24: 505-514. 
MENON, M. KRISHNA, 1933. The life histories cf decapod Crustacea from Madras. Bull, of 

the Madras Gov. Mus., Neiv Ser., 3 : 1-45. 
SMITH, SIDNEY I., 1877. The early stages of Hippa talpoida, with a note on the structure of 

the mandibles and maxillae in Hippa and Rcmipes. Trans. Conn. Acad. Sci., 

3: 311-342. 



FREE AMINO ACIDS IN SOME AQUATIC INVERTEBRATES 1 

JOHN W. SIMPSON, 2 KENNETH ALLEN AND JORGE AWAPARA 
Department of Biology, The Rice Institute, Houston, Texas 

Free amino acids and many of their derivatives are found present in tissues of 
all invertebrates so far studied. The distribution of amino acids seems to follow 
a pattern characteristic for the species. Compounds such as taurine were found 
in very high concentration in a number of invertebrates. As early as 1904, Kelly 
reported that Mytilus edulis has as much as 1.6 per cent taurine. Other inverte- 
brates contain also large amounts of taurine (Henze, 1905; Mendel, 1904; Kossel 
and Edlbacher, 1915; Okuda, 1920; Ackermann et al, 1924; Ackermann, 1935; 
Lewis, 1952; and Kermack et al., 1955). The function of taurine in invertebrates 
is not known at all, and its mode of formation remains to be elucidated. 

The distribution of free amino acids in a number of invertebrates has been 
studied by Camien et al. (1951), Duchateau and Florkin (1954), Duchateau et al. 
(1952), and Giordano et al. (1950). The amino acids were determined micro- 
biologically. Camien et al. found very high concentrations of glycine in muscles of 
Homarus vulgaris and Maia squinado and suggested that the role of glycine along 
with other amino acids was to regulate osmotic pressure. Kermack, Lees and Wood 
(1955) made an extensive study of the non-protein constituents of the lobster. 
They found that a large portion of the non-protein nitrogen was accounted for as 
free alpha-amino nitrogen. The remainder was distributed between trimethylamine 
oxide, glycine betaine, taurine and volatile bases. 

The object of the present study was to determine the pattern of distribution of 
free amino acids and related substances and to establish a correlation between the 
pattern of distribution and species and/or environment. The results reported here 
indicate that such differences do exist in relation to environment and species. 

MATERIALS AND METHODS 
Animals investigated 

The invertebrates investigated include representatives of the following phyla: 
Coelenterata, Arthropoda, Mollusca, and Echinodermata. The specimens were 
taken from their environment, rapidly frozen and maintained in this state just 
prior to analytic procedures (not more than a one-month period). Table I lists 
these organisms according to phylum and class and shows the location and habitat 
from which the specimens were taken. 

Extraction 

Immediately after thawing, the whole organism was quickly weighed and the 
nitrogenous substances extracted with 80 per cent ethanol according to the pro- 

1 This work was supported by grants from the Robert A. Welch Foundation, Houston, 
Texas, and the National Institutes of Health, U. S. Public Health Service. 

2 Part of these data was taken from the M.A. Thesis of John W. Simpson. 

371 



372 JOHN W. SIMPSON, KENNETH ALLEN AND JORGE AWAPARA 



TABLE I 

Organisms studied and their habitat 



Organism* 


Location of 
collection 


Habitat 


Coelenterata 






Anthozoa 






Bitnodosoma cavernata 


PA 


W 


Arthropoda 






Crustacea 






Penacus aztecus 


AB 


vv 


Clibinarius vittatus 


SLP 


E 


Pagurus pollicaris 


AB 


W 


Mollusca 






Gastropoda 






Oliva sayana 


SLP 


E 


Polinices duplicata 


AB 


W 


Thais haemastoma haysae 


AB 


W 


Busy con perversum 


AB 


W 


Fasciolaria distans 


PA 


W 


Siphonaria lineolata 


PA 


E 


Pelecypoda 






Lithophaga bisulcata 


PA 


W 


Crassostrea virginica 


SLP 


W 


Area umbonata 


PA 


W 


Volsella demissus granosissimus 


EBL 


E 


Cephalopoda 






Loligtincula brevis 


AB 


W 


Echinodermata 






Holothuroidea 






Thyone sp. 


