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Full text of "The Biological bulletin"

THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 



JOHN M. ANDERSON, Cornell University 
JOHN B. BUCK, National Institutes of Health 
SALLY HUGHES-SCHRADER, Duke University 

LIBBIE H. HYMAN, American Museum of 

Natural History 

SHINYA INOUE, Dartmouth College 

J. LOGAN IRVIN, University of North Carolina 



L. H. KLEINHOLZ, Reed College 

JOHN H. LOCHHEAD, University of Vermont 

ROBERTS RUGH, Columbia University 

WM. RANDOLPH TAYLOR, University of 

Michigan 

ANNA R. WHITING, Oak Ridge National 

Laboratory 

CARROLL M. WILLIAMS, Harvard University 



DONALD P. COSTELLO, University of North Carolina 
Managing Editor 



VOLUME 129 

JULY TO DECEMBER, 1965 




Printed and Issued by 

LANCASTER PRESS, inc. 

PRINCE & LEMON STS. 
LANCASTER, PA. 



11 

IHi BlOLOGK \i I' 1 LLETIN i> issued six times a \ear at llu 
Lancaster Press. Inc.. 1 'rince and Lemon Streets. Lancaster, I'enn 
sylvania. 

Snb.scriptions and similar matter should be addressed to The 
Biological I'.ulletin. Marine I !i( (logical Laboratory. Woods Ilole, 
Massachusetts. Agent for (ireat liritain : \\'heldon and Wesley, 
l.imiled. _'. .> and 4 Arthur Street. Xe\v Oxford Street, London, 
\\ . C. -. Single nnmlier>. S.v75. Subscription per volume (three 
issues . S'.00. 

Coinmunii-ations relative to manuscripts should be sent to Dr. 
Monald \\ C'ostello. Marine lliolog-ical Laboratory, Woods Hole, 
Massachusetts, between June 1 and September 1, and to Dr. 
Donald I 1 . Costello, P.O. Box 429, Chapel Hill, North Carolina. 
during the remainder of the year. 



Second-class postage paid at Lancaster, Pa. 



LANCASTER PRESS, INC., LANCASTER, PA. 



CONTENTS 



No. 1. AUGUST, 1965 

PAGE 

Annual Report of the Marine Biological Laboratory 1 

ARNOLD, JOHN M. 

The inductive role of the yolk epithelium in the development of the squid, 
Loligo pealii ( Lesueuer ) 72 

BROWN, FRANK A., JR., AND YOUNG H. PARK 

Phase-shifting a lunar rhythm in planarians by altering the horizontal mag- 
netic vector 7" 

DEAN, JOHN MARK, AND F. JOHN VERNBERG 

Effects of temperature acclimation on some aspects of carbohydrate me- 
tabolism in decapod Crustacea S7 

FENG, S. Y. 

Pinocytosis of proteins by ovster leucocvtes 95 

GLYNN, PETER W. 

Active movements and other aspects of the biology of Astichopus and 
Leptosynapta (Holothuroidea ) 106 

GOODBODY, I VAX 

The biology of Ascidia nigra (Savigny). III. The annual pattern of 
colonization 129 

JONES, JACK COLVARD, AND RONALD E. WHEELER 

Studies on spermathecal filling in Aedes aegypti (Linnaeus). I. Descrip- 
tion ' 134 

KOZLOFF, EUGENE N. 

New species of acoel turbellarians from the Pacific Coast 151 

LOHAVANIJAYA, PRASERT, AND EMERY F. SwAN 

The separation of post-basicoronal areas from the basicoronal plates in the 
interambulacra of the sand dollar, Echinarachnius parma ( Lamarck i . . . . 167 

MENZEL, R. WINSTON, AND MARGARET Y. MENZEL 

Chromosomes of two species of quahog clams and their hybrids 1S1 

SAKAI, YOSHI T. 

Studies on the ooplasmic segregation in the egg of the fish, Oryzias latipes. 
III. Analysis of the movement of oil droplets during the process of ooplas- 
mic segregation 1 S ( > 

ZEIN-ELDIN, ZOULA P., AND DAVID V. ALDRICII 

Growth and survival of postlarval Penaeus aztecus under controlled con- 
ditions of temperature and salinity 199 

No. 2. OCTOBKK, 1 l >(>5 
BARNES, ROBERT D. 

Tube-building and feeding in chaetoplerid polychartes 217 

CARLSON, ALBERT D. 

Factors affecting firefly larval luminescence 234 



iv CONTENTS 

G WILLIAM, G. F. 

The mechanism of the shadow retle: in Cirripedia. II. Photoreceptor cell 
response, second-order responses, and motor cell output 244 

HALTOX, IX \\".. AND |. I',. JKXXI 

Ohservations on tin- nutrition of monogenetic trematodes 257 

HoRircm, SHIKO. \.\i> CHARLES I-'.. LANE 

Digestive en/yines of the crystalline- style of Strombus gigas Linne. 1. 
Cellulate and some other carbohydrases 273 

JONES. JACK C'OLVAUD 

The heniocUr- of Rhodnius prolixus Stal 282 

KRIVAXKK. JKKOMK (X. AND l\oi;i.\ C. KRIYAXEK 

Evidence for transaminase activity in the slime mold, Dictyostelium dis- 
coideum Kaper 295 

MENDO/A. GUILLERMO 

The ovary and anal processes of "Characodon" eiseni, a viviparous cypri- 
nodont teleost from Mexico 303 

MrscAiixK. LEONARD, AXD HOWARD M. LENIIOFF 

Symbiosis of hydra and algae. II. Effects of limited food and starvation 

on growth of symbiotic and aposymbiotic hydra 316 

I\ IK MANX. |oiix (I. 

The development of eggs of the screw -worm fly Cochliomyia hominivorax 
(Coquerel) (Diptera: Calliphoridae ) to the blastoderm stage as seen in 
whole-mount preparations 329 

SCHELTEMA. RUDOLF S. 

The relationship of salinity to larval survival and development in Nas- 
sarius obsoletus (Gastropoda) 340 

SKINNER, DOROTHY M., DONALD J. MARSH AND JOHN S. COOK 

Physiological salt solution for the land crab, Gecarcinus lateralis 355 

STRAIX, I IAROLD H. 

Chloroplast pigments and the classification of some siphonalean green algae 

of Australia 366 

S-rrxKARi), HORACE W., AXD FRED E. Lux 

A microsporidian infection of the digestive tract of the winter flounder, 
Pseudopleuronectes americanus 371 

Ab>tracts of jiajx-rs jiresented at the Marine Biological Laboratory 388 

\o. 3. DECEMBER, 1965 

AIKU.O, EDWARD. AND GIAXCARLO GIMDERI 

Distribution and function of the branchial nerve in the mussel 431 

AKOV, SIIOSIIAXA 

'ihibition of blood digestion and oocyte growth in Aedes aegypti by 

5-fluorouracil 43 ( ' 

Ax. . JOHN MAXWKU. 

011 visceral regeneration in sea .stars. 111. Regeneration of the 

h in \stcrias forbesi I I )esor I 454 

!'. \IMII. 1 . ( ,. 

The nature oi he action of ions as inductors. 471 



CONTENTS v 

ECKSTEIN, B.. AND M. SPIRA 

Effect of sex hormones on gonadal differentiation in a cichlid, Tilapia 
aurea 482 

Fox, SISTER ALICE MARIE 

Effect of inhibitors on active transport by turtle intestinal segments 490 

GUTKNECHT, JOHN 

Ion distribution and transport in the red marine alga, Gracilaria foliifera 4^5 

GRANT, WILLIAM C, JR., AND GEORGE COOPER. IV 

Behavioral and integumentary changes associated with induced meta- 
morphosis in Diemyctilus 510 

HKXDRICKX, ANDREW G., AND ROBERT HANXLIK 

Developmental stages of the bob-white quail embryo ( Colinus virginianus ) 523 

JONES, JACK COLVARD, AND RONALD E. WHEELER 

Studies on spermathecal Filling in Aedes aegypti (Linnaeus). II. Ex- 
perimental 532 

LOOSANOFF, VICTOR L. 

Gonad development and discharge of spawn in oysters of Long Island 
Sound 546 

MlLLOTT. X., AND W. G. LYNN 

Further studies on the effect of phenylthiourea on pigmentation by me- 
lanin in amphibians 562 

STEPHENS. GROVER C.. JOHN F. VAN PILSUM AND DORRIS TAYLOR 

Phylogeny and the distribution of creatine in invertebrates 573 

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

Interactions of diurnal and tidal rhythms in the fiddler crab. Uca pugnax 582 



August, 1Q65 
Vol. 129, No. 1 

THE 



ERRATA 

Page 16 PHYSIOLOGY 

II. INSTRUCTORS 

delete WILLIAM F. HARRINGTON 

H YEAR 

Page 17- MARINE BOTANY 1 

II. INSTRUCTORS 4 

delete FRANK E. ROUND 

6 

Page 18 INVERTEBRATE ZOOLOGY 

9 

III. ASSISTANTS . . 

delete W. BRUCE HUNTER lents 23 

add STEPHEN C. BROWN, University of Michigan 35 

35 
Page 20- MARINE ECOLOGY . 37 

III. LABORATORY ASSISTANTS 37 

delete MARGARET C. LLOYD 
delete BARRY M. HEATFIELD 

add DONALD C. GORDON, University of Rhode Island 

add CONRAD GEBELEIN, The Johns Hopkins University 64 

65 

Page 34 The following Invertebrate Zoology students should carry the 
asterisk indicating they also completed the Post-Course Research 
Program, July 28-August 31 : 

BENNETT, JUDITH ANN, Syracuse University 

BOLENDER, ROBERT PAUL, Columbia University 

HALL, BARBARA SUE, College of St. Mary of the Springs 

HINE, CHARLES RISK, Lafayette College 

HUNTER, WILLIAM BRUCE, University of California, Santa Barbara Avenue, New 

LANGRETH, SUSAN GRANT, University of Chicago 

NUTT, JOHN GORDON, JR., Rice University 

RUNDLES, CHARLOTTE, Duke University :w York 

WALTERS, DAVID ROYAL, Harvard University ^e of Medicine 

WHISNANT, BETTY LYNN, Duke University 

York 5, New 



Copyright () 



Vol. 129, No. 1 August, 1965 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



THE MARINE BIOLOGICAL LABORATORY 
SIXTY-SEVENTH REPORT, FOR THE YEAR 1964 -SEVENTY-SEVENTH YEAR 

I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 14, 1964) 1 

II. ACT OF INCORPORATION 4 

III. BYLAWS OF THE CORPORATION 4 

IV. REPORT OF THE DIRECTOR 6 

Addenda : 

1 . Memorials 9 

2. The Staff 14 

3. Investigators, Lalor, Lillie and Grass Fellows, and Students . . 23 

4. Fellowships and Scholarships 35 

5. Training Programs 35 

6. Tabular View of Attendance, 1960-1964 37 

7. Institutions Represented 37 

8. Evening Lectures 39 

9. Evening Seminars 40 

10. Members of the Corporation 41 

V. REPORT OF THE LIBRARIAN 64 

VI. REPORT OF THE TREASURER . 65 



I. TRUSTEES 

GERARD SWOPE, TR., Chairman of the Board of Trustees, 570 Lexington Avenue, New 

York 22, New York 

ARTHUR K. PARPART, President of the Corporation, Princeton University 
JAMES H. WICKERSHAM, Treasurer, 791 Park Avenue, New York 21, New York 
PHILIP B. ARMSTRONG, Director, State University of New York, College of Medicine 

at Syracuse 
ALEXANDER T. DAIGNAULT, Assistant Treasurer, 7 Hanover Street, New York 5, New 

York 
GEORGE W. DE VILLAFRANCA, Clerk of the Corporation, Smith College 

1 
Copyright 1965, by the Marine Biological Laboratory 



MARINE BIOLOGICAL LABORATORY 

EMERITI 

\\~ILLIAM R. AMBERSON, Marine Biological Laboratory 

C. LALOR BURDICK, The Lalor Foundation 

C. LLOYD CLAFF, Randolph, Massachusetts 

W. C. CURTIS, 504 West Mount Avenue, Columbia, Missouri 

PAUL S. GALTSOFF, Woods Hole, Massachusetts 

E. B. HARVEY, Woods Hole, Massachusetts 

M. H. JACOBS, University of Pennsylvania 

CHARLES W. METZ, Woods Hole, Massachusetts 

CHARLES PACKARD, Woods Hole, Massachusetts 

A. C. REDFIELD, Woods Hole, Massachusetts 

CARL C. SPEIDEL, Randolph-Macon Woman's College 

A. H. STURTEVANT, California Institute of Technology 

ALBERT SZENT-GYORGYI, Marine Biological Laboratory 

TO SERVE UNTIL 1968 

E. G. BUTLER, Princeton University 
A. C. CLEMENT, Emory University 
ARTHUR L. COLWIN, Queens College 

DONALD P. COSTELLO, University of North Carolina 

JAMES D. EBERT, Carnegie Institution of Washington 

DOUGLAS A. MARSLAND, Marine Biological Laboratory 

ROBERTS RUGH, Columbia University, College of Physicians and Surgeons 

H. BURR STEINBACH, University of Chicago 

TO SERVE UNTIL 1967 

LESTER G. BARTH, Columbia University 

JOHN B. BUCK, National Institutes of Health 

AURIN M. CHASE, Princeton University 

SEYMOUR S. COHEN, University of Pennsylvania, School of Medicine 

TERU HAYASHI, Columbia University 

LEWIS KLEIN HOLZ, Reed College 

ALBERT I. LANSING, University of Pittsburgh 

S. MERYL ROSE, Tulane University 

TO SERVE UNTIL 1966 

F. A. BROWN, JR., Northwestern University 

F. D. CARLSON, The Johns Hopkins University 

SEARS CROWELL, Indiana University 

W. D. McELROY, The Johns Hopkins University 

C. LADD PROSSER, University of Illinois 

E. A. SCHARRER, Albert Einstein College of Medicine 
SISTER FLORENCE MARIE SCOTT, Seton Hill College 
WILLIAM RANDOLPH TAYLOR, University of Michigan 

TO SERVE UNTIL 1965 

ERIC G. BALL, Harvard Medical School 

D. W. BRONK, The Rockefeller Institute 
MAC V. EDDS, JR., Brown University 
RUDOLF KEMPTON, Vassar College 

I. M. KLOTZ, Northwestern University 

ARNOLD LAZAROW, University of Minnesota Medical School 

MORRIS ROCKSTEIN, University of Miami 

GEORGE WALD, Harvard University 



TRUSTEES 

STANDING COMMITTEES 
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES 

ARTHUR K. PARPART, ex officio, Chairman H. BURR STEINBACH, 1967 

GERARD SWOPE, JR., ex officio TERU HAYASHI, 1966 

JAMES H. WICKERSHAM, ex officio WILLIAM D. MCELROY, 1966 

PHILIP B. ARMSTRONG, ex officio ERIC G. BALL, 1965 

E. G. BUTLER, 1967 SEARS CROWELL, 1965 

THE LIBRARY COMMITTEE 

KEITH PORTER, Chairman C. LADD PROSSER 

ERIC G. BALL MORDECAI GABRIEL 

JAMES LASH STANLEY WATSON 
SEYMOUR S. COHEN 

THE APPARATUS COMMITTEE 

ALBERT I. LANSING, Chairman ARNOLD LAZAROW 

CLIFFORD HARDING WILLIAM D. MCELROY 

DAVID POTTER L. I. REBHUN 

THE SUPPLY DEPARTMENT COMMITTEE 

RUDOLF KEMPTON, Chairman SEARS CROWELL 

W. J. ADELMAN WALTER HERNDON 

HARRY GRUNDFEST FRANK M. FISHER 

HOWARD A. SCHNEIDERMAN GEORGE SCOTT 

THE INSTRUCTION COMMITTEE 

TERU HAYASHI, Chairman MAIMON NASATIR 

A. C. CLEMENT BOSTWICK KETCHUM 

LEWIS KLEINHOLZ DE\VITT STETTEN 
ROGER O. ECKERT 

THE BUILDINGS AND GROUNDS COMMITTEE 

EDGAR ZWILLING, Chairman J. WOODLAND HASTINGS 

E. G. BUTLER J. W. GREEN 

DANIEL GROSCH MELVIN SPIEGEL 
MAC V. EDDS, JR. 

THE RADIATION COMMITTEE 

P. M. FAILLA, Chairman H. BURR STEINBACH 

S. J. COOPERSTEIN ROBERTS RUGH 

DAVID SHEMIN GEORGE SZABO 
PAUL R. GROSS 

THE RESEARCH SPACE COMMITTEE 

WILLIAM D. MCLROY, Chairman H. BURR STEINBACH 

TERU HAYASHI EDGAR ZWILLING 

THE COMMITTEE FOR NOMINATION OF OFFICERS 

SEARS CROWELL, Chairman E. G. BUTLER 

ERIC G. BALL TERU HAYASHI 

WILLIAM D. MCLROY H. BURR STEINBACH 



4 MARINE BIOLOGICAL I. AM( >K ATORY 

II. ACT OF INCORPORATION 
No. 31/0 

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 1). 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 ; 

Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- 
setts, do hercb\ 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. 

U' it ness 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 Commomvcalth. 



III. BYLAWS OF THF CORPORATION OF THE MARINE 
BIOLOGICAL LABORATORY 

(Revised August 15, 1963) 

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 Chairman of the Board of 
Trustees, President, Director, Treasurer and Clerk. 

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

Y. 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 bylaws, no notice of the Annual Meeting need be given. Notice of any special 
meeting of members, howi-ver, 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. 



BYLAWS OF THE CORPORATION 5 

VII. The Annual Meeting of the Trustees shall be held promptly after the Annual 
Meeting of the Corporation at the Laboratory in Woods Hole, Massachusetts. Special 
meetings of the Trustees shall be called by the Chairman, 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 Chairman, the President, the 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 
continue to serve as Trustee until the next Annual Meeting of the Corporation, where- 
upon his office as regular Trustee shall become vacant and be filled by election by the 
Corporation and he shall become eligible for election as Trustee Emeritus for life. The 
Trustees ex officio and Emeriti shall have all the rights of the Trustees, except that 
Trustees Emeriti 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 Chairman of the Board of Trustees who shall be elected 
annually and shall serve until his successor is selected and qualified and who shall also 
preside at meetings of the Corporation. They shall elect a President of the Corporation 
who shall also be the Vice Chairman of the Board of Trustees and Vice Chairman of 
meetings of the Corporation, and who shall be elected for a term of five years and shall 
serve until his successor is selected and qualified, except that such term shall not run 
beyond the Annual Meeting of the Board following his 65th birthday ; candidates over the 
age of 65 shall be elected on an annual basis. They shall appoint a Director of the 
Laboratory for a term not to exceed five years, provided the term shall not exceed 
one year if the candidate has attained the age of 65 years prior to the date of the appoint- 
ment. They may choose such other officers and agents as they may think best. They 
may fix the compensation and define the duties of all the officers and agents ; and may 
remove them, or any of them except those chosen by the members, at any time. They 
may fill vacancies occurring in any manner in their own number or in any of the 
offices. The Board of Trustees shall have the power to choose an Executive Committee 
from their own number, and to delegate to such Committee such of their own powers 
as they may deem expedient. They shall from time to time elect members to the 
Corporation upon such terms and conditions as they may think best. 

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

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



o MAK1XK BIOLOGICAL LABORATORY 

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

X I II. These bylaws may be altered at any meeting of the Trustees, provided that the 
notice of such meeting shall state that an alteration of the bylaws will be acted upon. 

RESOLUTIONS ADOPTED AT TRUSTEE MEETING AUGUST 16, 

1963 K\ KCUTIVE COMMITTEE 

I. RESOLVED: 

(A) The Executive Committee is hereby designated to consist of ten members as 
follows: e.v officio members who shall be the Chairman of the Board of Trustees, 
President, Director and Treasurer; six additional Trustees, two of whom shall be 
elected by the Board of Trustees each year, to serve for a three-year term. 

(B) The President shall act as Chairman of the Executive Committee and the 
Chairman of the Board of Trustees as Vice Chairman. A majority of the members 
of the Executive Committee shall constitute a quorum and a majority of those present 
at any properly held meeting shall determine its action. It shall meet at such times 
and places and upon such notice and appoint such sub-committees as the Committee 
shall determine. 

(C) The Executive Committee shall have and may exercise all the powers of the 
Board during the intervals between meetings of the Board of Trustees except those 
powers specifically withheld from time to time by the Board or by Law. 

(D) The Executive Committee shall keep appropriate minutes of its meetings, and 
its actions shall be reported to the Board of Trustees. 

II. RESOLVED: 

The elected members of the Executive Committee shall be constituted as a standing 
"Committee for the Nominations of Officers," responsible for making nominations at 
the annual meeting of the Corporation and of the Board of Trustees, for candidates to 
fill each office as the respective terms of office expire. (Chairman of the Board, Presi- 
dent, Director, Treasurer, and Clerk.) 



IV. REPORT OF THE DIRECTOR 

To: Tin-. TRUSTEES OF THE MARINE BIOI.OCICAI. LABORATORY 
Gentlemen : 

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

1. Facilities Developments 

During the past year a number of changes and developments in our facilities 
were completed under a grant provided by the National Science Foundation. Over 
the winter of 1963-64 the library stacks were repainted and fluorescent lighting 
installed which brightened up the stacks most satisfactorily. The glass floors in 
the stacks were overlaid with plywood, on top of which rubber tile was installed, 
getting away from the hazard of breaking of the glass flooring and also giving a 



REPORT OF THE DIRECTOR / 

quiet walking surface. New quarters were provided for the library staff, directly 
off the card catalogue room. The reading room has been extended to include the 
area formerly occupied by the library staff, providing additional space for reference 
material as well as additional reading space. 

A centralized instrument laboratory was provided by remodeling rooms 109 
and 110 in the Lillie Building where large pieces of apparatus for the general use 
of investigators and students can be supervised by a qualified technical assistant. 
A suite of laboratories centrally located on the second floor of the Lillie Building 
has been remodeled for electron microscopy. Included is a general service labora- 
tory, facilities for two electron microscopists, and darkrooms for loading and devel- 
oping film. These laboratories and instruments are under the supervision of an 
expert electron microscopist. 

Also, under the National Science Foundation Grant a new collecting boat, the 
Ciona, was constructed, a 40-foot vessel powered by 180-hp. General Motors diesel 
engine. This boat has proved to be exceptionally seaworthy and well adapted to 
the Laboratory's service. The vessel was built by the Brownell Boatyard of Matta- 
poisett, after a design by Eldredge-Mclnnis, naval architects. 

The old Cayadetta dock, extending 140 feet out into Great Harbor in front of 
the Laboratory, was rebuilt and was used daily throughout the summer by the 
Cap'n Bill III, which unloaded its catch of bottom fish and squid directly into 
mobile sea water tanks on the dock. These facilities provided by the National 
Science Foundation Grant have contributed significantly to improved operations 
in these areas. In addition the Laboratory made important modification of two 
of the boats to better adapt them for biological collecting. The cruising speed of 
the Limulus has been stepped up from 10 to 16 knots, extending her range. Also, 
a new mast and boom, operating off of a motor driven winch, has improved the 
operation significantly. Extensive modifications in the interior arrangements of 
the Lhinihis and Dolphin have contributed to the ease, effectiveness, and safety in 
the operation of these two boats. 

2. Ford Foundation Grant 

In 1963 the Planning Committee was instructed by the Executive Committee 
to explore ways of funding its long-range plans which had previously been accepted 
by the Executive Committee. This plan included additional housing for investi- 
gators and students, new laboratory facilities for the courses and a laboratory- 
equipped survey boat for the Systematics-Ecology Program. The needs of the 
Laboratory were spelled out in detail and a request for a grant of $2,500,000 toward 
this program was made to The Ford Foundation. The plan envisions an instruc- 
tional building which will provide quarters for the regular courses and the Syste- 
matics-Ecology Program, a building of 65,000 square feet to be built at an estimated 
cost of $2,700,000. Also included in the program is a dining hall-dormitory to 
replace the present dining hall and the old residences currently used to house stu- 
dents. Included in the dormitory will be 125 double bedrooms. The estimated 
cost of this facility is $1,700,000. Also in the grant request was $200,000 for addi- 
tional cottages in the Devil's Lane tract and $100,000 for the Systematics-Ecology 
biological survey boat. 



S M \RL\l-; BIOLOGICAL LABORATORY 

\Ye were delighted to receive notification in June of a grant of $2,500,000 by 
The Ford Foundation to the Laboratory. Of this, $300,000 is an outright grant 
in the amounts requested for additional cottages, particularly for younger investi- 
gators, and also funds for the Systematics-Ecology survey vessel. The remainder 
of the grant, $2,200,000. covers half the estimated cost of the instructional building 
and the dining hall-dormitory, so matching funds must be obtained in a like amount. 

A topographical survey of the free area of the Devil's Lane Tract was imme- 
diately made, access roads laid out and construction of cottages promptly started. 
Some additional funds were obtained from individual subscribers, so that 24 cot- 
tages have been completed and will be ready for summer occupancy in 1965. The 
contributors included Mrs. F. Newton Harvey, Mrs. Gary N. Calkins, Mrs. Samuel 
O. Mast and The Gra.ss Foundation. Also, an extensive play area, readily accessi- 
ble to both cottage colonies, has been laid out, providing recreation facilities for 
children of all ages as well as adults. 

Plans for the new Systematics-Ecology survey vessel have been completed and 
construction will soon be started. This vessel will be 65 feet long, of steel con- 
struction and equipped with the most modern equipment and gear for survey work. 

Plans are being developed by the firm of Pierce, Pierce and Luykx for the 
instruction building and the dining hall-dormitory. The instruction building will 
be located on the north side of Center Street, east of the Apartment House. The 
site for the dining hall-dormitory has not yet been selected. In the meantime the 
Planning Committee is exploring various sources of additional funds to match the 
grant from The Ford Foundation toward these two facilities. 

3. Ricliard K. Mellon Foundation Grout 

The Laboratory \vas most fortunate in receiving a grant of $50,000 from the 
Richard K. Mellon Foundation, toward the cost of construction of the instruction 
building. "\Ye were very much gratified by this support from one of our Woods 
Hole neighbors. 

4. Rand Bequest 

The Laboratory was the beneficiary of a bequest this past year by the will of 
Mrs. Herbert \Y. Rand, in memory of her husband. Professor Herbert W. Rand, 
of Harvard University. Professor Rand first came to the Marine Biological Lab- 
oratory in 1923, and became a member of the Corporation in 1928. He resided in 
Falmouth after his retirement at Harvard in 1942 until his death. 

5. Personnel 

Deborah Lawrence Harlow was a member of the Library staff at the Marine 
I'.iological Laboratory for 40 years, retiring at the end of 1964. She joined the 
staff in 1925 as secretary to the Librarian. Mrs. Harlow became thoroughly 
acquainted with the operation of the Library over a period of years and succeeded 
Mrs. Montgomery as Librarian in 1948. During her tenure as Librarian, the 
number of journals to which the Laboratory subscribed increased from 1200 to 
1717, the number of volumes in the Library from 56,000 to well over 100,000. 
Although these are striking increases in numbers, Mrs. Harlow will always be re- 



REPORT OF THE DIRECTOR 9 

membered for her very effective management of the Library, for her cooperative- 
ness and for the relaxed manner in which she furthered the library work of the 
scientists and students at the Laboratory. Mr. Harlow, for 17 years head of the 
machine shop, and expert in his field, retired at the same time. 

1. MEMORIALS 

WINTHROP JOHN VANLEUVEN OSTERHOUT 
By THEODORE SHEDLOVSKY 

On August 2. 1871. Winthrop John Vanleuven Osterhout was born in Brooklyn, 
New York, the son of a Baptist minister whose ancestors came to America in the 
seventeenth century from the town of Osterhout in Holland. On April 9th of this year, 
Dr. Osterhout died in New York in his ninety-third year. In accordance with his 
wishes his ashes are buried in the cemetery of the Church of St. James the Less in 
Philadelphia, among four descendants of Benjamin Franklin, an ancestor of his widow, 
Marian Invin Osterhout. Many of us who knew him personally, and the Marine Bio- 
logical Laboratory, where he spent well over half the summers of bis long life, mourn 
his death. It seems to mark the passing of an important period in the history of bio- 
logical science, a period which bridged the nineteenth and twentieth centuries. At Woods 
Hole, as elsewhere, quantitative experimentation and important new ideas were supple- 
menting or displacing the traditional descriptive methods of research in biology. 

Here, at the Marine Biological Laboratory, a number of dedicated biologists, who 
were already eminent or were soon to become so, carried on their researches during 
the summer and influenced students an enterprise which is happily continuing. Among 
these dedicated biologists we find the names of Jacques Loeb, T. H. Morgan, Frank and 
Ralph Lillie, E. B. Wilson, E. G. Conklin, Walter Carrey, A. P. Mathews, Ivey Lewis, 
and, of course, W. J. V. Osterhout. Let us examine briefly his history as a man of 
science. 

While still an undergraduate student at Brown University, in 1892 young Osterhout 
came to the Marine Biological Laboratory, where W. A. Setchell introduced him to 
research in botany. He started work and soon found that the four spores in a red alga, 
Aquardhiclla tenero, each of which could produce a new plant, were able to combine and 
form a single plant. A year later, Osterhout had received the A.B. degree from Brown 
(I believe he was the class poet) and was again at Woods Hole, but now as Setchell's 
assistant in the Botany Course. Together, while collecting in Nobska Pond, they found 
Xitclla, but physiological experiments on this interesting material came only consider- 
ably later. 

After a year in Bonn, Germany, with the eminent plant cytologist, Eduard Stras- 
burger (1895-6) Setchell brought Osterhout to the University of California where he 
earned the Ph.D. degree in 1899 and met Jacques Loeb in 1902. Learning of Loeb's 
work on animal cells he began to make similar studies on plant cells and did so with 
considerable success. Among other things, he was much interested in Loeb's observa- 
tions on ionic antagonism, such as exists between monovalent and divalent or trivalent 
cations, and he used effectively the measurement of electrical conductance in such experi- 
ments with plant cells. Acquaintance with Loeb soon ripened into a great, life-long 
friendship. While still in California, Osterhout got to know Hugo de Vries and Svante 
Arrhenius. There, in those early years of the century, he doubtless participated in many 
discussions of matters scientific, philosophic, as well as honestly convivial. 

In 1909, Osterhout left the University of California as Associate Professor and 
moved to Harvard as Assistant Professor, to become Professor in 1913. When Loeb, 



10 M. \RI.\K BIOLOGICAL LABORATORY 

who was a member of The Rockefeller Institute for Medical Research, died in 1924, 
Dr. Osterhout was invited by the Director, Dr. Flexner, to accept membership in the 
Institute. This invitation he accepted. At the Institute he was given a substantial 
department of general physiology and a small laboratory in Bermuda for work on Falonia 
and Halicystis. In -\Y\v York he was joiiu-d by Drs. Marian Irwin, Lawrence Blinks, 
and, a little later, by S. E. Hill, W. Stanley and several others. Interested, as he always 
was, in a physico-chemical approach to biological problems he arranged for D. A. Mac- 
Innes to form a physical-chemical group, affiliated with his department. L. G. Longs- 
worth and I soon joined that group. 

After his return east from California, and while he was still at Harvard, Osterhout 
again became intimately associated with the Marine Biological Laboratory and remained 
so until just a few years ago. He had been a Trustee since 1910. Those of us who 
had the privilege of knowing him here at Woods Hole, in Cambridge, or in New York, 
will fondly recall scientific discussions with him, which often took the form of Socratic 
dialogues, general conversations which were seldom trivial and were usually well sea- 
soned with wit and wisdom. We remember him as a gentleman, in the best and most 
accurate sense of the word, always with dignity but never with pomp or without a subtle 
warmth. We shall miss him; not only the scientist, botanist, physiologist, but also the 
mentor, the councillor, the friend. I speak not only for myself but also, I am certain, 
for many others. 

Osterhout was a superb teacher. Although I did not have the good fortune of being 
one of his students at Harvard, I know that his influence in attracting young people to 
research in biology was great. He had a gift for devising beautiful experimental demon- 
strations which were presented with a persuasive but dignified enthusiasm for the subject 
that inspired many of his graduate students to undertake productive careers in research. 

\\hat were his main contributions to science? Here in the Marine Biological Lab- 
oratory Library, there are about 270 cards in the W. J. V. Osterhout file. These in- 
clude references to his early work in cytology, salt antagonism, osmotic studies and 
other physico-chemical aspects of plant cells and plant cell models. Perhaps his principal 
work was on permeability aspects and electrical properties of single plant cells. He was 
very early in accounting for the active transport of ions by a molecular carrier mecha- 
nism. To show this he constructed cell models which exhibited active transport with 
carrier molecules passing through non-aqueous membranes. For example, aqueous tri- 
choloracetic acid and pure water, separated by a layer of guaiacol in the bottom of a 
U-tube, showed the water apparently moving against its chemical potential gradient. 
Water movement and water absorption interested him greatly and formed one of the 
subjects of his work with Mrs. Osterhout into the evening of his scientific life. 

IVginning with his early experiments in California on the relation of electrical 
conductivity of plant cells and ionic antagonisms, the bio-electric phenomena in living 
cells had always held his active interest. This traditionally controversial field has been 
so from the time of Volta and Galvani, through the period of phase boundary potentials 
'crsiis diffusion potentials, and even remains so today in the present era of biochemical 
and biophysical euphoria. Such a field is not an easy one to explore, but as Osterhout 
said of Loeb, "He did not select problems because they were easy, but because of their 
importance. His courage sprung largely from his faith in the materialistic concep- 
tion. . . ." 

While at Harvard, his extensive electrical studies on Lcnninuria led in 1922 to the 
book, "injury, Recovery and Death in Relation to Conductivity and Permeability" This 
book stimulated other investigators by its novelty of concept and method of interpretation 
involving consecutive reactions. Throughout his life, Osterhout stimulated biologists 
to engage in meaningful quantitative experiments, and physical chemists and physicists 
to consider the problems presented by the living cell. 



REPORT OF THE DIRECTOR H 

Questions of photosynthesis, respiration, oxidation and related topics had received 
attention in his publications. In particular, mention may be made of his demonstration 
of photo induction through a striking observation with A. R. Haas (1918) in which 
lie noted that when the marine plant, Viva, was transferred from darkness to light the 
rate of photosynthesis was increased. 

I have already mentioned the important concept of carrier molecules, so much invoked 
today. It should also be noted that Osterhout pioneered in the concept of the steady- 
state as against equilibrium in accounting for the kinetics of penetration of substances 
into living cells, and, of course, no self-respecting student of molecular biology today 
will deny at least some knowledge of irreversible thermodynamics. But Osterhout's 
influence in general physiology was even greater than the sum of his papers and of his 
personal contacts with other investigators and students. I refer to the Journal of Gen- 
eral Physiology. He was, with Jacques Loeb, co-editor of this journal from its begin- 
ning. Let me quote from his own words in the "Outline of the History of the Journal 
of General Physiology," written in 1955 : "Dr. Jacques Loeb and I realized the need for 
a journal to promote the study of general physiology. Dr. Flexner agreed to publish 
the Journal from The Rockefeller Institute for Medical Research beginning in 1918. 
Dr. Loeb and I were the sole editors until he died in 1924. The statement, 'Founded by 
Jacques Loeb' was placed on the cover of the Journal and a memorial volume was pub- 
lished in his honor. Dr. John H. Northrop and Dr. William J. Crozier joined the 
editorial board in 1924 after Loeb's death. For about twenty-two years Dr. Northrop, 
Dr. Crozier and I were the only editors. In 1946 Dr. Wallace O. Fenn joined us. For 
about thirty-seven years each editor read every paper submitted." 

We shall miss Dr. Osterhout. But, for many of us, memory will often be refreshed 
when we see the Journal of General Physiology, when we visit the Marine Biological 
Laboratory, when we think of Nitella, Valonia, Halicystis, Laminaria, or when we 
recall the wit and wisdom which so often emanated from him to inspire us in so 
many ways. 

ELIOT R. CLARK 
By SEARS CROWELL 

Dr. Eliot R. Clark was born November 13, 1881, in Shelburne, Massachusetts. He 
received his A.B. degree from Yale University in 1903, and the M.D. degree from The 
Johns Hopkins University Medical School in 1907. He was on the staff of the Depart- 
ment of Anatomy at The Johns Hopkins University and carried on postdoctoral studies 
at the Universities of Munich and Krakow. From 1914 to 1922 he was a professor at 
the University of Missouri, and from 1922 to 1926 at the University of Georgia. In 
1926 he became Head of the Department of Anatomy at the University of Pennsylvania, 
a post which he held until 1947. He became Professor Emeritus in 1950 and a guest 
investigator at the Wistar Institute. He received an honorary Sc.D. from Washington 
and Jefferson College in 1940. During the past seven years a serious heart condition 
prevented much physical activity but he retained an alert mind and lively interest in the 
affairs of Woods Hole. His death came instantly on November 1, 1963 in his home in 
Philadelphia. 

His association with the Marine Biological Laboratory began in 1909 when he 
became a member of the Corporation and also met Eleanor Linton, who was to become 
his wife two years later. He served as a Trustee of the Laboratory from 1930 to 1946. 

Mrs. Clark's father was Dr. Edwin Linton, a distinguished parasitologist, who worked 
at the Bureau of Fisheries. Dr. and Mrs. Linton devoted much time and attention to 
the Marine Biological Laboratory Club and the Clarks carried on this tradition. Dr. 
Clark served as Secretary-Treasurer in 1918 and 1919, and later as President. Both of 



12 MARINE BIOLOGICAL I. \I5OKATOKV 

the Clarks were active in various fund-raising affairs for the Marine Biological Lab- 
oratory Club and Tennis Club. 

In the Wood> I loir community tin- (.'larks were involved with the choral group and 
in support of the Penzance 1 'layers. Later, as their children became sailing enthu- 
siasts, the Clarks contributed much to the Woods Hole Yacht Club. Dr. Clark was 
acting Commodore during World War II, and Commodore from 1947 to 1950. 

Apart from his connections with Woods Hole, Dr. Clark was most active profes- 
sionally in the American Association of Anatomists, serving as their Secretary-Treasurer 
for the periods of 1938-1942 and 1943-1946. He was largely instrumental in building 
up the excellent collection of research motion pictures, now housed in the Wistar Insti- 
tute, lie served for many years as Chairman of the Committee on Motion Pictures of 
the American Association of Anatomists. He reviewed films submitted by investigators 
and wrote brief descriptions of each accepted. He compiled the lists of these for pub- 
lication in the Anatomical Record. 

The majority of Eliot Clark's papers, many of them with Mrs. Clark as co-author, 
deal with problems of the circulatory system. Although he regarded himself as an 
anatomist, much of his work is developmental and physiological. By about 1930 he 
and his associates had perfected the technique of implanting windows in the ears of 
rabbits. With this technique he was able to study microscopically the development of 
blood vessels, lymphatics, nerves, epidermis, and various implanted tissues. Older 
workers at the Marine Biological Laboratory will recall with pleasure the demonstra- 
tions presented by the Clarks at the scientific meetings of the Laboratory. 

Dr. Clark's careful attention to detail, thoroughness, and devotion characterized both 
his scientific work and his services to the scientific community, the Marine Biological 
Laboratory, and Woods Hole. 

FRANK PATTENGILL KNOWLTON 
By WALTER S. ROOT 

Frank P(attengill) Knowlton, the son of Charles Fox and Mary (Pattengill) Knowl- 
ton, was born in Hollard Patent, New York, on June 17, 1875. He received the A.B. 
degree from Hamilton College in 1896 and the M.A. degree from the University of 
Michigan in 1897. FVom 1897-1900 he was an Instructor in Physiology and Embryol- 
ogy at the College of Medicine, Syracuse University. During this period he also 
studied medicine, receiving the M.D. degree in 1900. He served successively at Syra- 
cuse as Lecturer in Physiology, 1900-1906; Associate Professor, 1906-1908; Professor 
1908-1946; and Emeritus Professor of Physiology since 1946. As student and teacher 
he spent 49 years at Syracuse. I remember his remarking one day as he surveyed a 
new class that teaching the sons of former students can be taken in one's stride, but 
that when the grandsons appear, one feels older. 

Dr. Knowlton spent the years of 1911-1912 at Cambridge University and at Uni- 
versity College, London. The studies carried out at this time were concerned with 
the relation of colloids to diuresis, the effects of stimulating the 8th and 9th spinal nerve 
roots upon the toad bladder, the sugar consumption of the isolated mammalian heart, 
the sugar consumption of the normal and diabetic heart, and the nature of pancreatic 
diabetes. They were published in four papers in the Journal of Physiology, one in the 
Proceedings of the Royal Society and one in the Zcitsclirijt fiir Physiologic. Of this 
work, perhaps the most important was the development and use of the heart-lung prep- 
aration in collaboration with the great physiologist, Ernest II. Starling. Knowlton was 
fortunate in having the opportunity of working in England at a time when an impressive 
group of men were active. Among these may be mentioned F. Gowland Hopkins, John 
Scott Ilaldane, Charles Sherrington, Joseph Barcroft, John Newport Langley, T. R. 



REPORT OF THE DIRECTOR 13 

Elliott, William Bayliss, Walter Fletcher, E. A. Sharpey-Schafer, James Mackenzie, 
Henry Head, Thomas Lewis and others. 

Knowlton maintained a continuing interest in renal physiology, carbohydrate metabo- 
lism and diabetes mcllitus. In the latter part of his professional life he was active in 
studying the physiology of inhibition. The published papers on this subject are con- 
cerned with reciprocal inhibition in the earthworm, peripheral inhibition in arthropods, 
peripheral neuromuscular augmentation in the heart of Limulus polyphemus, inhibition 
in the cardiac ganglia of Limulus, the dual control of crustacean muscle, and inhibition 
of the turtle heart, and of turtle atria. 

Many of the publications in the field of comparative physiology were carried out in 
the Marine Biological Laboratory of which he was a Trustee from 1932 to 1946, and a 
Trustee Emeritus since 1946. As in his English experience, Knowlton was active in 
Woods Hole at a time when the Laboratory housed many distinguished scientists. 

In 1902 Dr. Knowlton married Clara Avis Roberts. It was a congenial partnership, 
and their home was always a pleasant place to visit. They had one child, a daughter, 
Catherine Morilla (now Mrs. Lucius Foote), who like her father is a medical graduate 
of Syracuse. A grandson, Knowlton Foote, is currently a graduate student in Bio- 
chemistry at the Syracuse Medical Center. 

Dr. Knowlton was a member of the Physiological Society of Great Britain, and the 
American Physiological Society, the annual meeting of which he attended regularly. 
He was active in the Western New York Section of the Society for Experimental 
Biology and Medicine for many years. He was also a member of a number of social 
and honorary societies (Delta Upsilon, Nu Sigma Nu, Sigma Xi, Alpha Omega Alpha, 
Phi Kappa Phi). 

Dr. Knowlton was a modest man. He was always a kind and considerate companion. 
A mutual friend wrote to me recently that he had died on October 30, 1963, without 
pain or suffering. I liked the statement that his end was much as his life had been, 
peaceful and orderly. 

IVEY FOREMAN LEWIS 
By CARL C. SPEIDEL 

Ivey Foreman Lewis was born on August 31, 1882, in Raleigh, North Carolina. 
Eighty-one years later he died in Charlottesville, Virginia, on March 16, 1964, after 
having been associated with the University of Virginia for nearly 50 years. He is 
survived by a son, Ivey Foreman Lewis, Jr., of Hampton, and a daughter, Margaret 
Elliott Lewis of Charlottesville. 

Ivey Lewis graduated from the LTniversity of North Carolina, receiving the degrees 
of A.B. in 1902 and M.S. in 1903. He was awarded the Ph.D. degree by The Johns 
Hopkins University in 1908. For his published dissertation, entitled "The Life History 
of GriffitJisia bornetiana" he was given the Walker Prize by the Boston Society of 
Natural History. He studied abroad in 1908 at the University of Bonn and at the Naples 
Zoological Station. During the academic year 1905-1906 and again after returning from 
Naples, he was Professor of Biology at Randolph-Macon College at Ashland, Virginia. 
During this period he made the acquaintance of Margaret Hunter, of Ashland, who 
became his wife in 1909. Three years later he left Randolph-Macon College to be 
Assistant Professor of Botany at the University of Wisconsin. In 1914 he was made 
Professor of Botany at the University of Missouri. The following year he was ap- 
pointed Miller Professor of Biology and Agriculture at the University of Virginia, 
where he served as Professor, and also as Dean from 1934 on, until his retirement in 
1953. 