SLP 


W 


Asteroidea 






Luidia clathrata 


SLP 


W 



PA Port Aransas, Texas 
AB Aransas Bay, near Rockport, Texas 
SLP San Luis Pass, Galveston Island, Texas 
EBL East Beach Lagoon, Galveston Island, Texas 
W -Collected directly from marine waters 
E Collected during a period of exposure 

* We are indebted to Mr. Howard Lee from the Texas Game and Fish Commission, Rock- 
port, Texas, for permitting the use of equipment necessary for acquisition of organisms. 

cedure of Awapara (1948). The amino acids extracted are not produced during 
the extraction procedure by proteolytic cleavage. Extractions were carried out 
using live organisms under conditions such as to prevent any enzymatic activity. 
The live organisms were ground with 80 per cent ethanol as indicated above. 
Others were frozen and then thawed. Extracts were also prepared as described. 
Analysis of the extracts revealed that no change had occurred as a result of freezing 
and thawing. 



FREE AMINO ACIDS 



373 



Fractionation of extractives 

Using ion-exchange chromatography the nitrogenous extractives were separated 
into basic, acidic and neutral substances. Basic substances were separated from 
neutral and acidic substances on Amberlite CG-50, type 2 H + , with a screen grading 
of approximately 200 (passing 200 mesh). The tissue extracts were placed in a 
beaker containing one gram of the resin and shaken for thirty minutes to allow 
equilibration with the resin. This suspension was placed in a small column 



TABLE II 
Amino acids identified 



Organism 


Al 


B-A1 


Gly 


Ar 


As 


Glu 


Tau 


Gla 


Pr 


OH-Pr 


Thr 


Tyr 


Aspr 


His 


Coelenterata 






























Anthozoa 






























Bunodosoiita cavernata 


+ 





+ 


+ 


+ 


+ 


+ 


+ 




















Arthropoda 






























Crustacea 






























Penaeus aztecus 


4- 


4. 


4- 


-f 


-(- 


4- 


4- 


4- 


4- 








4- 


4- 


4- 


Clibinarius vittatus 


4. 





-f 


-f 


-|- 


4- 


4- 


4- 


4- 





4- 


4- 


4- 


4- 


Pagurus poll i car is 


+ 





+ 


+ 


+ 


+ 


+ 


+ 


+ 





+ 


+ 


+ 


+ 


Mollusca 






























Gastropoda 






























Oliva say an a 


-j- 





4- 


+ 


+ 


4- 


4- 


4- 




















Polinices duplicata 


4- 





4- 


+ 


+ 


4- 


4- 


4- 


4- 


+ 














Thais haemastoma haysae 


-j- 





4- 


+ 


4- 


4- 


4- 


4- 


4- 


4- 





4- 


4- 


4- 


Busycon perversum 


4- 





4- 


+ 


+ 


4- 


4- 


4- 


+ 


+ 








4- 


+ 


Fasciolaria distans 


4- 





4- 


+ 1 


4- , 


4- 


4- 


4- 








4- 


+ 


4- 


4- 


Siphonaria lineolata 


4- 





+ 


+ 


4- . 


4- 


+ 


4- 


+ 


4- 





+ 


4- 





Pelecypoda 










, 




















Lithophaga bisulcata 


+ 


+ 


4- 


+ 


4- 


4- 


+ 


+ 




















Crassostrea virginica 


4- 


+ 


+ 


+ 


4- 


4- 


4- 


4- 


4- 





4- 











Area umbonata 


4- 


4- 


4- 


+ , 


4- 


4- 


4- 


4- 


4- 





4- 








4- 


Volsella demissus 






























granosissimus 


4- 


4- 


+ 


+ 


4- 


+ 


+ 


+ 


4- 





4- 


4- 





4- 


Cephalopoda 






























Loligunciila brevis 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 











Echinodermata 






























Holothuroidea 






























Thyone sp. 