For many years Dr. Lewis was associated with the Woods Hole Marine Biological 
Laboratory. He was an Instructor in Botany in 1907 and again from 1910 through 



14 MARINE BIOLOGICAL LABORATORY 

1917. From 1918 through 1927 he was in charge of botanical instruction, and also 
served as Trustee and member of the Executive Committee. In 1928 he was a Carnegie 
Fellow at the Dry Tortugas Laboratory and in 1929 a Professor at the Hopkins Marine 
Station of Stanford University. From 1933 until 1946 he was Director of the Uni- 
versity of Virginia's newly established summer station at Mountain Lake, Virginia. 

Dr. Lewis founded the Association of Virginia Biologists in 1920. This gave rise 
to the Virginia Academy of Science in 1923. The following year he was elected its 
first President. For eight years he was a member of the Division of Biology and Agri- 
culture of the National Research Council and served as Chairman from 1933 through 
1936. He was President of the American Society of Naturalists (1939), President of 
the American Biological Society (1942), and President of the Botanical Society of 
America (1949). 

In 1947 he received an honorary degree of Doctor of Science from the University 
of North Carolina. In 1959, six years after his retirement from active duty, he was the 
recipient of the University of Virginia's Thomas Jefferson Award. 

Dr. Lewis's research interests were primarily in the field of algology. In addition 
to his prize-winning doctorate thesis cited above, his publications include papers dealing 
with the algae of the Woods Hole and Charlottesville regions, the vegetation of Shackle- 
ford Bank, North Carolina, and the pollen of the Dismal Swamp of Virginia and North 
Carolina. 

One of his papers, entitled "The Flora of Penikese, Fifty Years After," published 
in Rhodora in 1924, is of historic interest to the Marine Biological Laboratory. This 
is a survey similar to one made in 1873 by David Starr Jordan, a member of Agassiz's 
Laboratory which was located on Penikese Island. Jordan's results were published in 
the American Naturalist in 1874. Dr. Lewis served as editor of the 1924 survey which 
was made cooperatively by the students and staff of the course in Botany at the Marine 
Biological Laboratory. Agassiz's Laboratory on Penikese is regarded as a kind of fore- 
runner of the Woods Hole Marine Biological Laboratory. 

For recreation Dr. Lewis greatly enjoyed the numerous collecting trips made as a 
part of the course in Botany. He also enjoyed the game of tennis and was an enthu- 
siastic and proficient player. His figure was a familiar one on the Mess Hall court. 
He always regretted that his duties in connection with the Mountain Lake Station in 
Virginia made it necessary for him to terminate his regular summer attendance at 
Woods Hole. 

Ivey Lewis's influence on the Marine Biological Laboratory was a beneficent one. 
In the most literal sense of the expression, he was a gentleman and a scholar. Those 
of us who knew him well miss him greatly. 

2. THE STAFF 
EMBRYOLOGY 

I. INSTRUCTORS 

JAMES D. EBERT, Director, Department of Embryology, Carnegie Institution of Wash- 
ington, in charge of course 

DONALD D. BROWN, Staff Member, Department of Embryology, Carnegie Institution 
of Washington 

ALLISON L. BURNETT, Associate Professor of Biology, Western Reserve University 

ROBERT L. DE HAAN, Staff Member, Department of Embryology, Carnegie Institution 
of Washington 

THOMAS J. KING, Head, Department of Embryology, Institute for Cancer Research, 
Philadelphia 



REPORT OF THE DIRECTOR 



15 



JAMES W. LASH, Assistant Professor of Anatomy, University of Pennsylvania 
AARON A. MOSCONA, Professor of Zoology, University of Chicago 

II. LABORATORY ASSISTANTS 

C. B. KIMMEL, The Johns Hopkins University 
DAVID E. KOHNE, Purdue University 



J. D. EBERT 
T. J. KING 
T. J. KING 
R. L. DE HAAN 

A. J. COULOMBRE 
E. ZWILLING 

S. SIMPSON 

J. LASH 
A. MOSCONA 
A. MOSCONA 
A. MOSCONA 
H. RUBIN 
M. ROSENBERG 
P. MARCUS 

A. L. COLWIN 

M. STEINBERG 
A. BURNETT 

A. BURNETT 
A. BURNETT 

J. D. EBERT 
J. W. LASH 
J. W. LASH 
K. R. PORTER 
K. R. PORTER 
E. HADORN 
T. GALL 
T. J. KING 

R. L. DE HAAN 
E. HADORN 

D. D. BROWN 
P. GROSS 

E. BELL 

T. R. COLLIER 

I. DAVID 

A. MONROY 



III. LECTURES 

Perspectives in developmental biology 

Teleosts I 

Teleosts II 

Cell movements and morphogenesis 

The morphogenetic interaction of the tissues of the eye 
during development 

Morphotypic diversity vs. histiotypic identity 

Tissue interactions and morphogenesis in lizard tail re- 
generations 

The induction of chondrogenesis in vitro 

Sponges I 

Sponges II 

Tissue reconstruction from dissociated cells 

The malignant transformation of animal cells by viruses 

Some applications of surface physics to cell biology 

Dynamics of plasma membrane modification in virus-in- 
fected and normal cells 

Role of the gamete membranes in fertilization 

Adhesive selectivity in intercellular reactions 

A model of growth for hydroids and tubules of higher 
organisms 

The role of neoblasts in the maintenance of form of 
hydroids 

Dedifferentiation and redifferentiation of somatic cells in 
Hydra an analysis of polymorphism 

Developmental aspects of immunity 

Ascidians I : General embryology 

Ascidians II: Metamorphosis 

Developmental cytology I 

Developmental cytology II 

Developmental aspects of pleiotropic effects of genes 

The nucleic acids of giant chromosomes 

The developmental capacity of nuclei transplanted from 
advanced embryos 

Comparative morphogenesis of annelids, molluscs and 
echinoderms 

Problems of differentiation and pattern formation in Dro- 
sophila blastemas 

Biochemistry of oogenesis and early development 

Microsymposium : biochemistry of early development 



16 MARINE BIOLOGICAL LABORATORY 

A. B. PARDKE Cell regulatory mechanisms I 

A. B. PARDEE Cell regulatory mechanisms II 

S. COHEN An epidermal growth-stimulating protein 

M. SINGER The nervous control of the regeneration of body parts in 

vertebrates 

V. HAMBURGER Xeurogenesis and the embryology of behavior 

K. MEYER Keratosulfates of cornea and cartilage 

G. W. COOPER Induction of somite chondrogenesis by cartilage and noto- 

chord : a correlation between inductive activity and 

cytodifferentiation 

H. SCHNEIDERMAN The hormonal control of insect development 

L. \\~EISS The structure of lymphatic tissue and its reaction in runt 

disease 

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

II. INSTRUCTORS 

J. WOODLAND HASTINGS, Professor of Biochemistry, University of Illinois, in charge 

of course 

E. A. ADELBERG, Professor of Microbiology, Yale University 
HARLYN HALVORSON, Professor of Bacteriology, University of Wisconsin 
SHINYA INOUE, Professor of Cytology, Dartmouth College 
K. E. VAN HOLDE, Professor of Physical Chemistry, University of Illinois 
FRED KARUSH, Professor of Microbiology, University of Pennsylvania 
WILLIAM F. HARRINGTON, Professor of Biology, The Johns Hopkins University 
HANS KORNBERG, Professor of Biochemistry, Leicester University, England 

III. LABORATORY ASSISTANTS 

GEORGE KISSIL, University of Wisconsin 

CAROLYN EBERHARD, University of California at Berkeley 

IV. LECTURES 

SKYMOUR COHKN The lethality of streptomycin and the stimulation of RNA 

synthesis 
A. J. Soi'ii IAXOPOULOS Protein denuturution and hydrogen-ion equilibria of lyso- 

zyme 

HOWARD SCIIACHMAN Macromolecular configurations 

GEORCI; WALD Human color vision 

R. K. CLAYTON Photosynthesis II : Physical aspects 

ROGER ECKKKT Excitation-response coupling: Bioelectric flash triggering 

in Noctiluca 
WILLIAM HAGTNS Early steps in the excitation of photoreceptors 



REPORT OF THE DIRECTOR 



17 



The bacterial endospore and the problem of biological 

dormancy 

Fine structure of muscle 
Studies on ciliary movement 
Studies on the spectral complexes between flavo-proteins 

and their competitive inhibitors 
Mechanism of hormone action 
Growth 

Microbial metabolism I 
Microbial metabolism II 
Microbial metabolism III 

Immunochemistry I. The interactions of immunoglobulins 
Immunochemistry II. The nature of immunoglobulins 
Immunochemistry III. The biosynthesis of immunoglobulins 
Applications of immunochemistry to problems in biology 
Phosphorescence properties of DNA complexes 
Synthesis and stability of messenger RNA 
Photosynthesis I : Biochemical aspects 

Transcription and translation of the bacterial chromosome 
Replication and transfer of bacterial DNA 
Some aspects of regulation of gene function 
Polarization microscopy : An approach to fine-structure 

analysis in living cells 
Dynamic aspects of the mitotic apparatus 
Molecular arrangement of DNA in the living sperm 
Microbial metabolism 

Protein structure III. Multichain proteins 
Protein biosynthesis I : In vivo 
Protein biosynthesis II : In vitro 
The control and timing of enzyme synthesis 
The bacterial chromosome : Structure and function 
The bacterial chromosome: Replication 
Enzymatic intermediates in the bacterial bioluminescent 

reaction 

Crystalline bioluminescent particles : The Gonyaulax sys- 
tem 

Some aspects of the mechanism of enzyme action 
Protein Structure I : Physical methods of investigation 
Protein Structure II : The folding of polypeptide chains 

MARINE BOTANY 

I. CONSULTANTS 

WILLIAM RANDOLPH TAYLOR, Professor of Botany, University of Michigan 
RICHARD C. STARR, Professor of Botany, Indiana University 

II. INSTRUCTORS 

WALTER R. HERNDON, Professor of Botany, University of Tennessee, in charge of course 

PHILIP W. COOK, Department of Botany, University of Vermont 

H. WAYNE NICHOLS, Assistant Professor of Botany, Washington University 

FRANK E. ROUND, Department of Botany, University of Bristol, England 

ROBERT T. WILCE, Assistant Professor of Botany, University of Massachusetts 



ALEX KEYNAN 

H. E. HUXLEY 
IAN GIBBONS 
C. VEEGAR 

RACHMIEL LEVINE 
ALBERT SZENT-GYORGYI 
HANS LEO KORNBERG 
HANS LEO KORNBERG 
HANS LEO KORNBERG 
FRED KARUSH 
FRED KARUSH 
FRED KARUSH 
LAWRENCE LEVINE 
IRVIN ISENBERG 
CYRUS LEVINTHAL 
R. K. CLAYTON 
E. A. ADELBERG 
E. A. ADELBERG 
K. C. ATWOOD 
S. INOUE 

S. INOUE 
S. INOUE 

HANS LEO KORNBERG 
K. E. VAN HOLDE 
HARLYN HALVORSON 
HARLYN HALVORSON 
HARLYN HALVORSON 
E. A. ADELBERG 
E. A. ADELBERG 
J. W. HASTINGS 

J. W. HASTINGS 

WILLIAM P. JENCKS 
K. E. VAN HOLDE 
K. E. VAN HOLDE 



18 MAKIXI-: UlOLOr.lCAL LABORATORY 

III. LABORATORY ASSISTANTS 

RUSSELL G. RHODES, Department of Botany, University of Tennessee 
ERNEST NEAL, Department of Botany, University of Tennessee 

IV. COLLECTOR 
MARTHA HODGE, l'ni\er>ity of Michigan 

INVERTEBRATE ZOOLOGY 

I. CONSULTANTS 

I'. A. MROWN. JR., Morrison Professor of Biology, Northwestern University 
I.IBBIE H. IIv.MAN, American Museum of Natural History 
CLARK P. READ, Professor of Biology, Rice University 
ALFRED C. REDFIELD, Woods Hole Oceanographic Institution 

II. INSTRUCTORS 

\Y. D. RUSSELL HUNTER, Professor of Zoology, Syracuse University, in charge of course 

GEORGE HOLZ, Professor of Microbiology, State University of New York, Upstate Medi- 
cal Center 

ROGER MILKMAN, Associate Professor of Zoology, Syracuse University 

IRWIN W. SHERMAN, Assistant Professor of Biology, University of California at River- 
side 

ALLAHVERDI FARM AN FARM AIAN, Professor of General Physiology, Pahlavi University, 
Shiraz, Iran 

ERIC L. MILLS, Assistant Professor of Biology, Queen's University, Kingston, Ontario, 
Canada 

FRANK M. FISHER, Assistant Professor of Biology, Rice University 

SEARS CROWELL, Professor of Zoology, Indiana University 

III. ASSISTANTS 

JOHN H. BUSSER, University of Rhode Island 

\Y. I'.RUCE HUNTER, University of California at Santa Barbara 

IV. LECTURES 

ROU.K D. MILKMAN 1'rotochordata II 

JOHN J. LEE The study of living Foraminifera in the laboratory 

GEORGE HOLZ The nature of the Protozoa 

GEORGE HOLZ Mastigophora 

GEORGE HOLZ Rhizopodea and Actinopodea 

<". IORGE HOLZ Foraminifera 

'ii'iRGE HOLZ Ciliophora 

I\. A. BOOLOOTIAN, Dialogue on aspects of ediinoderm physiology 

A. FARMAXFARMAIAN 

W. D. R. HUNTER An approach to zooplankton 

A. FARMANFARMAIAN The echinoderms I 

Introduction: C'rinoidea and Asteroidra 



REPORT OF THE DIRECTOR 



10 



IRWIN W. SHERMAN 
A. FARMANFARMAIAN 
A. FARMANFARMAIAN 
ROGER D. MILKMAN 
ERIC L. MILLS 

W. D. RUSSELL HUNTER 
ERIC L. MILLS 
ERIC L. MILLS 

LUIGI PROVASOLI 
ROBERT HESSLER 

ERIC L. MILLS 
FRANK FISHER 
FRANK FISHER 
W. D. RUSSELL HUNTER 

CLARK P. READ 
IRWIN W. SHERMAN 
IRWIN W. SHERMAN 

IRWIN W. SHERMAN 
ERIC L. MILLS 

ROGER D. MILKMAN 
FRANK FISHER 
FRANK FISHER 
W. D. RUSSELL HUNTER 

W. D. RUSSELL HUNTER 
W. D. RUSSELL HUNTER 

SEARS CROWELL 
SEARS CROWELL 
SEARS CROWELL 
SEARS CROWELL 
ROGER D. MILKMAN 



Physiological studies on malarial parasites 

The echinoderms II : Holothuroidea 

The echinoderms III : Echinoidea and Ophiuroidea 

Protochordata I 

Arthropoda II : Larvae, lines and limbs further introduc- 
tion to the Crustacea 

Molluscs leave the sea (physiological variation and evo- 
lution) 

Arthropoda III : Feeding in Crustacea Cephalocarida, 
Branchiopoda, and Mystacocarida 

Arthropoda IV: Feeding in Crustacea Copepoda, Cirri- 
pedia, and Malacostraca 

External metabolites in sea water 

Arthropoda V: Functional morphology of jaws and other 
things in the Crustacea 

Arthropoda VI : Pycnogonida and Xiphosurida 

Ectoprocta and Entoprocta 

Aschelminthes 

Mollusca IV : Functional morphology in Cephalopoda and 
minor groups 

Physiology of parasitic flatworms 

Annelida I : Introduction, reproduction and development 

Annelida II : Settling of larvae, regeneration, feeding and 
locomotion 

Annelida III: Respiration, osmoregulation, neuromuscular 
system and behavior 

Arthropoda I : Introduction to the Crustacea : The biology 
of limbs and exoskeleton 

Porifera II 

Platyhelminth.es I : Turbellaria and Trematoda 

Platyhelminthes II : Cestoda and Rhynchocoela 

Mollusca I : General molluscan organization functioning 
of mantle cavity in Gastropoda 

Mollusca II : Gastropoda mantle cavity and feeding mech- 
anisms in Bivalvia 

Mollusca III : Adaptations in bivalves aspects of general 
physiology of Gastropods and bivalves 

Cnidaria I : General diversity in hydroids 

Cnidaria II : Hydroid morphogenesis 

Cnidaria III : Other Cnidaria physiology of Cnidaria 

Ctenophora 

Porifera I 



MARINE ECOLOGY 
I. CONSULTANTS 

MELBOURNE R. CARRIKER, Marine Biological Laboratory 
BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution 
EDWIN T. MOUL, Rutgers University 
JOHN H. RYTHER, Woods Hole Oceanographic Institution 



20 



MAKIXE BIOLOGICAL LABORATORY 



II. INSTRUCTORS 

\V. ROWLAND TAYLOR, Department of Oceanography and the Chesapeake Bay Institute, 

The Johns Hopkins University, in charge of course 
HOWARD L. SANDERS, Woods Hole Oceanographic Institution 
LAWRENCE B. SLOBODKIN, Department of Zoology, University of Michigan 
RICHARD A. BOOLOOTIAN, Department of Zoology, University of California at Los 

Angeles 
OTTO KINNE, Biologische Anstalt Helgoland 

III. LABORATORY ASSISTANTS 

MARGARET C. LLOYD, University of Michigan 

BARRY M. HEATFIELU, University of California at Los Angeles 



W. ROWLAND TAYLOR 

\Y. ROWLAND TAYLOR 

W. ROWLAND TAYLOR 

W. ROWLAND TAYLOR 

W. ROWLAND TAYLOR 

YV. ROWLAND TAYLOR 

M. R. CARRIKER 

J. H. RYTHER 

D. MENZEL 

W. R. TAYLOR 
B. H. KETCHUM 

E. R. BAYLOR 
H. L. SANDERS 
II. L. SANDERS 

R. SCHELTEMA 

H. L. SANDERS 
ERIC L. MILLS 

J. B. PF.ARCE 
S. A. WAINRIGHT 
J. A. HELLEBUST 
OTTO KTNNE 

L. I'ROVASOLI 
L. PROVASOLI 
OTTO KINNE 

L. I'RMVASOT.I 
' )TTO KIN.NE 



IV. LECTURES 

Organisms and their environment experimental approaches 
The marine environment, its chemistry and physics 
Phytoplankton I : Diatoms 
Phytoplankton II: Dinoflagellates 
Primary productivity by phytoplankton I 
Primary productivity by phytoplankton II 
The Systematics-Ecology Program at the Marine Biologi- 
cal Laboratory 

Geographical variations in productivity 
Production and utilization of dissolved organic material in 

the oceans 

Physiology of migrating littoral diatoms 
Nutrient cycles in the sea 

Sea surface chemistry and the distribution of organisms 
Animal-sediment relationships 
A study of a marine benthic community 
Problems in benthic larval ecology 
Salinity, hydrography and the distribution of estuarine 

animals 
Ecology of a crustacean sibling species pair, or systematics 

unashamed 

Temporal and spatial distribution of mytilid associations 
Biology of reef corals 

Excretion of organic compounds by marine algae 
The effects of temperature on marine and brackish water 

organisms 
Culturing marine algae I : (Joint lecture with the Botany 

Course) 
Culturing marine algae II: (Joint lecture with the Botany 

Course) 
Effects of temperature and salinity on the hydroid, Cordylo- 

f>!n>ru cuspid 

External metabolites in sea water 
Kffects of temperature, salinity and oxygen on the fish. 

Cyprinodan iihicitltirins 



REPORT OF THE DIRECTOR 21 

A. FARMANFARMAIAN Temperature and salinity tolerance limits of the West 

Coast purple sea urchin 

OTTO KINNE Non-genetic adaptation to temperature and salinity in 

marine and brackish water organisms 

L. SLOBODKIN Ecological tautologies 

L. SLOBODKIN Fecundity, mortality and reproductive value 

L. SLOBODKIN Experimental population growth 

L. SLOBODKIN Energy and animal populations 

L. SLOBODKIN The strategy of evolution 

R. A. BOOLOOTIAN Food requirements and distribution of marine organisms : 

general considerations 

R. A. BOOLOOTIAN Types of food utilization by marine organisms with em- 

phasis on feeding adaptions 

R. A. BOOLOOTIAN Reproductive biology of marine organisms : General pat- 

terns 

R. A. BOOLOOTIAN Factors influencing the regulation of reproductive cycles 

R. A. BOOLOOTIAN Reproductive physiology of the purple sea urchin, Strongy- 

loccntrotus pitrpuratus 

Wednesday Evening Lecture Series, jointly sponsored by Marine Ecol- 
ogy, Invertebrate Zoology and Comparative Physiology : 

N. D. MARSHALL Some aspects of the biology of benthic deep-sea fishes 

J. H. WELSH Serotonin : Its occurrence in nature and its multiple bio- 

logical roles 
CARROLL M. WILLIAMS Light, brains, and metamorphosis 

B. C. ABBOTT Excitation-contraction coupling and mechanical responses 

in relation to muscle function 



SYSTEMATICS-ECOLOGY PROGRAM 

THE STAFF 

Director: MELBOURNE R. CARRIKER 

Resident Systematist : VICTOR A. ZULLO 

Resident Ecologist : ROBERT H. PARKER 

Postdoctoral Fellows and Research Associates: MARVIN CANTOR, JOHN C. H. CARTER, 

DAVID C. GRANT, JACK B. PEARCE, KAY W. PETERSEN, THOMAS J. M. SCHOPF, 

JOSEPH SIMON, EDMUND H. SMITH, GERALD E. WALSH 
Visiting Investigators in Residence: RICHARD A. BOOLOOTIAN, LOUISE BUSH, DUANE 

HOPE, E. T. MOUL 

Secretaries: SAN LINEA WEAVER, VIRGINIA SMITH 
Artists: RUTH VON ARX, DIANE JOHNSON 
Captain of Research Vessel : JAMES P. W. OSTERGARD, JR. 
Research Assistants: KAY CRAM, ANDREW L. DRISCOLL, J. STEWART NAGLE, PETER J. 

OLDHAM, PETER E. SCHWAMB, DIRK VAN ZANDT, HILARY M. WILLIAMS, JUNE 

THOMAS, VILIA TURNER 

I. SEMINARS (winter not included) 

ALBERT SZENT-GYORGYI Contraction of muscle 

JOHN H. RYTHER U. S. Biological Program of the Indian Ocean Expedition 

VICTOR A. ZULLO Keys to marine invertebrates of the Woods Hole region 



22 



MARINE BIOLOGICAL LABORATORY 



JACK B. PEARCE 

JAMKS ROSS 

JOSEPH L. SIMON 

RICHARD A. BOOLOOT IAN- 
ERIC L. MILLS 
WALTER R. UKRXDOX 

MARVIN CANTOR 
MARVIN CANTOR 
DAVID C. GRANT 

COPELAND MACCLINTOCK 

MELBOURNE R. CARRIKER 
JACK B. PEARCE 

DONALD F. SQUIRES 
ROBERT H. PARKER 
THOMAS J. M. SCHOPF 

KAY W. PETERSEN 
ROBERT R. HESSLER 
DARYL SWEENEY 



A preliminary report on the MytHns ednlis association in 
Ouicks Hole, Kli/al>eth Islands, Massachusetts 

Electrometric measurements of activities of ions and gases 
as applied to measurements in animals and of sea water 

Reproduction and larval development in the spionid poly- 
chaete, Spio setosa 

Aspects of reproductive biology of echinoderms 

The biology oi an amphipod crustacean sibling species pair 

Some approaches to taxonomic revision in chlorophycean 
algae 

Adaptation in a flagellate protozoan 

Metabolic adaptation in a flagellate protozoan 

Specific diversity in an intertidal community 

Microstructure of the shell in Gastropoda 

An aerial overview of the major marine habitats of the 
Cape Cod region 

A pilgrimage to Ellerslie (P.E.I., Canada) and its sur- 
rounding benthic communities 

Fossil coral thickets 

The 1958-59 Downwind Expedition to Easter Island 

Conodonts of the Trenton Group (Ordovician) in New 
York and Southern Ontario, Canada 

On the origin of Metazoa 

Derocheilocaris typicus revisited 

What good is dopamine; clams; biochemical evolution? 



THE LABORATORY STAFF 
HOMER P. SMITH, General Manager 



Miss JANE FESSENDEN, Acting Librarian 
CARL O. SCHWEIDENBACK, Manager, Supply 

Department 
IRVINE L. BROADBENT, Office Man 



ROBERT KAHLER, Superintendent, 
Buildings and Grounds 

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



JERAL OFFICE 



EDWARD J. BENDER 
MRS. VIVIEN B. BROWN 
MRS. FLORENCE S. BUTZ 
MRS. MARION C. CHASE 



MRS. VIRGINIA HKAXDENI'.UKC 
MKS. LKNOR \ Josi MI 



LIBRARY 



MRS. JUDITH A. KECK 
MRS. ANN W. LOOMIS 
MRS. VIVIAN I. MANSON 
Miss KATHERINE M. TRACY 



ALBERT K. NEAL 
MRS. DORIS T. RICKI-K 



MAINTENANCE oh' BUILDINGS AND GROUNDS 



ADAMS 
KI.DON P. ALLEN 
BERNARD F. CAVANAUGH 
MANUEL P. DUTRA 

S I AX LEY C. El.DKKDCK 

( i ARUM. R V. GAYTON 



KOP.KKT GUNNING 
DONALD B. LEIIY 
l\ M.PII H. LEWIS 
KrssKi.L F. LEWIS 
I IKXRY !". POTTER 
ROBERT H. WALKER, JR. 



REPORT OF THE DIRECTOR 

DEPARTMENT OF RESEARCH SERVICE 

GAIL M. CAVANAUGH Miss MARGARET E. SCOTT 

LOWELL V. MARTIN FRANK E. SYLVIA 

SUPPLY DEPARTMENT 

ARNO J. BOWDEN PAUL SHAVE 

DAVID H. GRAHAM BRUNO F. TRAPASSO 

MRS. E. GREEN JOHN J. VALOIS 

ROBERT W. HAMPTON HALLETT S. WAGSTAFF 
ROBERT O. LEHY 

DINING HALL AND HOUSING 

ROBERT T. MARTIN, Manager, Food Service 
MRS. ELIZABETH KUIL, Supervisor, Dining Room 
MRS. ELLEN T. NICKELSON, Supervisor, Dormitories 
ALAN G. LUNN, Supervisor, Cottage Colony 

3. INVESTIGATORS : LALOR, LILLIE AND GRASS FELLOWS ; STUDENTS 
Independent and Beginning Investigators, 1964 

ABBOTT, BERNARD C, Professor of Physiology and Biophysics, University of Illinois 

ADELBERG, EDWARD A., Professor and Chairman, Department of Microbiology, Yale University 

ADELMAN, WILLIAM J., JR., Associate Professor of Physiology, University of Maryland 

ALLEN, ROBERT DAY, Associate Professor of Biology, Princeton University 

ANDERSON, EVERETT, Professor of Zoology, University of Massachusetts 

ARMSTRONG, PHILIP B., Chairman, Department of Anatomy, State University of New York, 

College of Medicine at Syracuse 

ARNOLD, JOHN M., Lerner Marine Laboratory of the American Museum of Natural History 
AUCLAIR, WALTER, Assistant Professor of Zoology, University of Cincinnati 
AUSTIN, C. R., Member of External Scientific Staff, Medical Research Council of Great Britan 
BAKER, ROBERT F., Radiation Physicist, Brown University 
BANG, FREDERIK B., Professor of Pathobiology, The Johns Hopkins University 
BARDACH, JOHN E., Professor of Zoology, University of Michigan 
BARLOW, ROBERT B., The Rockefeller Institute 
EARTH, L. G., Professor of Zoology, Columbia University 
BARTH, L. J., Professor of Zoology, Barnard College 
BAUER, G. ERIC, Profesor of Anatomy, University of Minnesota 

BAUMANN, FRITZ, National Institute of Neurological Diseases and Blindness, National In- 
stitutes of Health 

BAYLOR, MARTHA BARNES, Marine Biological Laboratory 
BEATTY, RICHARD ALAN, Senior Principal Scientific Officer, Agricultural Research Council 

Unit of Animal Genetics, Great Britain 

BELAMARICH, FRANK A., Assistant Professor of Biology, Boston University 
BELL, EUGENE, Associate Professor of Biology, Massachusetts Institute of Technology 
BENNETT, M. V. L., Associate Professor of Neurology, Columbia University, College of 

Physicians and Surgeons 

BIGGERS, JOHN D., Professor of Reproductive Physiology, University of Pennsylvania 
BILLIAR, REINHART B., Research Fellow, Harvard Medical School 

BINSTOCK, LEONARD, Electronic Engineer (Instrumentation), National Institutes of Health 
BOOLOOTIAN, RICHARD A., Associate Professor of Zoology, University of California, Los 

Angeles 

BRANDT, PHILLIP W., Assistant Professor of Anatomy, Columbia University 
BRANHAM, JOSEPH M., Assistant Professor of Biology, Oglethorpe University 



24 MARINE BIOLOGIC \l. LABI iRATORY 

BRINLEY, F. J., Assistant Professor of Physiology, Tlie Johns Hopkins School of Medicine 

BROWX, DOXALD D., Staff Member in Biochemistry, Carnegie Institution of Washington 

BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University 

BRYAXT, S. H., Associate Professor of Pharmacology, University of Cincinnati 

BRZIN, MIRO, Senior Research Associate, University of Ljubljana 

BURCH, HELEN B., Associate Professor of Pharmacology, Washington University 

BURNETT, ALLISON L., Associate Professor of Biology, Western Reserve University 

CANTOR, MARVIN H., Postdoctoral Fellow, Systematics-Ecology Program, Marine Biological 

Laboratory 

CARLSON, FRANCIS D., Chairman and Professor of Biophysics, The Johns Hopkins University 
CARRIKER, MELBOURNE R., Director, Systematics-Ecology Program, National Institutes of 

Health 

CARTER, JOHX. Postdoctoral Fellow, Systematics-Ecology Program, Ford Foundation 
CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College, The City University of New 

York 

CHILD, FRANK M., Assistant Professor of Zoology, University of Chicago 
CLAFF, C. LLOYD, Treasurer and Chief Investigator, Single Cell Research Foundation, Inc. 
CLARK, ELOISE E., Assistant Professor of Zoology, Columbia University 
CLEMENT, A. C., Professor of Biology, Emory University 

COLE, KEXNETH S., Chief, Laboratory of Biophysics, NINDB, National Institutes of Health 
COLWIN, ARTHUR L., Professor of Biology, Queens College, The City University of New York 
COLWIN, LAURA HUNTER, Lecturer in Biology, Queens College, The City University of New 

York 

COOK, PHILIP WILLIAM, Assistant Professor of Botany, University of Vermont 
COOPERSTEIX, SHERWIN J., Associate Professor of Anatomy, Assistant Dean, Medical School, 

Western Reserve University 

COPELAND, EUGENE, Chairman and Professor of Zoology, Tulane University 
COSTELLO, DONALD PAUL, Kenan Professor of Zoology, University of North Carolina 
CROWELL, SEARS, Professor of Zoology, Indiana University 
DEHAAX, ROBERT L., Research Embryologist, Carnegie Institution of Washington 
DE LORENZO, A. J., Director, Anatomical and Pathological Research Laboratories, The Johns 

Hopkins University School of Medicine 
DETTBARN, WOLF-DIETRICH, Assistant Professor of Neurology, Columbia University, College 

of Physicians and Surgeons 

DE VILLAFRANCA, GEORGE W., Professor of Zoology, Smith College 
DISCHE, ZACHARIAS, Professor of Biochemistry, Columbia University, College of Physicians 

and Surgeons 

DUNHAM, PHILIP B., Assistant Profesor of Zoology, Syracuse University 
EBERT, JAMES D., Director, Department of Embryology, Carnegie Institution of Washington 
ECKERT, ROGER, Assistant Professor of Zoology, Syracuse University 
EDDS, MAC V., JR., Chairman and Professor of Medical Science, Brown University 
EHRENSTEIN, GERALD, Physicist, National Institutes of Health 
ESKRIDGE, ROSEMARY WAITE, Special Research Assistant of Biochemistry, State University 

of New York at Huffalo 

EVOY, WILLIAM H., Grass Fellow, University of Oregon 

FAILLA, PATRICIA MCQ.EMEXT, Associate Biophysicist, Argonne National Laboratory 
FARMANFARMAIAN, ALLAIIVERDI, Professor of General Physiology, Pahlavi University, Shiraz, 

Iran 

FISHER, FRANK M., JR., Assistant Professor of Biology, Rice University 

FISHMAX, Louis, Assistant Research Professor, New York University College of Dentistry 
FREEMAN, ALAN R., Postdoctoral Trainee Fellow USPHS, Columbia University 
Fi:r.MF.XTO, ANTONIO S., Postdoctoral Fello\v, University of Maryland 
FUJIO, HAJIME, Department of Microbiology, University of Pennsylvania Medical School 
FUORTES, M. G. F., Head, Section on Ncurophysiology, NINDB, National Institutes of Health 
!ii 1 1 PAN. EDWIN J., Assistant Professor of Neurophysiology, Harvard Medical School 
GARCIA, HORATIO A., Grass Fellow, Columbia University 
GEILENKIRCIIKX, W. I.. M.. I.crtnn-r, University of I'tivrlit. Holland 



REPORT OF THE DIRECTOR 25 

GELFANT, SEYMOUR, Associate Professor of Zoology, Syracuse University 

GERMAN, JAMES L., Ill, Director and Associate Professor of Division of Human Genetics, 

Cornell University Medical School 

GIBBO'NS, I. R., Assistant Professor, Biological Laboratories, Harvard University 
GILBERT, DANIEL L., Physiologist, National Institutes of Health 

GIMENEZ, MAXIMO, Grass Fellow, Columbia University, College of Physicians and Surgeons 
GLADE, RICHARD W., Chairman and Associate Professor of Zoology, University of Vermont 
GOLDMAN, LAWRENCE, Postdoctoral Fellow, Columbia University, College of Physicians and 

Surgeons 

GRANT, DAVID, Research Associate, Systematics-Ecology Program 

GRANT, PHILIP, Program Director, Developmental Biology, National Science Foundation 
GREEN, JONATHAN, Postdoctoral Research Fellow, The Johns Hopkins University 
GREY, HOWARD, The Rockefeller Institute 
GROSCH, DANIEL S., Professor of Genetics, North Carolina State of the University of North 

Carolina at Raleigh 

GROSS, PAUL R., Associate Professor of Biology, Brown University 
GRUNDFEST, HARRY, Professor of Neurology, Columbia University, College of Physicians and 

Surgeons 

GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College 
HADORN, E., Director, Zoologisch-Vergl. Anatomisches Institut, Der Universitat Zurich, 

Switzerland 

HAGINS, WILLIAM A., NIAMD, National Institutes of Health 
HALVORSON, H. O., Professor of Bacteriology, University of Wisconsin 
HARDING, CLIFFORD V., Associate Professor of Physiology, Columbia University, College of 

Physicians and Surgeons 

HAROSI, FERENC, Electronics Engineer, The Rockefeller Institute 
HASTINGS, J. WOODLAND, Professor of Biochemistry, University of Illinois 
HAYASHI, TERU, Chairman and Professor of Zoology, Columbia University 
HEGYELI, ANDREW, Institute for Muscle Research, Marine Biological Laboratory 
HERNDON, WALTER R., Head and Professor of Botany, University of Tennessee 
HERVEY, JOHN P., Senior Electronics Engineer, The Rockefeller Institute 
HIGASHINO, SHOJI, Research Associate in Physiology, Gunma University, Japan and Columbia 

University 

HIGGINS, DON CHENEY, Assistant Professor of Internal Medicine, Yale University 
HIRAMOTO, YUKIO, Assistant Professor at Misaki Marine Biological Station, University of 

Tokyo 
HOLZ, GEORGE G., Chairman and Professor of Microbiology, State University of New York, 

Upstate Medical Center 
HOPE, WILLIAM DUANE, Associate Curator, Division of Marine Invertebrates, United States 

National Museum 
HOSKIN, FRANCIS C. G., Assistant Professor of Neurology, Columbia University, College of 

Physicians and Surgeons 

HUMPHREYS, TOM, Assistant Professor of Biology, Massachusetts Institute of Technology 
HUNTER, W. D. RUSSELL, Professor of Zoology, Syracuse University 
HUVER, CHARLES W., Assistant Professor of Anatomy, University of Illinois 
INOUE, SHINYA, Chairman and Professor of Cytology, Dartmouth Medical School 
ISENBERG, IRVIN, Institute for Muscle Research, Marine Biological Laboratory 
JANOFF, AARON, Assistant Professor of Pathology, New York University School of Medicine 
JOHNSON, FRANK H., Professor of Biology, Princeton University 

KAMINER, BENJAMIN, Institute for Muscle Research. Marine Biological Laboratory 
KANE, ROBERT E., Assistant Professor of Cytology, Dartmouth Medical School 
KARLIN, ARTHUR, Research Associate, Columbia University, College of Physicians and 

Surgeons 

KARASAKI, SHUICHI, Biology Division, Oak Ridge National Laboratory 
KARUSH, FRED, Professor of Microbiology, University of Pennsylvania Medical School 
KATZ, GEORGE M., Electronics Engineer, Columbia University, College of Physicians and 

Surgeons 
KALEY, GABOR, Assistant Professor of Pathology, New York University 



26 MAkiXK BIOLOGICAL LABORATORY 

KEMPTON, RUDOLF T., Professor of Zoology, Vassar College 

KEOSIAN, JOHN, Professor of Biology, Rutgers The State University 

KESSEL, RICHARD G., Assistant Professor of Zoology, State University of Iowa 

KING, THOMAS J., Head, Department of Embryology, The Institute for Cancer Research 

KINXE, OTTO, Director and Professor of Biologische Anstalt Helgoland, Hamburg-Altona, 

Palmaille, 9, Germany 

KLEIN HOLZ, LEWIS H., Professor of Biology, Reed College 

KORNBERG, HANS LEO, Head, Professor of Biochemistry, University of Leicester, England 
KRASSNER, STUART AT., The Rockefeller Institute 

KRISHNAKUMARAN, A., Postdoctoral Research Fellow, Western Reserve University 
KUFFLER, STEPHEN W., Professor of Neurophysiology, Harvard University 
LANSING, ALBERT, Professor of Anatomy, University uf Pittsburgh 

LASH, JAMES W., Assistant Professor of Anatomy, University of Pennsylvania Medical School 
LASTER, LEONARD, Chief of Gastroenterology Unit, National Institute of Arthritis and Metabolic 

Diseases 

LAZAROW, ARNOLD, Head and Professor of Anatomy, University of Minnesota 
LECAR, HAROLD, Physicist, National Institutes of Health 

LERMAN, SIDNEY, Associate Professor of Ophthalmology & Assistant Professor of Bio- 
chemistry, University of Rochester School of Medicine & Dentistry 
LEVINE, LAWRENCE, Professor of Biochemistry, Brandeis University 
LEVY, MILTON, Chairman and Professor of Biochemsitry, New York University College of 

Dentistry 

LIEBMAN, PAUL A., Assistant Professor of Physiology, University of Pennsylvania 
LOEWENSTEIN, WERNER R., Associate Professor of Physiology, Columbia University, College 

of Physicians and Surgeons 
LOPEZ, ENRIQUE, Research Associate in Neurology, Columbia University, College of Physicians 

and Surgeons 

LORAND, L., Professor of Chemistry, Northwestern University 

LOVE, WARNER E., Associate Professor of Biophysics, The Johns Hopkins University 
MAcNiCHOL, EDWARD F., Professor of Biophysics, The Johns Hopkins University 
MAGGIO, RACHELE, Assistant Professor, Institute of Comparative Anatomy, University of 

Palermo, Italy 

MAHLER, HENRY R., Professor of Chemistry, Indiana University 
MALKIN, LEONARD I., Research Associate of Biology, Brown University 
MARCHALONIS, JOHN, The Rockefeller Institute 

MARMASSE, CLAUDE, The Commonwealth Fund, Marine Biological Laboratory 
MARSLAND, DOUGLAS, Research Professor, New York University 
McELROY, W. D., Professor of Biology, The Johns Hopkins University 
MELLON, DEFOREST, JR., Assistant Professor of Biology, University of Virginia 
MENDELSON, MARTIN, Professor in Physiology, New York University 
METZ. CHARLES B., Professor of Biologj in the Institute for Space Biosciences, Florida State 

University 

MILEIJI, Ku AKIIO, Reader in Biophysics, University College of London, England 
MILKMAN, ROGER DAWSON, Associate Professor of Zoology, Syracuse University 
MILLER, FAITH S., Assistant Professor of Anatomy, Tulane University 
MILLER, JAMES A., Professor and Chairman of Anatomy, Tulane University 
MOIILER, J. D., Associate Professor of Zoology, Oregon State University 
MOHRI, HIDEO, Research Associate of Biology, University of Tokyo 
MONROY, ALBERTO, Professor of Comparative Anatomy, University of Palermo, Italy 
MOORE, JOHN W., Associate Professor of Physiology, Chief, Laboratory of Cellular Neuro- 
physiology, Duke University 

MOSCONA, A. A., Professor of Zoology, University of Chi( 
Mn.i.ixs, L. J., Professor of Biophysics, University of Maryland 
NACE, PAUL FOLEY, Professor, Research Unit in Molecular Biology, McMaster University, 

Canada 

NAQAI, REIKO, Research Associate of Biology, Princeton University 

NAKAMURA, YUTAKA, Research Fellow, Columbia University, College of Physicians and 
Surgeons 



REPORT OF THE DIRECTOR 

NASATIR, MAIMON, Assistant Professor and Assistant to the Dean, Pembroke College 
NELSON, LEONARD, Associate Professor of Physiology, Emory University 
NICHOLS, H. WAYNE, Assistant Professor of Botany, Washington University at St. Louis 
NVBORG, WESLEY L., Professor of Physics, University of Vermont 

OKAZAKI, KAYO, Research Associate, Tokyo Metropolitan University ; University of Penn- 
sylvania 
PARKER, ROBERT H., Resident Ecologist, Systematics-Ecology Program, National Science 

Foundation 

PARPART, ARTHUR K., Chairman and Professor of Biology, Princeton University 
PEARCE, JOHN BODELL, Research Associate, Systematics-Ecology Program, National Science 

Foundation 

PERSON, PHILIP, Chief, Special Dental Research Laboratory, VA Hospital, Brooklyn 
PORTER, KEITH R., Head and Professor of Biological Laboratories, Harvard University 
RABIN, HARVEY, Assistant Professor of Pathology, The Johns Hopkins University 
RASMUSSEN, ROSEMARY CROCKETT, Research Associate, State University of New York, Up- 
state Medical Center 

READ, CLARK P., Professor of Biology, Rice University 
REBHUN, LIONEL L, Associate Professor of Biology, Princeton University 
REPORTER, MINOCHER, Carnegie Institution of Washington 

REUBEN, JOHN P., Assistant Professor of Neurology, Columbia University, College of Phy- 
sicians and Surgeons 

RICE, ROBERT V., Senior Fellow, Mellon Institute 

ROSE, S. MERYL, Professor of Experimental Embryology, Tulane University 
ROSENBERG, PHILIP, Assistant Professor in Neurology, Columbia University, College of 