+ 





+ 


+ 


+ 


+ 


4- 


+ 




















Asteroidea 






























Luidia dathrata 


-j- 





4. 


-f 


4- 


4. 


4. 


4. 


































1 















Legend : 



+ Present in readily detectable amounts 

Not present in readily detectable amounts 



Al alanine Ar arginine Tau taurine OH-Pr hydroxyproline 

B-A1 beta alanine As aspartic acid Gla glutamine Thr threonine 

Gly glycine Glu glutamic acid Pr proline Tyr tyrosine 

Aspr asparagine His histidine 



374 JOHN W. SIMPSON, KENNETH ALLEN AND JORGE AWAPARA 

(20 X 1 cm.) containing an additional gram of the resin. The neutral and acidic 
compounds were washed from the resin with 25 cc. water and the basic substances 
were eluted from the column with 25 cc. 4 N HAc. (Awapara, Davis and Graham, 
1959). After removal of the basic substances, taurine and other sulfonic acids 
were separated from the neutral and acidic amino acids on a column of Dowex-50 
H + . The water wash from the Amberlite was passed slowly through a column con- 
taining one gram of Dowex-50 H + . Taurine was obtained by washing with 25 cc. 
water and the neutral and acidic amino acids eluted from the resin with 25 cc. 4 N 
NH 4 OH. Each fraction was evaporated to dryness on a steam bath and brought 
to a 1-cc. volume with water. 




23 



PENAEUS 



Alanine 

Arginine 

i Aspartic 




CLIBINARIUS 



PAGURUS 



FIGURE 1. 



Estimated concentration of six amino acids in the tissues of three crustaceans 
Penaeus aztecus, Clibinarius vittatits and Pagurus pollicaris. 



Paper chromatography 

Amino acids of the acidic and neutral fractions were separated by two-dimen- 
sional ascending paper partition chromatography. Whatman number 3 MM. filter 
paper was used and the solvent system employed was phenol-water (72.5 per cent 
phenol) and 2,4-ltitidine- water (62 per cent) in the second direction. The basic 
amino acids were separated by one-dimensional ascending paper partition chroma- 
tography using butanol, acetic acid and water (4:1:1, by volume) as the solvent. 



FREE AMINO ACIDS 



375 



The amino acid spots were revealed by dipping the paper in a solution of 0.5 per 
cent ninhydrin in absolute ethanol ( \v/v ) . 

Identification of amino acids 

Amino acids were identified by : 1 ) their ninhydrin color and position on 
chromatograms according to previously prepared maps of known substances, and 
2) their presence in a particular fraction. 

Arginine was further identified by the Sakaguchi reaction, using the alpha 
napthol reagent described by Acher and Crocker (1952), and histidine by the 
Pauly's reaction using the sulfanilic acid reagent described by Smith (1958). 



Alanine Glycine 

|M Arginine $$j Glutamic 

Taurine 



Aspartic 



SIPHONARIA 




FASCIOLARIA 




15 



BUSYCON 



FIGURE 2. Estimated concentration of six amino acids in the tissues of three gastropods 
Siphonaria lincnlutu, [ : (isciolaria ilistans and Busycon pcrr 



The identification of /2-alanine and taurine was supported by preparing chromato- 
grams on paper treated with basic cupric carbonate (Block ct a!.. 1958). These 
two substances moved to their respective positions on both treated and untreated 
papers. 

Glutamine was further identified by subjecting an aliquot of the extract to 
hydrolysis with 6 N HC1. Chromatograms of the hydrolysed extracts showed the 
disappearance of this spot ; further, these same chromatograms showed an increase 
in the glutamic acid spot. 



376 JOHN VV. SIMPSON, KENNETH ALLEN AND JORGE AWAPARA 

The identification of the asparagine spot was substantiated by elution from un- 
developed chromatograms with subsequent acid hydrolysis and re-chromatography 
of the eluate. Chromatograms of the hydrolysate showed the absence of the spot 
and the appearance of a spot which corresponded in position and color with the 
aspartic acid standard. 