Physicians and Surgeons 
ROSENKRANZ, HERBERT S., Assistant Professor of Microbiology, Columbia University, College 

of Physicians and Surgeons 

ROSLANSKY, JOHN D., Institute for Muscle Research, Marine Biological Laboratory 
ROTH, JAY S., Professor of Biochemistry, University of Connecticut 
RUGH, ROBERTS, Associate Professor of Radiology (Biology), Columbia University 
RUSTAD, RONALD C, Associate Professor of Biology, Western Reserve University 
SANDERS, HOWARD L., Research Associate in Ecology, Woods Hole Oceanographic Institution 
SATO, HIDEMI, Assistant Professor of Cytology, Dartmouth Medical School 
SAUNDERS, JOHN W., Chairman and Professor of Biology, Marquette University 
SCHNEIDERMAN, HOWARD A., Chairman and Professor of Biology, Western Reserve University 
SCHMEER, SISTER M. ROSARII, Co-Director of Research, St. Mary of the Springs 
SCHNITZLER, RONALD MICHAEL, Research Fellow, University of Vermont 
SCHOPF, THOMAS J. M., Postdoctoral, Systematics-Ecology Program, Ford Foundation 
SCHUEL, HERBERT, Postdoctoral Fellow, Northwestern University 
SCOTT, ALLAN C., Professor of Biology, Colby College 

SCOTT, SISTER FLORENCE MARIE, Chairman and Professor of Biology, Seton Hill College 
SCOTT, GEORGE T., Chairman and Professor of Biology, Oberlin College 
SENFT, JOSEPH P., USPHS Postdoctoral Fellow, University of Maryland 
SHAW, CHARIS, Research Associate, Tulane University 
SHEMIN, DAVID, Professor of Biochemistry, Columbia University 

SHERMAN, IRWIN W., Assistant Professor of Zoology, University of California, at Riverside 
SICHEL, F. J., Chairman and Professor of Physiology and Biophysics, College of Medicine, 

University of Vermont 

SIMON, JOSEPH L., Sandeen Memorial Fellow, University of Florida 
SIMPSON, SIDNEY B., JR., Postdoctoral Fellow, Department of Anatomy, Western Reserve 

Medical School 

SINGER, IRWIN, Research Associate, National Institute of Mental Health 
SJODIN, RAYMOND A., Associate Professor of Biophysics, University of Maryland 
SLATER, CLARKE ROTHWELL, Grass Fellow, University College, London, England 
SLOBODKIN, LAWRENCE B., Professor of Zoology, University of Michigan 
SMELSER, GEORGE K., Professor of Anatomy, Columbia University 
SMITH, EDMUND H., National Institutes of Health Fellow, Systematics-Ecology Program 



MARINE BIOLOGICAL LABORATORY 

SMITH, THOMAS GRAVES, Research Neurophysiologist, NINDB, National Institutes of Health 
SMITH, WILLIAM ROY, School of Hygiene & Public Health, The Johns Hopkins University 
SPEIDEL, CARL C, Professor of Anatomy, University of Virginia 
SPINDEL, WILLIAM, Professor of Chemistry, Rutgers University 
SPIRTES, M. A., Associate Professor of Pharmacology, Hahnemann Medical College 
STEINBACH, H. BURR, Chairman and Professor of Zoology, University of Chicago 
STRITTMATTER, PIIILIPP, Associate Professor of Biochemistry, Washington University 
STU.XKAKD, HORACE W., Research Associate, American Museum of Natural History 
SURGENOR, DOUGLAS M., Dean of Medical School, Chairman of Biochemistry, State University 

of New York at Buffalo 

SzAB6, GEORGE, Assistant Professor of Anatomy, Harvard Medical School 
SZEXT-GYORGYI, ALBERT. Director and Chief Investigator, Institute for Muscle Research, 

Marine Biological Laboratory 

TAKATA, MITSURU, Assistant Professor of Physiology, Duke University 
TAKENAKA, TOSIIIKUMI, National Institutes of Health 
TASAKI, ICHIJI, Acting Chief, Laboratory of Neurobiology, NIMH, National Institutes of 

Health 
TAYLOR, PETER, Postdoctoral Fellow, Systematics-Ecology Program, National Science 

Foundation 
TAYLOR, ROBERT E., Associate Chief, Biophysics Laboratory, NINDB, National Institutes of 

Health 
TAYLOR, WALTER ROWLAND, Assistant Professor of Oceanography, The Johns Hopkins 

University 

TENCER, RENEE, Assistant, University of Brussels, Brussels 
TILNEY, LEWIS G., Postdoctoral Fellow, Harvard University 
TORCH, REUBEN, Associate Professor of Zoology, University of Vermont 
TRAVIS, DAVID M., Assistant Professor of Pharmacology & Therapeutics, University of Florida 

College of Medicine 

TRINKAUS, J. P., Professor of Biology, Yale University 
TROLL, WALTER, Associate Professor, New York University Medical Center, Institute of 

Industrial Medicine 

TWEEDELL, KENYON S., Associate Professor of Biology, University of Notre Dame 
USHERWOOD, PETER N. R., Research Associate, Columbia University, College of Physicians 

and Surgeons 

VAN HOLDE, K. E., Associate Professor of Chemistry, University of Illinois 
VAN VUNAKIS, HELEN, Associate Professor of Biochemistry, Brandeis University 
\Y.\i.D, GEORGE, Professor of Biology, Harvard University 

WALLACE, ROBIN A., Research Associate, Biology Division, Oak Ridge National Laboratory 
WALSH, GERALD EDWARD, Postdoctoral Research Associate, Systematics-Ecology Program, 

National Science Foundation 
WATKINS, DUDLEY T., Research Fellow, Department of Anatomy, Western Reserve Medical 

School 

:, If. MARGUERITE, Associate Professor, Research Assistant, Gouchcr College and North- 
western University 
WEISS, LEON, Associate Professor of Anatomy, The Johns Hopkins University School of 

Medicine 

WICHTERMAN, RALPH, Professor of Biology, Temple University 
WILCE, R. T., Assistant Professor of Botany, University of Massachusetts 
WILSON, WALTER L., Associate Professor of Physiology and Biophysics, University of Vermont 

College of Medicine 
WINTERS, ROBERT W., Professor of Pediatrics & Career Scientist, Columbia University, College 

of Physicians and Surgeons 

WYTTENBACII, CHARLES R., Assistant Professor of Anatomy, University of Chicago 
ZIGMAN, S., Instructor in Biochemistry, University of Rochester 
ZIMMERMAN, ARTHUR M., Assistant Professor of Pharmacology, State University of New 

York, Downstate Medical Center 
/i i i.o, VICTOR A., Assistant Director, Resident Systematist. Systematics-Ecology Program, 

Ford Foundation 



REPORT OF THE DIRECTOR 29 

Lalor Fellows, 1964 

RICHARD ALAN BEATTY, Senior Fellow, Agricultural Research Council Unit of Animal 

Genetics, United Kingdom 
JOSEPH M. BRANHAM, Oglethorpe University 
YUKIO HIRAMOTO, Misaki Marine Biological Station, Japan 
CHARLES W. HUVER, University of Illinois 
RACHELE MAGGIO, University of Palermo, Italy 
HIDEO MOHRI, University of Tokyo, Japan 
ROBERT W. WINTERS, Columbia University, College of Physicians and Surgeons 

Lillie Fellow, 1964 
E. HADORN, Der Universitat Zurich, Switzerland 

Grass Fellows, 1964 

NEIL DAVIDSON, State University of New York, Downstate Medical Center, Brooklyn 

WILLIAM H. EVOY, University of Oregon 

HORACIO A. GARCIA, Columbia University, College of Physicians and Surgeons 

MAXIMO GIMENEZ, Columbia University, College of Physicians and Surgeons 

RICARDO MILEDI, Forbes Lecturer, University College, London 

CLARKE ROTHWELL SLATER, University College, London 

Research Assistants, 1964 

ACQUAVIVA, PATRICIA ANN, Seton Hill College 

ALTSHULER, BERNARD, New York University Medical Center 

ANONELLIS, BLENDA C., Western Reserve University 

APICELLA, JAMES V., University of Pittsburgh 

ARDWIN, LINDSAY, S., Columbia University ^ 

ARMSTRONG, JUDY, Western Reserve University 

ARONSON, WENDY S., Vassar College 

ASHWORTH, JOHN MICHAEL, Leicester University, England 

BAIRD, SPENCER L., Institute for Muscle Research 

BARNHILL, ROBERT, Capitol Radio Engineering Institute 

BERRIEN, JUDI, Princeton University 

BIKLE, DANIEL, Harvard University 

BLAIR, MARION H., McMaster University, Canada 

BLUMENTHAL, DANIEL S., Oberlin College 

BOLLINGER, M. SUSAN, University of Maryland 

BRADY, FRANCINE, Syracuse University 

BREVER, ANTHONY CARL, Princeton University 

BRUNGARD, JOANNE, Harvard Medical School 

BURGER, RICHARD, Princeton University 

GARDEN, GEORGE ALEXANDER, III, Columbia University, College of Physicians and Surgeons 

CHAFFEE, RICHARD B., JR., Syracuse University 

CHAGNON, SUZANNE, University of Vermont 

CHANY, AMOS HWEI-CHEH, Columbia University 

CHASIS, JOEL ANNE, New York University School of Medicine 

GROUSE, FRANCES W., Biologische Anstalt Helgoland 

DANIELS, CHARLES, Duke University 

DAVIDSON, NEIL, State University of New York 

DE LUCA, MARLENE, The Johns Hopkins University 

DIMINO, PATRICIA, Columbia University 

DOANE, MARSHALL G., University of Maryland School of Aledicine 

DUMONT, JAMES N., University of Massachusetts 

EISENBERG, HENRY W., Columbia University 

FEDOROFF, NINA, Syracuse University 



30 MARINE BIOLOGICAL LABORATORY 

FISHER, EI.I.EN D. B., Columbia University 

FITZJARRELL, AUSTIN T., Tulane University 

FORAN, ELIZABETH H., Smith College 

FREEMAN, SUSAN G., Columbia University 

Fu, KAREN, Columbia University, College of Physicians and Surgeons 

GALTON, VIRGINIA, Harvard Medical School 

GEBELEIN, CONRAD DENNIS, The Johns Hopkins University 

GEDMINTAS, DANA, University of Chicago 

GOTTDIENER, DONNA, Vassar College 

GRAMSS, ELISE, Institute for Muscle Research 

HARRIS, EDWARD M., Duke University 

HARVEY, JENETTE, Johns Hopkins School of Medicine 

HECHTER, MICHAEL, Columbia University 

HECKMAN, JAMES D., Princeton University 

HEGAB, EL-SAYED, Tulane University 

HITCHCOCK, SUSAN M., Columbia University, College of Physicians and Surgeons 

HODES, BARTON L., Jefferson Medical College 

JAFFEE, STEPHEN, New York University School of Medicine 

JOHNSON, KURT E., The Johns Hopkins University 

KAUFMAN, ROBERT G., Columbia University 

KEHLENBECK, EDNA, Syracuse University 

KILEJIAN, ARAXIE, Rice University 

KIMBALL, FRANCES, Reed College 

KIRSCHBERG, GORDON J., Columbia University, College of Physicians and Surgeons 

LARSEN, WILLIAM J., Wesleyan University 

LESTER, GORDON JAMES, University of Minnesota 

LEVIXE, MARILYN, Western Reserve University 

MACNAMARA, GAEL R., Columbia University, College of Physicians and Surgeons 

MAZIA, JUDITH ANN, University of Chicago 

McGiLVRAY, JEAN MARIE, 'Dartmouth Medical School 

McENANEY, BARBARA, Marquette University 

MEISMER, DONALD M., University of Cincinnati 

MILLER, SANDRA M., University of Maryland 

MITTENTHAL, JAY E., The Johns Hopkins University 

MOHL, ROBERT L., Hahnemann Medical College 

MOSSER, JERRY L., Indiana University and The Rockefeller Institute 

MUNDAY, JOHN C., University of Illinois 

MUNRO, GEORGE F., University of Rochester 

MUNRO, JUDITH L., University of Rochester 

MURPHY, ANNE M., University of Maryland 

NEWMAN, BROOKE, Institute for Muscle Research 

OLMSTED, CHARLES E., University of Chicago 

P AIM RE, MARVE, State University of New York, Downstatc Medical Center 

PANNY, SUSAN R., Columbia University 

PAWELEK, JOHN M., Brown University 

POWERS, EARL G., University of Cincinnati 

RASMUSSEN, LEIF, Carlsberg Foundation 

RAVITZ, MELVYN J., University of Vermont 

RAY, PATRICIA, Seton Hill College 

REALE, VINCENT F., Princeton University 

RICHMOND, ARTHUR P., Single Cell Research Foundation, Inc. 

ROBERTSON, C. W., American Museum of Natural History 

ROSENBLUTH, RAJA, Institute for Muscle Research 

SANDER, GRETA, Princeton University 

SCHACHTER, MsRi, Columbia University 

SETLOW, PETER, Brandeis University 

SINDELAR, WILLIAM, Western Reserve University 

SLOANE, ELEANORE M., Mellon Institute 



REPORT OF THE DIRECTOR 31 

SLOANE, MOLLA R., Wellesley College 

THOMAS, JUNE M., University of California, Los Angeles 

TOBEY, JOHN H., Maine Vocational Technical Institute 

TRAVIS, JEANNE D., University of Florida 

TRENHAFT, PAUL STEVEN, Oberlin College 

TSUKIDATE, JIUNICHI, Raskins Laboratories 

TUCKER, ROBERT W., Dartmouth Medical School 

TURNER, VILIA G., University of California, Los Angeles 

TUTUNJIAN, JEAN, Columbia University, College of Physicians and Surgeons 

UEHARA, MARGARET H., University of Hawaii 

VAN PRAAG, DINA, New York University 

VASQUEZ, CARMEN, University of Michigan 

WALDBAUM, MARK, Hahnemann Medical College 

WASSERMAN, ELEANOR, Brandeis University 

WEINER, BEVERLY, Harvard University 

WILSON, LOUISE P., Wellesley College 

YUYAMA, SHUHEI, Western Reserve University 

ZOLLINGER, WILLIAM K., JR., University of Pittsburgh Medical School 

Library Readers, 1964 

ATWOOD, KIMBALL C, Professor of Microbiology, University of Illinois 
BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School 
BERNE, ROBERT M., Professor of Physiology, Western Reserve University 
BRIDGMAN, ANNA JOSEPHINE, Chairman and Professor of Biology, Agnes Scott College 
BUTLER, ELMER G., Osborn Professor of Biology, Princeton University 
CARBON, JOHN A., Research Associate, Department of Biochemistry, Abbott Laboratories 
CHASE, AURIN M., Professor of Biology, Princeton University 
CLARK, ARNOLD M., Professor of Biology, University of Delaware 

COHEN, SEYMOUR S., Chairman, Department of Therapeutic Research, University of Penn- 
sylvania School of Medicine 
DAVIS, BERNARD D., Head, Department of Bacteriology and Immunology, Harvard Medical 

School 

EDER, HOWARD A., Professor of Medicine, Albert Einstein College of Medicine 
GABRIEL, MORDECAI L., Professor of Biology, Brooklyn College 
GINSBERG, HAROLD S., Chairman, Department of Microbiology, University of Pennsylvania 

School of Medicine 

GREEN, JAMES W., Professor of Physiology, Rutgers University 
HANDLER, PHILIP, Professor of Biochemistry, Duke University 
HESSLER, ANITA YOUNG, Marine Biological Laboratory 

HODES, ROBERT, Research Associate, Department of Neurophysiology, The Mount Sinai Hospital 
ISSELBACKER, KURT J., Chief, Gastrointestinal Unit, Massachusetts General Hospital and 

Assistant Professor of Medicine, Harvard Medical School 

JACOBS, M. H., Emeritus Professor of General Physiology, University of Pennsylvania 
KALTENBACH, JANE COUFFER, Assistant Professor of Zoology, Mount Holyoke College 
KASHA, MICHAEL, Director, Institute of Molecular Biophysics, Florida State University 
KLEIN, MORTON, Professor of Immunology, Temple University Medical School 
LEIGHTON, JOSEPH, Professor of Pathology, University of Pennsylvania School of Medicine 
LEVINE, RACHMIEL, Chairman, Department of Medicine, New York Medical School 
LEVINTHAL, CYRUS, Professor of Biophysics, Massachusetts Institute of Technology 
LINEAWEAVER, THOMAS H., Ill, Marine Biological Laboratory 
MARKS, PAUL A., Associate Professor of Medicine, Columbia University, College of Physicians 

and Surgeons 
MATEYKO, GLADYS MARY, Associate Professor of Biology, Washington Square College, New 

York University 

MEYER, KARL, Professor of Biochemistry, Columbia University 
MOULTON, JAMES M., Associate Professor of Biology, Bowdoin College 



MA K I XI-. BIOLOGICAL LABORATORY 

NASON, ALVIN, Professor of Biology, Associate Director. McCollum-Pratt Institute, The 

Johns Hopkins University 

NEEDLEMAN, SAUL B., Senior Research Biochemist, Abbott Laboratories 
XIIVIKOKF, ALEX B., Research Professor, Albert Einstein College of Medicine 
XmvoTXY, ALOIS H., Professor of Immunochemistry, Temple University School of Medicine 
OVERTON, JANE H., Associate Professorial Lecturer in Biology, University of Chicago 
RAPPORT, MAURICE M., Professor of Biochemistry, Albert Einstein College of Medicine 
ROWLAND, LEWIS P., Associate Professor of Neurology, Columbia University, College of 

Physicians and Surgeons 

RUSSELL, HENRY D., Museum of Comparative Zoology, Harvard University 
SPIEGEL, MELVIX, Associate Professor of Biology, Dartmouth College 
SPRAGUE JAMES M., Professor of Anatomy, University of Pennsylvania 
STETTEN, MARJORIE R., Research Professor, Rutgers Medical School 

SUDAK, FREDERICK N., Assistant Professor of Physiology, Albert Einstein College of Medicine 
S \VANSON, CARL P., William D. Gill Professor of Biology, The Johns Hopkins University 
SZENT-GYORGYI, Andrew G., Professor of Biophysics, Dartmouth Medical School 
WAINIO, WALTER, Professor of Biochemistry, Rutgers The State University of New Jersey 
WARNER, ROBERT C., Professor of Biochemistry, New York University School of Medicine 
WHEELER, GEORGE E., Associate Professor of Biology, Brooklyn College 
WILSON, THOMAS HASTINGS, Associate Professor of Physiology, Harvard Medical School 
YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate 

Medical Center 

ZACKS, SUMNER I., Assistant Professor of Neuropathology, Pennsylvania Hospital, Uni- 
versity of Pennsylvania 
ZORZOLI, ANITA, Chairman, Professor of Physiology, Vassar College 

Students, 1964 

All students listed completed the formal course program, June 17-July 27. Asterisk 
indicates students completing Post-Course Research Program, July 28-August 31. 

ECOLOGY 

*ADAMSON, JEAN M., Allegheny College 

ALLESSIO, MARY L., University of Colorado 

AVERY, PATRICIA P., Wheaton College 
*BARVENIK, FRANK W., University of Connecticut 
*BUCHSBAUM, VICKI M., Stanford University 
*CALDER, WILLIAM ALEXANDER, JR., Duke University 

GJESSING, HELEN WITTON. College of the Virgin Islands 
*HEATFIELD, BARRY MARK, University of California 

JONES, MEREDITH HOWE, Boston University 

JONES, NANCY GALE, Oberlin College 

KOETZER, KENNETH L., University of Rhode Island 
*LLOYD, MARGARET C., Bryn Mawr Colic- r 

MAYO, CHARLES A., Ill, Dartmouth College 

OrixN, SISTER GENEVIEVE, Catholic University of America 
*REA, INA K., Indiana University 
*RICHARDSON, W. NORMAN, Earlham College 

WHITE, JOSEPH JAMES, University of Illinois 

EMBRYOLOGY 

BARIL, EARL FRANCIS, University of Connecticut 

BERRILL, MICHAEL, McGill University 
*CONNEI.L, CAROLYN, Indiana University 
*DicK, MIRIAM, Brandeis University 



RKI'OKT OF Til 1C DIRKCTOU 

*GAEDE, LnRov LEWIS, Rensselaer Polytechnic Institute 
*GOLDIZEN, VERNON CLAIRE, Western Reserve University 
''GOULD, MEREDITH C., Stanford University 

HAYASHI, MASAKI, University of Illinois 
*HEIDGER, PAUL ML CLAY, JR., Tulane University 
*HELD, WILLIAM ALLEN, Marquette University 

INSELBURG, JOSEPH, Brandeis University 

KAPLAN, STANLEY, University of Miami 

*Kopp, LOWELL ELLIS, Massachusetts Institute of Technology 
*LARSEN, LYNDELL LOUISE, The Rockefeller Institute 

MORTENSEN, RICHARD, Purdue University 
*PERCUS, JEROME KENNETH, New York University 

PRINGLE, JOHN ROBERT, Harvard University 
*READ, MARGARET TYLER, Harvard University 
*REIGART, JOHN ROUTT, II, Dartmouth Medical School 

ROGERS, MARY ELIZABETH, Yale University 

BOTANY 

BURG, CAROL ANN, University of Connecticut 
*BYTNAR, PATRICIA ANN, Seton Hill College 

CONNER, CLARICE MARIE, University of Wisconsin 
*HOLT, BUFORD REID, University of Tennessee 
*HOWELL, STEPHEN H., The Johns Hopkins University 

KEVIN, SISTER M. PETRA, Fordham University 

KIES, ROBERT LUDWIG, University of Erlangen, Germany 

KOCHERT, GARY DEAN, Indiana University 
*LEE, THOMAS F., Clark University 

McLEAN, ROBERT J., University of Connecticut 
*PRINCE, JEFFREY S., University of Massachusetts 
*RAMUS, JOSEPH STEPHEN, University of California, Berkeley 

SMITH, JOYCE EILEEN, Cornell University 

STROTHER, JOHN LANCE, Washington University, St. Louis 
*TRERICE, ELIZABETH MABEL, Dalhousie University, Halifax, Nova Scotia 
*URBAN, PAUL, Tufts University 

WAER, RICHARD DENNIS, University of Arizona 
*WEBER, JILL LOUISE, Vassar College 
*WiLcox, ROBERT STIMSON, University of Michigan 

PHYSIOLOGY 

*BARBOUR, STEPHEN DAVID, Temple University 

*BIBER, MICHAEL PETER, University of Chicago Medical School 

*CAROLAN, ROBERT MILLS, Dartmouth Medical School 

*CRAIG, NESSLY COILE, University of Pennsylvania 

^CONVERSE, CAROLYN ANN, Brown University 

*ELFBAUM, STANLEY GOODMAN, Northwestern University 

*ETZLER, MARILYNN EDITH, Washington University, St. Louis 
GAZITH, JOSEPH, Vanderbilt University 
GIBERMAN, ELDAD, Massachusetts Institute of Technology 

*GOLD, LAWRENCE MARSHALL, University of Connecticut 

*HATLING, DONNA LYNNE, Columbia University 

*HAUSCHKA, PETER VOORHESS, Amherst College 
HAYTLER, PETER G., E. I. duPont de Nemours & Company 

*JURAS, DANUTE, Marquette University 

*KUBAI, DONNA FAROLINO, University of Wisconsin 

*LATTMANN, EATON EDWARD, The Johns Hopkins University 



MARINE BIOLOGICAL LABORATORY 

*LLOYD, DAVID ALBERT, University of Illinois 

*MANDEL, MORTON, Stanford University School of Medicine 

*NICHOLSON, ANNE, University of Pennsylvania School of Medicine 
DERAZUMNEY, CKLIA ESTER FREDA, University of Pennsylvania School of Medicine 
ROTHEN STEIN, ARTHUR STANLEY, Rutgers, The State University 

*SPARKS, HARVEY V., Harvard Medical School 
SWITZER, SAM, Albert Einstein College of Medicine 

*TERANDO, SISTER MARY LORETTA, Saint Louis University 

*TERRELL, KATHLEEN Lois, Columbia University, College of Physicians and Surgeons 

*\VARD, JOHN CLIVE, The Johns Hopkins University 

*\\'ARD, RAYMOND LELAND, University of California, Livermure 

*WECHSLER, JAMES ALAN, Yale University 

*WEINBERG, ERIC S., The Rockefeller Institute 

*WHITE, ERIC S., Dartmouth Medical School 

INVERTEBRATE ZOOLOGY 

ALLEN, JEFFREY CHARLES, Oberlin College 

APPLEBAUM, RICHARD, Columbia University 

BARTIZAL, FREDERICK JOSEPH, Beloit College 

BENNETT, JUDITH ANN, Syracuse University 

BOLENDER, ROBERT PAUL, Columbia University 

BOYD, CARL M., Dalhousie University, Halifax, Nova Scotia 

CHANE, PAULA FRANCES, Tulane University 

COGGESHALL, RICHARD EDWIN, Harvard Medical School 

COTMAN, CARL WAYNE, Wesleyan University 

DENNAKER, GERMAINE SUZANNE, Morgan State College 

FISCHER, BARBARA ANN, St. Louis University 

HALL, BARBARA SUE, College of St. Mary of the Springs 

HINE, CHARLES RISK, Lafayette College 

HUNTER, WILLIAM BRUCE, University of California, Santa Barbara 

JAMPOL, LEE MERRILL, Yale University 

JOHNSON, KURT EDWARD, The Johns Hopkins University 

KAUFMAN, ROBERT GORDON, Columbia University 

KOERING, MARILYN J., University of Wisconsin 

Koo, HELEN PING-CHING, University of Minnesota 

LANGRETH, SUSAN GRANT, University of Chicago 

MEADOWS, ROBERT T., Syracuse University 

NOLLEN, PAUL MARION, Purdue LIniversity 

NUTT, JOHN GORDON, JR., Rice University 

PAGE, CHARLES HENRY, Yale University 

PAWALEK, JOHN MASON, Brown University 

PETTIT, BARBARA, Marquette University 

REUSS, CECILIA MONICA, Marquette University 

ROBINSON, CAROLYN ANNE, Clark University 

RUNDLES, CHARLOTTE, Duke University 

STINE, DEBORAH CLARE, Wilson College 

TOTH, STEVEN EDWARD, Bowling Green State University 

TRACY, SUSAN FRANCES, University of Massachusetts 

WALCOTT, BENJAMIN, University of Oregon 

WALDRON, INGRID LORE, University of California, Berkeley 

WALTER, MARY A., Ripon College 

WALTERS, DAVID ROYAL, Harvard University 

WARD, OSCAR GARDIEN, JR., Purdue University 

;ER, BETSKV ANN, Drew University 
WHISNANT, BETTY LYNN, Duke University 
ZEIMEN, SISTER MARIA GORETTI, Catholic University of America 



REPORT OF THE DIRECTOR 



35 



4. FELLOWSHIPS AND SCHOLARSHIPS, 1964 

Lucretia Crocker Scholarship : 

VICKI M. BUCHSBAUM, Ecology Course 
BUFORD R. HOLT, Botany Course 

Edwin Linton Memorial Endowment of the 
Washington and Jefferson College: 

PATRICIA ANN BYTNAR, Botany Course 

Turtox Scholarship Fund : 

JOHN BUSHNELL 

5. TRAINING PROGRAMS 
FERTILIZATION AND GAMETE PHYSIOLOGY TRAINING PROGRAM 



I. INSTRUCTORS 

C. B. METZ 

C. R. AUSTIN 
JOHN BIGGERS 
ALBERTO MONROY 
LEONARD NELSON 

II. ASSISTANTS 
RACHELE MAGGIO 

III. STUDENTS 

R. BERKELEY 
J. F. FALLON 
L. E. FRANKLIN 
M. S. GOROVSKY 
R. HALLBERG 
G. S. HAND, JR. 
S. HAUSCHKA 

D. L. HESSEL 
B. HORWITZ 
M. R. LURIE 

D. MOORE 

M. C. REPORTER 
N. M. SCHULKIND 
A. E. S. SMITH 

E. L. STERN 
D. T. SULLIVAN 

IV. LECTURES 

D. SZOLLOSI 
R. A. BEATTY 
R. MAGGIO 

H. MOHRI 

P. M. BHARGAVE 

Y. HIRAMOTO 

R. RlKMENSPOEL 

R. YANAGIMACHI 
R. C. RUSTAD 
L. WEISS 



Florida State University, in charge of program 
Cambridge University, England 
University of Pennsylvania 
University of Palermo, Italy 
Emory University 



University of Palermo, Italy 



University of Pennsylvania 

Marquette University 

Florida State University 

University of Chicago 

The Johns Hopkins University 

University of North Carolina 

The Johns Hopkins University 

The Johns Hopkins University 

Emory University 

University of Miami 

University of Pennsylvania 

Carnegie Institution of Washington 

New York University, School of Medicine 

California Institute of Technology 

University of Chicago 

The Johns Hopkins University 



Ultrastructural Studies on Fertilization and the Gametes 

Genetic Effect on Gametes 

Activation of Protein Synthesis in the Sea 

Urchin Egg at Fertilization 

Mitochondrial Functions of Bull Spermatozoa 

Ribonucleic Acid and the Amino Acid 

Incorporation in Spermatozoa 

Mechanical Properties of the Protoplasm of the Sea Urchin 

Biophysical Approaches to Problems of Spermatozoan Motility 

The Hamster as a Material for the Study of Fertilization 

Radiation Effects in Sea Urchin Gametes 

Interactions Between Cells Making Contact 



36 



MARINE BIOLOGICAL LABORATORY 



I. INSTRUCTORS 

S. W. KUFFLER 
E. J. FURSHPAN 
J. G. NlCHOLLS 

II. ASSISTANTS 



NEUROl'lIVSIOLOGY TRAINING PROGRAM 



Harvard Medical School, in charge of program 
Harvard Medical School 
Harvard Medical School 



R. Bosler Harvard Medical School 

(No lectures given only seminars) 



III. STUDENTS 

J. M. CAMHI 

A. M. FRIEDLANDER 
J. HARVEY 

J. S. McREYNOLDS 

R. PIPKIN 
P. STERLING 

B. WlCKELGREN 



I. INSTRUCTORS 

L. KLEINHOLZ 
B. C. ABBOTT 

A. JANOFF 
G. KALEY 

B. ZWEIFACH 



Harvard Medical School 

University of Pittsburgh 

The Johns Hopkins University 

Harvard Medical School 

Harvard Medical School 

Western Reserve University 

Massachusetts Institute of Technology 

COMPARATIVE PHYSIOLOGY TRAINING PROGRAM 



Reed College, in charge of program 
University of Illinois 
New York University 
New York University 
New York University 



II. ASSISTANTS 
F. KIMBALL 

J. C. MUNDAY 



Reed College 
University of Illinois 



III. STUDENTS 
E. A. ASHBY 

G. M. CONNELL 
G. A. COTTRELL 

P. J. DOWD 
C. R. JONES 
W. R. KEM 
M. J. PAR 
P. STERN 



University of Texas 
Indiana University 
Harvard University 
Vestibular Laboratory 
Fordham University 
University of Illinois 
LIniversity of Minnesota 
University of Michigan 



IV. LECTURES 

L. KLEINHOLZ 
G. KALEY 

A. JANOFF 

B. C. ABBOTT 

F. A. BROWN, JR. 

C. READ 
R. ALLEN 

G. COTTRELL 



Ncurosecretion and Endocrine Physiology 

Cardiovascular Physiology 

Comparative Aspects of Lysosome Function & Comparative Aspects 

of Leucocyte Physiology 

K\ citation-Contraction Coupling and Relaxing Factor in Muscle 
A Unified Clock Theory 

Comparative Aspects of Membrane Transport 
Cell Movement 
Binding of Biologically-Active Substances 



REPORT OF THE DIRECTOR 



37 



6. TABULAR VIEW OF ATTENDANCE, 1960-1964 



1960 

INVESTIGATORS TOTAL 458 

Independent 273 

Library Readers 50 

Research Assistants 135 

STUDENTS TOTAL 122 

Invertebrate Zoology 49 

Embryology 20 

Physiology 28 

Botany 18 

Ecology 13 

TRAINEES TOTAL 

Nerve-Muscle 

Comparative Physiology 

Fertilization and Gamete 

TOTAL ATTENDANCE 580 

Less persons represented in two categories 2 



1961 

458 

256 

49 

151 

130 
40 
21 
28 
19 
22 



1962 

494 

279 

56 

159 

121 

38 

20 

28 

20 

15 



1963 1964 

490 512 

261 273 

51 47 

178 192 



124 
40 
20 
28 
20 
16 



578 
INSTITUTIONS REPRESENTED TOTAL , 144 



By Investigators 



83 



By Students 61 

SCHOOLS AND ACADEMIES REPRESENTED 

By Investigators 5 

By Students 2 

FOREIGN INSTITUTIONS REPRESENTED 14 

By Investigators 11 

By Students 3 



586 
1 

585 

132 

107 

70 



3 


28 
21 

7 



615 

4 

611 

118 

81 
57 



3 
2 

31 
17 
14 



614 

5 



7. INSTITUTIONS REPRESENTED, 1964 



Abbott Laboratories 

Agnes Scott College 

Albert Einstein Medical School 

Allegheny College 

American Museum of Natural History 

Amherst College 

Argonne National Laboratory 

Arizona, University of 

Beloit College 

Boston University 

Bowdoin College 

Bowling Green State University 

Brandeis University 

Brooklyn College 

Brown University 

California, University of, Los Angeles 

California, University of, Berkeley 

California, University of, Livermore 

California, University of, Santa Barbara 

Capitol Radio Engineering Institute 

Carnegie Institution of Washington 

Catholic University of America 

Chicago, University of, Medical School 

Chicago, University of 



Cincinnati, University of 

Cornell University 

Cornell University, Medical College 

Dartmouth College 

Dartmouth Medical School 

Delaware, University of 

Drew University 

Duke University 

duPont de Nemours & Company 

Earlham College 

Emory University 

Florida State University 

Florida, University of 

Fordham University 

Goucher College 

Hahnemann Medical School 

Harvard University 

Harvard University Medical School 

Haskins Laboratories 

Hawaii, University of 

Illinois, University of 

Indiana University 

Institute for Muscle Research 

Iowa State University 



126 
40 
20 
30 
19 
17 

30 
7 
7 

16 

668 

7 



609 661 

120 140 

83 117 

73 23 

4 



21 32 

15 28 

6 4 



38 



MAKIXK BIOLOGICAL LABORATORY 



Jefferson Medical College 
Johns Hopkins University, The 
Johns Hopkins University School of 

Medicine, The 
Lafayette College 
Lerner Marine Laboratory, of the American 

Museum of Natural History 
Maine Vocational Technical Institute 
Marquette University 
Maryland, University of 
Massachusetts General Hospital 
Massachusetts Institute of Technology 
Massachusetts, University of 
Mellon Institute 
Miami, University of 
Michigan, University of 
Minnesota, University of 
Missouri, University of, Medical School 
Morgan State College 
Mount Holyoke College 
Mount Sinai Hospital, The 
National Institutes of Health 
New York State University, College of 

Medicine at Brooklyn 
New York State University, College of 

Medicine at Syracuse 
New York University, Bellevue Medical 

Center 

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

College 
North Carolina State of the University of 

North Carolina at Raleigh 
North Carolina, University of 
Northwestern University 
Notre Dame, University of 
Oak Ridge National Laboratory 
Oberlin College 
Oglethorpe University 

Oregon Regional Primate Research Center 
Oregon State University 
Oregon, University of 
Pembroke College 
Pennsylvania, University of 



Pennsylvania Medical School, University of 

Pittsburgh, University of 

Princeton University 

Purdue University 

Queens College 

Reed College 

Rensselaer Polytechnic Institute 

Rhode Island, University of 

Rice University 

Ripon College 

Rochester, University of, School of Medicine 

and Dentistry 
Rockefeller Institute, The 
Russell Sage College 
Rutgers, The State University 
Saint Louis University 
Seton Hill College 

Single Cell Research Foundation, Inc. 
Smith College 
South Florida College 
Stanford University 

Stanford University, School of Medicine 
State University of New York at Buffalo 
Swarthmore College 
Syracuse University 
Temple University 
Tennesee, University of 
Tufts University 
Tulane University 
Vassar College 
Vermont, University of 
Veterans Administration Hospital 
Vanderbilt University 
Virginia, University of 
Washington University, at St. Louis 
Wellesley College 
Wesleyan University 
Western Reserve University 
Western Reserve University, School of 

Medicine 
Wheaton College 
Wilson College 
Wisconsin, University of 
Yale University 



FOREIGN INSTITUTIONS REPRESENTED, 1964 



Agricultural Research Council of Great 

Britain 
Anatomisches Institut, der Universitat Zurich, 

Switzerland 

Biologische Anstalt Helgoland, Germany 
Brussels University, Brussels 
Dalhousie University, Halifax 
Erlangen, University of, Germany 
Gunma University, Japan 
Leicester University, England 



Ljubljana University, Yugoslavia 
Medical Research Council of Great Britain 
McMaster University, Canada 
Pahlavi, University of, Shiraz, Iran 
Queen's University, Ontario, Canada 
Tokyo, University of, Japan 
University College, London, England 
University of Palermo, Italy 
Utrecht, University of, Holland 



REPORT OF THE DIRECTOR 



39 



SUPPORTING INSTITUTIONS, AGENCIES, AND INDIVIDUALS 



Abbott Laboratories 

Associates of the Marine Biological 

Laboratory 

Atomic Energy Commission 
CIBA Corporation 
The Commonwealth Fund 
Josephine B. Crane Foundation 
Dr. William D. Curtis 
The Ford Foundation 
Dr. and Mrs. David W. Gaiser 
The Grass Foundation 
Mr. and Mrs. William H. Greer, Jr. 
Dr. Ethel Browne Harvey 
Mr. and Mrs. George F. Jewett, Jr. 



The Lalor Foundation 

Mrs. Grace T. Mast 

Olin Matheson Charitable Trust 

National Institutes of Health 

National Science Foundation 

Office of Naval Research 

The Rockefeller Foundation 

Schering Foundation, Inc. 

Scientific American, Inc. 

Mr. Gerard Swope, Jr. 

The Upjohn Company 

Wallace Laboratories 

Mr. James H. Wickersham 



8. FRIDAY EVENING LECTURES, 1964 

July 3 

HANS LEO KORNBERG Anaplerotic Sequences in Microbial Metabo- 

The University of Leicester lism : Their Significance and Regulation 

July 9, Thursday 

RICARDO MILEDI Localization of Acetylcholine-Receptors and 

University College, London Cholinesterase in Muscle Fibres, Part I 

Alexander Forbes Lecturer at the MBL 
July 10 

RICARDO MILEDI Localization of Acetylcholine-Receptors and 

Cholinesterase in Muscle Fibres, Part II 
July 17 

IRWIN R. KONIGSBERG Clonal Analysis of Myogenesis 

Carnegie Institution of Washington 
July 24 

SIDNEY W. Fox Experimental Geosynthesis and a Theory of 

The Florida State University Cellular Origins 

July 31 

MELBOURNE R. CARRIKER Hard Tissue Destruction by Marine Predatory 

MBL Gastropods 

August 7 

SOL SPIEGELMAN The Transcription and Translation of Genetic 

University of Illinois Messages 

August 14 

R. ALAN BEATTY The Gamete as a Microorganism 

University of Edinburgh 

Senior Lalor Fellow at the MBL 
August 21 

ERNST HADORN New Patterns of Differentiation Arising in 

University of Zurich Permanent Cultures of Drosophila Cells in 

F. R. Lillie Fellow at the MBL vivo 

August 28 

KENNETH D. ROEDER What a Moth's Ear Tells its Nervous System 

Tufts University about Bats 



40 MARINE BIOLOGICAL LABORATORY 

9. TUESDAY EVENING SEMINARS, 1964 
July 7 

H. SCHUEL Isolation of Muscle-Relaxing Particles with 

L. LORAND the Zonal Centrifuges 

R. SCHUEL 

J. S. NAGLE Differential Sorting of Shells in the Swash 

Zone 

C. C. SPEIDEL Deviations in Motility of Developing Sea 

R. H. CHENEY Urchins Induced by Irradiation (film) 

July 14 

J. COHEN The Transfer of Melanin Granules from Me- 

G. SZABO lanocytes into Malpighian cells of the Mam- 

malian Epidermis (illustrated with timelapse 
photography) 

S. KARASAKI The Sites of Nuclear RNA Synthesis during 

Amphibian Embryogenesis 

R. ALAN BEATTY Density Gradient Media for Spermatozoa 

July 21 

S. HIGASHINO Analysis of Biological Excitable Membrane by 

Means of Voltage-Current-Time Characteristics 

A. M. ZIMMERMAN Further Studies on Incorporation of H 3 Thymi- 

L. SILBERMAN dine in Arbacia Eggs Under Hydrostatic Pres- 

sure 

D. MARSLAND High Pressure Reversal of the Anti-Mitotic 

Effects of Heavy Water (D O) 
July 28 

A. JANOFF Production of Inflammatory Changes in the 

B. ZWEIFACH Micro-Circulation by Cationic Proteins Ex- 

tracted from Lysosomes 

H. SATO Condensation of the Sperm Nucleus and Orien- 

S. INOUE tation of DNA Molecules during Spermiogene- 

sis in Loligo pealii 
P. PERSON Cartilage in a Marine Polychaete Eudistylia 

August 4 

W. E. LOVE Microheterogeneity in the Hemoglobin from 

Individual Sea Lampreys 
Y. HIRAMOTO Further Studies on the Cell Division without 

Mitotic Apparatus in Sea Urchin Eggs 
A. B. NOVIKOFF GERL, its Form and Function in Neurons of 

Rat Spinal Ganglia 
August 1 1 

D. BOWNDS The Reaction of Rhodopsin and its Derivative 

G. WALD with Sodium Borohydride 

S. ZIGMAN \ Cold-Precipitable Protein in the Dogfish 

S. LERMAN Lens 

August 18 

\Y. AUCLAIR On the Chromosome Number of Arbacia 

V. ZULLO Re-evaluation of the Late Cenozoic Cirriped, 

"Tamiosoma" Conrad 

R. PARKER Preliminary Quantitative Study of Small-Scale 

A. DRISCOLL Environmental and Faunal Variability 

J. S. NAGLE 
K. LUKAS 



REPORT OF THE DIRECTOR 41 

10. MEMBERS OF THE CORPORATION, 1964 



Life Members 

ADOLPH, DR. EDWARD F., University of Rochester, School of Medicine & Dentistry, 

Rochester, New York 

BRODIE, MR. DONALD, 522 Fifth Avenue, New York 18, New York 
COLE, DR. ELBERT C., 2 Chipman Park, Middlebury, Vermont 
COWDRY, DR. E. V., 4580 Scott Avenue, St. Louis 10, Missouri 
CRANE, MRS. W. MURRAY, 820 Fifth Avenue, New York 21, New York 
HESS, DR. WALTER, 286 North Fairview Avenue, Spartanburg, South Carolina 
HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge 38, 

Massachusetts 

IRVING, LAURENCE, University of Alaska, College, Alaska 

JACOBS, DR. M. H., Department of Physiology, University of Pennsylvania, Phila- 
delphia 4, Pennsylvania 

LOWTHER, DR. FLORENCE, Barnard College, New York 21, New York 
MACDOUGALL, DR. MARY STUART, Mt. Vernon Apartments, 423 Clairmont Ave- 
nue, Decatur, Georgia 

MALONE. DR. E. F., 6610 North llth Street, Philadelphia 26, Pennsylvania 
MEANS. DR. J. H., 15 Chestnut Street, Boston Massachusetts 
MEDES, DR. GRACE, 303 Abington Avenue, Philadelphia 11, Pennsylvania 
MOORE, DR. J. PERCY, RD No. 1, Box 437, Chapel Hill, North Carolina 
PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana 
PLOUGH, DR. H. H., Amherst College, Amherst, Massachusetts 
PORTER, DR. H. C., University of Pennsylvania, Philadelphia 4, Pennsylvania 
SCOTT, DR. ERNEST L., Columbia University, New York 21, New York 
SCHRADER, DR. SALLY, Duke University, Durham, North Carolina 
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., 2768 Egypt Road, Audubon, Pennsylvania 
WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut 

Regular Members 

ABELL, DR. RICHARD G., 55 East 2nd Avenue, New York 28, New York 
ADELBERG, DR. EDWARD A., Department of Microbiology, Yale University, New 

Haven, Connecticut 06520 
ADELMAN, DR. WILLIAM J., Department of Physiology, University of Maryland 

Medical School, Baltimore 1 , Maryland 

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

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

New Jersey 08540 
ALSCHER, DR. RUTH, Department of Physiology, Manhattanville College, Purchase, 

New York 



42 M \KINE BIOLOGIC \1. LABORATORY 

AMATNIEK, DR. ERNEST, 34 Horner Avenue, Hastings-on-the-Hudson, New York 

AMBERSON, DR. WILLIAM R., Katy Hatchs Road, Falmouth, Massachusetts 

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

ANDERSON, DR. RUBERT S., Medical Laboratories, Army Chemical Center, Mary- 
land 

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

ARNOLD, DR. JOHN MILLER, Department of Zoology, Iowa State University, Ames, 
Iowa 50010 

ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory, 
Oak Ridge, Tennessee 37831 

ATWOOD, DR. KIMBALL C, 702 West Pennsylvania Avenue, Urbana, Illinois 

AUCLAIR, DR. WALTER, Department of Biological Sciences, University of Cincin- 
nati, Cincinnati, Ohio 45221 

AUSTIN, DR. COLIN RUSSELL, Delta Regional Primate Research Center, Covington, 
Louisiana 70433 

AUSTIN, DR. MARY L., 506Vo North Indiana Avenue, Bloomington, Indiana 

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

BAITSELL, DR. GEORGE A., Osborn Memorial Laboratories, Yale University, New 
Haven, Connecticut 06520 

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

BALLARD, DR. WILLIAM W., Department of Biological Sciences, Dartmouth College, 
Hanover, New Hampshire 

BALTUS, DR. ELYANE, Laboratoire de Morphologic Animale, 1850 Chaussee de 
Wavre, Bruxelles 16, Belgique 

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

BARD, DR. PHILLIP, The Johns Hopkins Medical School, Baltimore 5, Maryland 

BARTH, DR. L. G., Marine Biological Laboratory, Woods Hole, Massachusetts 
02543 

BARTH. DR. LUCENA, Marine Biological Laboratory, Woods Hole, Massachusetts 
02543 

BARTLETT, DR. JAMKS II., Department of Physics. University of Illinois, Urbana, 
Illinois 

BAUER, DR. ERIC G., Department of Anatomy, University of Minnesota, Minne- 
apolis, Minnesota 

BAYLOR, DR. E. R., Woods Hole Oceanographic Institution, Woods Hole, Massa- 
chusetts 02543 

BAYLOR, DR. MARTHA B., Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 02543 

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

BKCK, DR. L. V., Department of Pharmacology, Indiana University, School of Ex- 
perimental Medicine, Bloomington, Indiana 



REPORT OF THE DIRECTOR 43 

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

BELL, DR. EUGENE, Department of Biology, Massachusetts Institute of Technology, 
Cambridge 39, Massachusetts 

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

BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet 
Briar, Virginia 24595 

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

BERMAN, DR. MONES, National Institutes of Health, Institute of Arthritis & 
Metabolic Diseases, Bethesda, Maryland 20014 

BERNE, DR. ROBERT M., Department of Physiology, Western Reserve University, 
Cleveland 6, Ohio 

BERNHEIMER, DR. ALAN W., New York University College of Medicine, New 
York, New York 10016 

BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne State University 
College of Medicine, Detroit 7, Michigan 

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

BEVELANDER, DR. GERRIT, Dental Branch, Medical Center, University of Texas, 
Houston, Texas 77025 

BIGELOW, DR. HENRY B., Museum of Comparative Zoology, Harvard University, 
Cambridge, Massachusetts 02138 

BISHOP, DR. DAVID W., Department of Embryology, Carnegie Institution of Wash- 
ington, 115 West University Parkway, Baltimore 10, Maryland 

BLANCHARD, DR. K. C., The Johns Hopkins Medical School, Baltimore 5, Mary- 
land 

BLOCK, DR. ROBERT, 518 South 42nd Street, Apt. C-7, Philadelphia 4, Pennsyl- 
vania 

BLUM, DR. HAROLD F., Department of Biology, Princeton University, Princeton, 
New Jersey 08540 

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

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

BOELL, DR. EDGAR J., Osborn Zoological Laboratories, Yale University, New 
Haven, Connecticut 06520 

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

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

BOREI, DR. HANS G., Department of Zoology, University of Pennsylvania, Phila- 
delphia 4, Pennsylvania 

BOWEN, DR. VAUGHN T., Woods Hole Oceanographic Institution, Woods Hole, 
Massachusetts 02543 

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

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



44 MARINE BIOLOGICAL LABORATORY 

BRINLEY, DR. F. J., JR., Department of Physiology, The Johns Hopkins Medical 

School, Baltimore 5, Maryland 
BRONK, DR. DETLEV W., The Rockefeller Institute. (>(>lh 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 60201 
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, Maryland 20014 " 
BULLOCK, DR. T. H., Department of Zoology, University of California, Los Angeles 

24, California 
BURBANCK, DR. WILLIAM D., Emory University, Box 15134, Atlanta, Georgia 

30333 
BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilmington, 

Delaware 

BURKENROAD, DR. M. D., 3169 Bremerton Place, La Jolla, California 92037 
BUTLER, DR. E. G., Department of Biology, Box 704, Princeton University, Prince- 
ton, New Jersey 08540 
CANTONI, DR. GUILLIO, National Institutes of Health, Mental Health, Bethesda, 

Maryland 20014 

CARLSON, DR. FRANCIS D., Department of Biophysics, The Johns Hopkins Uni- 
versity, Baltimore, Maryland 21218 

CARPENTER, DR. RUSSELL L., Tufts University, Medford 55, Massachusetts 
CARRIKER, DR. MELBOURNE R., Systematics-Ecology Program, Marine Biological 

Laboratory, Woods Hole, Massachusetts 02543 
CASE, DR. JAMES, Department of Biological Sciences, University of California, 

Santa Barbara, California 
CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York Avenue, 

New York 21, New York 
CHAET, DR. ALFRED B., Department of Biology, American University, Washington 

16, D. C. 
CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical 

School, Coral Gables, Florida 
CHANG, DR. JOSEPH J., Inst. f. physikal. Chemie an der Techn. Hochscule, Aachen, 

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

New Jersey 08540 
CHENEY, DR. RALPH 11., Biological Laboratory, Brooklyn College, Brooklyn 10, 

New York 
CHILD, DR. FRANK M., Department of Zoology, University of Chicago, Chicago 37, 

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



REPORT OF THE DIRECTOR 45 

CLARK, DR. A. M., Department of Biological Sciences, University of Delaware, 

Newark, Delaware 19711 
CLARK, DR. ELOISE E., Department of Zoology, Columbia University, New York, 

New York 10027 
CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, New 

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

Massachusetts 02138 
CLELAND, DR. RALPH E., Department of Botany, Indiana University, Bloomington, 

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

Georgia 
COHEN, DR. SEYMOUR S., Department of Biochemistry, University of Pennsylvania 

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

Maryland 20014 

COLLETTE, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts 
COLLIER, DR. JACK R., Department of Biology, Rensselaer Polytech. Institute, 

Troy, New York 

COLTON, DR. H. S., Box 699, Flagstaff, Arizona 
COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, Long 

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

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

Hanover, New Hampshire 

COOPERSTEIN, DR. SHERWIN J., School of Dental Medicine, University of Con- 
necticut, Hartford, Connecticut 06105 

COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine 
CORNMAN, DR. IVOR, Department of Zoology, University of West Indies, Mona, 

Kingston, Jamaica 
COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 27515 

COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro- 
lina, Chapel Hill, North Carolina 27515 
CRANE, MR. JOHN O., Woods Hole, Massachusetts 02543 
CRANE, DR. ROBERT K., Department of Biochemistry, The Chicago Medical School, 

2020 West Ogden Avenue, Chicago 12, Illinois 

CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire 
CROUSE, DR. HELEN V., Institute for Molecular Biophysics, Florida State Uni- 
versity, Tallahassee, Florida 32306 
CROWELL, DR. SEARS, Department of Zoology, Indiana University, Bloomington, 

Indiana 
CSAPO, DR. ARPAD L, The Rockefeller Institute, 66th Street and York Avenue, 

New York 21, New York 

CURTIS, DR. MAYNIE R., Box 3215, University Branch, Coral Gables 46, Florida 
CURTIS, DR. W. C., 504 Westmont Avenue, Columbia, Missouri 



46 MARINE BIOLOGICAL LABORATORY 

DAIGNAULT, MR. ALEXANDER T., W. R. Grace & Company, 7 Hanover Square, 
New York 5, New York 

DAN, DR. JEAN CLARK, Misaki Biological Station, Misaki, Japan 

DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan 

DANIELLI, DR. JAMES F., Department of Medicinal Chemistry, University of 
Buffalo School of Pharmacy, Buffalo 14, New York 

DAVIS, DR. BERNARD D., Harvard Medical School, 25 Shattuck Street, Boston 15, 
Massachusetts 

DAWSON, DR. A. B., Biological Laboratories, Harvard University, Cambridge, 
Massachusetts 02138 

DAWSON, DR. J. A., 129 Violet Avenue, Floral Park, Long Island, New York 

DEANE, DR. HELEN W., Department of Pathology, The Albert Einstein College 
of Medicine, New York 61, New York 

DETTBARN, DR. WOLF-DIETRICH, Department of Neurology, Columbia University, 
College of Physicians & Surgeons, New York, New York 10032 

de VILLAFRANCA, DR. GEORGE W., Department of Zoology, Smith College, North- 
ampton, Massachusetts 

DILLER, DR. IRENE C, Institute for Cancer Research, Fox Chase, Philadelphia, 
Pennsylvania 19111 

DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania 

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

DOOLITTLE, DR. RUSSELL F., Department of Biology, University of California, 
La Jolla, California 

DOWBEN, DR. ROBERT, Department of Biology, Massachusetts Institute of Tech- 
nology, Cambridge, Massachusetts 

DURYEE, DR. WILLIAM R., Department of Pathology, George Washington Uni- 
versity School of Medicine, Washington 7, D. C. 

EBERT, DR. JAMES DAVID, Department of Embryology, Carnegie Institution of 
Washington, 115 West University Parkway, Baltimore 10, Maryland 

ECKERT, DR. ROGER OTTO, Department of Zoology, Syracuse University, Syracuse, 
New York 13210 

EDDS, DR. MAC V., JR., Department of Biology, Brown University, Providence 12, 
Rhode Island 

EDER, DR. HOWARD A., The Albert Einstein College of Medicine, New York 61, 
New York 

EDWARDS, DR. CHARLES, Department of Physiology, University of Minnesota, 
Minneapolis 14, Minnesota 

EICHEL, DR. HERBERT J., Department of Biological Chemistry, Hahnemann Medi- 
cal College, Philadelphia, Pennsylvania 

ELSEN, DR. HERMAN, Department of Medicine, \Yashington University, St. Louis, 
Missouri 

ELLIOTT, DR. ALFRED M., Department of Zoology, LIniversity of Michigan, Ann 
Arbor, Michigan 

ESSNER, DR. EDWARD S., Sloan-Kettering Institute for Cancer Research, Rye, New 
York 



REPORT OF THE DIRECTOR 47 

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

Iowa 
FAILLA, DR. PATRICIA M., Argonne National Laboratory, Radiological Physics 

Division, Argonne, Illinois 60440 
FARMANFARMAIAN, DR. ALLAHVERDI, Professor of General Physiology, Faculty 

of Medicine, Pahlavi University, Shiraz, Iran 

FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France 
FAWCETT, DR. D. W., Department of Anatomy, Harvard Medical School, Boston 

15, Massachusetts 
FERGUSON, DR. F. P., National Institute of General Medical Sciences, National 

Institutes of Health, Bethesda, Maryland 20014 
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, Louisiana 70118 
FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, 

Richmond, Virginia 
FISHER, DR. FRANK M., JR., Department of Biology, Rice University, Houston, 

1, Texas 
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, 16 Divinity Avenue, Cambridge, Massachusetts 
FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois, 

Urbana, Illinois 

FREYGANG, DR. WALTER J., JR., 6247-29th Street, Washington 15, D. C. 
FRIES, DR. ERIK F. B., Box 605, Woods Hole, Massachusetts 02543 
FUORTES, DR. MICHAEL C. F., (NINDB), National Institutes of Health, Bethesda, 

Maryland 20014 
FURSHPAN, DR. EDWIN J., Department of Neurophysiology, Harvard Medical 

School, Boston 15, Massachusetts 

FURTH, DR. JACOB, 99 Fort Washington Avenue, New York 32, New York 
FYE, DR. PAUL M., Woods Hole Oceanographic Institution, Woods Hole, Massa- 
chusetts 02543 
GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn 10, 

New York 
GAFFRON, DR. HANS, Department of Biology, Institute of Molecular Biophysics, 

Florida State University, Tallahassee, Florida 32306 

GALL, DR. JOSEPH G., Department of Zoology, Yale University, New Haven, Con- 
necticut 06520 

GALTSOFF, DR. PAUL S., Bureau of Commercial Fisheries, Woods Hole, Massa- 
chusetts 02543 



48 MARINE BIOLOGICAL LABORATORY 

GERMAN, DR. JAMI.S LAFAYETTE, III, Department of Pediatrics and Medicine, 

Cornell University Medical College, 1300 York Avenue, New York 21, New 

York 
GILBERT, DR. DANIEL L., Laboratory of Biophysics, NINDB, National Institutes 

of Health, Bethesda. Maryland 20014 
OILMAN, DR. LAUREN C, Department of Zoology, University of Miami, Coral 

Gables 46, Florida 

GINSKHRG, DR. HAROLD S., Department of Microbiology, University of Pennsyl- 
vania School of Medicine, Philadelphia 4, Pennsylvania 
GOLDSMITH, DR. TIMOTHY H., Department of Zoology, Yale University, New 

Haven, Connecticut 06520 
GOLDSTEIN, DR. LESTER, Department of Zoology, University of Pennsylvania, 

Philadelphia 4, Pennsylvania 
GOODCHILD, DR. CIIAUNCEY G., Department of Biology, Emory University, Atlanta 

22, Georgia 
GOTSCHALL, DR. GERTRUDE Y., 315 Kast (>8th Street, Apt. 9-M, New York, New 

York 10021 
GRAHAM, DR. HERBERT, U. S. Fish and Wildlife Service, Bureau of Commercial 

Fisheries, Woods Hole, Massachusetts 02543 
GRAND, MR. C. G., Cancer Institute of Miami, 1155 N. YY. 15th Street, Miami, 

Florida 
GRANT, DR. PHILIP, National Science Foundation, 1921 Constitution Avenue, 

Washington 25, D. C. 
GRAY, DR. IRVING F., Department of Zoology, Duke University, Durham, North 

Carolina 27706 
GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New 

Brunswick, New Jersey 08903 

GREEN. DR. JONATHAN PASCAL, Department of Biology, Brown University, Provi- 
dence 12, Rhode Island 

GREEN, DR. MAURICE, Department of Microbiology, Saint Louis University Medi- 
cal School, St. Louis, Missouri 
GREGG, DR. JAMES II., Department of Biological Sciences, University of Florida, 

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

Carolina 27706 
GREIF, DR. ROGF.R L., Department of Physiology, Cornell University Medical 

College, New York 21, New York 
GRIFFIN, DR. DONALD F., Biological Laboratories, Harvard University, Cambridge, 

Massachusetts 02138 
GROSCH, DR. DANIKL S., Department of Genetics, Gardner Hall, North Carolina 

State College, Raleigh, North Carolina 
GROSS, DR. PAUL, Department of Biology, Brown University, Providence 12, Rhode 

Island 
GRUNDFEST, DR. HAKKY, Department of Neurology. Columbia University College 

of Physicians & Surgeons, New York 32, NYw York 

Gi [THAN, DR. RITA, Department of Biology, Brooklyn College, Brooklyn 10, 
Xew York 



REPORT OF THE DIRECTOR 49 

GWILLIAM, DR. G. F., Department of Biology, Reed College, Portland, Oregon 
97202 

HAJDU, DR. STEPHEN, National Institutes of Health, Bethesda, Maryland 20014 

HALL, DR. FRANK G., Department of Physiology, Duke University Medical School, 
Durham, North Carolina 27706 

HALVORSEN, DR. HARLYN O., Department of Bacteriology, University of Wisconsin, 
Madison, Wisconsin 53706 

HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St. 
Louis, Missouri 63130 

HAMILTON, DR. HOWARD L.. Department of Biology, University of Virginia, 
Charlottesville, Virginia 

HANCE, DR. ROBERT T., RR No. 3, 6609 Smith Road, Loveland, Ohio 

HARDING, DR. CLIFFORD V., JR., Oakland University, Rochester, Michigan 

HARNLY, DR. MORRIS H., Washington Square College, New York University, New 
York 3, New York 

HARTLINE, DR. H. KEFFER, The Rockefeller Institute, 66th Street and York Ave- 
nue, New York 21, New York 

HARTMAN, DR. FRANK A., Ohio State University, Hamilton Hall, Columbus, Ohio 

HARTMAN, DR. P. E., Department of Biology, The Johns Hopkins University, 
Baltimore 18, Maryland 

HARVEY, DR. ETHEL BROWNE, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 02543 

HASTINGS, DR. J. WOODLAND, Department of Biochemistry, University of Illi- 
nois, Urbana, Illinois 

HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo 
3, New York 

HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institution of 
Oceanography, University of California, La Jolla, California 

HAYASHI, DR. TERU, Department of Zoology, Columbia University, New York, 
New York 10027 

HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesley 81, Massachusetts 

HAYWOOD, DR. CHARLOTTE, Box 14, South Hadley, Massachusetts 

HENDLEY, DR. CHARLES D., 615 South Avenue, Highland Park, New Jersey 

HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina, 
Chapel Hill, North Carolina 27515 

HERNDON, DR. WALTER R., Office of the Dean, University of Tennessee, Knoxville, 
Tennessee 

HERVEY, MR. JOHN P., Box 735, Woods Hole, Massachusetts 02543 

HESSLER, DR. ANITA Y., Marine Biological Laboratory. Woods Hole, Massa- 
chusetts 02543 

Hi ATT, DR. HOWARD H., Department of Medicine, Harvard Medical School, Boston 
15, Massachusetts 

HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio 

HIRSHFIELD, DR. HENRY I., Department of Biology. Washington Square Center, 
New York University, New York 3, New York 

HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge, 
Massachusetts 02138 



50 MARINE BIOLOGICAL LABORATORY 

HODES, DR. ROBERT, Department of Pediatrics, The Mount Sinai Hospital, New 

York 29, New York 

HODGES, DR. CHARLES, IV, Department of Biology, Temple University, Phila- 
delphia 22, Pennsylvania 
HOFFMAN, DR. JOSEPH, National Heart Institute, National Institutes of Health, 

Bethesda, Maryland 20014 
IIoi.i.AKXDER, DR. ALEXANDER, Biology Division, Oak Ridge National Laboratory, 

Oak Ridge, Tennessee 37831 
Ilor.z, DR. GEORGE G., JR., Department of Microbiology, State University of New 

York, Upstate Medical College, Syracuse, New York 13210 
HOPKINS, DR. HOYT S., 59 Heatherdell Road, Ardsley, New York 
HOSKIN, DR. FRANCIS C. G., Department of Neurology, Columbia University, 

College of Physicians & Surgeons, New York 32, New York 
HUNTER, DR. FRANCIS R., University of the Andes, Calle IS-a, Carrera 1-E, 

Bogota, Colombia, South America 
HUNTER, DR. W. D. RUSSELL, Department of Zoology, 110 Lyman Hall, Syracuse 

University, Syracuse, New York 13210 
HURWITZ, DR. J., Department of Molecular Biology, The Albert Einstein College 

of Medicine, New York 61, New York 
HUTCH ENS, DR. JOHN E., Department of Physiology, University of Chicago, 

Chicago 37, Illinois 
HYDE, DR. BEAL B., Department of Botany, University of Texas, Austin, Texas 

78703 
HYMAN, DR. LIBBIE H., American Museum of Natural History, Central Park 

West at 79th Street, New York 24, New York 
INOUE, DR. SHINYA, Department of Cytology, Dartmouth Medical School, Hanover, 

New Hampshire 
ISENBERG, DR. IRVIN, Institute for Muscle Research, Marine Biological Laboratory, 

Woods Hole, Massachusetts 02543 

ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts 02543 
ISSELBACKER, DR. KURT J., Massachusetts General Hospital, Boston, Massachusetts 
JANOFF, DR. AARON, Department of Pathology, New York University School of 

Medicine, 550 First Avenue, New York, New York 10016 
TENNER, DR. CHARLES E., Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 27515 
JOHNSON, DR. FRANK IT.. Department of Biology, Princeton University, Princeton, 

New Jersey 08540 
JONES, DR. E. RUFEIN, JR., Department of Biological Sciences, University of 

Florida, Gainesville, Florida 
JONES, DR. RAYMOND F., Department of Biology, State University of New York, 

Stony Brook, Long Island, New York 11733 

JOSEPHSOX, DR. R. K., Department of Zoology, University of Minnesota, Minne- 
apolis 14, Minnesota 
KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachusetts 

02543 

KARAT, DR. E. A., Neurological Institute, Columbia University, College of Phy- 
sicians & Surgeons, New York 32, New York 



REPORT OF THE DIRECTOR 51 

KALEY, DR. GABOR, Department of Pathology, New York University School of 
Medicine, 550 First Avenue, New York, New York 10016 

KAMINER, DR. BENJAMIN, Institute for Muscle Research, Marine Biological 
Laboratory, Woods Hole, Massachusetts 02543 

KANE, DR. ROBERT E., Department of Cytology, Dartmouth Medical School, Han- 
over, New Hampshire 

KARUSH, DR. FRED, Department of Microbiology, University of Pennsylvania 
School of Medicine, Philadelphia 4, Pennsylvania 

KAUFMANN, DR. B. P., Department of Zoology, University of Michigan, Ann 
Arbor, Michigan 

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

KEMPTON, DR. RUDOLF T., Department of Zoology, Vassar College, Poughkeepsie, 
New York 

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

KETCHUM, DR. BOSTWICK H., Woods Hole Oceanographic Institution, Woods 
Hole, Massachusetts 02543 

KEYNAN, DR. ALEXANDER, Institute for Biological Research, Ness Ziona, Israel 

KILLE, DR. FRANK R., State Department of Education, Albany 1, New York 

KIND, DR. C. ALBERT, Department of Zoology, University of Connecticut, Storrs, 
Connecticut 

KINDRED, DR. J. E., Box 1873, University of Virginia, Charlottesville, Virginia 

KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa 

KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca, 
New York 

KINNE, DR. OTTO, Director, Biologische Anstalt Helgoland, 2 Hamburg-Altona, 
Palmaille 9, Germany 

KISCH, DR. BRUNO, 71 Maple Street, Brooklyn 25, New York 

KLEIN, DR. MORTON, Department of Microbiology, Temple University, Phila- 
delphia, Pennsylvania 

KLEINHOLZ, DR. LEWIS Ii., Department of Biology, Reed College, Portland, Ore- 
gon 97202 

KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston, 
Illinois 60201 

KOLIN, DR. ALEXANDER, Department of Biophysics, University of California Medi- 
cal 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 

KRANE, DR. STEPHEN M., Massachusetts General Hospital, Boston 14, Massa- 
chusetts 

KRASSNER, DR. STUART MITCHELL, The Rockefeller Institute, New York 21, New 
York 

KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, College 
Park, Maryland 



52 MARINE I'.lol OGICAL I. VBOR VTORY 

KKKIG, DR. WENDELL I. S., 303 East Chicago Avenue, Chicago, Illinois 

Kui-n.LR, I)u. STEPHEN \\'., Department of I'hannacology, Neurophysiological 
Laboratory, Harvard Medical School, Boston 15, Massachusetts 

Ki'NiTZ, DR. MOSKS, The Rockefeller Institute, 6(>th Street and York Avenue, 
New York 21, New York 

LAMY, DR. FRANCOIS, Department of Anatomy, University of Pittsburgh School 
of Medicine, Pittsburgh 1.3, Pennsylvania 

LANCEFIELD, DR. D. E., (Jueens College, Flushing, New York 

LANCEFIELD, DR. REBECCA C., The 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 School 
of Medicine, Pittsburgh 13, Pennsylvania 

LASH, DR. JAMES W., Department of Anatomy, University of Pennsylvania School 
of Medicine, Philadelphia 4, Pennsylvania 

LAI FKR, DR. JlAxs, Department of Biology, The Johns Hopkins University, Balti- 
more, Maryland 21218 

LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts- 
burgh 13, Pennsylvania 

LAWLER, DR. H. CLAIRE, Department of Biochemistry and Neurology, Columbia 
University, College of Physicians & Surgeons, New York 32, New York 

LAVIN, DR. GEORGE I., 6200 Norvo Road, Baltimore 7, Maryland 

LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota School 
of Medicine, Minneapolis 14, Minnesota 

LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford Medical School, Palo 
Alto, California 

LEE, DR. RICHARD E., Cornell University College of Medicine, New York 21, 
New York 

LEFEVRE, DR. PAUL G., University of Louisville School of Medicine, Louisville, 
Kentucky 

i,i ii MANN, DR. FRITZ, /oologi.schc Institut, University of Berne, Berne, Switzer- 
land 

I.KVINE, DR. RACIIMIEL. Department of Medicine, New York Medical School, 5th 
Avenue and 106th Street, New York 29, New York 

LEVY, DR. MILTON, Department of Biochemistry, New York University School of 
Dentistry, New York 10, New York 

LEWIN, I )R. RALPH A., Scripps Institution of Oceanography, La Jolla, California 

Li \vis, I )R. IIi.k.MAN WILLIAM, Genetic Biology Program, National Science 
Foundation, Washington 25, D. C. 

LING, DR. GlLBi UT, 307 Berkeley l\oad, Merion, Pennsylvania 

LITTLE, DR. E. P., 21<> Highland Street, West Newton, Massachusetts 

LLOYD, DR. DAVID P. ('., The Rockefeller Institute, (><>th Street and York Avenue, 
New York 21, New York 

LoCHHEAD, DR. JOHN II., Department of /oology. University of Vermont, Burling- 
ton, Vermont 

Loi B, DR. R. F., 950 Park Avenue, New York 2S. New York 



REPORT OF THE DIRECTOR 

LOEWENSTEIN, DR. WERNER R., Department of Physiology, Columbia University, 
College of Physicians & Surgeons, New York 32, New York 

LOFTFIELD, DR. ROBERT B., Massachusetts General Hospital, Boston 14, Massa- 
chusetts 

LONDON, DR. IRVING M., Department of Medicine, The Albert Einstein College 
of Medicine, New York 61, New York 

LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans- 
ton, Illinois 60201 

DE LORENZO, DR. ANTHONY, Anatomical and Pathological Research Laboratories, 
The Johns Hopkins Hospital, Baltimore 5, Maryland 

LOVE, DR. WARNER E., 1043 Marlau Drive, Baltimore 12, Maryland 

LUBIN, DR. MARTIN, Department of Pharmacology, Harvard Medical School, 
Boston 15, Massachusetts 

LYNCH, DR. CLARA J., The Rockefeller Institute, 66th Street and York Avenue, 
New York 21, New York 

LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America, 
Washington 17, D. C. 

MAAS, DR. WERNER K., New York University College of Medicine, New York, 
New York 10016 

MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 136 
Harrison Avenue, Boston, Massachusetts 

MAHLER, DR. HENRY R., Department of Biochemistry, Indiana University, Bloom- 
ington, Indiana 

MANWELL, DR. REGINALD D., Department of Zoology, Syracuse University, Syra- 
cuse, New York 13210 

MARKS, DR. PAUL A., Presbyterian Hospital, Columbia University, College of 
Physicians & Surgeons, New York 32, New York 

MARSHAK, DR. ALFRED, Tulane University Medical School, New Orleans, Louisi- 
ana 

MARSLAND, DR. DOUGLAS A v 48 Church Street, Woods Hole, Massachusetts 
02543 

MARTIN, DR. EARL A., 682 Rudder Road, Naples, Florida 33940 

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

MAZIA, DR. DANIEL, Department of Zoology, University of California, Berkeley 
4, California 

McCANN, DR. FRANCES, Department of Physiology, Dartmouth Medical School, 
Hanover, New Hampshire 

McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School, 
Philadelphia 4, Pennsylvania 

McDoNALD, SISTER ELIZABETH SETON, Department of Biology, College of Mount 
St. Joseph, Mt. St. Joseph, Ohio 

MCDONALD, DR. MARGARET R., Waldemar Medical Research Foundation, Sunny- 
side Boulevard and Waldemar Road, Woodbury, Long Island, New York 

MCELROY, DR. WILLIAM D., Department of Biology, The Johns Hopkins Uni- 
versity, Baltimore, Maryland 21218 



54 MARINE BIOLOGICAL LABORATORY 

MEINKOTH, DR. NORMAN A., Department of Biology, Swarthmore College, 
Swarthmore, Pennsylvania 

METZ, DR. CHARLES B., Institute of Molecular Evolution, School of Environ- 
mental and Planetary Sciences, University of Miami, Coral Gables 46, Florida 

METZ, DR. CHARLES W., P.ox 714, Woods Hole, Massachusetts 02543 

MIDDLEBROOK, DR. ROBERT, Department of Physiology, Dartmouth Medical Center, 
Hanover, New Hampshire 

MILKMAN, DR. ROGER D., Department of Zoology, Syracuse University, Syracuse, 
New York 13210 

MILLER, DR. J. A., |R., Department of Anatomy, Tulane University Medical School, 
New Orleans 18, Louisiana 

MILXE, DR. LORUS, J., Department of Zoology, University of New Hampshire, 
Durham, New Hampshire 03824 

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, 
Palermo, Italy 

MOORE, DR. GEORGE M., Department of Zoology, University of New Hampshire, 
Durham, New Hampshire 03824 

MOORE, DR. JOHN A., Department of Zoology, Columbia University, 954 Schermer- 
horn, New York, New York 10027 

MOORE, DR. JOHN W., Department of Physiology and Pharmacology, Duke Uni- 
versity Medical School, Durham, North Carolina 27706 

MOORE, DR. R. O., Department of Biochemistry, Ohio State University, Columbus 
10, Ohio 

MORAN, DR. JOSEPH F., JR., Department of Biology, Russell Sage College, Troy, 
New York 

MORRILL, DR. JOHN B., JR., Department of Biology, Wesleyan University, Middle- 
town, Connecticut 06457 

MOSCONA, DR. A. A., Department of Zoology, University of Chicago, Chicago 37, 
Illinois 

MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick, 
New Jersey 08903 

MOUNTAIN, MRS. J. D., Charles Road, Mount Kisco, New York 

MULLINS, DR. LORIN J., Department of Biophysics, University of Maryland 
School of Medicine, Baltimore 1, Maryland 

MCSACCHIA, DR. XAVIER J., Department of Biology, Saint Louis University, St. 
Louis 4, Missouri 

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

XACE, DR. PAUL FOLEY, Department of Biology, Hamilton College, McMaster 
University, Hamilton, Ontario, Canada 

NACHMANSOIIN, DR. DAVID, Department of Neurology, Columbia University, 
College of Physicians & Surgeons, New York 32, New York 

NASATIR, DR. MAIMON, Department of P.otany. Brown University, Providence 12, 
Rhode Island 



REPORT OF THE DIRECTOR 55 

NASON, DR. ALVIN, McCollum-Pratt Institute, The Johns Hopkins University, 

Baltimore, Maryland 21218 

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

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

Seattle 5, Washington 

NICOLL, DR. PAUL A., Black Oak Lodge, RR No. 2, Bloomington, Indiana 
Niu, DR. MAN-CHIANG, Temple University, Philadelphia 22, Pennsylvania 
NOVIKOFF, DR. ALEX B., Department of Pathology, The Albert Einstein College 

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

York 10016 
ODUM, DR. EUGENE, Department of Zoology. University of Georgia, Athens, 

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

Mawr, Pennsylvania 

OSTERHOUT, DR. MARIAN IRWIN, 456 East 63rd Street, New York 21, New York 
PACKARD, DR. CHARLES, Woods Hole, Massachusetts 02543 
PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio 
PALMER, DR. JOHN D., Department of Biology, New York University, University 

Heights, New York 53, New York 

PARPART, DR. ARTHUR K., Department of Biology, Princeton University, Prince- 
ton, New Jersey 08540 
PASSANO, DR. LEONARD M., Department of Zoology, University of Wisconsin, 

Madison, Wisconsin 53706 
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, VA Hospital Special Dental Research Program. 

Brooklyn 9, New York 
PETTIBONE, DR. MARIAN H., Division of Invertebrate Zoology, U. S. National 

Museum, Washington 25, D. C. 
PHILPOTT, DR. DELBERT E., Department of Biochemistry, University of Colorado 

Medical Center, 4200 East Ninth Avenue, Denver 20. Colorado 
PICK, DR. JOSEPH, Department of Anatomy, New York University-Bellevue Medi- 
cal Center, New York. New York 10016 
PIERCE, DR. MADELENE E., Department of Zoology, Vassar College, Poughkeepsie, 

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

New York 10027 

POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut 
POTTER, DR. DAVID, Department of Neurophysiology, Harvard Medical School, 

Boston 15, Massachusetts 
PORTER, DR. KEITH R., Biological Laboratories, Harvard University, Cambridge, 

Massachusetts 02 138 



56 MARINE I'.IOLOGKAI. LABORATORY 

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

PROSSER, DR. C. LADD, Department of Physiology, Burrill Hall, University of 
Illinois, Urbana, Illinois 

PROVASOLI, DR. Lric.i. llaskins Lai (oratories, 305 East 43rd Street, New York 
17, New York 

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

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

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

RAPPORT, DR. M., Department of Biochemistry, The Albert Einstein College of 
Medicine, New York 61, New York 

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

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

READ, DR. CLARK P., Department of Biology, Rice University, Houston 1, Texas 

REBHUN. DR. LIONEL L, Department of Biology, Princeton University, Princeton, 
New Jersey 08540 

RECHNAGEL, DR. R. O., Department of Physiology, Western Reserve University, 
Cleveland 6, Ohio 

REDFIELD, DR. ALFRED C., \Voods Hole, Massachusetts 02543 

RF.NN, DR. CHARLES E., The Johns Hopkins University, 509 Ames Hall, Balti- 
more, Maryland 21 2 18 

REUBEN, DR. JOHN P., Department of Neurology, Columbia University, College 
of Physicians & Surgeons, New York 32, New York 

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

RICH, DR. ALEXANDER, Department of Biology, Massachusetts Institute of Tech- 
nology, Cambridge, Massachusetts 

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

RICHARDS, DR. A. GLENN. Department of Entomology, University of Minnesota, 
St. Paul, Minnesota 55101 

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

ROCKSTEIN, DR. MORRIS, Medical Research Building, 1600 N. W. 10th Avenue, 
Miami 36, Florida 

K'OMKK, DR. ALFRED S., Museum of Comparative Zoology, Harvard University, 
Cambridge, Massachusetts 02138 

koXKiN, DR. RAPHAEL R., Department of Biological Sciences, University of Dela- 
ware, Newark, Delaware 19711 

ROOT, DR. l\. \Y., Department of Biology, College of the City of New York, New 
York, New York 

ROOT, DR. \Y. S., Department of Physiology, Columbia I "iiiversily. College of 
Physicians & Surgeons, New York 32, New York 

l\osi . DR. S. MKRYL, Department of Anatomy, Tulanc I'liivcrsity, New Orleans, 
Louisiana 



REPORT OF THE DIRECTOR 

ROSENBERG, DR. EVELYN K., Department of Pathology, New York University- 
Bellevue Medical Center, New York, New York 10016 

ROSENBERG, DR. PHILIP, Department of Neurology, Columbia University, New 
York 32, New York 

ROSENBLUTH, Miss RAJA, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 02543 

ROSENKRANZ, DR. HERBERT S., Department of Microbiology, Columbia University, 
New York 32, New York 

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

ROSLANSKY, DR. JOHN, Marine Biological Laboratory, Woods Hole, Massachusetts 
02543 

ROTH, DR. JAY S., Department of Zoology and Entomology, University of Con- 
necticut, Storrs, Connecticut 

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

RUGH, DR. ROBERTS, Radiological Research Laboratory, Columbia University, 
College of Physicians & Surgeons, New York 32, New York 

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

RUSTAD, DR. RONALD C., Department of Radiology, Western Reserve University, 
Cleveland 6, Ohio 

RUTMAN, DR. ROBERT J., General Laboratory Building, 215 South 34th Street, 
Philadelphia, Pennsylvania 

RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole, 
Massachusetts 02543 

SAGER, DR. RUTH, Department of Zoology, Columbia University, New York, New 
York 10027 

SANBORN, DR. RICHARD C., Department of Biological Sciences, Purdue University, 
Lafayette, Indiana 47907 

SANDERS, DR. HOWARD L., Woods Hole Oceanographic Institution, Woods Hole, 
Massachusetts 02543 

SATO, DR. HIDEMI, Department of Cytology, Dartmouth Medical School, Hanover, 
New Hampshire 

SAUNDERS, DR. JOHN, Department of Biology, Marquette University, Milwaukee 
3, Wisconsin 

SAUNDERS, MR. LAWRENCE, West Washington Square, Philadelphia 5, Pennsyl- 
vania 

SAZ, DR. ARTHUR KENNETH, National Institutes of Health, Bethesda 14, Maryland 

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

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

SCHLESINGER, DR. R. WALTER, Department of Microbiology, Rutgers Medical 
School, New Brunswick, New Jersey 

SCHMIDT, DR. L. H., National Primate Center, University of California, Davis, 
California 



M \K1.\K IMOLOGICAL LABORATORY 

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

SCHMITT, DR. O. H., Department of Physics, University of Minnesota, Minne- 
apolis 14, Minnesota 

SCHNEIDERMAN, DR. HOWARD A., Department of Biology, Western Reserve Uni- 
versity, Cleveland 6, Ohio 

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

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

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

SCHUEL, DR. HERBERT, Department of Chemistry, Northwestern University, Evans- 
ton, Illinois 

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

SCOTT, DR. D. B. McNAiR, Lippincott Building, 25th and Locust Streets, Phila- 
delphia, Pennsylvania 19103 

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

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

SEARS, DR. MARY, Glendon Road, Woods Hole, Massachusetts 02543 

SELIGER, DR. HOWARD H., McCollum-Pratt Institute, The Johns Hopkins Univer- 
sity, Baltimore, Maryland 21218 

SENFT, DR. ALFRED W., Marine Biological Laboratory, Woods Hole, Massachu- 
setts 02543 

SEVERINGHAUS, DR. AURA E., 375 West 250th Street, New York 71, New York 

SHAPIRO, DR. HERBERT, 6025 North 13th Street, Philadelphia 41, Pennsylvania 

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

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

SHEMIN, DR. DAVID, Department of Biochemistry, Columbia University, New 
York, New York 10027 

SHERMAN, DR. I. W., Division of Life Sciences, University of California, River- 
side, California 92502 

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

SICHEL, MRS. FERDINAND, Department of Biology, Trinity College, Burlington, 
Vermont 

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

SIMMONS, DR. JOHN E., JR., Department of Biology, Rice University, Houston 1, 
Texas 

SLIFER, DR. ELEANOR H., 308 Lismore Avenue, Glenside, Pennsylvania 

SLOBODKIN, DR. LAWRENCE BASIL, Department of Zoology, University of Michi- 
gan, Ann Arbor, Michigan 

SMKLSER, DR. GEORGE K., Department of Anatomy, Columbia University, New 
York, New York 

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



REPORT OF THE DIRECTOR 59 

SMITH, MR. HOMER P., General Manager, Marine Biological Laboratory, Woods 
Hole, Massachusetts 02543 

SMITH, MR. PAUL FERRIS, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 02543 

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 

SPECTOR, DR. A., Howe Laboratories, Harvard Medical School, Boston 15, Massa- 
chusetts 

SPEIDEL, DR. CARL C., Department of Biology, Randolph-Macon Woman's College, 
Lynchburg, Virginia 

SPIEGEL, DR. MELVIN, Division of Biology, California Institute of Technology, 
Pasadena, California 91109 

SPINDEL, DR. WILLIAM, Department of Chemistry, Yeshiva University, Belfer 
Graduate School, Amsterdam Avenue and 186th Street, New York, New York 

SPIRTES, DR. MORRIS ALBERT, Department of Pharmacology, Hahnemann Medical 
College, Philadelphia, Pennsylvania 

S PRATT, DR. NELSON T., Department of Zoology, University of Minnesota, Minne- 
apolis 14, Minnesota 

SPYROPOULOS, DR. C. S., Building 9, Room 140, National Institutes of Health, 
Bethesda, Maryland 20014 

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

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

STEINBERG, DR. MALCOLM S., Department of Biology, The Johns Hopkins Uni- 
versity, Baltimore, Maryland 21218 

STEINHARDT, DR. JACINTO, Georgetown University, Washington 7, D. C. 