Estimation of amino acids 

Six amino acids which were consistently present were estimated quantitatively. 
Alanine, aspartic acid, glycine and glutamic acid were measured by the method of 
Awapara, Landua and Fuerst (1950) with the following modifications suggested 



E 

O) 



u 
O 

O 

c 



18 




60 



THAIS 



POUNICES 



Alanine 
||f Arginine 

i Aspartic 
Glycine 



: Glutamic 
T a u r i n e 




OLIVA 



FIGURE 3. Estimated concentration of six amino acids in the tissues of three gastropods : 
Thais haemastoma, Polhrices duplicata and Oliva sayanct. 

by Fow.den (1951) : 1) the areas of paper containing the spots were cut, placed in 
test tubes, and 0.2 cc. N NaOH added. The tubes were evacuated for three hours 
in a desiccator containing concentrated H 2 SO 4 ; 2) additional citrate was included 
in the ninhydrin reagent of Moore and Stein (1948) equivalent to the amount of 
NaOH added. 

Taurine was found to be the only ninhydrin-positive material present in the 
acidic fraction ; therefore, this substance was measured directly using the color 
reagent of Moore and Stein (1948). 



FREE AMINO ACIDS 



377 



Arginine was measured in the extracts by the method of Rosenberg ct al. 
(1956). We found no other Sakaguchi-positive material in the extracts as de- 
termined by paper chromatography. If other guanidine derivatives were present, 
their concentration was too low to measure. We can safely assume that nearly all 
the color in the Rosenberg reaction was due to arginine . 

RESULTS AND DISCUSSION 

In Table 11 are shown all the amino acids and related substances detected 
chromatographically. Other components were present but their identity was not 
established. In Figures 1 to 6 are shown the concentrations of six amino acids 



12- 



70 




Ip Alanine 
life Arginine 



Giycine 
y Glutamic 
Taurine 




ARCA 



VOLSELLA 



CRASSOSTREA 



FIGURE 4. Estimated concentration of six amino acids in the tissues of three pelecypods : 
Area umbonata, Volsella demissus granosissimus and Crassostrea virginica. 



which were found invariably in the species studied. The most striking feature is 
the high concentration of taurine and glycine in nearly all species studied. The 
glycine concentration is in many cases equal to that of taurine. The highest 
concentration of taurine was found in Penaeits actccus, but our value is still con- 
siderably lower than the value reported for Mytilus editlis. The mode of formation 
of taurine in invertebrates is not known at all. In mammals it is formed from 
cysteine after oxidation to cysteine sulfinic acid and decarboxylation of the latter 



378 JOHN W. SIMPSON, KENNETH ALLEN AND JORGE AWAPARA 

to hypotaurine (Awapara and Wingo, 1953). Hypotaurine is then oxidized to 
taurine. Hypotaurine has been reported only once in invertebrates ( Shibuya and 
Ouchi, 1957). The cysteine sulrmic decarboxylase has not been reported in 
invertebrates. There is the possibility that taurine is not produced by inverte- 
brates but acquired from their diet. Glycine, which is also present in very 
high amounts, could also be obtained from their diet. One objection to this possibil- 
ity is the wide range of variation in the taurine concentration of species from the 
same environment. 

Aspartic acid, glutamic acid, and alanine vary much less in concentration than 
taurine and glycine. These three amino acids are closely linked to the citric acid 




55 



Alanine 
HI Arginine 
l^i Aspartic 

Glycine 
X;X;X Glutamic 
III Taurine 



LITHOPHAGA 



LOLIGUNCULA 



FIGURE 5. Estimated concentration of six amino acids in the tissues of pelecypods 
Lithophage bisulcata and the cephalopod LoUguncnla brcvis. 

cycle and their concentration could be easily regulated by the reactions of the cycle. 
/?-alanine was found in some forms but not in others. The only coelenterate which 
we studied had none. It was found in some Mollusca but not in others. The same 
was true in the arthropods studied. The function of /?-alanine is again unknown. 
It exists usually as a moiety of carnosine which is known to exist in the muscle of 
vertebrates and many invertebrates. The problem of formation arises again, /?- 
alanine can be produced by the decarboxylation of aspartic acid, or by the hydrolytic 
breakdown of dihydrouracil. Similarly, /?-ammoisobutyric acid is formed by the 
hydrolytic breakdown of dihydrothymine. We found /?-aminoisobutyric acid in 



FKKE AMINO ACIDS 



379 



small amounts in Volcclla and in larger amounts in Mytilus. A somewhat curious' 
finding is that of asparagine in certain representatives of the Mollusca and Arthrop- 
nda, as shown in Table II. Asparagine is not often found in detectable amounts in 
mammals. 

Arginine was present in all the species studied. It probably resulted from the 
hydrolysis of arginine phosphate, a well-known phosphagen in invertebrates. 

Inasmuch as the taurine concentration appeared to vary with the environment 
we proceeded to study a number of fresh- water forms and also other marine forms 
for their taurine content. The results are shown in Table 111. The presence of 



12-- 



10- 



H| Arginine 

Alanine 
Aspartic 



Glycine 

|^ Alanine :: Glutamic 

Taurine 



14 



E 

0) 



8- 



o 

c. 

'i 4. 

a 




20 




BUNODOSOMA 



THYONE 



LUIDIA 



FIGURE 6. Estimated concentration of six amino acids in the tissues of the coelenterate 
Biitwdosoma cavcniata and the echinoderms Tliyouc sp. and f.nidia chithrnta. 



taurine by chromatography is detectable in concentrations as low as 0.1 micromole 
per gram. Our results indicate that taurine is either absent in the fresh-water 
forms of molluscs or it is present in concentrations lower than 0.1 micromole per 
gram. Every fresh-water and terrestrial mollusc studied showed no taurine 
whereas every form of invertebrate living in salt water or brackish water contained 
taurine in detectable and measurable amounts. 

At this stage of this study, it is not possible to attribute to taurine any definite 
role. But the evidence is suggestive that it plays a very important role as an 
osmoregulator. 



380 JOHN W. SIMPSON, KENNETH ALLEN AND JORGE AWAPARA 



TABLE III 

Tnnrine content in various molluscs 



Mollusca 
Gastropoda 

Lymnaea palustris 
Marisa cornuarietis 
Pomacea bridges! 

Rumina decollata 
Ota I a lac tea 
Mesodon thyroidus 
Bulimulus alternatus 

Murcx fulvcscens 
Littorina irrorata 
Oliva say ana 
Polinices duplicata 
Busy con perversum 
Siphonaria lineolata 
Fasciolaria distans 
Thais haemastoma hay sac 

Pelecypoda 

. 1 nadonta grandis 
Quadrula qnadruln 
Lampsilis sp. 
Elliptio sp. 

Rangia cuneatu 
Brachiodontcs recurvus 
Crassost/ra virginica 

Donax variabilis 
Venus mercenarid 
Dosinia discus 
Area incongrua 
A rca campechiensis 
Noetia ponderosa 

Cephalopoda 

Loliguncula brevis 



Environment 



Taurinc 



Fresh water 
Fresh water 
Fresh water 

Terrestrial 
Terrestrial 
Terrestrial 
Terrestrial 

Marine 
Marine 
Marine 
Marine 
Marine 
Marine 
Marine 
Marine 



Fresh water 
Fresh water 
Fresh water 
Fresh water 

Brackish-Fresh water 
Brackish- Marine 
Brackish-Marine 

Marine 
Marine 
Marine 
Marine 
Marine 
Marine 



Marine 



SUMMARY 

1. Free amino acids of 17 species of aquatic invertebrates were determined 
1>y chromatographic methods. 

2. Qualitative differences in some amino acids were detected in organisms of 
different species. The concentration of alanine, arginine, aspartic acid, glutamic 
acid, glycine and taurine was measured and significant differences recorded. 

3. Taurine was found in high concentration in all the marine organisms 
studied but was not found in several fresh-water and terrestrial organisms. 

4. The possible role of taurine and other free amino acids in aquatic organisms 
is discussed. 



FREE AMINO ACIDS 



LITERATURE CITED 



381 



ACHER, R., AND C. CROCKER, 1952. Reactions colorees specifiques de 1'arginine et de la 

tyrosine realisees ap