STEPHENS, DR. GROVER C., Department of Biological Sciences, University of Cali- 
fornia, Irvine, California 92650 

STETTEN, DR. DE\YITT, JR., Rutgers University Medical School, New Brunswick, 
New Jersey 

STETTEN, DR. MARJORIE R., Rutgers University Medical School, New Brunswick, 
New Jersey 

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

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

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

STRAUSS, DR. W. L., JR., Department of Anatomy, The Johns Hopkins University 
Medical School, Baltimore 5, Maryland 

STREHLER, DR. BERNARD L., 4115 West View Road. Baltimore 18, Maryland 

STRITTMATTER, DR. PHILIPP, Department of Biological Chemistry, Washington 
University School of Medicine, St. Louis, Missouri 



60 MARINE BIOLOGICAL LABORATORY 

STUNKAKD, DR. HORACE \V., American Museum of Natural History, Central Park 
West at 79th Street, New York 24, New York 

STURTEYANT, DR. AT.FRF.D H., California Institute of Technology, Pasadena, Cali- 
fornia 9 1109 

SUDAK, DR. FREDERICK N., Department of Physiology, The Albert Einstein Col- 
lege of Medicine, New York 61, New York 

SULKIN, DR. S. EDWARD, Department of Bacteriology, University of Texas, 
Southwestern Medical School, Dallas, Texas 

SrssMAN, DR. MAURICE, Department of Biology, Brandeis University, Waltham, 
Massachusetts 

SWANSON, DR. CARL PONTIUS, Department of Biology, The Johns Hopkins Uni- 
versity, Baltimore, Maryland 21218 

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

SZABO, DR. GEORGE, Department of Dermatology, Massachusetts General Hospital, 
Boston 14, Massachusetts 

SzENT-Gy<">RCYi, DR. ALBERT, Institute for Muscle Research, Marine Biological 
Laboratory, Woods Hole, Massachusetts 02543 

SZENT-GYORGYI, DR. ANDREW G., Department of Cytology, Dartmouth Medical 
School, Hanover, New Hampshire 

TASAKI, DR. ICHIJI, Laboratory of Neurobiology (NIMH), National Institutes of 
Health, Bethesda, Maryland 20014 

TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology (NINDB), National 
Institutes of Health, Bethesda, Maryland 20014 

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

TAYLOR, DR. W. ROWLAND, Department of Oceanography, The Johns Hopkins 
University, Baltimore, Maryland 21218 

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

TRACY, DR. HENRY C., 3595 Mynders No. 3, Memphis, Tennessee 

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

TRAVIS, DR. D. M., Department of Pharmacology, University of Florida, Gaines- 
ville, Florida 

TRINKAUS, DR. J. PHILIP, Department of Zoology, Osborn Zoological Labora- 
tories, Yale University, New Haven, Connecticut 06520 

TROLL, DR. WALTER, Department of Industrial Medicine, New York University 
College of Medicine, New York 16, 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, Pasa- 
dena, California 91109 

URETZ, DR. l\or.i irr B., Department of Biophysics, University of Chicago, Chicago, 
Illinois 

VAX llot.Di, DR. KENSAI. EDWARD, Department of Chemistry, University of Illi- 
nois, Urbana, Illinois 

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



REPORT OF THE DIRECTOR 61 

VINCENT, DR. WALTER S., Department of Anatomy, University of Pittsburgh, 
Pittsburgh 13, Pennsylvania 

WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New 
Brunswick, New Jersey 08903 

WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge. 
Massachusetts 02138 

WARNER, DR. ROBERT C.. Department of Chemistry, New York University College 
of Medicine. New York, New York 10016 

WARREN, DR. LEONARD, Research Medical Officer, National Institutes of Health, 
Bethesda, Maryland 20014 

WATERMAN, DR. T. H., Department of Zoology, 272 Gibbs Research Laboratory. 
Yale University, New Haven, Connecticut 06520 

WATSON, DR. STANLEY WILLARD, Woods Hole Oceanographic Institution, Woods 
Hole, Massachusetts 02543 

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

WEISS, DR. LEON PAUL, Department of Anatomy, The Johns Hopkins Medical 
School, Baltimore 19, Maryland 

WEISS, DR. PAUL A., Laboratory of Developmental Biology, The Rockefeller Insti- 
tute, 66th Street and York Avenue, New York 21, New York 

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

WERMAN, DR. ROBERT, Institute of Psychiatric Research, Indiana University Medi- 
cal Center, 1100 West Michigan Street, Indianapolis 7, Indiana 

WHITAKER, DR. DOUGLAS M., 320 Good Hill Road, Kentfield, California 

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

WHITING, DR. ANNA R., 535 West Vanderbilt Drive, Oak Ridge, Tennessee 

WHITING, DR. PHINEAS, 535 West Vanderbilt Drive, Oak Ridge, Tennessee 

WICHTERMAN, DR. RALPH, Department of Biology. Temple University, Phila- 
delphia 22, Pennsylvania 

WICKERSHAM, MR. JAMES H., 791 Park Avenue, New York 21, New York 

WIERCINSKI, DR. FLOYD ]., Chicago Teachers College North, 5500 North St. Louis 
Avenue, Chicago 25, Illinois 

WIGLEY, DR. ROLAND L., U. S. Fish and Wildlife Service, Woods Hole, Massa- 
chusetts 02543 

WILBER, DR. C. G., Director, Marine Laboratory, University of Delaware, New- 
ark, Delaware 19711 

WILLIER, DR. B. H., Department of Biology, The Johns Hopkins University, Balti- 
more, Maryland 21218 

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

WILSON, DR. T. HASTINGS, Department of Physiology, Harvard Medical School, 
Boston 15, Massachusetts 

WILSON, DR. WALTER L., Department of Biology, Oakland I'niversity. Rochester, 
Michigan 

WINTERS, DR. ROBERT WAYNE, Department of Pediatrics, Columbia University. 
College of Physicians & Surgeons, New York 32, New York 



62 



MARINE BIOLOGICAL LABORATORY 



\YiTSc-in, DR. EMIL, Universitat Basel, Anatomisches Institut, Pestalozzistrasse 

20, Basel, Switzerland 
WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry, 

The Albert Einstein College of Medicine, New York 61, New York 
WRIGHT, DR. PAUL A., Department of Zoology, University of New Hampshire, 

Durham, New Hampshire 03824 
WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton, 

Massachusetts 
WYTTENBACH, DR. CHARLES RICHARD, Department of Anatomy, University of 

Chicago, Chicago 37, Illinois 
YXTKMA, DR. CHESTER 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 
ZIMMERMAN, DR. A. M., Department of Pharmacology, State University of New 

York, Downstate Medical Center, Brooklyn 3, New York 

ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings- 
ton, Rhode Island 
ZIRKLE, DR. RAYMOND E., Department of Radiobiology, University of Chicago, 

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

New York 

ZULLO, DR. VICTOR AUGUST, Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 02543 
ZWEIFACH, DR. BENJAMIN, New York University, Bellevue Medical Center, New 

York, New York 10016 
ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham 54, 

Massachusetts 

ASSOCIATE MEMBERS 



ALTON, MRS. BENJAMIN 
ANDRES, MRS. WILLIAM 
ARMSTRONG, MRS. PHILIP B. 
AUCLAIR, DR. AND MRS. WALTER 
BACON, MR. AND MRS. ROBERT 
BAKALAR, MR. AND MRS. DAVID 
BALL, MRS. ERIC G. 
BARBOUR, MRS. Lucius 11. 
BARROWS, MRS. ALBERT W. 
BARTOW, MR. AND MRS. CLARENCE W. 
BARTOW, MRS. FRANCIS D. 
BARTOW, MRS. PHILIP 
BEALE, MR. AND MRS. E. E. 
P.i.r.L, MRS. ARTHUR W. 
BIGELOW, MRS. ROBERT P. 
BRADLEY, DR. AND MRS. CHARLKS 
BROWN, DR. A\D MRS. F. A., JR. 
BROWN, MRS. THORNTON 
BI-RDICK, DR. C. LALOR 



BUTLER, DR. AND MRS. E. G. 

CAHOON, MRS. SAMUEL T., SR. 

CALKINS, MRS. GARY N. 

CALKINS, MR. AND MRS. G. N., JR. 

CAREY, Miss CORNELIA 

CARLTON, MR. WINSLOW G. 

CLAFF, MRS. C. LLOYD 

CLAFF, MR. MARK 

CLARK, MRS. JAMES B. 

CLARK, MRS. LEROY 

CLARK, MR. AND MRS. VAN ALAN 

CLOWES, MR. ALLEN W, 

CLOWES, MRS. G. H. A. 

CLOWES, DR. AND MRS. GEORGE IT. A., 

JR. 

I'OI.TON, MRS. IT. SEYMOUR 

CON K 1.1 N. Miss ISABEL 

("KAMKR, MR. AND MRS. IAN D. W. 

CRANE, MR. JOHN 



REPORT OF THE DIRECTOR 



63 



CRANE, MR. JOSEPH B., Foundation 

CRANE, Miss LOUISE 

CRANE, MR. STEPHEN 

CRANE, MRS. W. CAREY 

CRANE, MRS. W. MURRAY 

CROCKER, MR. AND MRS. PETER J. 

CROSSLEY, Miss DOROTHY 

CROSSLEY, MR. AND MRS. ARCHIBALD M. 

CROWELL, MR. AND MRS. PRINCE S. 

CURTIS, DR. AND MRS. WILLIAM D. 

DAIGNAULT, MR. AND MRS. A. T. 

DANIELS, MR. AND MRS. B. G. 

DANIELS, MR. AND MRS. F. H. 

DAY, MR. AND MRS. POMEROY 

DRAPER, MRS. MARY C. 

DREYER, MRS. F. A. 

DuBois, DR. AND MRS. A. B. 

ELSMITH, MRS. DOROTHY 

ENDERS, MRS. FREDERICK 

EVVING, MR. WILLIAM 

FAXON, DR. AND MRS. NATHANIEL W. 

FIRESTONE, MR. AND MRS. EDWIN 

FISHER, MRS. B. C. 

FORBES, DR. ALEXANDER 

FRANCIS, MR. LEWIS H., JR. 

GAISER, DR. AND MRS. DAVID W. 

GALTSOFF, MRS. PAUL S. 

GARFIELD, Miss ELEANOR 

GARLOCK, MR. AND MRS. ROBERT 

GlFFORD, MR. AND MRS. JOHN A. 

GlLCHRIST, MR. AND MRS. JOHN M. 

GILDEA, DR. MARGARET C. L. 
GILLETTE, MR. AND MRS. ROBERT S. 
GLAZEBROOK, MRS. JAMES R. 
GOLDMAN, DR. AND MRS. ALLEN S. 
GREEN, Miss GLADYS M. 
GREIF, DR. AND MRS. ROGER 
GREER, MR. AND MRS. WILLIAM H., JR. 
GULESIAN, MRS. PAUL J. 

GUREWICH, DR. AND MRS. V. 

HAMLEN, MR. AND MRS. J. MONROE 
HANNA, MR. AND MRS. THOMAS C. 
HARRINGTON, MR. AND MRS. R. D. 
HARVEY, DR. AND MRS. E. NEWTON, JR. 
HARVEY, DR. AND MRS. RICHARD 
HERVEY, MRS. JOHN P. 

HlRSCHFELD, MRS. NATHAN B. 

HOPKINS, MRS. RALPH H. 



1 TOUSTON, MR. AND MRS. HOWARD E. 

JKWETT, MR. AND MRS. G. F., JR. 
JONES, MR. AND MRS. DsWiTT C., JR. 
KAHN, DR. AND MRS. ERNEST 
KEITH, MRS. HAROLD C. 
ROLLER, DR. AND MRS. LEWIS R. 
LAWRENCE, MR. AND MRS. MILFORD R. 
LEMANN, MRS. LUCY BENJAMIN- 
LOBE, MR. AND MRS. JOHN 
LOEB, DR. AND MRS. ROBERT F. 

LOVELL, MR. AND MRS. HoLLIS R. 

MARSLAND, DR. AND MRS. D. A. 
MARVIN, MRS. WALTER TAYLOR 
MAST, MRS. S. O. 
MATHER, MR. FRANK J., Ill 
MAVOR, MRS. JAMES W. 

McCuSKER, MR. AND MRS. PAUL T. 

MCELROY, DR. AND MRS. W. D. 

McGlLLICUDDY, DR. AND MRS. JOHN J. 

MCKELVY, MR. JOHN E. 

McLANE, MRS. HUNTINGTON 

McViTTY, MRS. A. E. 

MEIGS, MR. AND MRS. ARTHUR 

MEIGS, DR. AND MRS. J. WISTER 

MINIS, MR. AND MRS. ABRAM J., JR. 

MITCHELL, MRS. JAMES McC. 

MITCHELL, MRS. PHILIP 

MIXTER, MRS. WILLIAM JASON 

MOSSER, MRS. BENJAMIN D. 

MOTLEY, MRS. THOMAS 

NEWTON, Miss HELEN K. 

NICHOLS, MRS. GEORGE 

NIMS, MRS. E. D. 

THE AARON E. NORMAN FUND, INC. 

PACKARD, MRS. CHARLES 

PARPART, DR. AND MRS. ARTHUR K. 

PARK, MR. MALCOLM S. 

PATTEN, MRS. BRADLEY 

PENNINGTON, Miss ANNE H. 

PHILIPPE, MR. PIERRE 

PUTNAM, MR. AND MRS. WILLIAM A., 

Ill 

REDFIELD, DR. AND MRS. ALFRED C. 
REZNIKOFF, DR. AND MRS. PAUL 
RIGGS, MR. AND MRS. LAWRENCE, III 
RIVINUS, MRS. F. M. 
ROGERS, MRS. CHARLES E. 
ROOT, DR. AND MRS. WALTER S. 



64 



MARINE BIOLOGICAL LAI'.OKATORY 



RUDD, MRS. H. W. DWIGHT 
RUGH, DR. AND MRS. ROBERTS 
SANDS, Miss ADELAIDE G. 
SAUNDERS, MR. AND MRS. LAWRENCE 
SCHWARTZ, MRS. VICTOR B. 
SmvERicK, MRS. ARTHUR 
SINCLAIR, MR. AND MRS. \Y. RICHARD- 
SON 

SMITH, MRS. HOMER P. 
Si '!:! DEL, MRS. CARL C. 
STONE, MR. AND MRS. LEO 
STONE, DR. WILLIAM, JR. 
STONE, MRS. SAMUEL M. 
STRAUSS, MR. AXD MRS. DONALD B. 
STUNK ARD, MRS. HORACE 
SWIFT, MR. E. KENT, JR. 
SWOPE, MR. DAVID 



SWOPE, MR. AND MRS. GERARD, JR. 
SWOPE, Miss HENRIETTA H. 

SZENT-GYORGYI, DR. ALBERT 

TOMPKINS, MR. AND MRS. B. A. 
WARREN, DR. AND MRS. SHIELDS 
WEBSTER, MRS. EDWIN S. 
WHITELY, Miss MABEL W. 
WHITELY, MR. AND MRS. GEORGE C., 

JR. 

WHITNEY, MRS. GEORGE 
WlCKERSHAM, MRS. JAMES H. 
WILHELM, DR. HAZEL S. 
WILSON, MRS. EDMUND B. 
WILSON, DR. MAY G. 
WOLFE, DR. CHARLES 

WOLFINSOHN, MRS. W. 

YNTEMA, MRS. CHESTER L. 



V. REPORT OF THE LIBRARIAN 

The summer of 1964 saw many physical changes in the Library. The older 
section of the stacks was repainted, new tile flooring put down on all five floors, and 
lighting changed completely to a fluorescent system. New lights were placed on 
all tables in the wing and the reserve desks now contain file cabinets. A large 
catalog room, three new offices for the staff, and a Xerox room were added to the 
existing space. Also, for the first time, all of our most valuable books were brought 
together in a Rare Books Room, which is lined with floor-to-ceiling paneled shelv- 
ing and contains as of this date 517 volumes. This room is located within the 
offices and is available only while the staff is on duty. 

During the year we received and serviced 844 requests on Interlibrary Loan, 
and we requested 137 titles for our use here. In the Fall, 1545 volumes were sent 
to the bindery. A very generous gift of nearly 2000 reprints was received from 
Dr. William Randolph Taylor. The total number of bound volumes in the Library 
is now approximately 100,000. 

The Library holdings for this year are : 

Total Number of Serial Titles in Library 3567 

Number Received Currently 1917 

' )n Subscription 788 

( )n Exchange (with Biol. Bull., Coll. Reprints) 874 

< n Gift Basis 255 

Total Number of Hooks in I .ibrary 15,384 

Number Added in I'M ' -!(>_' 

Received I'Yom Book Exhibit 101 

Total Number of Reprints 231,351 

Xnml.er Added in I'M 3351 



I AXE FESSENDEN, 

Librarian 



REPORT OF THK TREASl'RKR 65 

VI. REPORT OF THE TREASURER 

The market value of the General Endowment Fund and the Library Fund at 
December 31. 1964, amounted to $2.370.890 as against book value of $1,235,860. 
This compares with values of $2.155.4X9 and $1,242,89(>. respectively, at the end 
of the preceding year. The average yield on the Securities was 3.51% of tin 
market value and 6.74% of the book value. The total uninvested principal cash 
in the above accounts as of December 31, 1964. was $5,595. 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. 1964 was $5X0,077 
with uninvested principal cash of $2,210, the market value at December 31, 1963, 
being $390.026. The book value of the securities in this account was $562,547 on 
December 31, 1964, compared with $315,196 a year earlier. The average yield on 
market value was 3.6% and 4.6% of book value. 

The proportionate interest in the Pool Fund Account of the various Funds as 
of December 31, 1964. is as follows : 

Pension Funds 23.098% 

General Laboratory Investment 32.2<>4' , 

Other : 

Bio Club Scholarship Fund 923' , 

Rev. Arsenius Boyer Scholarship Fund 1.130' < 

Gary N. Calkins Fund 1.059% 

Allen R. Memharcl Fund 206% 

F. R. Lillie Memorial Fund 3.566% 

Lucretia Crocker Fund 3.860' . 

E. G. Conklin Fund 652% 

Jevvett Memorial Fund 343' '/ 

M. If. Jacobs Scholarship Fund 4(>5' - 

Anonymous Gift 1 .221 % 

Herbert W. Rand Fellowship 23.326' \ 

Mellon Foundation 7.8X7' , 

Donations from the MBL Associates for 1964 were $4,830.00 as compared \vith 
$4,295.00 for 1963. Unrestricted gifts from foundations, societies and companies 
amounted to $14,575. 

During the year, we administered the following grants: 

Investigators Training .I//. 1 /, Institutional 

13 NIH 3 NIT I 4 Nil I 

3 NSF 2 NSF 3 NSF 

1 Ford 3 ONR 

1 ONR 1 A EC 

1 Commonwealth 1 Ford 

19 M 



66 MARINE BIOLOGK ^LABORATORY 

The rate of overhead on grants to investigators is 20%, based on the amount 
expended. The overhead on these Brunts for this year amounted to $81,239 as com- 
pared with $5(>.4 ( >4 for the preceding year. 

The Lillie Fellowship Fund, with a market value of $114,293 and a hook value 
of $92,893. as well as the investment in General Biological Supply House with 
book value of SI 2.70(\ is carried in the Balance Sheet item "Other Investments." 

The General biological Supply House fiscal year ended June 30. 1964, and had 
a profit after taxes >f S30 l| .<>51 as compared to $241,616 in 1963 and $302,657 in 
1 ( '<>2 and $302.S51 in WM and $314,034 in 1960. 

In the fiscal year 1 ( ><>4 the Marine Biological Laboratory received dividends 
from the General Biological Supply House of $63,500 as against $42,164 in 1963, 
$38.100 in 1962. $33.020 in 1%1. and $30,480 in I960. 

Following is a statement of the auditors: 
I'd ///< 'J'nistccs oj Marine Hioloc/ical Laboratory, U'oods Hole, Massachusetts: 

\Ye have examined the balance sheet of Marine Biological Laboratory as at 
December 31. 1904, the related statement of operating expenditures, income and 
current fund and statement of funds for the year then ended. Our examination 
was made in accordance with generally accepted auditing standards, and accord- 
ingly included such tests of the accounting records and such other auditing proce- 
dures as we considered necessary in the circumstances. We examined and have 
reported on financial statements of the Laboratory for the year ended December 
31. 19(,4. 

In our opinion, the accompanying financial statements present fairly the assets, 
liabilities and funds of Marine Biological Laboratory at December 31, 1964 and 
1963 and the results of its operation for the years then ended on a consistent basis. 

The supplementary schedules included in this report were obtained from the 
Laboratory's records in the course of our examination and in our opinion, are 
fairly stated in all material respects in relation to the financial statements, taken as 
a whole. 

I 'oston, Massachusetts 
March 26, 1W>5 

LYBRANI>, Ross BROS. & MONTC.OMKRY 
JAMKS If. WICKHRSHAM, 

Treasurer 



REPORT OF THE TREASURER 67 

MAR I XI-: BIOLOGICAL LABORATORY 

BALANCE SHEETS 
December 31, 1964 and 1963 

Investments !<Jf>4 10f>3 



Investments held by Trustee: 

Securities, at cost (approximate market quotation 1964 

$2,370,890) ................................ . $1,235,860 $1,242,896 

Cash. 5,595 431 



1,241,455 1,243,327 

Investments of other endowment and unrestricted funds: 

Pooled investments, at cost (approximate market quotation 1964 

$579,161) less $5,728 temporary Investment of current fund 

cash 479,344 309,468 

Other investments 118,888 120,424 

Cash 39,522 14,083 

Accounts receivable 10,664 21,131 



$1,889,873 $1,708,433 



Plant Assets 

Land, buildings, Library and equipment (note) 5,136,289 4,931,472 

Less allowance for depreciation (note) 1,378,887 1,313,162 



3,757,402 3,618,310 

Construction in progress 109,215 

Short-term investments, at cost 192,360 



$4,058,977 $3,618,310 



Current Assets 

Cash 79,637 100,991 

Temporary investment in pooled securities 5,728 5,728 

U. S. Treasury bills, at cost 96,045 74,324 

Accounts receivable (U. S. Government, 1964 $84,793 ; 1963 $95,142) 138,489 140,825 

Inventories of specimens and Bulletins 33,401 45,288 

Prepaid insurance and other 6,844 7,062 



$ 360,144 $ 374,218 



68 MAUIXK 1:101. ()<;K \i. I.AHOK 

MARINE BIOLOGICAL LAB( )RAT( )RY 

BAI.AM i-. SIIKI-:TS 
December 31, 1964 and 1963 

l\ndir:i'inent Funds l<><->4 I'HtJ 

I ndowmenl funds gixen in trust for beneht ot the Marine Biological 

Laboratorv. 81,241,455 si, 243, 327 



Endowment funds for awards and scholarships: 

Principal.. 295,710 12(>,980 

Unexpended income. 14,289 12.077 

309,999 139,057 

rnre-trii ted funds functioning as ciulowment . . . . 206,378 206,378 

Retirement fund. 123,298 108,481 

I'ook-d in\ c-i mrnt> accumulated gain 8,743 11,190 



81,889,873 $1,708,433 



Plant Funds 

Funds expended for plant, less retirements 5,245,504 4,931,472 

I .e-^ allowance for depreciation charged thereto 1,378,887 1,313,162 



3,866,617 3,618.310 

1 nexprnded plant funds. 192,360 



$4,058,977 $3,618,310 



Cm-rent Liabilities and Funds 

Accounts payable and accrued expeu-.es. 33,315 62,763 

Advance Mibscriptiou, 10,926 10,362 

I'nexpended grants research.. 99,000 110,433 

I Hexpended balances of gifts for designated purposes 17,995 13,531 

Current fund 1'>S,908 177,129 



360,144 $ 374,218 



\ ole. The Laboratory lias since Janu.u v 1 , 1916, provided for reduction ol book amounts ol 
])laut assets and funds in\-ested in plant at annual rates ranging from 1 % to 5% of the original cost 
ol the assets. 



RKl'ORT Ol-' THK TRKASURER 6<) 

MAR I NIC BIOI.OC.ICAI. LABORATORY 

STATEMENTS OF ( )IM-:K ATFNC; KXPHXIHTIRKS, INOIMK AND (YKKKNT I-VND 
Years Funded December 31, 1964 and 1963 



Operating Expenditures 
Research and accessory services - 5 


1964 

5 293,396 


1963 

$ ? 73 3^3 


Instruction 


160,820 


147,163 


Library and publications (including book purchases 1964, $24,304; 
1963, $25,628) 


80,416 


77,922 


1 'irect costs on research grants . 


493,890 


484 640 


I )irert costs on institution support grants . . 


150,450 


129.64 7 








Administration and general 


1,178,972 
IP, 453 


1,112,700 
97,339 


Plant operation and maintenance 


139,684 


11 1,441 


Dormitories and dining 


1 75,696 


174,030 


Additions to plant from current income 


5 045 


7 793 








Less depreciation included in plant operation and dormitories and 
dining above but charged to plant funds ... 


1.611,850 
67,437 


1 ,503,303 
67,47? 










1,544,413 


1,435,831 


Income 

Research fees 


88,240 


113,024 


Accessory services (including sales ot biological specimens 1964, 
$42,051 1963, $38,019) 


114,181 


103,398 


I nstruction fees .... 


28,470 


28,461 


Library fees, Bulletins, subscriptions and other 


49,393 


43,952 


Dormitories and dining income 


133 759 


129486 


Grants for support of institutional activities: 
Instruction and training 


138,540 


131,564 


Support services . 


150,450 


129,642 


General 


109,219 


113,853 


Reimbursements and allowances for direct and indirect costs on specific 
research grants 


575,129 


541,134 


Gifts used for current expenses 


22,135 


29,285 


Investment income used for current expenses 


156,676 


134,591 










1,566,192 


1 .498,390 


Kxcess current income 


21,779 


62,559 


Current fund balance January 1 


177.129 


114,570 









Current fund balance December 31 . . $ 198,908 $ 177,129 



70 



MARIXK HIOI.fH.H \l. I. . \MORATOK V 



M. \RI\T. BIOLOGICAL L.\B( )RATORY 



Si \TKMKNT OF Fl'NDS 



Year Kndod December 31, 1%4 



(Hits and Invest- I 'ted for Other 

.fun may Oilier men! Current Expendi- 

1, l"t>4 Receipts Income Expenses hires 

Invested funds. . $1,708,433 $ 187,132 $164,448 $ 153,890 $16,250 

Unexpended plant 

funds.. 452,800 1,575 262,015 

Unexpended research 
jrrants.. . $_110.4.U 1,110,905 1,122,338 

Unexpended gilts tor 

designated purposes. 8 13,531 29,687 22,135 3,088 

Current fund . . $ 177,129 21,779(1) 

$1,802,303 $166,023 $1,298,363 $281,353 

Gifts.. 198,447 

Grant fur facilities. . . . 452,800 

Grants for rex-arch, 
training and 
support.. 1,110,905 

Appropriated from cur- 
rent income and 
other 22,722 

Net loss on sale ol 

securities (4,350) 

Excess of current in- 
come over expendi- 
tures.. 21,779(1) 

$1,802.303 

Expended for con- 
struction and 
renovation of 
facilities 262,015 

Scholarship awards 4,124 

Payments to 

pensioners. . . 1 2,126 

Other. 3,088 



Balance 
December 
31, 1964 

$1,889,873 
$ 192,360 
$ 99,000 

$ 17,995 
$ 198,908 



S281.353 



REPORT OF THE TREASURER 



71 



MA RIM: BIOLOGICAL LABORATORY 

SlMMAKY OF IXVF.STMIXTS 
I )r(Vllll)lT .11, 



Securities held by Trustee: 

General endowment fund: 

U. S. Government securities 

Corporate bonds 

Preferred stocks 

Common stocks 

General Educational Board 
endowment fund: 

U. S. Government securities 

Other bonds 

Preferred stocks 

Common stocks 

Total securities held by 

Trustee 

Investments of other endowment 
and unrestricted funds: 
Pooled investments: 

U. S. Government securities 

Corporate bonds 

Common stocks 

Less temporary investment 
of current fund cash. . 



Cost 

; 92,310 

528,638 

54,422 

358,452 

1,033,822 



1,235,860 



61,005 
125,736 
298,331 
485,072 



(5,728) 
170.344 
Other investments: 

U. S. Government securities 27,947 

Other bonds 15,031 

Preferred stocks 3,728 

Common stocks 58,887 

Real estate 13,295 

11 8, "888 

Total investments of 
other endowment and 
unrestricted funds. . . $ 598,232 

Total investment income 

Custodian's fees charged thereto. . . . 

Investment income distributed to 
funds 

Plant investments: 

U. S. Treasury bills, due 3/4/65 . 80,085 

Bank of New York acceptances, 

due January, 1965 112,275 

$ 192,360 

Current investments: 

U. S. Treasury bills, due March 4 

and October 31, 1965 $ 96,045 

Temporary investment in pooled 

securities. . . $ 5,728 



% of 
Total 

8.9 

51.1 

5.3 

34.7 
100.0 



18,086 8.9 

107,310 53.2 

15,641 7.7 

61,001 30.2 

202,038 100.0 



12.6 
25.9 
61.5 

100.0 



Market 
Quotations 

$ 91,086 

522,095 

53,200 

1,306,140 

1,972,521 



17,836 
109,091 

15,100 
256,342 



$2,370,890 



59,730 
125,370 
394,061 



%of 
Total 

4.6 

26.5 

2.7 

66.2 

100.0 



4.5 
27.4 

3.8 
64.3 



398,369 100.0 



10.3 
21.7 

68.0 



579,161 100.0 



Investment 

Income 

1964 

$ 4,449 

21,177 

2,186 

42,950 

70,762 



1,221 

4,106 

700 

6,508 



83,297 



2,308 
4,782 
7,489 

74^79 

(251) 
14.328 

1,312 

749 

130 

65,576 

67.767 

82,095 

165,392 
(944) 

164,448 
684 

891 

1,575 

2,686 

251 

2,937 
$168,960 



THE [NDUCTIVE ROLE OF THE YOLK EPITHELIUM IX THE 

DEVELOPMENT OF THE SQUID, LOI.Kio 

PEALI] (LESUEUR) 1 - ; 

'i IHN M. ARXOLD 

ii' l-iio!i>iiiciil Laboratory, H'onds Hole. Massachusetts 02^-13, and the I)cf>arlincnl of 
/.oolou\, I'lih't'rsifv of Minnesota, Minneapolis, Minnesota 55/.v :: 



While a fairlv large body of information exists on the descriptive aspects of 
the embrvologx of the cephalopods, relatively little experimental work has been 
done on these animals. These embryos seem superficially to be ideally suited to 
embryological analysis since they are large, abundant, and are relatively easy to 
obtain (Arnold, 1 () (>2>. ( >ne of the major difficulties has been the inability to 
work with embryos outside of the chorion. Ranzi (1931) tried to raise Sepia 
officinalis in sea water but had little success. He was able to isolate various organs 
and parts of the embryos of Sepia by operating through the chorion. and concluded 
that isolated organs and tissues were capable of self-differentiation. This technique 
severely limited operative procedures, and he was forced to use rather late stages, 
( Xaef stage XII : - Arnold stage 2(> ) in which most of the organ primordia were 
rather well formed. 

In the summer of 1 ( ">1 it was possible to devise culture techniques by which 
the dechorionated embryos would survive and develop normally. This has allowed 
some experimental analysis of young embryos of the common Atlantic coast squid, 
l.i >l it/i i pealii. 

MATERIALS AND METHODS 

Since the techniques used in this study have not been fully described, it is 
necessary to give a detailed account of the procedures used. Most of the experi- 
ments were performed in ritro in a culture medium made of three basic components: 
whole adult squid blood, sterile sea water, and an antibiotic stock solution. The 
blood was drawn under sterile conditions from the vena cava of the adults and 
stored frozen in 3-ml. glass lubes. Before use it was thawed and diluted with an 
equal volume of sterile sea water, To this was added about ]' ', of an antibiotic 
Mock solution composed of 0.5'r streptomycin, 0.05 r / phenol red, and 50,000 units 
of potassium penicillin <i in double-distilled water which had been saturated with 
sulfadia/ine. The embryos were cultured in glass vessels made expressly for this 
purpose. Each of these vessels had a volume of about 0.1 ml. and had two small 
depressions in the bottom lor holding tin- embryos. This culture vessel was placed 

' This work is a portion of a tin-sis submitted in partial fulfillment of the Ph.D. require- 
ments of the Department ot /oology, I niversity of Minnesota. 

- 1'art of this \\ork was done while the author was the recipient of an X.S.I'". 1 'redortoral 

hellow ship. 

( in-rent addles : Department ot Zoology and Kntomolo<>y, Iowa State I'liiversity, \rnes, 
[owa, ?onln. 

72 



INDUCTION IX LOLIGO 

in a covered preparation dish to which a few drops of sterile sea water had been 
added to saturate the atmosphere and maintain osmotic equilibrium with the medium 
in the vessel. 

The embryos were prepared for culture by stripping off the outer tunics of the 
egg string with the fingers, cutting up the denuded string and transferring indi- 
vidual embryos in their chorions through two changes of sterile sea water. The 
chorion was then torn open and the embryo washed twice in sterile sea water. The 
operative procedures described below were performed and the embryo was then 
transferred to the freshly prepared medium. The embryos were incubated at 18 C". 
(0.5 C.) and examined twice daily. The medium was changed every second 
day when the experiments exceeded this length of time. 

Under these conditions the embryos appeared to develop completely normally 
when compared with control embryos still in their chorions and in normal sea 
water. The cultured embryos seemed to develop slightly faster than the controls, 
possibly because of greater availability of oxygen due to the lack of the chorion. 
Some embryos were maintained for 178 hours in culture with no apparent adverse 
effects. If the medium was not changed at least every three days, development 
would slow down. This effect was reversed by the addition of fresh medium. 

Parts of the stage H> and 17 embryos (Arnold, 1965) were removed with stain- 
less steel needles before culturing. At these stages, the cephalopod embryo con- 
sists essentially of an outer layer of cells (future body of the embryo), a layer of 
greatly flattened cells (yolk epithelium), and a central mass of yolk. The various 
organ primordia could be identified by the thickened nature of the outer layer of 
cells (columnar z<s. cuboidal cells) and the asymmetrical nature of the egg. Most 
of the operations involved removal of the parts of the eye, but the same confirming 
experiments were also done on the otocyst, arms and funnel folds. Three different 
types of operations were performed. The first involved the removal of the whole 
eye primordium, including some of the underlying yolk. This isolate would round 
up and the yolk would become incorporated within the center. These isolates sur- 
vived quite well in the culture medium and underwent considerable differentiation. 
The second operation separated the outer layer of cells from the yolk epithelium. 
This was accomplished by gently rubbing the cells with the blunt edge of a needle 
until the cells became loosened and somewhat sticky. The outer layer of cells could 
then be caught on the point of a needle and pulled off in a sheet. A second needle 
was used to cut the sheet of cells well beyond the organ primordium. \Yith practice, 
about one-fourth to one-third of the total surface of the outer cells could be removed 
and the embryo transferred to culture medium with only a few minutes exposure 
to sterile sea water. Sections of unstripped and stripped embryos are shown in 
Figures 1 and 4. In a few cases the eye anlage was removed by the technique 
described below for dissociating cells. A large number of embryos was examined 
and appropriate ones were selected. In the third operation the outer layer of cells 
was stripped and a small portion of the yolk epithelium removed. This was rather 
hard to accomplish since the embryos tended to lose volk from the wound, which 
mechanically inhibited wound healing. However, enough cases were successful so 
interpretable results were obtained. In a few cases masses of cells were grafted 
onto the freshly stripped yolk epithelium. 'I hese cells were obtained bv cutting 
up an egg string and shaking it in sea water adjusted to pH 5 with 1 \ T HC1 



74 



1 




10 








|''K;CRKS 1-10. 



[NDUCTION IX l.ni.K.n 75 

until the egg-jelly dissolved. ( )nce the egg-jelly was completely dissolved and 
the embryos inside their chorion were completely free, the embryos were shaken 
violently for about seven to ten minutes. The outer layer of cells was dissociated 
from the embryos, and usually single cells or small groups of cells resulted. The 
embryos were immediately examined, and those which appeared to be well disso- 
ciated were selected. At stage 17 the dissociated cells would reaggregate and form 
small solid clumps in about 15 minutes. These small clumps would fuse until 
about seven to fifteen aggregates remained in each chorion. Although these clumps 
would remain alive for extended periods, no organ or tissue differentiation was 
ever observed. Newly formed aggregates (one to two hours after dissociation ) 
were stained in a solution of neutral red (0.01^) and grafted onto the freshly 
stripped yolk epithelium. The aggregates stuck quite readily and tended to flatten 
out on the surface of the embryo. The aggregates remained visible and were 
easily distinguished from the cells of the host embryo by their dye content. 

RESULTS 

\\hen the whole eye primordium was removed, together with some of the sur- 
rounding tissue and underlying yolk, the resultant isolate rounded up with the yolk 
on the inside. These isolates differentiated quite normally and complete organs 
were formed (Figs. 2 and 3). The wound on the donor embryo lost a portion of 
yolk, but eventually the edges of the wound closed, development continued, and the 
unoperated organs differentiated normally. There was no evidence of any regen- 
eration or replacement of the organs removed (Fig. 5). A total of 25 operations 
was performed on stage 17, and 13 operations on stage 16 embryos. In all cases 
the results of these operations were the same. The possibility of inhibition of 
development due to operative trauma was checked by a series of sham operations 
in which either less than the whole eye primordium was removed or a few cuts 
were made in the surface of the embryo. In these cases there was loss of yolk at 
the site of the wound but after healing normal development ensued. 

Removal of the outer layer of cells resulted in a different pattern of develop- 
ment. The isolated cells usually formed a small clump that showed no further 
differentiation and lost the characteristics formerly possessed. These small clumps 
of cells did not survive well, possibly due to the trauma of surgery. Of those that 

FIGURE 1. Section of the eye region of a stage 17 embryo. Note the yolk epithelium and 
the columnar nature of the outer layer of cells. Epon 812, Azure II-methylene blue; 660 X. 

FIGURE 2. Isolated organs of the embryo shown in Figure 5. Both the outer layer <>t cells 
and the yolk epithelium were isolated; ca. 190 X. 

FIGURE 3. Lower section of the same isolate as in Figure 2. In culture 45 hours. 

FIGURE 4. Section of the yolk epithelium after removal of the outer layer of cells. One 
yolk epithelium cell has rounded up abnormally above the rest of the yolk epithelium; 660 X. 

FIGURE 5. Donor embryo for Figures 2 and 3. Note lack of organs on one side. 

FIGURE 6. Regenerated otocyst after removal of the outer layer of cells; 900 X. 

FIGURE 7. Control eye for Figure 10; 725 X. 

FIGURE 8. Section through the retina of a stripped embryo onto which dissociated-reaggre- 
gated cells were grafted. In culture 40 hours; 1000 X. 

FIGURE 9. Dissociated-reaggregated cells after 55 hours. Note the lack of any tissue 
differentiation; 1000 X. 

FIGURE 10. Regenerated eye after removal of only the outer layer of cells. Compare with 
Figure 7. 



76 

did survive, no evidence of differentiation could he detected. Tin- wound produced 
In this operation was closed In migration ol the surrounding outer layer of cells 
Over the denuded yolk epithelium. In coxering the yolk epithelium the cells would 
cut off any small hlehs of yolk that occasionally resulted from small punctures 
accidentally produced in the \olk epithelium during the operation. That these cells 
of the outer layer actually migrated over the wound rather than grew there was 
demonstrated In the speed at which the covering took place (one to two hours) 
as well as In a series of careful ohservations during wound closure. A total of 
2\ stage 17 and If) stage 1<> emhryos was operated on in this fashion and all were 
cultured for at least 45 hours. In all of these cases the cells which migrated over 
the stripped yolk epithelium differentiated with the structures that would have 
normally differentiated in that site. In some cases (8) one-third of the total outer 
layer of cells was removed, vet the resultant emhryos had all of their organs when 
examined and sectioned after approximately 48 hours in culture (Figs. 7 and 10). 
There seemed to he a slight retardation of development of the replaced organs hut 
otherwise the cour.se of development corresponded exactly to that of the unoperated 
side of the embryo. The eve. otocyst, arms and funnel folds could he thus stripped 
off and replaced In the cells normally destined to form other organs (c.c/.. Fig. 6). 

Since the ahove results would implicate the yolk epithelium as possibly haying 
a causal role in the differentiation of the oyerlying cells, two other experiments 
were attempted to test this hypothesis. The first of these involved stripping off 
the outer layer of cells of stage 17 emhryos and removing a small portion of the 
yolk epithelium. This was not easily accomplished because the yolk epithelium 
was easily torn. I lowever, in two cases the yolk epithelium in the future region of 
the otocyst was successfully removed and development was carefully followed. In 
this case closure of the wound proceeded as before and differentiation of the organs 
followed but the embryo lacked the otocyst on the operated side. The second 
test involved grafting dissociated-reaggregated cells onto the freshly denuded yolk 
epithelium. Normally these aggregated cells survived in the culture medium for 
extended periods and gradually died and disintegrated. In no case did any of the 
aggregates resulting from well dissociated embryos ever show any interpretable 
differentiation (Fig. 9). When these cells were grafted onto the denuded yolk 
epithelium, they stuck unite readily and spread out slightly. The position and ex- 
tent of the grafted cells could be easily ascertained by the dye they contained. 
When sectioned it appeared that those cells in contact with the yolk epithelium 
differentiated into rather normal-looking tissue which corresponded to the location 
on the embryo (Fig. 8). In the case' of the primordial retina, the cells became 
columnar in appearance, underwent mitosis in the characteristic position, and ap- 
peared to be well incorporated in the host organ. In eight successful cases, parts 
of four gratis unquestionably became incorporated into the developing retina. In 
the remaining case the results were not as clearcut because of yolk leakage at the 
site of the graft. 

The possibility of the yolk playing a significant specific causal role in the dif- 
ferentiation was eliminated by experimentally removing up to one-half of the yolk 
of the embryo. Despite this rather large loss in volume the embryos differentiated 
normally but were reduced in size, particularly in the yolk sac region. These 
results agree with those obtained by ( >kada ( 1"J7) on l.o/ii/n hlcckcri. 



ixnrcTiox ix LOLK.O 77 

DISCUSSION 

The results reported have led the author to the conclusion that the cells of tin- 
outer layer are indeterminate in their fate unless influenced In the underlying yolk 
epithelium. This appears to tit the classic ideas of embryonic induction, with the 
yolk epithelium being the inductor and the outer layer of cells responding to its 
inductive influence. This can he stated with certainty for the eye and otoc\>t and 
as probable for some of the other organs (funnel folds, arms and gills). Organs 
other than these remain to be tested, but preliminary experiments indicate that tlicii 
development is of the same general nature. It appears, therefore, that all of the 
organs of the embryonic body are laid out in inductive areas of the yolk epithelium 
and are fairly localized. Tbis can be visualized as a "morphogenetic inductive 
map" in which developmental information is transferred to the highly labile or in- 
different cells that overlie it. One of the rather unique features of this inductive 
map is its two-dimensional nature. Unlike many other systems in which induction 
occurs within a mass of heterogeneous tissue, the inductor in this case is present as 
a sheet. This system, therefore, should be more amenable to experimental analyst 
because the presumptive areas of the embryo can be rather easily localized and 
subjected to direct experimental analysis. Preliminary experiments with chemical 
treatment of the denuded yolk epithelium offer encouragement along these lines. 

The problem of how to fit these results with the classical ideas of mosaic de- 
velopment of the molluscs is an open question. It is obvious that part of the 
embryo (the yolk epithelium ) is rather rigidly fixed in its fate while the outer layer 
of cells is quite labile and subject to the inductive influences of the yolk epithelium. 
Just when and how this developmental information arises is still unknown but 
preliminary experiments indicate that the egg cortex of this embryo is also rather 
rigidly patterned and the morphogenetic patterns would then be referred ultimately 
to the ovary. Obviously, further work along these lines is indicated. 

I would like to thank Drs. K. C. Shaw and A. L. Allenspach for reading this 
manuscript. Special thanks are due to Dr. Nelson T. Spratt. Jr. for his help and 
guidance with this work. 

SUMMARY 

1. Techniques for the in i'itro culture of dechorionated Loligo pcalii embryos, 
involving the use of whole adult squid blood, sterile sea water, and antibiotics, 
have been devised. Essentially normal development will take place in this culture 
medium. 

2. The embryos in stages 10 and 17 are composed essentially of three com- 
ponents: an outer layer of cells, an inner cellular yolk epithelium, and the central 
mass of yolk. \Yhen the outer layer of cells and the yolk epithelium are isolated 
together, normal histogenesis and development will occur. \\ hen the outer layer 
of cells is isolated by itself, no differentiation occurs. This leads to the conclusion 
that the yolk epithelium induces the outer layer of cells to differentiate. This con- 
clusion was upheld by grafting and deletion experiments. 

3. The role of the yolk epithelium, therefore, may be in acting as a "morpho- 
genetic inductive map." 



JOHN M. \K\OI.D 

I I II R \ i QRE CITED 

ARNOLD, I. M.. I'>n2. Mating behavior and social structure in Lulii/o pculii. Hint. Hull.. 123: 

53-57. 
AKXOLD, I. M., 1%5. Xonnal cmhryunir stages of the squid, Lolif/o pcalii (Lesueur). Jiinl. 

Bull., 128: 24-32. 
\ \i r. A., 1 ( '2>\ Die C'e]ilialnp ( uli-i:. Monographie 35. Fauna e Flora del (iolfo di Xapoli, 

Vol. 1. 
OKADA, ^"., 1^27. \"ersui-lic iilier die \\irkung der Dotterwegnahme am meroblastischcn FA. 

Zool. Ans., 73: 2SO- 2S4. 
I\\\/i, S., l')31. Sviluppo di parti isolate di enihrioni di Cefalopcxli. 1'nhh. Stu::. Znol. 

\tipoli. 11: 104-146. 



PHASE-SHIFTING A LUNAR RHYTHM IX PLAXARIAXS B 
ALTERING THE HORIZOXTAL MAGNETIC VECTOR 1 

FRANK A. BROWN, JR. AND YOUNG H. PARK 
Department of Biological Sciences, Northwestern University, r.i'anxton, Illinois 



It has been demonstrated that planarian worms directed initially northward 
in the late morning hours in an unvarying field-pattern of illumination will displav 
a synodic monthly variation in their tendency to alter their course (Brown, 1962, 
1963). From about September to March, the worms veer maximally to the left 
at new moon and to the right at full moon. During March and April the monthly 
rhythm typically becomes gradually transformed into a semi-monthly one with 
maxima in right-veering at both new and full moon, and left- veering at the moon's 
quarters. 

The natural synodic month owes its existence, of course, to the periodic inter- 
ference between the solar-daily and lunar-daily cycles, and hence it is reasonable 
to presume that the worms in the generation of their synodic monthly periodism, 
which remains phase-synchronized in a characteristic temporal relationship to the 
geophysical cycles, are depending upon responses to pervasive geophysical varia- 
tions possessing both of these two important natural physical frequencies. 

This monthly phenomenon was investigated under each of three experimental 
conditions on mornings during November through January, 1960-61. The con- 
ditions were: (1) controls in the unmodified ambient field, (2) augmenting tin- 
natural horizontal magnetic vector to 4.0 gauss, and (3) imposing a 4.0-gauss 
horizontal field in an east-west orientation. The monthly rhythm was conspicu- 
ously present in the natural field, absent in the augmented north-oriented magnetic 
field, and present but reduced in amplitude in the east-directed field. During these 
studies the observations of path direction on any given morning for each of the 
above conditions required an average of about 20 minutes to permit about 50 
worm-paths to be recorded as an assay of the influence on that particular morning 
of each individual experimental condition. 

The more recent demonstrations of after-effects of exposures of planarians in 
experimentally altered horizontal magnetic vectors with descriptions of their prop- 
erties, together with suggestive variations in response dependent upon time of ex- 
posure to the experimental fields (Brown, 1 ( ><>5; Brown and Park, 1965), raised 
questions concerning what might be relationships of these effects and after-effects 
to influences of experimentally altered weak magnetic fields on the monthly rhythm 
of the worms. 

The following report describes the results of an attempt to learn more concern- 
ing influences of experimental changes in strength and direction of the horizontal 

1 This study was aided by grants from the National Science Foundation ((J-15008) and 
the National Institutes of Health ( CIM-07405), and by a contract with the Office of Naval 
Research (1228-30). 

79 



> s <> I-'K AXK A. I!K()\\ X, JR. \XD YOUNG II. I'AKK 

magnetic vector upon the monthlv variations in orientational tendencies and upon 
their generating mechanism. 

M. \TKKI.\I.S AND MKTHODS 

The data which were used in this investigation were the same as those employed 
for determining the duration of the after-effects of reversed horizontal magnetic 
vectors ( lirown and Park. 1 ( ">5 I. The data were obtained over the seventh-month 
period from ( )ctober S. l'X>3. through April 30, 1964. The detailed methods by 
which these data were derived and the primary reductions to which they were 
subjected have been described in the earlier report. 

In essence, uniformly distributed over the 206-day period of the study, 3(>0 
series of observations were made, in each of which series an observer determined 
the average rate- of turning of north-oriented worms for each of the ten 5-minute 
intervals of a continuous 50-minute period. For the first 10 minutes the worms 
were in the natural ambient geophysical field. Then daring the next three con- 
secutive 5-minute intervals, the worms remained continuously exposed to an ex- 
perimentally reversed horizontal magnetic vector of 0.05 gauss. The final five 
5-minute intervals followed return of the worms to the natural ambient field. 
Similar! v, uniformly distributed over the same 206-dav period, 357 50-minute series 
ot observations were being made, just like the preceding series except that the 
reversed horizontal magnetic vector-strength was 4.0 gauss. Since an average 
of about 14 worm-paths could be recorded during a 5-minute period, about 50,000 
individual worm-paths were recorded during the study. The mean path for each 
5-minute interval while in the reversed magnetic field and following its removal 
was expressed as difference from the mean path for the initial 10-minute "controls." 
to obtain a measure of the response to the reversed fields. The response data were 
then reduced to mean responses to each field strength for each 5-minute interval 
for each of 41 consecutive 5-dav periods of the study. Five-day means were used 
in order to reduce the size' of the influence of day-to-dav variations in uncontrolled 
^eophvsical factors. 

For purposes ot the present analysis, these data permitted one to determine the 
torm ot the mean monthlv variation in the path of the initial "controls," and the 
form ot am mean monthly variations which might be present in the turning re- 
sponses ot the worms to each of the- two reversed field strengths. It was, linallv. 
possible to follow and characterize any after-effects on such rhvthmic responses 
which might tend to persist following the removal of the experimental fields. 

Farlier studies had indicated that monthlv variations occur in response to vcrv 
weak magnetic fields. To disclose any monthly variation in response to the ex- 
perimental fields ot this studv and in the 1 after-effects, the data were used in the 
following manner. The mean response for each 5-dav interval was treated as if it 
were a value obtained on that particular dav over which the interval was centered. 
Mnis. each 5-day group could be ascribed a specific dav relative to new moon in 
the natural lunar monthlv cycles The mean response was then calculated for X, 
approximately equally spaced 3- or 4-dav intervals over the lunar cvcle. The in- 
tervals were centered on ihe times in the monthlv cvcles which are illustrated 
in ! igure 1 . 



PHASE-SHIFTING A RHYTHM BY MAGNETISM 



81 



H 



or 




UJ 

O 



cr 
LU 

L_ 
L_ 







A 



B 




c 






J L 



FM 



N M 



FM 



FIGURE 1. A. The variation in the difference between responses to the 4.0-gauss and the 
0.05-gauss reversed horizontal vectors, as related to the phase of moon. B. The same difference, 
but for the first IS minutes after removal of the magnets. C. The same difference, from 15 to 
25 minutes after removal of the magnets. 



82 



FRANK A. I'.K'MU \", JR. AND YOUNG H. PARK 



RESULTS 

In Figure 1A is plotted the mean response of the 4.0-gauss worms relative 
to that of the 0.05-gauss ones during the 15 minutes of exposure to the experi- 
mental fields, as a function of phase of moon. A monthly variation is clearly sug- 
gested, with a maximum difference occurring over full moon and minimum, even 
possibly a reversal of the difference, over new moon. The comparable variations 
of relative paths in the two field-strengths with phase of moon during the first 15 
minutes following removal of the experimental field, and during the last 10 minutes 
after removal, are shown in Figures IB and 1C, respectively. A monthly variation 
appears to persist for a time hut to have disappeared nearly completely by 20 to 




NM 



FM NM 

PHASE OF 



FM 



NM 



MOON 



FIGURE 2. Variation in mean path of the worms (controls) with phase of moon during 
their initial 10-minute period while in the natural ambient geophysical field. The mean monthly 
cycle is repeated. 

25 minutes after the field removal. The rate of the disappearance of the monthly 
variation in this difference strikingly coincides with the rate of loss of the general 
after-effect reported earlier (Brown and Park, 1965). 

The results pertaining to general after-effects which have been described pre- 
viously had suggested that during this 7-month study a mean, overall response to 
the two experimental field strengths occurred chiefly for the reversed 4.0-gauss 
one. However, evidence was advanced to indicate that the worms also displayed 
a very definite response to the 0.05-gauss reversed field but that the character of 
the response varied with time more strikingly than did response to the 4.0-gauss 
field, even changing in sign. The results suggested an annual variation. From 
the present study it became evident upon examining separately the responses to 
the two strengths of the magnetic fields, together with their subsequent after-effects, 
that response to the 0.05-gauss field varies as a function of elongation of the moon. 



PHASE-SHIFTING A RHYTHM BY MAGNETISM 



83 



To learn exactly what effect the magnet might have, it was necessary first to learn 
the nature of the monthly variation of the initial controls. The presence and 
characteristics of such a rhythm in worms had previously been shown and char- 
acterized for morning hours. For the present study about 40% of the results 
were obtained, instead, in the afternoon. Determination of the relationship be- 




FM 



NM 



FM FM 



NM 



FM FM 



NM 



FM 



FIGURE 3. A and A'. The variation in response to the 0.05- and 4.0-gauss reversed 
horizontal vectors, respectively, in relation to moon phase during the first five minutes of field 
application (solid line), the second five minutes (dotted line) and the third five minutes (dashed 
line). B and B'. The corresponding relationships for the first three S-minute intervals follow- 
ing removal of the magnets. C and C'. The corresponding relationships for the fourth 5- 
minute interval (solid line) and the fifth one (dashed line) following experimental-field 
removal. 



tween phase of moon and means of the paths of the initial control-worms (Fig. 2) 
disclosed a mean monthly pattern of variation with a maximum right-turning near 
first quarter of the moon and maximum left-turning just prior to new moon. 

The mean response to the reversed 0.05-gauss field (Fig. 3 A) was an immedi- 
ate gross reversal of phase of this normal monthly variation. Now, maximal 
right-turning occurred just prior to new moon. During the 15 minutes immedi- 
ately subsequent to the removal of the experimental field (Fig. 3B) the amplitude 
of this 180 phase-shifted monthly variation slightly increased and its form be- 
came somewhat altered. Only relatively minor changes in the mean monthly 
pattern then seem to have ensued through the 20- and 25-minute observation 



84 FRANK A. BROWN, JR. AND YOUNG H. PARK 

times (Fig. 3C). The new cycle. 180-shifted relative to the initial one, had 
persisted in an extraordinary manner. 

The mean response to the reversed 4.0-gauss field was a definite and immediate 
displacement of the mean worm paths to the right of the controls and a slight 
change in the monthly pattern ( Fig. 3A' I. Although there was a beginning of an 
inversion of the monthly cycle, in that maximum right-turning occurred near third 
quarter of the moon, there was lacking the striking immediate, essentially complete, 
inversion that was observed for the response to the 0.05-gauss field. Despite, 
also, quite a different transient pattern from that observed for the weaker, 0.05- 
gauss, field during the 15 minutes immediately subsequent to magnet removal 
(Fig. 3B') the increased general right-turning behavior gradually subsided and a 
persistently altered monthly pattern of response became evident (Fig. 3C). The 
patterns of monthly variation in response witnessed during the 20- and 25-minute 
periods were quite similar to those observed for the corresponding periods following 
the removal of the 0.05-gauss reversed field, and again were essentially 180- 
shifted relative to the phase of the initial control pattern. 

A disappearance of all differences between the patterns of the monthly variation 
following the magnetic response and the pattern of the initial controls would 
have been expected if the shifted rhythm had gradually been lost over the 25- 
minute post-magnet period. And yet, the worms from both magnetic series (Fig. 3C 
and 3C') now displayed, in common, a monthly pattern of difference from the 
initial controls which resembled astonishingly a cycle 180 "-phase-shifted from that 
one which was present before the submission to the 15 minutes of the geographically 
reversed experimental magnetic field. The relationship, relative to nciv moon 
for persisting effect, now resembled closely the former relationship to full moon 
for the initial controls (compare Fig. 2 with Fig. 3C and 3C'). 

Since the observations in these experiments had been made only during 7 or 
8 daytime hours, between about 9 AM and 5 PM, the monthly rhythm of the controls 
could be considered as reflecting a lunar-daily variation in the orientational re- 
sponse of the worms, when north-directed, to the ambient geophysical field, and 
after magnetic field-reversal as a 180 -phase- shifted lunar-daily variation in 
response. 

DISCUSSION 

The results of this study extend our knowledge of responses of planarians to 
altered very weak horizontal magnetic fields to include another kind of response. 
This one is a phase-shifting of a lunar rhythm by an abruptly reversed horizontal 
vector. This response .appears to be independent of vector strength at least over 
a range from about 1/3 to about 23 times the earth's local horizontal vector, the 
range in this investigation. This response appears to relate geomagnetic vector 
direction in some manner to phase angle in the biological lunar rhythm. 

Fven by the end of 25 minutes following removal of the experimental fields 
the relation between mean path and elongation of moon had displayed no tendency 
to return to its initial state, llad return occurred, the two relationships depicted 
in Figures 3( ' and 3( v would have exhibited no characteristic monthly pattern of 
variation. Instead, a monthly variation of closely the same character is present 
for the 20- and 25-minute response patterns following the 0.05- and 4.0-gauss 



PHASE-SHIFTING A RHYTHM BY MAGNETISM 85 

reversed fields. This pattern resembles, even in striking details in its form, the 
monthly pattern of variation of the initial controls. Even amplitude is similar. 
It is, however, 180 out of phase with the controls. The worms thus appear to 
have superimposed on their initial state a fully comparable monthly pattern with 
respect to full moon that as initial controls they had had with respect to new moon. 
In other words, the experimentally reversed magnetic fields, whether 0.05- or 
4.0-gauss, had, after the very different transient states during and immediately 
following the reversed field, come to effect a common pattern of lunar-monthly 
variation in response to the natural ambient geophysical field. 

In a previously reported study (Brown, 1962) a 4-gauss horizontal magnetic 
field parallel to the axis of N-directed planarians had abolished a distinct unimodal 
monthly variation which was present when the worms were in the natural ambient 
field. The current study indicates that a S-directed 4-gauss field, under com- 
parable conditions, tends to convert the natural unimodal monthly variation in 
orientational behavior at least initially into a bimodal one ( Fig. 3 A' ) , suggestively 
a transitional state between the initial cycle and the 180-shifted, essentially uni- 
modal one which ultimately appears following return of the worms to the natural 
environment. At the same time this study indicates that far less conspicuous 
transient alterations result from application of a 0.05-gauss reversed field, judging 
from the general similarity between the relationships depicted in Figures 3A 
through C. The weaker field appeared, therefore, to be the more efficient in 
effecting the phase shift. 

The slightly increased amplitude of the monthly variation evident immediately 
following the 0.05-gauss field (Fig. 3B), and the initial delay in reaching the full 
cycle range following exposure to the 4.0-gauss field (Fig. 3B'), suggest a 
hypothesis that following exposure to horizontal vectors weaker than the earth's, 
animals are transiently hypersensitive to the earth's vector, and following exposure 
to vector fields stronger than the earth's, transiently hyposensitive. 

The explanation of the mean monthly variation in the response to the 4.0-gauss 
field when expressed as difference from response to the 0.05-gauss one, and its 
gradual decay following removal of the experimentally reversed horizontal mag- 
netic vector, have now become apparent. This reflected the difference in influence 
on the monthly rhythm between the responses to the two strengths of experimental 
fields. The immediate phase-shift in response to the 0.05-gauss field, together 
with the initial, only partial, shift as response to the 4.0-gauss one, resulted in a 
monthly variation in the difference between them (Fig. 1A). This difference, 
though steadily diminishing during the first 15 minutes following removal of the 
experimental fields (Fig. IB), did not essentially disappear until 15 to 25 minutes 
had elapsed (Fig. 1C). The difference had practically vanished only after both 
experimental groups came ultimately to stabilize with the same 180-shifted 
monthly pattern. 

Perhaps the most interesting and potentially most significant finding of this 
study is the apparent association between phase angle of a spatial vector factor, the 
horizontal component of terrestrial magnetism, relative to other contemporary 
vector forces and the phase angle of a biological rhythmic variation. Although a 
number of kinds of observations have suggested an existence of some common de- 
nominators for the biological clock and biological compass mechanisms, this is the 



86 FRANK A. HROXVX. JR. AND YOUNG H. PARK 

first specific piece of experimental evidence that such a relationship may actually 
exist. While much r\ idrnce has been advanced in support of the conclusion that 
geographic orientation is influenced In phase-resettings of biological clocks, this 
is the first evidence that a biological rhythm itself can have its phase reset by 
altering the vector angle of any geographical field component. 

The observed monthly variation in orientational tendencies of N-directed 
planarians is a mosaic of the activities of numerous individual members sampled 
from day to day from a very large population. The phase-reversal which is de- 
scribed here had clearly become essentially fully effected for the 0.05-gauss worms, 
even in time to be fully evident during the first 5-minute test assays of the worm- 
samples from the population over the 7-month period of study. This is hardly 
the type of behavior expected of an independent internal physico-chemical oscillator 
system as it became phase-entrained to some altered relationship. There is, obvi- 
ously, no time in the phenomenon to permit conventional physiological transients. 
Rather, it is more plausibly like an abrupt reversal in the character of the response 
of the experimentally treated worm population to an external cyclic geophysical 
pattern. Hence, this capacity for virtually instantaneous reversal in rhythm phase 
appears to constitute further suggestive evidence for extrinsic timing of biological- 
clock systems. 

SUMMARY 

1. It has been shown that a 180 shift in the direction of the horizontal vector 
of magnetism will effect a 180 shift in the phase of the monthly rhythm in geo- 
graphical orientation in planarians. 

2. The shift is essentially completed immediately when the reversed field has a 
strength of 0.05-gauss, or about 1/3 the earth's natural horizontal vector strength. 

3. When the experimentally reversed magnetic field has a strength of 4.0-gauss, 
more than 20 times the earth's, the complete reversal of the rhythm requires as 
long as 15 minutes, following the removal of the experimental fields, during which 
time characteristic transients intervene. 

4. Implications for the biological-clock problem of this demonstrated relation- 
ship between vector direction of one geophysical component, relative to the others, 
and phase relationship of a fundamental biological rhythm are discussed. 

LITERATURE CITED 

BROWN, F. A., JR., 1962. Response of the planarian, Dii(/cxiti. and the protozoan, l\inuuciium, 

to very weak horizontal magnetic fields. Bin!. Hull., 123: 264-281. 
BROWN, F. A., JR., 1963. An orientational response to weak gamma radiation. Biol. Bull., 125: 

206-225. 
llrowN, F. A., JR., 1965. Effects and after-effect* on planarians of reversals of the horizontal 

magnetic vector. Nature (in press). 
BROWN, F. A., JR., AND V. 11. I'AKK, 1965. Duration of an after-effect in planarians following 

a reversed horizontal magnetic vector. Hint. Hull., 128: 347-355. 



EFFECTS OF TEMPERATURE ACCLIMATION ON SOME ASPECTS 
OF CARBOHYDRATE METABOLISM IN DECAPOD CRUSTACEA 1 

JOHN MARK DEAN 2 AND F. JOHN VERNBERG 

Duke University Marine Laboratory, Beaufort, North Carolina, and Duke University, 

Durham, North Carolina 

Temperature is one of the environmental factors to which the organism must 
adjust if it is to exist successfully in its habitat. In temperate-zone forms the 
severe temperature extremes of mid-wiuter and late summer may result in a shift 
of metabolic processes, tending to compensate for these extremes. However, 
tropical forms living in a relatively constant thermal environment do not have to 
contend with such extremes and their metabolic processes may not show a com- 
pensatory shift (Vernberg, 1962). Thus the temperature effects on an animal 
may be reflected in its physiology. Past investigations of temperature effects on 
populations have concentrated upon comparisons of rate functions such as oxygen 
consumption, ciliary activity, heart beat, and thermal limits of tissues and/or 
whole organisms (Bullock, 1955). It is to be expected that the ability to exist 
at an environmental temperature is expressed in the physiological and biochemical 
responses of the animal. The nature of these responses to temperature may vary 
with species or stage of the life cycle. Thus, not only is there a variation in the 
rate function of metabolic change with temperature adaptation, but the nature 
of the metabolic reaction or pathway may be altered (Ekberg, 1958; Hochachka 
and Hayes, 1962). The present research has been concerned with possible varia- 
tions in carbohydrate metabolism with temperature acclimation. Several differ- 
ent species of crabs have been studied, using physiological parameters such as 
blood glucose, the total reducing sugar in the blood and hepato-pancreas glycogen 
levels. Also included was a qualitative analysis of blood carbohydrates using 
chromatographic techniques. 

MATERIAL AND METHODS 

Glucose oxidase (Huggett and Nixon, 1957) was used for the determination 
of blood glucose and the classic Folin-\Yu method (1920) for the total reducing 
sugars. Glycogen was determined by the phenol-sulfuric acid method of Mont- 
gomery (1957). Chromatography of blood carbohydrates was done with ascend- 
ing, descending and two-dimensional flow on Whatman #1 and #3 filter paper. 
Various solvents were used, with the best resolution obtained with n-butanol, ethyl 
alcohol, acetic acid and water in an 8:2:1:3 mixture by volume. The papers 
were washed in the solvent system prior to spotting the unknown. Sprays for 
the analysis of unknown carbohydrates included silver nitrate and sodium hydroxide, 

1 Supported by National Science Foundation Grant G-12888 to F. J. Vernberg. 

2 Present address : Biology Department, Pacific Northwest Laboratory, Battelle Memorial 
Institute, Richland, Washington. 

87 



88 



JOHN M. DEAN \\D F. JOMX VERNBERG 



aniline hydrogen phthalate, iodine vapor, I'-anisidine HC1 and triphenyl-tetrazolium 
chloride. Blood samples were collected from different sites in the various species 
of crabs used: I 'en put/Untor. U. uihia.v and U. pitt/na.r were sampled in the 
second segment proximal to the cheliped ; while Callinectes sapidus were sampled 
from the sinus at the base of the fifth pereiopod and for Cancer irronilns, Libinia 
einari/inata, Panopcns licrl'sth and J/rj///>/v mercenaria, directly from the heart. 
Males only were used for analysis to reduce the possible effects of the hormonal 
factors associated with the reproductive cycle in the female (Dean and Vernberg, 



CHROMATOGRAPHIC PRESENCE ( + ) OR ABSENCE 



OF CARBOHYDRATES IN BLOOD 



Callinectes sapidus 
Cancer i r r o r a t u s 
Libinia emarginata 
Menippe mercenaria 
Panopeus herbst i i 

Uca m i nax 

(Early Spring) 

Uca m inax 

(Autumn) 

Uca m inax 

(10). "(18). (280) 

Uca p u g i I a t o r 



Uca pugi lator 

(Early Spring) 

Uca pugi lator 
(Autumn) 

Cancer magister 1 

anD 
Hemigrapsus nudus 

Orconectes vi r i I is? 



Ma Itotetraose 
Maltotr iose 
Maltose 
Glucose 



Meenakshi and Scheer 
McWhinnie and Sailer 



Galactose 



Man nose 



Fucose 



TABI.K I 



Galactan 
Derivative 



1964). For chromatography, the blood was deproteinized by heating in a boiling- 
water bath for one minute and the supernatant fraction obtained after ceutrifuga- 
tion was desalted (I)owex \( I-501-XX ) ; the resulting effluent was taken to dry- 
ness, dissolved in water to give a concentration equivalent to about 20 micrograms 
per microliter of dried material and standards were run with each unknown (Mc- 
\Vhinnie and Sailer, 1'^iOj. l\ f and R g values were calculated for comparison 
with values obtained by other workers. In acclimation experiments, other than 
the chromatography, I ': pin/Hn/or were used. These animals were acclimated 
to a given temperature lor a minimum of three weeks. They were kept under 
a uniform 14 hours light and 10 hours dark photoperiod and the water was main- 



TEMPERATURE ACCLIMATION IN CRABS 89 

tained at a constant salinity of 30/{ c and changed every 36 hours on an 8 AM, 8 
PM, 8 AM schedule. The diet consisted of Clark's fish pellets. This maintenance 
procedure resulted in a very low mortality in the laboratory animals. For the 
eyestalk experiments, the eyestalks were removed at their base and the wound 
closed with a cold cauterizer. 



30 



o> 

Q_ 



20 



0> 

o 

ID 

O 

T3J 
O 

o 

CO 



10 



o 21 Day Acclimated 
Uca pugilator 

+ 21 Day Acclimated 

F uerto R ican Uca rapax 



10 



15 



20 



25 



30 



Temperature. C 

FIGURE 1. Blood glucose in acclimated crabs. 

RESULTS AND DISCUSSION 

The qualitative chromatography of blood carbohydrates, as seen in Table I, 
in seven species of crabs gave similar results. All species have maltotetraose, 
maltotriose, maltose, galactose, glucose and a galactan derivative. Some differ- 
ences occur in the appearance of mannose and lucose in some species. Mannose 
is present in Menippe and early-spring Uca pugilator and possibly in Libinia, 
autumn Uca mina.\- and Callinectes. Fucose is definitely present in Menippe and 
possibly in Libinia and autumn Uca inina.i". Glucose-6-phosphate is known to be 
present in the blood of these crabs from other work done in this laboratory. These 
results compare favorably with those obtained by Hu (1958) for the shore crab, 



JOHN M. I) KAN A.VD F. JOHN VERNBERG 



Hcmi(jni[>sus nndns and Cancer, and the work of McWhinnie and Sailer (1960) 
on the fresh-water crayfish, Orconcctcs. Fairbairn (1958), using disc chromatog- 
raphy, has demonstrated trehalose in the tissues of several crustaceans. Using 
colorimetric techniques, we detected trace amounts of trehalose in the blood of 
several species and a higher amount was found in the blood of Libinia. However, 
these results are quite variable. Samples of blood of three species of Uca ac- 
climated to different temperatures have been analyzed by chromatography. No 
qualitative differences could be seen in the blood carbohydrates. 

Results of blood glucose and total reducing sugar levels of crabs acclimated to 
different temperatures would indicate that the concentration of glucose in blood is 
depressed at the lower temperatures (Fig. 1). A minimum of 15 individual sam- 
ples was used in the determination of each point, and the figures show the mean 
value and standard error. Blood glucose is usually 20-25% of the total reducing 
sugar value of the blood. This ratio does not seem to vary significantly with 



o> 



cn 

E 



o> 

i/i 
O 



35 



30 



25 



20 



15 



o 
o 



10 



Uca m i nax Accl i mated 
90 Days 







\ 







10 



20 



30 



Temperature, C 

FIGUKK 2. J! Idod glucose in acclimated crabs. 



TEMPERATURE ACCLIMATION IN CRABS 



91 



30 



o> 

Q_ 

cr> 



20 



10 



O 
_0 

CO 



+ A 
X 
O T 



A 

V 



0+ 
X 



T 
fi 







10 20 



Temperature, C 



30 



FIGURE 3. Blood glucose in crabs (X U. pugilator field animals, + U. pugilator 21 
day acclimation, O U. minax 21 day acclimation, T U. ininax 90 day acclimation, A 
U. rapax, CD U. pugnax), 

acclimation. Also, a tropical species (Uca rapax), acclimated at the same tem- 
perature as temperate species, has similar blood glucose values. Apparently labora- 
tory acclimation had results similar to natural field conditions because 2 laboratory- 
acclimated U. pugilator had a low blood glucose value, as did the newly emerged 
crabs in early March. Short periods of acclimation to higher temperatures resulted 
in a higher blood glucose level, and long-term acclimation to high temperature, as 
in U. minax, showed an even more marked increase. However, U. minax shows 
much the same response as U. pugilator (Fig. 2). Long-term acclimation, in this 
case three months, resulted in a trend to temperature-dependent blood glucose 
values. The level for field animals emerging early in the spring and acclimating 
animals followed the patterns seen in U. pugilator. 

Uca pugnax, a temperate-zone form, fits the range of blood glucose values for 
the other species (Fig. 3) and U. rapax, which is a tropical species, is also in the 
same general range. Thus, it may be seen that there is a general trend to low 



92 



.101 IX M. 1)K\\" AND F. JOHN VERNBERG 



glucose ;it low temperature and an upward trend at higher temperatures. 
The data indicate a plateau for blood glucose around the optimum temperature 
range of the animal, which is a pattern similar to that seen in respiration experi- 
ments (Vernberg, 1^59). 



o> 

Q_ 
O1 



O> 

un 
O 
<_> 

13 

O 



o 
o 



CO 



50 



40 



30 



20 



10 



o 







+ Blood Glucose in 10 
Acclimated Animals 

o B lood Glucose in 30 
Acclimated Animals 



10 



Days 



FIGUKK 4. Kffivt of f.-islin.u on MMM.I uhu-Mse in [7. fiitgilator. 



TEMPERATURE ACCLIMATION IN CRABS 



93 



25 



20 



Q_ 

CD 



15 



oo 
O 



= 10 



-o 

O 

o 

CO 



+ Blood Glucose in 10 
Accl i mated Animals 

o Blood Glucose in 30 
Acclimated Animals 



T 

+ 



T 

+ 

i 



T 
J+ 










1 



5 



10 



Days 

FIGURE 5. Effect of eyestalk removal on blood glucose in U. pngllator. 

Qualitatively there is no major change in blood carbohydrates with acclimation. 
However, there may be quantitative differences, as shown by the lower blood 
glucose values obtained with animals acclimated to low temperatures. 

We were interested also in the effects of diet and hormones in relationship to 
the carbohydrate metabolism of the animal. Crabs were well acclimated for 21 
days at 10 and 30. They were then fasted and sampled on days 0, 1, 5 and 10. 
Unlike Libinia, which shows no change in blood sugars with fasting or eyestalk 
removal (Kleinholz and Little, 1949), it may be seen in Figure 4 that the blood 
glucose in the fasting 30 U. pugilator decreased consistently with time while the 
fasted 10 animals were fairly constant. The hepato-pancreas glycogen levels 
followed this same pattern. 

Eyestalk removal during the intermolt stage will induce molting in the crab 
except at lower temperatures where temperature acts as a molt inhibitor (Passano, 
1960). During ecdysis, a period of high physiological activity, several dramatic 
changes in the carbohydrate metabolism occur. To induce molting, eyestalks were 
removed from 10 and 30 acclimated U. pugilator fed ad libitum. Samples were 
taken on days 0, 1, 3, 5 and 10. Under these conditions, which initiate the molt 
sequence of events, there is an extremely rapid drop in the blood glucose level of 
the 30 animals (Fig. 5). This remains at a low level for a period of time and 
then begins a slow increase. However, the 10 animals did not show this change 



94 JOHN M. DEAN AND F. JOHN VERNBERG 

in blood glucose level, did not molt, or initiate proecdysis, and had a high mortality 
rate. There is no significant change in the hepato-pancreas glycogen values in 
the 10 animals but the 30 crabs showed a typical buildup in hepato-pancreas 
glycogen as molt approaches, and rapid decline with ecdysis. 

The authors are grateful to Miss Rosemary McCarthy for her fine technical 
assistance and the J. R. Clark Co., Salt Lake City, Utah for supplying the diet. 

SUMMARY AND CONCLUSIONS 

The preceding work on carbohydrate metabolism in Uca would suggest the 
following : 

First, with temperature acclimation there is no qualitative shift in carbohydrate 
metabolic pathways, but rather there may be quantitative variations. Second, at 
low temperature the animal reduces its energy output to a minimal level. This 
may be related to the energy demands of the molt cycle. It would seem that even 
though sufficient carbohydrate reserves are present at low temperature, there may 
be variations in hormone levels which would affect the molt cycle. These physio- 
logical characteristics correlate well with field observations and the general ecology 
of the fiddler crab. However, generalities cannot be made for all Crustacea as 
there are obvious differences between genera. 

LITERATURE CITED 

BULLOCK, T., 1955. Compensation for temperature in the metabolism and activity of Poikilo- 

therms. Biol, Rev., 30: 311-342. 
DEAN, J. M., AND F. J. VERNBERG, 1964. Variations in the blood glucose level of Crustacea. 

Comp. Biochem. Physiol. (in press). 
EKBERG, D. R., 1958. Respiration in tissues of goldfish adapted to high and low temperatures. 

Biol. Bull., 114: 300-316. 
FAIRBAIRN, D., 1958. Trehalose and glucose in Helminths and other invertebrates. Canad. J. 

Zool.,36: 787-795. 
FOLIN, O., AND H. Wu, 1920. A system of blood analysis Supplement 1. A simplified and 

improved method for determination of sugar. J. Biol. Chem., 41: 367-374. 
HOCHACHKA, P. W., AND F. R. HAYES, 1962. The effect of temperature acclimation on path- 
ways of glucose metabolism in the trout. Canad. J. Zool., 40: 261-270. 
Hu, A. S., 1958. Glucose metabolism in the crab Hcmigrapsus nudus. Arch. Biochem. Bio- 

phys., 75 : 387-395. 
HUGGETT, A. ST. C., A. \D D. A. NIXON, 1957. Enzymatic determination of blood glucose. 

Biochem. J., 66: 12 P. 
KLEINHOLZ, L. H., AND B. C. LITTLE, 1949. Studies on the regulation of the blood sugar in 

crustaceans. I. Normal values and experimental hyperglycemia in Libinia emarginata. 

Biol. Bull., 96: 218-227. 
MEENAKSHI, V. R., AND B. T. SCIIEER, 1961. Metabolism of glucose in the crabs Cancer 

magistri and IJciini/nipsnx nitdns. Comp. Biochem. Physio!., 1: 110-122. 
McWniNNiE, M. A., AND SK. P. N. SALLER, 1960. Analysis of blood sugars in the crayfish 

Orconcctes ririlis. Comp. Bun-hem. Physiol., 1: 110-122. 

Md\ I(,()\IKKV, R., l ( >57. The (It-termination of glycogen. Arch. Biochem. Biophys. 67: 378-386. 
I '.\- -A NO, L. M., 1960. ]. ueruture blockage of molting in I'ca puqnax. Biol. Bull.. 

118: 129-136. 
VERNBERG, F. J., 1959. Studies on the physiological variation between tropical and temperate 

zone fiddler crabs of the genus Ccn. II. Oxygen consumption of whole organisms. 

liiol. Bull., 117: 163-184. 
VERNBERG, F. J., 1962. Comparative physiology: Latitudinal effects on physiological properties 

of animal populations. Ann. A'<v. Physiol., 24: 517-546. 



PINOCYTOSIS OF PROTEINS BY OYSTER LEUCOCYTES l > -> 3 > * 

S. Y. FENG 
Department of Zoology, Rutgers, The State University, New Brunswick, N. J. 

The term "pinocytosis" (cell drinking) was first introduced by Lewis (1931). 
This same phenomenon, however, was observed earlier by Metchnikoff (1901) in 
mammalian leucocytes in an aseptic exudate induced by a 10% gelatin solution, 
and also by Edwards (1925) in amoebae when certain simple salts were added to 
their culture medium. Later Mast and Doyle (1934) found that, in the presence 
of albumin or calcium gluconate solutions, Amoeba proteus and a number of related 
species also display pinocytic activities. After the publication of these papers, 
pinocytosis was virtually unheard of for nearly 20 years. Recently this old subject 
has been investigated with renewed vigor by using new techniques : e.g., electron 
microscopy, fluorescence microscopy, interference microscopy, radioisotope and 
fluorescent dye labelling, and serological methods. A brief review of the literature 
reveals that pinocytosis has been demonstrated for mammalian cells, in tissue cul- 
tures, such as polymorphs (Bessis and Bricka, 1952), macrophages (Lewis, 1931), 
erythroblasts, normoblasts, reticulocytes and certain pathological erythrocytes 
(Bessis and Breton-Gorius, 1957^), sarcoma cells (Lewis, 1937), ascites tumor 
cells (Easty, Ledoux and Ambrose, 1956) and HeLa cells (Rose, 1955) ; for 
protozoa such as A. proteus (LAjys, 1937), Chaos chaos (Holter and Marshall, 
1954; Brandt, 1958), Plasmodininiopliurae and P. bcrghei (Rudzinska and Trager, 
1957, 1959) ; and for blood element^ of invertebrates, e.g., elaiocytes of the coelomic 
fluid of echinoderms (Holter, 1959) and phagocytes of planarians (Rosenbaum and 
Rolon, 1960). V* 

Oyster leucocytes are known to ingest a variety of participate materials (Yonge, 
1926; Takatsuki, 1934; Stauber, 19*0; Tripp, 195Sa, 1958b, 1960; Feng, 1962). 
In the present study, the in vivo and in vitro uptake of proteins by oyster leucocytes 
was demonstrated by using serological techniques and fluorescence microscopy. 

MATERIALS AND METHODS 
* 

1. Oysters, aquaria and sea ivater 

The oysters and sea w-ater used in this study were collected from the Navesink 
River, near Red Bank and trie Shrewsbury River at Highlands, New Jersey, re- 

1 Part of a thesis submitted to the graduate faculty of Rutgers, The State University, in 
partial fulfillment of the requirements for the degree of Doctor of Philosophy. 

2 This investigation was supported in whole by Public Health Service Research Grant 
AI-00781, from the National Institute of Allergy and Infectious Diseases of the National 
Institutes of Health. 

3 In the oyster the terms amebocyte, leucocyte, and phagocyte are used interchangeably, as 
suggested by Stauber (1950). 

* Present address : Oyster Research Laboratory, Rutgers, The State University, New 
Brunswick, N. J. 

95 



96 S. Y. FENG 

spec-lively. I'yrex battery jars, measuring 25 X 25 cm., were used as aquaria to 
hold oysters. The oysters were kept m sea water of 20%e throughout the experi- 
ment. Constant aeration was maintained in each aquarium. Water was changed 
at least twice dailv. 

2. Preparation of ovsters (or injection and bleeding 

Exposing the heart for injection and bleeding was achieved by following the 
established procedure of Feng (1965). 

3. Preparation, injection, and detection of inocnla 

a. Bovine hemoglobin solution 

A 5 % crystalline bovine hemoglobin solution was prepared in a diluent of oyster 
plasma which was drawn and pooled from 10 oysters. Desired amounts of this 
solution were injected into oysters via the ventricular route. 

The reduction of bovine hemoglobin from the blood stream by the oyster was 
followed colorimetrically by sampling the heart blood at appropriate intervals over 
a period of 154 minutes. The presence of bovine hemoglobin within the leucocytes 
after injection was detected visually by the pink coloration of the washed sedi- 
mented blood cells. 

b. Diphtheria antitoxin 

Horse antibody to diphtheria toxin, kindly supplied by Dr. R. J. DeFalco. 
Director of the Serological Museum, Rutgers, The State University, was injected 
into 10 oysters in volumes of 0.2 ml. per animal (3125 units per ml. of antiserum). 
Pooled blood samples were taken from the oysters in the amount of 0.2 ml. per 
oyster at 10 minutes, 1, 2, 4 and 8 hours. Oyster leucocytes were separated from 
the plasma by centrifugation and washed in several changes of sea water. The 
cells were ruptured by grinding them with fine sand and the extract was recon- 
stituted to 2.0 ml. with O.S5'/f saline. The sand was removed by centrifugation. 

The presence of the diphtheria antiserum in both the plasma and leucocyte 
saline extract was detected by reacting them with diphtheria toxoid, using a modi- 
fied procedure of Ramon titration. The procedure of ordinary Ramon titration 
(Boyd, 1956) consists of mixing constant dilutions of toxoid with varying dilu- 
tions of antiserum or vice versa. The amount of toxoid which gives most rapid 
flocculation with one standard unit of antiserum is first determined. The amount 
is designated as the Lf unit. Then unknown antisera can be titrated against this 
standardized toxoid. The time required for the first flocculent precipitate to occur 
is referred to as Kf. or flocculation time. The modified procedure devised for this 
work is carried out under the condition that the Lf units of both the toxoid and 
antiserum are known. When materials containing unknown units of toxoid or 
antiserum are added to the above system, the flocculation time will change ac- 
cordingly. 

I'erhaps the points made above are best illustrated in the following example: 

Till..- No. 1234 

Toxoid (50 Lf /ml.) 0.5 ml. 0.5 ml. 0.5 ml. 0.5 ml. 

\ntisc rum (312.5 units/ml.) 11.00 ml. 0.08ml. 0,10ml, 0.12ml. 

Kf (minutes) 35 



PINOCYTOSIS BY OYSTER LEUCOCYTES ( J7 

Tube No. 2 is the first one to show the flocculent precipitate after 35 minutes incu- 
bation at 40 C. If 0.5 ml. oyster plasma containing an unknown amount of anti- 
serum was mixed with the toxoid in each of the above four tubes and then the usual 
amounts of antiserum added, there could be the following possible consequences: 
the Kf of Tube No. 2 could be prolonged, shortened or unchanged depending on 
the amount of antiserum contained in the oyster plasma; or Tube No. 3 could be 
the first one to show the flocculent precipitate. If the latter case took place, the 
unknown might be solved as follows: 50 Lf X 0.5 + X == 312.5 X 0.10, where X 
represents the unknown Lf units contained in 0.5 ml. of oyster plasma. 

c. Rhodamine-labelled proteins 

Conjugation of crystalline human gamma globulin, albumin fraction V and 
Li nut Ins serum with Lissamine Rb 200 was carried out as specified by Chadwick, 
McEntegart and Nairn (1958). The conjugated proteins were brought to pH 
7.8 and normal tonicity by dialyzing overnight against filtered sea water (20%e). 
The final concentration of these solutions was 2.5 gm. per 100 ml. 

The fluorescence equipment used in the present study is manufactured by 
Reichert. It consists of a regular monocular microscope, with a high pressure 
mercury arc (HBO 200) as source of excitation. When viewed under the UV 
microscope with Schott BG-12 (3 mm.) and Corning-5840 as primary filters and 
Eastman Kodak WA-15 as secondary filter, the rhodamine conjugates exhibit a 
brilliant orange fluorescence which is readily distinguished from the blue-green 
intrinsic fluorescence of oyster leucocytes and other tissues. 

RESULTS 

1. Bovine hemoglobin 

Three oysters (A, B and C) were injected with 0.3, 0.2 and 0.15 ml. of a 5% 
bovine hemoglobin solution, respectively. The different rates of disappearance of 
hemoglobin from the heart blood shown by the three oysters reflect differences in 
the amount of inoculum received, since the three oysters were comparable in size 
(Fig. 1). According to Figure 1, 130, 63 and 48 minutes were required to reduce 
50% of the injected hemoglobin by oysters A, B and C, respectively. 

It was noticed that nearly all samples of oyster whole blood taken 30 minutes 
after injection contained some pink leucocytes. The leucocytes retained their pink 
coloration even after several gentle washings in filtered sea water. 

2. Diphtheria antitoxin 

Since the ionic concentration and other constituents of oyster plasma and 
leucocyte saline extract are probably quite different from those of mammalian sera, 
a series of controls designed to test the effects of oyster plasma on the toxoid- 
antitoxin system was first initiated. The result (Table I) indicates that adding 
0.5 ml. of 0.85% saline to Control A increases the Kf almost 7\% (line B). Addi- 
tion of either oyster plasma or leucocytes saline extract to the toxoid-antitoxin 
system greatly retards the reaction ; in certain instances the Kf is 2 to 5 times 
longer than the control (compare B with C; E with F and G). Superficially, the 



98 



S. Y. FENG 



inhibitory effect of oyster plasma appears to be stronger than that of leucocyte 
saline extract. The importance of this difference will remain unanswered until 
information concerning the precise protein content, ionic composition and other 
constituents in a unit volume of oyster plasma and leucocyte saline extract becomes 



100- 
8 80 

DO 



S 60 



40 



o 
oo 



.0 

I 20 

0? 
x> 



10 




oyster A 
B o 
CA 



20 40 



60 80 100 
Minutes 



120 140 160 



FIGURE 1. 



The reduction of intracardially injected bovine hemoglobin from the heart blood 

of three oysters. 



available. The data also suggest that the Kf is a function of the concentration of 
reagents. The toxoid of Controls I',, C and D contains 25 Lf units, while the anti- 
scrum of Control !'.. !' and G is 125 Lf units. Consequently, the Kf for the latter 
group is 3 to 12 times shorter than that of the former group. 1 lenee. wherever a con- 
siderable shortening of Kf occurs in the experimental group, as contrasted with the 
proper control, it is implied that this shortening of Kf is probably due to the pres- 



PINOCYTOSIS BY OYSTER LEUCOCYTES 



99 



ence of an unknown amount of Lf units in the oyster plasma and leucocyte saline 
extract. 

Based upon the above considerations, it is found that the presence of diphtheria 
antiserum in the oyster plasma could be detected 10 minutes to four hours after 
the injection, while in the leucocyte saline extract it probably lasted two hours. 
However, the subsequent negative results do not necessarily suggest that the 
injected material is degraded but may merely indicate that the concentration of 
this material becomes too low to be detected by this procedure. 

TABLE I 

The detection of diphtheria antiserum (horse) in oyster plasma and leucocyte saline 

extract, expressed in terms of flocculation lime (Kf) by using a modified 

Ramon titration procedure 





Flocculation time (Kf) in minutes 




System 




10 min. 


1 hr. 


2 hr. 


4 hr. 


8 hr. 


Remarks 






















Experimental samples 




A. T-A 


35 





, 











T (constant) = 25 


B. T-S-A 


60 

















Lf/0.5 ml. 


C. T-Op-A 


120 





- 


53 


65 


* 


A (variable) = 312.5 


D. T-O1-A 


68 








48 


71 


65 


Lf/ml. 


E. A-S-T 


5 

















A (constant) = 125 


F. A-Op-T 


25 


5 


30 











Lf/0.04 ml. 


G. A-O1-T 


20 


** 


8 











T (variable) = 300 
















Lf/ml. 



T, A, S, Op and Ol represent diphtheria toxoid, diphtheria antiserum, saline, oyster plasma 
and oyster leucocyte saline extract, respectively. The saline used is a solution of 0.85% NaCl. 
* No reaction was noticed after three hours. 
** Sample lost. 



3. Rhodaniine-labcllcd proteins 

a. The effect of concentration on the uptake of rhodamine-labelled human gamma 
globulin by oyster leucocytes 

Four rhodamine-labelled human gamma globulin solutions : 2.5, 0.25, 0.025 
and 0.0025 gm.%, were used in the experiment. One drop of the above solutions 
was mixed with four drops of oyster blood on a slide. The drops of protein solu- 
tion and oyster blood were delivered by a tuberculin syringe with a 30-gauge needle. 
Thus, on the slide the leucocytes were exposed to rhodamine-labelled human gamma 
globulin solutions with the following final concentrations : 0.5, 0.05, 0.005 and 
0.0005 gm.%. After the leucocytes were exposed to the solution for a specified 
period (5, 15, ... 60 minutes), the excess protein solution was removed by sev- 
eral gentle washings with filtered sea water. The preparation was sealed with a 
Vaselined coverglass and viewed under the UV microscope. Each sample con- 
sisted of three such preparations. The number of leucocytes containing orange-red 



100 



S. Y. FENG 



fluorescent dots in at least 100 leucocytes was used to calculate the per cent pinocy- 
tosjs by the leucocvtes. 1'oth e\|)eriinents were carried out at 23 C. 

The results shown in Figure 2 surest that in both experiments the leucocytes 
require longer time to take up protein from a less concentrated rhodamine-labelled 
human stamina ulobulin solution than from a more concentrated solution. It is 
noticed that there are differences between experiments but the general order of 
concentration effect on the rate of uptake of the protein solution by leucocytes is 
consistent. Kor instance, to reach the level of 20% pinocytosis by the leucocytes 



lOOi Effect of concentration 




5xlO"'gm% 



-2 
n5xlO gm% 



Exp. A 23 C. 




A5xl6 gm% 

-4 
o5xl(Tgm% 

5xlO~ gm% 



-2 
5xlO gm% 



-3 



gm% 



-4 
oSxIO gm% 







10 20 30 40 50 60 
Minutes 



I'll,! R] The effect of concentration and time of exposure on the pinocystoMs of rliod- 

amine-labelled human gamma ^lohulin l>y oyster K-ucdcyte.s. I ; ,acli jioint on the .uraph rrprcM-nt> 
the median of three samples. 



PINOCYTOSIS BY OYSTER LEUCOCYTES 101 

in Experiment A, 2, 15 and 25 minutes are required for 0.5, 0.05 and 0.005 gm.%, 
respectively; for 0.0005 gm.%, less than 5% of the leucocytes showed pinocytosis 
at the end of 50 minutes. In Experiment B for comparable concentrations of pro- 
tein solution, the relative rate of uptake by the leucocytes is in general lower than 
that of Experiment A, i.e., 5 and 22 minutes for 0.5 and 0.05 gm.%, respectively. 
Also at the end of one hour less than \5%. and 5% of the leucocytes showed pinocy- 
tosis in 0.005 and 0.0005 gm.%. Efforts were made to render conditions as com- 
parable as possible in the two experiments. However, the number of leucocytes 
per drop of oyster blood as delivered by the tuberculin syringe with a 30-gauge 
needle was not determined for the two experiments. It is suspected that some 
of the differences in the rate of uptake in the two experiments might result from 
unequal numbers of leucocytes in the drops of oyster blood used. 

b. The effect of temperature on the uptake of rhodamine-labelled human gamma 
globulin by oyster leucocytes 

Two experiments were performed at 10 and 24 C., respectively. In con- 
ducting the experiment at 24 C., the experimental procedure was similar to that 
of Experiment A and B in the above study. For the experiment carried out at 
10 C., special procedures were followed in order to maintain the leucocytes, in- 
oculum and instruments used in this experiment at the same temperature. The 
temperature of a refrigerator was adjusted so that the temperature was 10 C. on 
the bottom shelf where the inoculum and a tuberculin syringe with 30-gauge needle 
were stored overnight. Fresh oyster heart blood was obtained by bleeding the 
animal at room temperature. Four drops of oyster blood were placed on each of 
the 15 slides. They were immediately stored in the refrigerator in a moist chamber 
to prevent excessive evaporation. At the end of 30 minutes, one drop of 2.5 gm.% 
rhodamine-labelled human gamma globulin was added to each of the 15 leucocyte 
preparations. At each preclesignated time interval, three leucocyte preparations 
were removed from the refrigerator, washed, sealed and examined as described for 
Experiments A and B reported above. 

Figure 3 indicates that the uptake of the rhodamine-labelled human gamma 
globulin (0.5 gm.%) by the leucocyte is considerably faster at 24 than at 10 C. 
In order to attain the level at which 50% of the leucocytes show pinocytosis, 12 
and 28 minutes were required for the leucocytes held at 24 and 10 C, respectively. 

c. Distribution of rhodamine-labelled proteins in the tissue of oysters 

Three groups of oysters (A, B and C), each consisting of 8 oysters, were used 
for the injection of rhodamine-labelled human albumin, human gamma globulin 
and Lunnlus serum protein, respectively. Each oyster was injected with 0.2 to 
0.4 ml. of the above labelled proteins. A fourth group (D) with 8 uninjected 
oysters served as a control. The experiment was performed with the temperature 
ranging from 18 to 20 C. Sampling was made at the following intervals: 15 
minutes, 1, 2 and 4 hours, and 1, 2, 4 and 6 days. At the above intervals, four 
oysters, one from each of the four groups, were sacrificed by removing the shell. 
The whole oyster was cut into three pieces through the following regions : oral 
hood, visceral mass and adductor muscle. These pieces of oysters were wrapped 



102 



S. Y. FENG 



separately with aluminum foil, dropped into liquid nitrogen for quick freezing and 
-lured iu the refrigerator at '.. to be sectioned with a freezing microtome. 

The sections placed on glass slide> were fixed briefly with lO^o formalin in sea 
water. They were then dehydrated in three changes of dioxane of four minutes 
each and mounted in non-fluorescent medium. The distribution of fluorescent 
proteins in tissues was ascertained by noting (1) the presence or absence of inocu- 
lated substances free or pinocytosed in selected lumina (blood vessels, intestine, 
digestive diverticulum. etc. ), and (2) migration of protein-laden leucocytes through 
various epithelia. 



ioo 



80" 



60- 



" 40- 



o 

.1 20- 

Q_ 



Effect of temperature 
5xlO~ l gm% 




24 C. 



10 C. 







10 20 30 40 50 
Minutes 



FicukK 3. The effect ot' temperature and time of exposure on the pinocytosis of rhodamine- 
labelled Inmian gamma globulin by oyster leucocytes. Each point on the graph represents the 
median of three samples. 



Regardless of the fact that the inocula consisted of three different protein so- 
lutions. no outstanding differences were found in their distribution or in the subse- 
quent elimination of the inoculated materials from the oyster. Thus, unless other- 
wise noted, the following results apply to all three inocula. The blood in the 
arterial and venous systems (anterior aorta, posterior aorta leading to the adductor 
muscle, hlood spaces in the muscle, small arteries in the visceral mass, blood spaces 
immediately adjacent to the stomach, intestine, style sac and rectum, sinuses in the 
mantle, medial gill axis vein and lateral palliobranchial veins) appeared to be filled 
with the labelled proteins 15 minutes after intracardial injection. At no time 
during the experiment were labelled protein^ detected in the circumpallial arteries. 



PINOCYTOSIS BY OYSTER LEUCOCYTES 103 

In the subsequent samples, the general pattern of distribution of the labelled 
protein was essentially similar to that of the 15-minute sample described above. 
The only noticeable change was that the amount of free labelled proteins in the 
lumina of blood vessels and spaces decreased with time ; this is indicated by the 
gradual reduction of orange-red fluorescence in these areas. Concurrently with 
the above observation, it was noticed that some of the labelled proteins outlined the 
blood spaces as though adsorbed to the cells delimiting these spaces. The labelled 
materials were found to be pinocytosed by leukocytes in the peri-intestinal region 
within one hour post-injection. Migration of the protein-laden leucocytes across 
the arterial wall was not observed in all samples. However, migration of protein- 
laden leucocytes across the epithelia of stomach, intestine, rectum, digestive 
diverticula and mantle facing the palp was first noticed 15 minutes after injection 
of labelled human gamma globulin. This process did not commence in the oysters 
inoculated with labelled human albumin and Limn I us serum protein until two 
hours post-injection. Rejecta and dejecta collected from the aquarium 24 hours 
post-injection showed numerous leucocytes containing rhodamine-labelled proteins. 
This observation constitutes further evidence for the elimination of some of the 
injected protein solutions. Epithelial linings of mantle facing the shell and promyal 
chamber were only occasionally used by the protein-laden leucocytes as exits. 
No protein-laden leucocytes were observed to traverse the wall of gonoducts and 
nephridial tubules. Protein-laden leucocytes were also seen in the external lining 
of the heart and the parietal pericardium. Although protein-laden leucocytes were 
only very rarely seen in the lumina of stomach, intestine, digestive diverticula and 
rectum, the observation that large numbers of protein-laden cells were found in 
the dejecta has led the writer to believe that the rare occurrence of such cells in 
these regions might be due to the fast emptying time of the digestive tract and the 
concentration represented by the formation of dejecta. 

DISCUSSION 

The distribution and the sites of elimination of the injected rhodamine-labelled 
proteins in oyster tissues in general do not differ significantly from those obtained 
by the injection of other participate materials (Stauber, 1950; Tripp, 1958a, 1958b, 
1960). Only the wide distribution of the labelled proteins and the earlier com- 
mencement of migration of protein-laden leucocytes were in contrast with the find- 
ings of Stauber and Tripp. For example, migrations of protein-laden cells were 
first encountered in the epithelia of stomach, intestine and digestive diverticula 15 
minutes to two hours post-injection (18 to 20 C.), whereas the process in the 
same area was observed 8 days post-injection in oysters injected with India ink 
at 12 to 21 C. (Stauber, 1950) and 2 to 5 days in oysters injected with yeast 
cells and vegetative Bacillus mycoides at 17 1 C. (Tripp, 1960). The evidence 
indicates that migration of host leucocytes through epithelial surfaces is a normal 
physiological process. However, under experimental conditions, this process could 
be accentuated or retarded, depending on the size and number of particles phago- 
cytosed or pinocytosed by the leucocytes and the susceptibility of particles to intra- 
cellular digestion. Lack of protein-laden leucocytes migrating through the arterial 
wall could be ascribed in part to the relatively early wide distribution of the 



104 S. Y i 1'NG 

labelled proteins to the terminal branches of the circulatory system and, therefore, 
occlusion of the major vessels, if it occurs, is probably very transient. 

On the basis of the preseni findings and observations made by other investi- 
gators, pinocytosis as displayed '>> various tissue cells and certain protozoa is now 
well established. In the oyster, the pinocytic activity of leucocytes appears to be 
primarily defensive, since protein-laden leucocytes are seen to traverse the 
epithelium of the mantle and intestine on their way to exterior. In P. lophnrac 
and P. bcnjhci. pinocytosis is a means of securing nutrient (Rudzinska and Trager, 
1957, 1959). However, the significance of this process is still unclear in amoebae 
and malignant cells. Lewis (1931) assumed that digestion occurs within the 
pinocytosis vacuoles of amoebae. Recent studies indicate that low molecular 
weight sub>taiices. e.g., glucose and methionine, carried inside the amoebae by 
pinocytosis are probably utili/.ed ( Chapman- Andresen and Holter, 1955; C'hap- 
man-Andresen and I'rescott. 105f>), while the fate of high molecular weight ma- 
terials, e.g., proteins, is still unknown. In the free-living amoebae, such as C. 
chaos, possessing a trait such as pinocytosis probably enhances the survival of the 
species, especially during the period when the external environment becomes hyper- 
tonic to the amoeba's "milieu interieur." Hence, it is possible that pinocytosis 
in the free-living amoeba could be employed as a defense mechanism against de- 
hydration. Parasitic amoebae, on the other hand, are bathed constantly in a me- 
dium of tissue debris, red cells, bacteria and plasma, and one may infer that these 
organisms enrich themselves by employing pinocytosis and phagocytosis simul- 
taneously in securing both soluble and participate materials in the abscess. In 
conclusion, pinocytosis may occur as a means of defense, of obtaining nutrients or 
as a cytopathological manifestation of malignant cells. 

The author is deeply indebted to Dr. L. A. Stauber for his numerous sugges- 
tions, helpful criticisms and generous support of this work. Special thanks are 
due to the late Professor T. |. Murray, and Drs. M. Solotorovsky and G. Kemp 
of the Department of bacteriology for their cooperation in the use of freezing 
microtome and UV microscope. 

SUMMARY 

In vivo and in vitro studies indicated that bovine hemoglobin, diphtheria anti- 
serum, rhodamine-labelled human gamma globulin, human albumin fraction V and 
Liinuliis serum proteins were pinocytosed by oyster leucocytes. The presence of 
the various proteins within the leucocytes was detected by visual inspection of the 
washed sedimented leucocxte^, serological techniques and fluorescence microscopy. 
When the proteins were injected into the living oyster via the ventricular route, 
they were readily removed by migration of protein-laden leucocytes through 
epithelial surfaces to the exterior. The rate of in vitro uptake of rhodamine- 
labelled human gamma globulin bv the leucootcs was a function of the ambient 
temperature and the concentration of the protein solution in which they were 
bathed. 

LITER XTl'KK <TI I.I i 

BESSIS, M.. \M> M. BRICKA, l''5J. Aspect dynamiqedes cellules <lu sang. Son etude par la 
microcinematographie en omtraste <le phase. AYr/ir d'hematologic, 7: 407-435. 



PINOCYTOSIS BY OYSTER LEUCOCYTES 105 

BESSIS, M., AND J. BRETON-GORIUS, 1957. Iron particles in normal erythrohlasts and normal 

and pathological erythrocytcs. /. Bio{>hys. Biochcm. Cytol., 3: 503-504. 
Bovn, W. C., 1956. Fundamentals of Immunology. 3rd edition, Interscience Publishers, Inc., 

New York. 
BRANDT, P. W., 1958. A study of the mechanism of pinocytosis. E.\-p. Cell Res., 15: 300- 

313. 
CHADWICK, C. S., M. G. MCNTEGART AND R. C. NAIRN, 1958. Fluorescent protein tracers, 

a simple alternative to fluorescein. Lancet, 274: 412-414. 
CHAPMAN-ANDRESEN, C., AND H. HOLTER, 1955. Studies in the ingestion of C 14 glucose by 

pinocytosis in Chaos chaos. Exp. Cell Res. (Suppl.), 3: 52-63. 
CHAPMAN-ANDRESEN, C., AND D. M. PRESCOTT, 1956. Studies on pinocytosis in the amoebae 

Chaos chaos and Amoeba proteus. C. R. Trav. Lab. Carlsberg, 30: 75-78. 
EASTY, D. M., L. LEDOUX AND E. J. AMBROSE, 1956. The action of ribonuclease on neoplastic 

growth. III. Studies by interference microscopy. Biochiin Biophys. Acta, 20: 528- 

537. 
EDWARDS, J. G., 1925. Formation of food-cups in Amoeba induced by chemicals. Biol. Bull., 

48:236-239. 
FENG, S. Y., 1962. The response of oysters to the introduction of soluble and particulate 

materials and the factors modifying the response. Dissertation Abstract, 23(6) : 2099- 

2100. 
FENG, S. Y., 1965. Heart rate and leucocyte circulation in Crassostrea mrginica (Gmelin). 

Biol. Bull, 128: 198-210. 
HOLTER, H., 1959. Pinocytosis. In Internat. Rev. Cytol., edited by G. H. Bourne and J. F. 

Danielli, 8:481-504. 
HOLTER, H., AND J. M. MARSHALL, JR., 1954. Studies in pinocytosis in the amoeba Chaos 

chaos. C. R. Trav. Lab. Carlsberg, 29: 7-27. 

LEWIS, W. H., 1931. Pinocytosis. Bull. Johns Hopkins Hosp., 49: 17-27. 
LEWIS, W. H., 1937. Pinocytosis by malignant cells. Amcr. J. Cancer, 29: 666-679. 
MAST, S. O., AND W. L. DOYLE, 1934. Ingestion of fluid by Amoeba. Protoplasma, 20: 555- 

560. 

METCHNIKOFF, E., 1901. L'immunite dans les Maladies Infectieuses. Masson et Cie, Paris. 
ROSE, G. G., 1955. A varient pinocytic cell (Vp of Gey's strain HeLa) produced and stimu- 
lated by human serum nutrient. Texas Rep. Biol. Med., 13: 475-489. 
ROSENBAUM, R. M., AND C. I. ROLON, 1960. Pinocytosis in phagocytes of planarians. Anat. 

Rec., 137: 389. 
RUDZINSKA, M. A., AND W. TRACER, 1957. Intracellular phagotrophy by malaria parasites: 

an electron microscope study of Plasmodimn lophurae. J. Protosool., 4: 190-199. 
RUDZINSKA, M. A., AND W. TRACER, 1959. Phagotrophy and two new structures in malaria 

parasite Plasmodimn berghci. J. Biophys. Biochem. Cytol., 6: 103-112. 
STAUBER, L. A., 1950. The fate of India ink injected intracardially into the oyster, Ostrea 

virginica Gmelin. Biol. Bull, 98: 227-241. 
TAKATSUKI, S., 1934. On the nature and functions of the amoebocytes of Ostrea cdulis. 

Quart. J. Micro. Sci., 76: 379-431. 
TKIPP, M. R., 1958a. Disposal by the oyster of intracardially injected red blood cells of 

vertebrates. Proc. Nat. Shellfish. Assoc., 48: 143-147. 
TRIPP, M. R., 1958b. Studies on the defense mechanism of the oyster, Crassostrea virginica. 

J. Parasitol., 44(4, sect. 2) 35-36. 
TRIPP, M. R., 1960. Mechanisms of removal of injected microorganisms from the American 

oyster, Crassostrea znrginica (Gmelin). Biol. Bui/., 119: 273-282. 
YONGE, C. M., 1926. Structure and physiology of the organs of feeding and digestion in 

Ostrea edulis. J. Mar. Biol. Assoc., 14: 295-386. 



ACTIVE MOVEMENTS AND OTHER ASPECTS OF THE BIOLOGY OF 
ASTICHOPUS AND LEPTOSYNAPTA (HOLOTHUROIDEA) 1 

PETER W. GLYNN 

Institute f Marine Biology. University of Puerto Rico, 
Wayaguez, 1'ncrto Rico 

The ability of certain benthic sea cucumbers to execute relatively rapid move- 
ments has not been gem-rally recognized or given adequate treatment in compre- 
hensive accounts of the Holothuroidea (e.g., Ludwig, 1892; Cuenot, 1948; Hyman, 
1955). In some bathypelagic species the performance of rapid progressive move- 
ments is regarded a normal means of locomotion. Ludwig (1892) and Hansen and 
Maclsen (1956) have noted the remarkable swimming movements, first observed 
by M. Sars (1868), of Juilliyplotcs natans (=Stichof>us natans), an aspidochirotid 
of the typically deep-sea family Synallactidae. Gilchrist (1920, p. 381) observed 
that ". . . some of the Holothurians procured in deep water off the South African 
coasts have the power of swimming about freely in the water by an undulatory 

movement of the body " He further surmised that ". . . such deep-sea 

Holothurians do not bury themselves in the soft mud of the floor of the ocean, 
but flit more or less readily over its surface." More recently, Hansen and Madsen 
(1956, p. 55) have suggested, "Probably a powder of swimming, though often 
awkward, may be attributed to a considerable number of Holothurians of the 
family Psychropotidae within the order Elasipoda and of the genera Bathyplotes 
and Paelopatides within the Synallactidae of the order Aspidochirota." These 
authors remarked that of the known bathypelagic holothurians, Galatheathnria 
uspera is probably the best adapted for active swimming, which in this species is 
effected through undulatory movements of the lateral brim much as in the swim- 
ming of the cuttlefish, Scf>ia. The elasipodid, Bcnthodytes typica, with a wide 
brim all around the body, is also well adapted for swimming. 

1 The portion of the study dealing with Astichopus was supported by National Science 
Foundation Grant GB-888; observations on Lcptosynapta were made by the author while 
employed with the California Cooperative Oceanic Fisheries Investigations. Appreciation is 
expressed for the aid rendered by the following persons and institutions: Charles E. Cutress, 
Smithsonian Institution, \Yashington, D. C., who supplied information on the swimming be- 
havior of Astichopus and made available pertinent literature; David L. Fawson, also of the 
Smithsonian Institution, who made available- pertinent literature and reviewed the manuscript; 
Alfred II. Hummel, SCUBA instructor at K'anu \ \\\- Force Base, Aguadilla, Puerto Rico, who 
assisted in the collrclion of specimens of Astichopus; Frank Fernandez, Research Assistant, 
Institute of Marine Biology, who helped with tin- fit-Id and laboratory studies; John Shoup, 
Bernice I'. Bishop Muslim, Honolulu, Hawaii, who helped with certain phases of the be- 
havior studies; Stan \Yiinherh-y, Geology Section, University of Puerto Rico, Mayagiiez, who 
aided in the analysis of sediment samples; I.uis M. Quiiiones-Rodriguez, Department of 
1'hy.sics, I'niversity of I'uerto Rico, Mayagnex, who .supplied monochromatic lamps and fdters. 
Credit is also due Elisabeth Deicliniann, Museum of Comparative Zoology, Harvard University 
and Kenneth R. Jl. Read, Division of General Education, Boston University, for criticizing 
the manuscript. 

106 



ACTIVE HOLOTHURIAN MOVEMENTS 107 

Of the typically bottom-living, non-pelagic sea cucumbers, only a small number 
of species were known to execute relatively rapid movements. All of these species 
are members of the family Synaptidae in the order Apodida. As originally re- 
ported by Nutting (1919), and quoted by Fisher in Deichmann (1926), Euaplu 
lap fa can swim to a limited extent. Costello (1946) has described well the 
active, scissor-like movements of the young of Leptosynapta albicans ( = in!ntcrcns), 
as first reported briefly by Clark (1907, p. 63). Recently, Hoshiai (1963) has 
observed undulatory swimming in the young of Ldbidoplax dnhia. 

The quick swimming movements observed in adults of the aspidochirotid 
Astichopits multifidus and Leptosynapta albicans are documented for the first time 
in the present communication. Further, a description of some other kinds of 
locomotory movements performed by Astichopits, which were formerly unknown 
in the Holothuroidea, is included in this paper. Also, other aspects of the biology 
of Astichopits are investigated in relation to the species' active movements, viz. 
the nature of its habitat, the reactions elicited as a result of alterations of the 
immediate environment (for example mechanical disturbances, temperature, light, 
salinity), and its toxicity. A description of the sinusoidal swimming behavior of 
adult Leptosynapta concludes the study. 

MATERIALS 

Astichopits nntltifidns (Sluiter. 1910) is a member of the order Aspidochirotida ; 
members of this group are characterized by possessing disk-shaped tentacles and 
respiratory trees. Examination of the structure of the gonad demonstrated that 
it occurs as two tufts, thus confirming that the species does belong to the family 
Stichopodidae (Deichmann, 1954). Astichopus, a monotypic genus in the West 
Indies, is easily recognized because it is very large and soft, with both dorsum 
and ventrum uniformly covered by hundreds of tube feet ; the dorsal tube feet are 
papillate (Figs. 1 and 4). The dorsum of all specimens examined was some shade 
of brown or gray, and exhibited a variable color pattern. Two individuals had 
a chocolate brown dorsum with numerous small (ca. 1 cm. diameter) scattered 
white spots ; the ventrum was also chocolate brown. Several specimens had a 
light brown dorsum, and one of these possessed in addition three large (3-5 cm. 
diameter), evenly spaced, chocolate brown spots. Lighter colored individuals 
tended to have a white ventrum. The tube feet and papillae were light in color, 
usually a translucent light yellow or light brown. Undisturbed, crawling Asti- 
clwpus demonstrated a range in total length of 29-46 cm. Specimens observed 
by Clark (1933) were somewhat larger, at least 45 cm. in length. Aggregates of 
numerous minute grains and scattered C-, S-. or O-shaped calcareous particles 
occur in the body wall (Deichmann. 1954). Three of the specimens collected in 
Puerto Rico and used as material in this study have been deposited in the Smith- 
sonian Institution. U. S. National Museum (Number E-10325). 

Few specimens of Astichopits have been collected previously; the largest 
number reported were brought up in trawl hauls made on the Campeche Bank 
in the Gulf of Mexico. According to H. Hildebrand (quoted in Deichmann, 1954), 
this species is an abundant form in this region. Several specimens were also col- 
lected at Port Antonio, Jamaica, by Clark (1933). Since these earlier occurrences, 
no other specimens have been reported from Jamaica (Fontaine, 1953). This 



108 



PETER W. GLYNN 



species has been reported I'rom . oilier localiu in the tropical western 

Atlantic, namely at Tortugas. Florida ( I )eichinann, 1963); the specimens found 
in Puerto Rico constitute a new record for this region. Because a dense popula- 
tion of this comparatively rare .species has been discovered in Puerto Rico, an ac- 
count of the habitat is given in the next section. 

Leptosynapta ullucans ( Selenka. 1S67), a well known California!! sea cucumber, 
belongs to the order . \podida. a group wholly lacking tube feet, and to the family 




FIGURE 1. Underwater photograph of Astichopits nntltifidits at the edge of a bed of the 
seagrass, Ifulnpliilii huilloiiix, in 15 m. of water at Crashboat Landing, Aguadilla (November 
20, 1964). Tlic length of this animal, as it is crawling in the picture, was approximately 35 
cm. Forward prngres.Mon i> toward the right; visible are the cloacal aperture at the rear end 
of the animal to the left and a lateral fringe of papillae bordering the ventrum. 



Synaptidae, whose members possess calcareous spicules in the form of anchors and 
anchor plates and tentacles with slender digits. The observations reported in this 
study were made on animals living in Monterey Bay, California. 

Limited studies were carried out on Synaptitlu Jiydrijonnis (Le Sueur, 1823), 
a viviparous member of the Synaptidae. This is an abundant species of the West 
Indian fauna, usually living a-vsociaied with algae. All specimens were collected 
from the red alga, Lanronia /v//>;7/.w/. \\hich grows on a sandy bottom in the shade 
of red mangroves (Rhizophora nnintjlc'} on the reef flat at Cayo Majimo, La 
I'arguera, I 'uerto Rico. 

P.eraii>e different methods were employed in the various experiments per- 
formed, the.se are discussed separately under the appropriate, sections to follow. 



ACTIVE HOLOTHURIAN MOVEMENTS 



109 



ASTICHOPUS MULTIFIDUS 



Habitat 



Astichopiis has been found at five different localities in the coastal waters of 
western Puerto Rico (Fig. 2). It is most abundant on the northwestern coast, 
and at Crashboat Landing, midway between Pta. Borinquen and Aguadilla, several 
specimens have been observed on numerous occasions throughout the year. The 
species has been seen at depths of 201-0 m. at different times near Isabela, Camuy 
and Arecibo, and probably occurs in favorable localities between these areas. 2 The 




Son Juan 




KEY 



Number of individuals actually 
collected 



O Presence observed 



Presence inferred 



FIGURE 2. Map of western district of Puerto Rico showing the localities where Astichopus 
has been collected (solid circles) and observed (open circles) from June, 1960, to October, 
1964. Its probable occurrence along the north coast is also indicated (dots). The small inset 
map of Puerto Rico shows the sector of the island examined. 

results of a typical collecting trip to obtain specimens of Astichopiis, conducted at 
Crashboat Landing on September 29, 1964, give an approximate indication of its 
abundance in this locality. Three individuals were found, two at a depth of 
20 m. and one at 10 m. (measured with a wrist depth gauge), by two SCUBA 
divers swimming abreast, searching a path 10 m. in width over a distance of 
about 1 km. 

A dense growth of the marine phanerogam, Halophila baiUonis, was present 
where Astichopiis was collected at 20 m. ; the other specimen was taken from the 
bare sandy bottom. An underwater photograph of a cucumber on a Halophila- 
covered bottom is shown in Figure 1. The four individuals of AsticJwpiis re- 
ported from La Parguera were found in the months of January, February, August 
and October at a depth of from 1 to 3 m., either on a bottom with a dense growth 
of turtle grass (Thalassia tcstudinum) , or nearby on the bare sand on the leeward 

2 Gary E. Branham and Alfred H. Hummel informed the author of the occurrence of 
Astichopiis at all localities from Pta. Borinquen east to Arecibo. 



110 PETER W. GLYNN 

side between the two inshore coral reefs, Cayo Caracoles and Cayo Majimo. The 
present observations on the bathymetric range of Astichopus support Deichmann's 
(1963) belief that this species normally lives in deeper water, occurring at shallow 
depths only sporadically. 

Other echinoderms observed commonly at Crashboat Landing on the sandy 
bottom between 30 and 45 m. of depth were the echinoids, Astro pyga magnified 
(Diadematidae) and M count vcntricosa (Clypeasteridae). Astropyga is not re- 
ported from La Parguera and Mcoina has been found there only infrequently. 

Physically and biologically the shore line from Aguadilla to Camuy is decidedly 
different from that in the vicinity of La Parguera on the south coast. Kaye (1959) 
has described the northwestern coast, from Aguadilla to Arecibo, as largely com- 
posed of a limestone cliff, occasionally interrupted by a narrow rocky or sandy 
bench, which often forms a firm surface where cementation of dunes has occurred. 
Mangrove forests border most of the coastline around La Parguera and shallow 
fringing and patch reefs, composed of such prominent coral species as Acropora 
palmata, Montastrea annnlaris and Poritcs poritcs var. fnrcata, are numerous 
(Almy and Carrion, 1963). 

Considerably stronger waves buffet the north coast of Puerto Rico than along 
the south shore, except during cyclonic disturbances from the southern quarter 
(Glynn, Almodovar and Gonzalez, 1964). The normally heavy surf along the 
north coast is a result of the following conditions : (a) this region is exposed di- 
rectly to the high seas generated across the Atlantic Ocean, (b) the island shelf 
is narrow with few offshore reefs and banks, and (c) the windward shore receives 
large swells resulting from storms in the North Atlantic during the winter season 
(Kaye, 1959). 

Substantial fresh-water discharge is also a prominent feature along the Atlantic 
seaboard of Puerto Rico. Because of the southern location of the north-south 
drainage divide, seven of Puerto Rico's 17 principal rivers, with an approximate 
drainage area of 1398 square miles or 64% of the total considered here, discharge 
at more or less regular intervals along the north coast (Arnow and Bogart, 1960). 
No permanent river system is present in the vicinity of La Parguera on the south 
coast. 

Surface sediment samples (upper 5 cm. stratum), collected from the sites 
where Astichopus was found in greatest abundance at Crashboat Landing and 
Cayo Caracoles, vary considerably in grain size and composition (Fig. 3). The 
median diameters and degrees of sorting (as indicated by phi standard deviation ; 
Tnman, 1952) for the Aguadilla and La Parguera samples were 0.212 mm., with 
1.1 phi-units and 0.392 mm., with 2.2 phi-units, respectively. The terrigenous 
fraction of the sample from Crashboat Landing contained mostly quartz and feld- 
spar with about 5 r f heavy minerals. Calcareous bioclastic materials, constituting 
97.2% of the dry weight of the sample from La Parguera. were nearly three times 
as great as at Aguadilla. Ifaliuicihi fragments were the principal constituents 
in the south coast sample, with the remainder composed of broken skeletons of 
other calcareous algae, the sessile foraminiferan. Hoinotrcnui nthnini, coral frag- 
ments, echinoid tests and spines and a varictv of other invertebrate hard parts. 
The more poorly sorted sediment sample from La I 'arguera might be explained by 
the high per cent composition of plate-like Halnucda fragments and the location 



ACTIVE HOLOTHURIAN MOVEMENTS 



111 



of the area on the Caribbean Sea, in the lee of the heavy swells and surf action 
of the Atlantic coast of Puerto Rico. Sediment analyses reported by Guillou 
and Glass (1957) confirm the divergent character of the substrata as revealed in 
the present study. Calcareous and non-calcareous materials were observed to be 
present in equal amounts in the beach sands from Aguadilla to Rio Camuy, while 
inshore sediments along the southwestern coast (all of south coast as shown in 
Figure 2) were predominantly calcareous. 



99 99 



2 95 



: 84 

^ 

I 50 



16 

5 




- Crasnboat Landing 
Terrigenous residue 62.5 



Calcium carbonate 
Organic matter 



368 
0.7 



b - Cayo Caracoles 
trace 

97.2 
2.8 



-1.0 1.0 3.0 5.0 

2.0 0.5 0.125 0.031 

Phi-units and grain size 



mm 



FIGURE 3. Plot on probability paper showing cumulative percent distributions of grain 
size in sediment samples from Astichopus habitats at Crashboat Landing, Aguadilla (a) and 
Cayo Caracoles, La Parguera (b). The per cent composition of the samples, based on dry 
weight, is tabulated in terms of terrigenous residue, calcium carbonate and organic matter con- 
tent. Grain size distribution was determined by standard sieve analysis, calcium carbonate 
and organic matter contents by the difference in weights obtained after ample treatment with 
HC1 and H 2 O 2 , respectively. The H 2 C>2 technique employed is outlined by Stevenson and 
Emery (1958). Terrigenous residue, as here denned, was that portion of the sample remaining 
after the above treatment. 

Movements 

Clark (1933) observed that Astichopus is a very active form and remarked 
(p. Ill) that it ". . . moved about more obviously than any other large holo- 
thurian I have ever watched." The various movements performed by Astichopus 
were studied in the field during daylight hours and in the laboratory during the 
day and at night. Animals maintained in captivity were kept in 200-liter 
DUROTEX (asbestos) troughs and 210-liter aquaria supplied with running sea 
water. Figure 4 illustrates some of the different movements observed, and the 
account of these follows. 

A slow crawl is the most frequent means of progression when the animal is 
left undisturbed (Fig. 4a). All specimens observed in the field were crawling 
over the sandy bottom, ingesting the substratum by means of the circlet of large 
tentacles surrounding the mouth. Examination of gut contents showed that there 



112 



I'KTKR \V. GLYXX 



is no apparent discrimination of particle size, and that large fragments of either 
living or dead plant material arc generally avoided. Two cucumbers, timed while 
crawling under laboratory conditions and not feeding, progressed at a mean rate 
of 0.25 and 0.15 in.. / min. ( Table [). Individuals observed in the field while 
feeding moved along at a slower rate. As indicated by the lengths measured in 
the two individuals included in Table 1 I. a disturbed animal will contract to about 
three-quarters of its crawling length. When the body is contracted the dermal 
papillae (dorsal tube feet) are frequently withdrawn into the body wall. The 




Fna'KK 4. I -"our distinct movements performed by .Istic/inpiis in captivity. The four 
photographs in each horizontal series demonstrate (a) crawling, (b) bounding, (c) rolling and 
iili an exploratory activity. Arrows in .-erics (a) through (c) indicate the direction of for- 
ward progression, vicuing the photographs in sequence from left to right. The inset scale 
in the first photograph in series (d) indicates approximately the sixes of the three different 
individuals illustrated. The actual lengths of the specimens, measured while crawling, were 
(a) 32 cm., (b) and (c) 30 cm., and (d) 46 cm. 

crawl is accomplished through the forward progression of a peristaltic wave 
originating from the posterior end of the animal. The posterior end is first ele- 
vated two to four cm. from the substratum and then the wave moves forward, 
forming a two-cm. -high arch between the ventrum and the underlying surface. 
Of the many tube feet uniformly distributed oxer the ventrum, the few attached to 
the substratum are detached momentarily as the peristaltic wave moves forward. 
Three peristaltic waves passed across the bodv of a 40-cm individual in a mean 
time of (o seconds. A second cycle is initiated by the time the first has moved over 
two-thirds of the body length. 



ACTIVE HOLOTHl'RIAN MO\ 'KM KNTS 



113 



Under certain conditions, to be discussed in the next section, the crawl will 
suddenly quicken to a walking or a bounding type of locomotion (Fig. 41)). A 
gradation in speed from crawling to bounding is clearly evident, but once one of 
the three modes of progression is executed it persists for some few minutes, \\~alk- 
ing speeds of three-quarters to one ni./min. are usual, whereas one and one-half 
to two m. /inin. are rates typical of the bounding movement (Table I). As in 
crawling, both walking and bounding are initiated by a peristaltic wave which 
moves forward along the body. However, the cycle is obviously more rapid and 
exaggerated at greater velocities. For example, in the bounding movement a wave 
passes along the entire length of the body in three to five seconds and there is no 
attachment of the tube feet to the bottom. As a wave terminates anteriorly, the 
forward end of the body is thrown upwards forcefully and at the same moment a 
new cycle begins at the hind end. Fven at the highest speed attained, Astichopus 

TABLE I 

Rates of progress-ion for the crawling, walking and bounding movements of 
Astichopus as observed in captivity 



Specimen 


Type of movement (m./min.) 


Crawling 


Walking 


Bounding 


Light brown individual; crawling length 
39 cm. ; contracted length 30 cm. 


0.23 
0.25 


0.62 
0.76 


1.72 
1.89 






0.26 


0.88 


1.98 






X 0.25 


0.75 


1.86 


Dark 
39 


brown individual; crawling length 
cm.; contracted length 32 cm. 


0.12 
0.16 


0.74 
0.90 


1.33 

1.43 






0.18 


1.01 


1.71 






X 0.15 


0.88 


1.49 



always maintained contact with the bottom. Indeed, it appears that this contact 
is necessary for the forward thrust, since animals pushed over on their sides and 
still bounding remain essentially stationary. Many of the dorsal papillae contract 
into the skin while the animal is walking or bounding. The total distance covered 
by one specimen while bounding in an aquarium was about 10 in. Fach time the 
animal reached the obstructing wall at the end of the aquarium it was turned around 
gently so as not to interfere with its forward progression. Animals walking or 
bounding tended to deviate little from a straight-line course. Only limited bound- 
ing movements have ever been observed in the field, i.e., of the order of one to one 
and one-half m. traversed at a time. 

A swimming-like response was observed by Cutress (personal communication) 
on one occasion. The animal actually left the bottom and progressed through the 
water in an undulatory manner. Further attempts were made to repeat the act, 
but they resulted only in the bounding movement already described. In the 
present study one individual on two different occasions was stimulated to bound 
in the field by subjecting the animal to a sudden change in the temperature of the 



114 I'KI EE W. GLYNN 

water (see section on environmental effects on movements), but the bounding 
movement did not lead to swimming. 

Rolling movements also occur frequently, and as shown in Figure 4c the body 
Ilexes into a I' during this activiu. A complete roll is carried out in unison along 
the whole length of the animal. About one-half of the dorsal papillae are con- 
tracted during this movement. ( )ne individual made several complete rolls in a 
mean time of five seconds. Rolling may last as long as five minutes, with the 
animal moving little from its original position. It became evident that all of the 
movements thus far described were less easily evoked in individuals recently stimu- 
lated and in some animals activity decreased in frequency and intensity with time 
maintained in captivity. 

An exploratory or searching activity was observed on only one occasion (Fig. 
4d). and this occurred in one of three individuals in the same aquarium imme- 
diately after transportation to the laboratory from the field. About one-third of 
the anterior portion of the body was elevated, and this swung frequently from side 
to side, describing an arc of approximately 40. A full swing to the left is shown 
in the second photograph in Figure 4d. The exploratory movements lasted for 
about 10 minutes and culminated in the erect posture illustrated in photographs 
three and four of Figure 4d. \Yith over one-half of the body in an upright position 
the animal bowed forward several times, forming an angle of 45 from the vertical. 

The five longitudinal retractor muscles, which must play an important role in 
all of the various movements, are very well developed in Astichopus as 1-2-cm.- 
broad bands running the length of the body. Comparable muscles in a closely 
related, sluggish form. Isoticlwpiis (== Stichopus*) badionotus, are thin and narrow. 
The body wall is tough and thick in both species, but soft and pliable in Astichopus 
and rigid in Isostichopus. Excessive muscular development has also been reported 
in the swimming sipunculid. Sipnnciilns natans (Fisher, 1954), a member of a 
typically benthic group. This species has developed strong longitudinal muscle 
bands and enormous wing-muscles. 

Environmental effects on inurements 

In order to learn how the various movements just described may be used to 
advantage by Asticliopits, several animals were stimulated in the field and in the 
laboratory to produce disturbing circumstances likely to be encountered by the 
species in its natural surroundings. Such conditions studied were various bodiK 
disturbances, alterations in the temperature of the water, reduction in salinity. 
effects of oxygen-deficient water and heha\ ior in numerous divergent light regimes. 
In all instances parallel control procedures demonstrated that the movements 
evoked were due to the particular conditions under investigation. 

Procedure involving mechanical stimulation was as follows. Two individuals 
were buried completely beneath the bottom sediments and elevated quickly (in 30 
seconds) toward the surface, over an ambient pressure gradient of one atmosphere. 

All of the animals responded by contracting initially, then after a minute or 
two began to crawl, (ientle handling of specimens normally produce's the same 
reaction. All of several individuals, turned oxer on their dorsa and sides, righted 
themselves immediately, in 3-5 seconds. 1>\ the rolling movement. A half- or 
quarter-roll only was performed to regain normal posture. Since current action 



ACTIVE HOLOTHURIAN MOVEMENTS 115 

on the bottom is relatively strong at Crashboat Landing, often swaying the animals 
from side to side down to a depth of 30 m., this quick, righting response is well 
suited to help maintain proper orientation. 

Sudden changes in sea water temperature, of the order of 3-4 C.. elicited the 
walking, bounding and rolling movements more effectively than any other environ- 
mental alterations investigated. To test the effects of temperature, animals were 
moved rapidly from one aquarium, at a lower or higher temperature, to another. 
In addition, some individuals were maintained in a slightly cooled or heated state 
in captivity, and then transferred rapidly to the field. The laboratory sea water 
temperatures ranged from 27.5 to 29.3 C. over the duration of the experiments. 
Coastal sea water temperatures at this time of the year (October) were in the 
range of 27-29 C. Different individuals of Astichopiis were subjected to the 
following typical changes in temperature: from 29.1 C. to 32.4 C., and from 
27.7 C. to 23.3 C. Essentially the same movements were performed when the 
animals were subjected to a temperature which was greater or less than the initial 
temperature. Immediate bounding was the most frequent response, lasting for 
one to three minutes. A rolling movement was less common, while walking was 
observed on relatively few occasions. Once a particular type of movement began 
it usually persisted until the animal slowed down to a crawl. 

The behavior of Astichopus under different conditions of lighting was investi- 
gated with the following light sources : natural daylight, house light (tungsten 
filament lamp), red light (infrared lamp), yellow light (sodium lamp), filtered 
green light, blue light (mercury lamp) and violet light (ultraviolet lamp). The 
light beam was directed so as to enter a 210-liter aquarium on one side from above; 
cucumbers were placed lengthwise along the edge of the lighted portion and the 
shadow of the dark side of the aquarium, which was covered with black cloth. 
Observations were made in a darkroom where the light intensities employed were 
equal to or less than 10 foot-candles. Light intensities up to 75 foot-candles were 
measured with a Western photoelectric cell (Model 703, sight meter) ; an estima- 
tion of greater illumination was obtained by the interposition of green filters over 
the photocell and by calibrating the Weston meter against a General Electric Mascot 
exposure meter (Type PR-35). 

Individuals showed a marked attraction to natural and artificial light of low 
intensity (5 foot-candles), by immediately crawling toward the source. No con- 
sistent phototactic attraction or repulsion could be discerned at higher light intensi- 
ties of 10, 20, 30, 50 and even 11.000 foot-candles (zenith sun at noontime in 
November). Usually, however, activity did increase at higher illuminations. In 
contrast to the usual photo-negative response of holothurians (Crozier, 1915) 
Astichopns crawled with equal frequency toward and away from the illuminated 
end of the aquarium. The phototactic behavior of Astichopus seemingly parallels 
that of Isostichopus, a form not apparently irritated by a strong source of illumina- 
tion. 

Monochromatic light was adjusted so that a maximum intensity of 10 foot- 
candles entered through one side of the aquarium. Astichopus demonstrated a 
strong, positive attraction to red, green, blue and violet light; individuals moved 
toward the light immediately by crawling and lingered in the region of greatest 
illumination. Only a modest attraction to yellow light was observed. 



116 PETER W. GLYNN 

.Istichopns did not demonstrate any clear tendencies in geotactic or thigmo- 
tactic behavior. Animals climbed readily up and down the vertical sides of con- 
tainers and surfaces inclined at various angles to the horizontal. ( )ccasionally 
individuals in the field were found alongside submerged pipes and pilings. Move- 
ments of confined laboratory animals indicate, however, that the association with 
solid objects may occur simply by chance wandering. 

Effects resulting from a reduction in salinity were observed in the laboratory 
in individuals transferred from the sea water in which they were maintained to 
tanks containing sea water diluted with tap water. The normal, mean, surface 
salinity on the inshore reefs of the south coast for November, when these particular 
studies were made, was around 34. SO'/, (unpublished data). Experimental dilu- 
tions were 31.00',, and 17.25'iV. Animals suddenly subjected to these conditions 
usually contracted, but occasionally executed a bounding motion. An equal in- 
tensity of reaction, in terms of speed and duration, was noted in both dilutions. 

Individuals of .-Isticlio^ns were also transferred to oxygen-deficient sea water- 
obtained directly over anaerobic mud on the floor of a mangrove canal. < )f tin- 
rapid movements a bounding response was elicited most frequently under these 
conditions; limited rolling was also observed. 

The possibility that the association of Astichopits with members of its own 
species or with other organisms may elicit active movements was also investigated. 
Individuals together in the same container behaved independently, i.e., they crawled 
about as if alone and tended to avoid contact with other cucumbers. Animals in- 
troduced into an aerated aquarium in which Astichopiis was previously living- 
showed no signs of excitation. The water in this case was recirculated through 
a filter instead of being replaced by fresh, running water. Simulated biting, by 
firm pinching of different regions of the body with the bare hand, was observed 
both in the field and in the laboratory. This evoked a typical contraction of the 
body with subsequent crawling after 1-2 minutes. Introduction of a fresh slime 
preparation from the skin of Lactoplirys bicandalis, a trunk-fish which regularly 
preys on the small holothurian, Microthele (-- Holotlntria) parrnla. in the imme- 
diate vicinity of Asticliopits also failed to produce any active movements. 

Two observations indicated that AsticJiopns might possibly possess a toxic 
substance that adversely .affects other organisms nearby. Petrochirus diogenes, a 
large scavenging and possibly predatory hermit crab (Randall, 1964) found in 
association with Asticliopus, tended to avoid the cucumber in captivity. In addi- 
tion, several fishes in the same aquarium with .Isticlio/^ns died after one cucumber 
eviscerated. 

Toxicity 

Yamanouchi (1955) has clearly demonstrated that numerous species of holo- 
thurians are toxic; of 27 species investigated, 24 contained a venomous substance. 
Xigrelli and (akowska ( 1 ( >60) reported that poisonous species are known in four 
of the five orders comprising the class; members of the deep-sea order Elasipodida 
have not been examined in this connection. The total number of species of cucum- 
bers known to be toxic to fishes is 30 24 of the.se live in the- Pacific Ocean, three 
in the Mediterranean and four in the West Indies (I'ahamas). One of the latter 



ACTIVE HOLOTHURIAN MOVEMENTS 1 1 7 

species occurs in the Mediterranean and in the West Indies. The toxic principle 
in the common Bahamian species. .Ictinopyga agassizi, has been identified as a 
saponin, a chemical structure previously unknown in the animal kingdom (Nigrelli 
ctal, 1955 ). 3 

Investigations were made of the possible adverse effects on other marine animals 
of (a) the water in which Astichopns had recently performed active movements, 
(b) its coelomic fluids, and (c) alcohol extracts of the body wall. Freshly killed 
cucumbers were used in all instances. The potency of Astichopns was assayed by- 
observing the effects produced in seven species representative of six different animal 
phyla: Coelenterata, Madreporaria Poritcs poritcs var. jurcata Lamarck, 1816; 
Annelida, Polychaeta Hcsionc proctocliona Schmarda, 1861 ; Mollusca, Pelecypoda 
Lima scabra Born, 1778; Arthropoda, Crustacea Mitlira.v ( Mitliracnlits} scalp- 
tiis (Lamarck, 1818) ; Echinodermata, Ophiuroidea Ophiothri.v ongulata Say, 
1825; Chordata, Tunicata Ectcinascidia turbinata Herdman, 1880; Chordata. 
Pisces Jcnkinsia laniprotacnia (Gosse, 1851). All of the invertebrate species 
were held in wide-mouth bowls with one liter of aerated sea water at a temperature 
of 28-29 C. ; Jcnkinsia was held in a circular bottle of 10 liters capacity. All 
invertebrates except Ectcinascidia were collected on the same day of an experi- 
ment from the south shore of Magueyes Island, in association with the fringing 
Poritcs poritcs var. furcata reef. Ectcinascidia was collected from mangrove roots 
bordering the canal which separates Magueyes Island from the mainland ; Jcnkinsia 
was netted near the shoreline of the same canal. Parallel control material, with 
at least the same number of individuals observed as in experimental, always accom- 
panied each experiment. 

Two 10-ml. samples of sea water, taken from separate 5-liter containers in 
which one Astichopus had rolled and another bounded, did not produce any visible 
effects on the test animals over a 24-hour period. 

An unsuccessful attempt was made to stimulate evisceration in three different 
individuals by firmly squeezing and pinching various areas of the body. Further, 
evisceration did not occur under the diverse experimental conditions to which 
Astichopus was subjected. Only two individuals eviscerated over a two-month 
period and the cause was not readily apparent. Much of the gut and the respira- 
tory trees were ejected ; Cuvieran organs are unknown in all Stichopodidae. For 
these reasons, it does not appear likely that evisceration is a normal defensive 
response. The body fluids tested for toxicity, then, were obtained by dissection 
from the coelomic cavity. Undiluted coelomic fluid, equal to a final concentration 
of 1000 ppm. (parts per million), was added to the water in which the test species 
were confined, and their reactions observed continuously for the first two hours 
after introduction and then at 4 hours and 8 hours. At the termination of the 
experiment animals were transferred to running sea water for a duration of 12 
hours in order to observe recovery. The various responses noted are summarized 
in Table II. 

Polyps of the scleractinian coral Poritcs contracted slightly in the first one-half 
hour and remained in this state for 8 hours. In 4-8 hours a mild lethargic re- 
sponse was observed in Hcsionc, Mitlira.v and Ophiothri.r; they all became less 

3 Studies on the chemical nature of the toxic agent are presently being carried out by the 
author in collaboration with Horace Graham, Department of Biology, University of Puerto 
Rico, Mayagiiez. 



118 



PETER W. GLYNN 



irritable to mild mechanical stimulation. Lima re-acted immediately by violently 
closing its valves; after about one hour they began to gape. By two hours the 
water became noticeably reddened and contained numerous small fragments of 
mantle tissues voided by the animals. Iictcinasciciia responded by slowly contract- 
ing until at 8 hours all individuals were dead ; cessation of heart beat was used as 
a criterion of death. Jenkinsni, the small clupeid, perished quickly; two fish were 

TABLE II 

( >l>scn'<itions af the effects on some marine animals of the body fluids of 
Astichopus at a concentration of 1000 parts per million 



Organism 


X umber 
observed 


Reactions at indicated time intervals 


Recovery 
(12 hrs.)** 


Jhr. 


1 hr. 


2 hrs. 


4 hrs. 


8 hrs. 


Coelenterata 
Parties parties 
var. furcata 


3 terminal 
branches, 
over 100 
polyps in 
each 


polyps 
slightly 

contracted 


same 


same 


same 


same 


yes 


Annelida 
Hesione proc- 
tochona 


3 


normal* 


normal 


normal 


loss of 
quick 
wriggling 
response 


same 


yes 


Mollusca 
Lima scabra 


5 


immediate 
and com- 
plete closure 
of vab < 


valves open 
in 2 indi- 
viduals 


all open; 
small bits of 
mantle 
tissue 
ejected 


same 


same 


yes 


Arthropoda 

Mithrax ( Mithra- 
citlus) sculpt us 


4 


normal 


normal 


normal 


loss of 
quick de- 
fensive 
reaction 
of chelae 


same 


yes 


Echinodermata 
Ophiothrix 
angulata 


5 


normal 


normal 


normal 


normal 


sluggish 


no 


Chordata-Tunicata 
Rcteinascidia 
turbinata 


1 colony of 
20 in- 
dividuals 


normal 


siphons 
slightly 
contracted 


same 


siphons and 
body wall 
contracted 


all < lead; si- 
phons and 
body wall 
greatly con- 
tracted ; no 
heart beat 





Chordata-Vertebrata 
Jenkinsia 
lamprotaenia 


5 


normal 


2 dead 


all dead 











* A normal reaction indicates that no difference could be discerned between the test animals and controls. Same 
applies to Tables III and IV. 

** Twelve hours in running sea water were allowed for recovery after the termination of the experiment. Same 
applies in Tables III and IV. 

dead at the end of the first hour and all five had succumbed in two hours. The five 
control individuals of Jenkinsia lived beyond the <S hours of the experiment. Re- 
covery occurred in four of the five surviving species; Ophiothrix died. 

Yamanouchi (1955) and Xigrelli and Jakowska ('1 ( '(>0) have extracted with 
hot ethanol an active, toxic principle from the body wall of various holothurians. 
Alcohol extracts obtained from Astichopus were prepared as follows. A one-inch 
square of the body wall was macerated in a mortar with 30-40 mesh quart/ sand. 
The mash and juices were then treated with 50 ml. of ( >5'/r. hot ethanol at 50-60 C. 
for 10 minutes. The extracts at concentrations of 1000 and 5000 ppm. were tested 



ACTIVE HOLOTHURIAN MOVEMENTS 



119 



after cooling to room temperature. The results are presented in Tables III and IV. 
The reactions of the test animals to alcohol extracts were very similar to those 
resulting from exposure to body fluids. Usually, however, a more severe reaction 
was evoked by the extract when equal to or at a greater concentration than the 
body fluids. The slightly contracted polyps of Poritcs after one hour were com- 
pletely withdrawn in two hours. Lethargy again occurred in Hesione, Mithrax 
and Ophiothrix. Additional effects observed after 8 hours were inflation of the 

TABLE III 

Observations of the effects </ some marine animals of an alcohol extract of the 
body wall of Astichopus at a concentration of 1000 parts per million 



Organism 


Number 
observed 


Reactions at indicated time intervals 


Recovery 
(12 hrs.) 


Jhr. 


1 hr. 


2 hrs. 


4 hrs. 


8 hrs. 


Coelenterata 


3 terminal 


normal 


polyps 


polyps 


same 


same 


no 


Poriles porites 


branches. 




slightly 


fully 








var. furcata 


over 100 




contracted 


contracted 










polyps in 
















each 














Annelida 


5 


normal 


normal 


normal 


loss of 


body in- 


yes 


Hesione proc- 










quick 


flated; 




tochona 










wriggling 


proboscis 














response 


everted 




Mollusca 


5 


active swim- 


valves par- 


small bits of 


moribund 


all dead 





Lima scabra 




ming fol- 


tially open; 


mantle 












lowed by 


tentacles 


tissue 












complete 


constricted 


ejected 












closure of 
















valves 












Arthropoda 


5 


normal 


normal 


normal 


loss of 


same 


yes 


Mithrax (Mithra- 










quick de- 






culns) sculptus 










fensive re- 
















action of 
















chelae 






Echinodermata 
Ophiothrix angulata 


5 


normal 


normal 


sluggish 


same 


autotomy 
of arms 


no 


Chordata-Tunicata 

Ecteinascidia 


1 colony of 
20 indi- 


normal 


siphons and 
body wall 


same 


same 


siphons and 
body wall 


no 


turbinala 


viduals 




slightly 






greatly 










contracted 






contracted 




Chordata-Vertebrata 


5 


erratic 


_ 


_ 








Jenkinsia 




swimming 












lamprolaenia 




followed by 
















death in 10 
















minutes 













body and eversion of the proboscis in Hesione and extensive autotomy of the arms 
in Ophiothrix. Violent swimming movements were executed by Lima in the first 
few minutes, followed by closure of the valves. Individuals began to open after 
1-2 hours ; the pallial tentacles were noticeably shortened and constricted ; red- 
dened water, apparently due to the leakage of blood through ruptured tissues, 
appeared after two hours. All specimens of Lima succumbed during the experi- 
ment. Ecteinascidia responded by severe muscular contraction and cessation of 
heart beat at a concentration of 5000 ppm. Rapid, erratic swimming commenced 
in Jenkinsia immediately, resulting in the death of all specimens within 10 minutes. 
The gills were reddened in fish subjected to a concentration of 5000 ppm., indi- 
cating hemorrhage of the capillaries. A similar cause of death was observed in 



120 



ITTKK W. GLYNN 



the killitish, Cyprinodon haconi, after exposure to an aqueous preparation of the 
toxic agent of .Ictinopytjd ai/assici ( Xigrelli, 1952). Five species survived and 
two recovered from exposure to the extract at 1000 ppm., whereas of the four 
survivng species at 5000 ppm. only the niajid crah, Mit/ira.v, recovered. 



TABLE IV 

of the effects on some marine animals of an alcohol extract of the body 
ictill nf A ^tichopus at a concentration of 5000 parts per million 



Organism 


Number 
observed 


Reactions at indicated time intervals 


Recovery 
(12 hrs.) 


Jhr. 


1 hr. 


2 hrs. 


4 hrs. 


8 hrs. 


Coelenterata 


3 terminal 


normal 


polyps 


polyps fully 


same 


same 


no 


Forties porites 


branches. 




slightly 


contracted 








var. furcata 


over 100 




contracted 












polyps in 
















each 














Annelida 


5 


normal 


normal 


less active 


sluggish; 


body in- 


no 


Hesione proc- 










loss of 


flated; 




tochona 










quick 


proboscis 














wriggling 


everted 














response 






Mollusca 


5 


active swim- 


valves 


2 dead; 


4 dead ; 


all dead 





Lima scnbra 




ming fol- 


tightly 


3 moribund; 


1 moribund 










lowed by 


closed 


valves par- 












complete 




tially open ; 












closure of 




small bits of 












valves 




mantle 
















tissue 
















ejected 








Arthropoda 


3 


normal 


normal 


normal 


loss of 


same 


yes 


\litlirnx ( Mithra- 










quick 






culus) sculptus 










defensive 
















reaction of 
















chelae 






Echinodermata 


5 


normal 


normal 


sluggish 


same 


autotomy 


no 


Ophiothrix 












of arms 




angulata 
















Chorda ta-Tunicata 


1 colony of 


normal 


siphons and 


all dead; 





___ 





Ecteinascidia 


20 indi- 




body wall 


siphons and 








turbinata 


viduals 




slightly 


body wall 














contracted 


greatly con- 
















tracted; no 
















heart beat 








Chordata-Vertebrata 


5 


erratic 


_ 


_ 


_ 








Jenkinsia 




swimming 












lamprolaenia 




followed bv 
















death in 11) 
















miniili-- , 
















Kills 
















reddened 













LKI'TOSVNAI'TA Al.BICANS 

< )n three different occasions over the period January-May, 1957, four adult in- 
dividuals of Leptosynapta ull'icdus were ohserved swimming at night light stations 
in Monterey Kay, California. During that time of year, night light stations were 
occupied at approximately weekly intervals. All observations were made on dark 
nights hetween ( > :()() and 1 1 :()() I'M, from a float secured to the Monterey Municipal 
Tier. The sea hottoni is sandy helow the float and ahout 7 in. dee]) (measured 



ACTIVE HOLOTHURIAN MOVEMENTS 



121 



from mean sea level ). 4 The direction of swimming was along the outer border of 
visibility illuminated by a 200-watt lamp suspended in the water about three m. 
away from the observer. 

The swimming animals described a sinusoidal path as they moved through the 
water near the surface (Fig. 5). It was not possible to determine whether the 
progressive waves of activity occurred in the lateral or dorso-ventral plane. While 
swimming the cucumbers were extended in length to about 20 cm., but when 
captured quickly contracted to about 5 cm. It is estimated that a complete wave 
passed from the head to the tail end in about two seconds, and that the animals 






5 CM 



FIGURE 5. Diagrams of the undulatory swimming movements performed by Leptosynapta in 

tin- field (a and b) and the more frequent appearance of the animal when crawling (c). 

moved through the water at a rate of one meter per minute. Individuals appeared 
to move in the direction of the tentacular crown, although this requires verification. 
Participation of the tentacles in swimming appeared insignificant. Hoshiai (1963) 
observed that Labidopla.v turns its anal end in the direction of movement. Like 
the swimming young of Leptosynapta (Costello, 1946) and Labidoplax, captured 
adult Leptosynapta did not void an appreciable amount of fecal material. Three 
specimens dissected open showed no signs of recent gonadal activity. 

All netted specimens were transported to the laboratory and maintained in an 
aquarium with running sea water. To induce swimming the same animals were 
treated in the following ways: (a) maintained in natural and total darkness, (b) 
subjected to sudden changes in light intensity (the most severe of these involved 
transfer from a dark room to direct sunlight), (c) dropped through a four-foot 
column of water, (cl) subjected to a sudden increase or decrease of water tempera- 

4 This area has not been re-examined since the construction of a nearby breakwater and 
small craft shelter. 



122 PETER \V. GLYNN 

ture (14 4 C.I. (e) subjected to electric shocks (three graded scries of voltages 
at 1, 10 and 100. each deli\ere<l al !re<|uencies of 1, 10 and 100 per second), (f) 
sul)jectecl to a sudden increase in concentration of acid (HC1) or base (NaOH) ; 
two to three drops of IS N HC1 and a saturated solution of NaOH (ca. 30 N) 
were introduced separately into one end of a small pan containing the cucumber. 
An attempt \vas always made to keep deleterious stimuli at a sublethal level. None 
of the above treatments elicited a swimming response. In most cases the cu- 
cumbers tended only to avoid the stimuli by contracting the body and tentacles. 

Since swimming movements have been reported for three species of apoclid 
holothurians, Syiniptnla hydriforwis, a readily available species belonging to the 
same group, was also investigated. Stimuli identical to those enumerated above 
were administered to several individuals of Syuaf>tula. In addition, a fresh slime 
preparation from the skin of the trunk-fish, Lactophrys bicaudalis. was introduced 
into an aquarium containing several individuals of Synaptula. The sea water tem- 
perature during the experiment was 28 4 C. As in Lef>tosyna[>ta, none of these 
treatments prompted a swimming or otherwise active response. The reactions 
were similar to those observed by Olmsted ( 1 ( '17). involving muscular contraction 
of various parts of the body. 

DISCUSSION 

It is of interest to contrast the habitat of Asticliopits in Puerto Rico with that 
portion of the Campeche Bank in the Gulf of Mexico where the species also occurs 
in abundance. Numerous specimens are commonly encountered in the shrimp 
grounds on the Campeche Bank, from a few fathoms down to a depth of 20 m. 
The location of the shrimp grounds in this region (Hedgpeth, 1954, p. 206). when 
compared with Lynch's (1954. p. 79) map of the sedimentary provinces, shows 
that the bottom deposits are essentiallv mudd\ . The usuallv muddv and estuarine 

/i* - 

conditions where shrimps are found along the Gulf coastal states, including a high 
organic matter content, contrast notably with the environment of the north coast 
of Puerto Rico. However, there is evidence that the faunal composition of t In- 
more southern shrimping grounds is decidedly different from that in the northern 
Gulf (Hedgpeth. 1 ( >5.}. 1954), perhaps reflecting significantly different physical 
and chemical properties of the environment as well. Kornicker ct al. (1959), who 
compiled a list of the biota of Alacran Reef, located near the center of the Campeche 
Bank and away from muddy deposits, did not report the presence of Astichopus 
or the seagrass, Halophila. 

The relationship between grain si/.e and the distribution of benthic feeding 
types suggests a further complication in an attempt to delineate the set of environ- 
mental factors favorable to the species. Sanders' ( T>5S ) findings, that clay is a 
good sediment correlate in the distribution of deposit-feeding organisms, would 
seem to apply where .Isticliopns occurs in very line sediments on the Campeche 
Bank. However, Me \ulty ct al. (1962), in agreement with the present findings, 
observed that deposit feeders were most abundant in sediments with a median grain 
size of about 0.25 mm. < >n the other band, individuals of Asticliopus with a dry 
body weight (excluding ingested materials i of the order of 100 gm. do not lie on 
the curve relating body si/.e to grain si/e proposed by McXulty and co-workers for 
deposit feeders. According to their findings, which show a linear relationship be- 



ACTIVE HOLOTHURIAN MOVEMENTS 123 

tween grain size and the cube root of dry tissue weight, one would expect AsticJwpus 
to occur in sediments with a median grain diameter in excess of 0.8 mm. The 
delicate and highly dendritic structure of the tentacles and the way these are em- 
ployed in ingesting fine sediments indicate that feeding may be more efficiently exe- 
cuted on fine-grained substrata. 

Deichmann (personal communication) has observed that many species of cu- 
cumbers go shorewards during the breeding time. However, the four individuals 
of Astichopus found in shallow water in La Parguera did not have the gonads in 
an active condition. Possible breeding in the late autumn or early winter is sug- 
gested by the occurrence of a 5-mm. AsticJwpus in shallow water in the Bahamas 
in May (found by C. Fernandez Mosher). La Parguera specimens were collected 
in the winter, summer and autumn seasons. The present scanty records indicate 
that the occurrence of Astichopus at shallow depths is not associated with breeding. 

The walking, bounding, rolling and erect exploratory movements of Astichopus 
represent newly-described activities for the benthic Holothuroidea. Even the 
relatively slow crawl of this highly active species is rapid compared with other 
forms. The mode of crawling in Parastichopus pari'hncnsis (Parker, 1921) re- 
sembles very closely that in Astichopus. but individuals of nearly equal size pro- 
gressed at a rate of only 1 m./15 min., or 0.07 m./min., equivalent to one-fourth 
to one-half the speed in Astichopus. Peristaltic waves pass along the bodies of the 
two species at the same rate, viz. at mean times of 63 seconds in P. parr'nncnsis and 
65 seconds in AsticJwpus. In Parastichopus one peristaltic wave at a time passes 
over the body ; in Astichopus a second cycle begins before the first has ended. 

The swimming movements of the species Bathyplotcs natans were described by 
M. Sars (Hansen and Madsen, 1956) as similar to those of swimming leeches. 
The snake-like bends of the body occurred in the dorso-ventral plane, not side-wise. 
Assuming that the progressive peristaltic waves were initiated from behind in 
Bathyplotcs (in leeches the waves pass back along the body from the head) it is 
possible that the swimming observed in Astichopus by Cutress is very similar to 
that reported by Sars. 

A speed of nearly 2 m./min. attained by Astichopus when bounding, approaches 
the greatest velocities observed among echinoderms, viz. comatulicl crinoids, 5 
m./min.; Ccntrostcphanus longispinus (echinoid), 2.1 m./min.; Crossaster papposus 
(asteroid), 2 m./min.; ophiuroids, 1.8 m./min. (Hyman, 1955). Prosobranch 
gastropods, which, like sea cucumbers, progress by means of peristaltic waves, are 
considerably slower; one rapid crawler, Thais nistica, attained a maximum velocity 
of 0.3 m./min. (Coomans, 1961). 

The fast movements performed by Astichopus may actually facilitate a more 
rapid adjustment to certain adverse conditions encountered naturally. As pointed 
out earlier, a rolling movement is used by the cucumber for stabilization in strong 
currents. The method by which a bounding movement could help Astichopus 
avoid conditions of high temperature stress is suggested by the following possible 
circumstances. Reactions to light and gravity indicate that Astichopus is capable 
of moving readily into shallower or deeper water. Also, this species is very 
sensitive to a sudden change in the sea water temperature; such a change will 
immediately evoke a bounding response. Protected, shallow bodies of water heat 
up considerably during mid-day low water phases of the tide in the summer. For 



124 i>|. r|.;u' ,\ . CI.VNN 



example, on one occasion in La I'arguera a temperature difference of 8 C. was 
observed between shoal water on the Ice side of a reef (36 C.) and the surf zone 
to windward (28 C. ) . A cucumber wandering into such heated shallows could 
possibly escape the high temperature if the bounding movements were executed 
and properly directed. Abrupt changes in temperature are probably not so fre- 
quently experienced where .-Isticliopns occurs at greater depths. Bathythermo- 
graph curves, obtained from stations located slightly less than one mile west of 
I'ta. Borinquen, show that a distinct thermocline occurs as deep as 90-120 in., with 
a temperature gradient of .V C'. over about 7 m. (Gilbert Hane, personal com- 
munication ) . 

Bounding sometimes resulted under experimental conditions when animals were 
suddenly subjected to reduced salinities and oxygen-deficient water. Although 
ample data are not available on the patterns of salinity and oxygen distributions 
around the river mouths on the north coast, aerial views show turbid river dis- 
charge extending seaward 2-3 miles and as streaks up to 5 miles along the coast. 
The effects of such conditions on populations of Astichopus will be the subject of a 
future investigation. 

Aside from an exploratory type of behavior, observed on only one occasion 
in captivity (in the presence of two other animals), active movements are not 
elicited in .Ist/clwpus through association with members of its own species. Ex- 
posure to the juices of a possible predator ( Lactophrys bicaudalis) also failed to 
arouse the cucumber. Sund (1^58) likewise could not demonstrate that certain 
supposed predatory starfishes were responsible for quick swimming movements 
performed by the actinian. Stoniplihi coccinca. 

Water in which .-Isticliopus had performed movements did not have a toxic 
effect on other animals, thus showing that increased activity does not cause the re- 
lease of a toxic substance. The coelomic fluid caused death in a species of brittle 
star, tunicate and fish at a concentration of 1000 ppm. However, the likelihood 
that body fluids are released naturally does not seem great, since Astichopus is not 
inclined to eviscerate even under extreme irritation. Alcohol extracts of the body 
wall proved to be more toxic than the coelomic fluids; five of the seven species 
tested perished at a concentration of 1000 ppm. Yamanouchi ( 1955) concluded that 
the poisons contained in holotlmrians apparently have little ecological significance. 
Though the toxic principle is confined largely to the tissues of the cucumber, a pos- 
sible role of influencing the activities of other animals through diffusion of trace 
amounts into the surrounding water, for example in averting potential predators, 
cannot be dismissed. 

C'ostello's (1'Mu) description of nocturnal swimming bv the young of Lcpto- 
\yiHipta has dispelled the common notion that this species passes its entire post- 
planktonic life completely buried in soft bottoms (Hyman, 1^55, p. 209). Moreover, 
the undulatory swimming movements performed by adult I.cptosvinipta show that 
Costello's suggestion is not true, namely that (1'Mn, p. ( >5 ) ". . . Lcptosvinipta 
>wims only during a limited period of Us voting adulthood." The scissor-like bodv 
flexures in the swimming young are entirely different from the sinusoidal undula- 
tions which move across the entire length of the body in adults. In addition, the 
movements of the young were described by Costello as more or less aimless, with 



ACTIVE HOLOTHURIAN MOVEMENTS 125 

a velocity of 5-6 cm./min.. whereas in adults swimming was directed and occurred 
at a velocity of about 1 in./min. 

Specimens of Labidoplax dnbia, like aduli Leptosynapta species, also swim in 
an undulatory manner (Hoshiai, 1963). Curiously, though, this species holds 
its anal end highest and toward the direction in which it is moving. Labidopla.r 
swims from early June to late July during any lunar phase, but only after darkness; 
swimming begins one hour after sunset and ends one hour before sunrise. By 
inducing several individuals to swim in the dark during the day, Hoshiai clearly 
substantiated Costello's idea that swimming in synaptids is a dark-conditioned 
phenomenon. Darkness and a variety of other experimental procedures did not 
elicit swimming in adult Leptosynapta. It does not seem likely that the swimming 
behavior is related to spawning, since the gonads showed no signs of being in a ripe 
condition or recently spent. Furthermore, Runnstrom observed that Lcptos\naphi 
albicans erects itself only part way out of the burrow when spawning (Hyman, 
1955, p. 176). In the light of present knowledge no definite statement can be made 
about the stimulus that evokes swimming in synaptids or the possible benefit received. 

SUMMARY 

1. Aspects of the biology of the aspidochirotid, Astichopus nnt/tifidus and the 
apodid, Lcptosyuapta albicans, studied in Puerto Rico and California, respectively, 
were investigated in relation to the active movements performed by these species. 

2. Astichopus is present in greatest abundance between 10 and 40 m. of depth 
on the northwestern coast of Puerto Rico. It often occurs in or near beds of the 
marine phanerogam, Halophila baillonis. Sandy beaches, cemented dunes, and 
beach rock, exposed to the heavy seas of the Atlantic Ocean, make up the shoreline 
of this region. Numerous large rivers loaded with terrigenous materials dis- 
charge on the north shore. The sediment on which Astichopus lives is compara- 
tively fine-grained (median diameter 0.212 mm.) and well sorted (o-^l.l); 
the terrigenous component is high (62.5%), calcareous bioclastic materials occur 
in substantial amounts (36.8%), and the organic matter content is low (0.7%). 
A smaller number of AsticJiopus has been collected from shallow water ( 1-3 m.) 
in the winter, summer and autumn at La Parguera on the south coast. 

3. In addition to a comparatively fast crawl, forward progression in Astichopus 
is executed by rapid walking and bounding movements, which in the latter case may 
approach a rate of 2 m./min. Rolling and exploratory movements are also per- 
formed by Astichopus. 

4. Mechanical stimulation usually causes Astichopus to contract for 1-2 minutes. 
Walking, bounding and rolling movements are elicited by sudden changes in the 
temperature of the water, of the order of 3-4 C. A positive phototactic response 
occurs at low light intensities (5 foot-candles); photota.xis increases at higher 
light intensities, but no definite negative response is apparent. A strong positive 
attraction to red, green, blue and violet light is evident at a low intensity of 10 
foot-candles. No clear tendencies were noted in geotactic or thigmotactic behavior. 
Bounding movements sometimes occurred when cucumbers were suddenly subjected 
to diluted sea water and oxygen-deficient water. Active movements were not 
evoked through the association of Astichopus with members of its own species or 
in the presence of other animals. 



126 PETHR W. GI.YXX 

5. Coelomic fluids and alcohol extracts of the body wall of Asticliofns are toxic 
to a variety of marine animals at concentrations of 1000-5000 ppm. It does not 
seem likely that a poison is released by the animal naturally, since the water in 
\vhich active movements are performed is non-toxic and evisceration occurs only 
rarely. 

6. Sinusoidal swimming movements wen- observed in adult Leptosynapta on 
three different occasions near the surface at night. Specimens subjected to a 
variety of experimental conditions in captivity failed to elicit the swimming re- 
sponse. Svnaptula Jivdrijonnis. a related West Indian species, did not swim either 
when subjected to diverse stimuli as with Leptosynapta or when exposed to the 
juices of a presumed predatory trunk-fish. 

LITERATURE CITED 

AI.MY, C. C., JR., AND C. C ARMH'X-ToRRES, 1963. Shallow-water stony corals of Puerto Rico. 

Caribbean J. Sci., 3: 133-162. 
. \RXOW, T.. AND D. B. BOGART, 1960. Water problems of Puerto Rico and a program of 

water-resources investigations. Trans. Second Caribbean Geol. Conf., pp. 120-129. 
CLARK, H. L., 1907. The apodous holothurians. A monograph of the Synaptidae and 

Molpadiidae. Smithsonian Contrib. Knowlcdfic, 35: 1-231. 
CLARK, H. L., 1933. A handbook of the littoral echinoderms of Porto Rico and the other West 

Indian islands. Sci. Surrey Porto Rico ]'iri/in Islands, 16: 1-147. 
COOMANS, 11. H., 1961. Experiments on the velocity of marine gastropods. Ann. Rept. Am. 

Malacol. Union, pp. 6-7 (abstract). 

COSTELLO, D. P., 1946. The swimming of Lcptosynapta. Biol. Bull., 90: 93-96. 
CROZIER, W. b, 1915. The sensory reactions of Holothnria siiriiiaiiiensis Ludwig. Zool. Jahrb. 

1'hyswl, 35:233-297. 
CUENOT, L., 1948. Anatomic, ethologie et systematique des echinodermes. Pp. 3-272. In: 

Pierre-P. Grasse (ed.), Traite de Zoologie. Echinodermes, Stomocordes & Procordes, 

Tome XL Masson et Cie., Paris, France. 

DEICHMANN, E., 1926. Report on the holothurians collected by the Barbados-Antigua Expedi- 
tion from the University of Iowa. I'nh. Iowa Studies Nat. Hist., 11: 9-31. 
DEICHMANN, E., 1954. The holothurians of the Gulf of Mexico. Pp. 381-410. In: P. S. 

Gait so ff (coord.), Gulf of Mexico, it* origin, waters, and marine life. Fisli. Bull. 89. 
DEICHMANN, E., 1963. Shallow water holothurians known from the Caribbean waters. Studies 

I'tiuna Curasao Other Caribbean Is., 14: 100-118. 

FISHER, W. K., 1954. A swimming Sipitncnlns. Ann. Man. -V<//. I fist., Ser. 12. 7: 238-240. 
FOXTAIXK, A., 1953. The shallow water ecliinodenns of Jamaica. Part IV. The sea-cucumbers 

(Class Holothurioidea) . Xat. Hist. Xotes (mimeo.), Nat. Hist. Soc. Jamaica, pp. 

29-33. 
GILCHRIST, J. I). F., V>2(1 rianktitthuria diaphana. (/en. et .v/>. ;/. Ouart. J . Micr. Sci., 64: 

373-382. 
GLYNX, P. \\'., L. R. .\i.\iom >YAK AND J. (i. GONZALEZ, 1%4. Fffects of hurricane Edith on 

marine life in La I'arguera, I'uerto Rico. Ctiril>hcan J. Sci.. 4: 335-345. 
(im.Lou, R. B., AXD |. I. (ii.Ass, 1 ( ;57. .\ reconnaissance study of the beach sands of I'uerto 

Rico. Geol. Survey Bull. 1042-1, pp. 273-305. 
I IAN SEN, B., AND F. J. MAIISKX, 1956. On two bathypelagic holothurians from the South 

China Sea, (Jalatheathuria n. g. asf'cra ( Therl ) and l-.n\puiastes </lohosa n. s]i. 

(nilathea Kept.. 2: 55-59. 
I II.IM.IM-.TII, J. YV., 1953. An introduction to the xoogeograiiliy of the nortlm estern Gulf of 

Mrxico with referi-ncc to the invertebrate fauna. I'uhl. fust. Mar. Sci.. 3: 107-224. 
HEDGPI rn, J. W., 1954. Bottom communities of the Gulf oi Mexico. T].. 203-214. In: P. S. 

Galtsoff (coord.), Gulf of Mexico, its origin, waters, and marine life, l-'isli. />//. 89. 
Ho in \T, T., 1963. Some olisci'vations on the swimming of Labidopla.v dnbia (Semper). 

null. Mar. Itwl Sta. Asamushi, 11: U>7 170. 



ACTIVE HOLOTHURIAN MOVEMENTS 127 

HYMAN, L. H., 1955. The Invertebrates. IV: Echinodcrmata. McGraw-Hill Book Co., Inc., 

N. Y., pp. 1-763. 
INMAN, D. L., 1952. Measures for describing the size distribution of sediments. /. Scd. Petrol., 

22: 125-145. 
KAYE, C. A., 1959. Shoreline features and Quaternary shoreline changes, Puerto Rico. Geol. 

Survey Prof. Paper 317-B, pp. 49-140. 
KORNICKER, L. S., F. BONET, R. CANN AND C. M. HosKiN, 1959. Alacraii Reef, Campeche 

Bank, Mexico. Pitbl. Inst. Mar. Sci., 6: 1-22. 
LTJDWIG, H., 1889-92. Die Seewalzen. Pp. 1-460. /;/: H. G. Bronn (ed.), Klassen und 

Ordnungen des Tierreichs. Echinodermen. 
LYNCH, S. A., 1954. Geology of the Gulf of Mexico. Pp. 67-86. In: P. S. Galtsoff (coord.), 

Gulf of Mexico, its origin, waters, and marine life. Fisli. Bull., 89. 
McXuLTY, J. K., R. C. WORK AND H. B. MOORE, 1962. Some relationships between the infauna 

of the level bottom and the sediment in south Florida. Bull. Mar. Sci. Gulf Caribbean, 

12:322-332. 
NIGRELLI, R. F., 1952. The effects of holothurin on fish, and mice with sarcoma 180. Zoologica, 

37 : 89-90. 
NIGRELLI, R. F., AND S. JAKOWSKA, 1960. Effects of holothurin, a steroid saponin from the 

Bahamian sea cucumber (Actinopyga agassisi), on various biological systems. Ann. 

New York Acad. Sci., 90: 884-892." ' 
NIGRELLI, R. F., J. D. CHANLEY, S. K. KOHN AND H. SOBOTKA, 1955. The chemical nature of 

holothurin, a toxic principle from the sea-cucumber (Echinodermata : Holothurioidea). 

Zoologica, 40: 47-48. 

NUTTING, C. C., 1919. Barbados-Antigua Expedition. Unii'. Iowa Studies \'at. Hist., 8: 1-274. 
OLMSTED, J. M. D., 1917. The comparative physiology of S\uaf>tula hydriformis (Lesueur). 

/. Exp. Zool, 24: 333-379. 
PARKER, G. H., 1921. The locomotion of the holothurian Stichopus panimensis [sic] Clark. 

/. Exp. Zool,, 33: 205-208. 
RANDALL, J. E., 1964. Contributions to the biology of the queen conch, Stroinlnis gigas. 

Bull. Mar. Sci. Gulf Caribbean, 14: 246-295. 
SANDERS, H. L., 1958. Benthic studies in Buzzards Bay. I. Animal-sediment relationships. 

Liiiinol. Occanog., 3: 245-258. 
SARS, M., 1868. Om nogle Echinodermer og Coelenterater fra Lofoten. Vidensk. Selsk. 

ForhandL, 1867: 1-7. 
STEVENSON, R. E., AND K. O. EMERY, 1958. Marshlands at Newport Bay, California. Allan 

Hancock Found. Pub., Occ. Pap., (20) : 1-109. 
SUND, P. N., 1958. A study of the muscular anatomy and swimming behaviour of the sea 

anemone, Stomphia coccinea. Quart. J. Micr. Sci., 99: 401-420. 
YAMANOUCHI, T., 1955. On the poisonous substance contained in holothurians. Publ. Seto 

Mar. Biol. Lab., 4: 184-202. 



THE BIOI.i HiY <>! \SUMIA \HiRA (SAVIGNY). [II. THE ANNUAL 

PATTERN OF COLONIZATION 1 

[VAN GOODBODY 

lh-f>(irtmcnt of '/.oniony, I'nhrrsity of the U'cst Indies, .fniiniicti 

In the first paper in this series (Goodhody, 1962) an account was given of the 
development and survival of a single ])opulation of ascidians at Tort Royal. 
Jamaica. In October. 1 ( '5 () . a second experiment was initiated with the object of 
comparing populations which settled and commenced life at different seasons of 
the year. The present paper deals solely with aspects of colonization and supports 
the view previouslv expressed that Ascidia n'ujra is a primary colonizer in the 
sessile community and only succeeds with difficulty in developing in the later stages 
of community succession. A subsequent paper will deal with survival in the same 
population. 

METHODS 

The methods used for obtaining populations were exactly the same as described 
previously (Goodbody, 1 ( ^>2). Panels of Tufnol with an available settlement area 
of 216 sq. in. on each side were suspended from rafts at the same locality and in 
the same manner as before; natural sessile communities, which included popula- 
tions of Ascidia ni</ni, were allowed to develop on the panels, which were inspected 
at monthly intervals. Altogether six separate populations were set up in this 
way and were designated A. B, C, D, E and F. Four panels were used for each 
population, two sited at four feet and two at eight feet below the sea surface. The 
populations were set up so that thev commenced life at intervals of two months over 
a period of a year; this was accomplished bv immersing the sets of panels at suc- 
cessive intervals as follows: 

Population l>aic<>l panel immersion 

A 1st October 1959 

15 1st DrrcmlxT 1'J.S'J 

C 1st I'Ybruury I960 

I) 1st April 1060 

K M June 1960 

!' Is1 August 1960 

After immersion even panel was examined and photographed at monthly intervals 
and a permanent record thus obtained of the fate of every individual ascidian. 
l\econU of settlement continued until March, 1 ( >(>2, and of survival until October, 
1 (| '2. Copulation C suffered damage and handling lo.ss in the winter of 1960/61 
and observations on it were abandoned alter January, 19(>1. 

' Sii]>i>i irtcd in part l>y a tyrant from tin- Xul'lirld Foumlatii ,n. 

128 



BIOLOGY OF ASCI DI A \K,k \ 



129 



THE PATTKRN OF COLONIZATION 

Table I shows the number of new colonizers recorded in each population in 
each month throughout its history. It should be noted, however, that no counts 
were made in the first month for populations A, B and F, so that in each of these the 
figure given for the second month is the grand total of new colonizers in the first 
two months after immersion. Furthermore it was not possible to make any ob- 
servations in November, 1960, so that the figures for December are totals for the 
period October to December. 

These data reveal several points concerning the ecology of +1. nic/ra. First, 
it will be seen that with the exception of population E (immersion date June, 1960) 
all the populations had dense colonization during the first two months after im- 
mersion but very few colonizers in subsequent months. In the earlier paper the 
same phenomenon was observed but it had to be assumed that the fall-off in the 
number of new colonizers was due to some form of competition and was not due 

TABLE I 

The number of new individuals appearing in each month within different populations of 
Ascidia nigra. Months given in parentheses indicate the month in which the panels 

were first immersed 



Month 


Population 


A 
(Oct., 1959) 


B 
(Dec., 1959) 


c 

(Feb., 1960) 


D 
(April, 1960) 


p 1 

(June, 1960) 


P 

(Aug., 1960) 


Dec., 1959 


147 












Jan., 1960 


10 












Feb. 


2 


463 










Mar. 





4 


199 








April 


1 


4 


48 








May 


1 


2 


7 


415 






June 


1 


3 


1 


101 






July 


1 


1 





5 


74 




Aug. 


6 








2 


12 




Sept. 


1 





13 


2 







Oct. 


6 





33 








187 


X ov. 




















Dec. 


8 


2 


37 


35 


80 


31 


Jan., 1961 


2 


11 


6 


5 


34 


4 


Feb. 


2 


2 




2 


16 


6 


Mar. 


1 


1 




4 


1 


2 


April 





2 




1 


6 


8 


May 










30 


2 


5 


June 










8 


3 


1 


July 










3 








Aug. 





2 




1 


4 


3 


Sept. 


1 


3 







7 


2 


Oct. 


4 


7 




5 


6 





Nov. 


4 


7 




9 


9 


9 


Dec. 


7 


5 




10 


3 


8 


Jan., 1962 


1 


8 




6 


12 


1 


Feb. 


2 


1 




3 


1 


2 


Mar. 










3 









IV \\ . .. 10DBODY 

to the absence of larvae for settlement. The present data prove this assumption 
'to have been correct. The fact that larvae were continually available for new 
colonization throughout the year is shown by the high density of colonizers occurring 
on the newly immersed panels of successive populations and yet, while these new 
settlements were occurring, the older populations were- receiving a negligible number 
of new colonizers. 

If we accept this view, that competition is responsible for the decline in new 
colonizers after the second month, we must enquire what form the competition takes 
and whether it functions by preventing new settlement or preventing growth of 
animals after metamorphosis. The development and succession in these com- 
munities have been briefly outlined elsewhere (Goodbody. 1963c) and will be dis- 
cussed in detail in a later paper. The important aspects of this development are 
that an earl\ Balanus/Enterovnorpha community, in which A. nu/ra colonizes, is 
rapidly overgrown by the colonial ascidian, Iiidcninum concliyliatniii (Sluiter), 
so that this ascidian and A. nit/ra become temporary dominants in the second serai 
stage. After a period of 7 to 12 months the Dideinintin fragments and retreats, and 
sponges, lamellibranchs and stolidobranch ascidians become climax dominants. The 
rapid decline in colonizers after the second month is almost certainly due to the 
rapid spread of l>. conchyliatum and, to a lesser extent, other colonial ascidians. 
These colonial ascidians overgrow and smother the smallest-sized young Ascidia 
iiit/ra ( Goodbody j l ( '(>3a) and also, by covering all available surface of the panel, 
effectively prevent any further settlement. Competition at this stage is therefore 
for space and not for food, those individuals of A. nigra which have successfully 
gained a foothold continuing to grow rapidly in association with the D. conchy- 
liatum. 

In the later stages of serai succession the problem becomes more complex and 
difficult to unravel. With certain exceptions noted below fresh colonization in the 
community continues at a low level even though the sheets of D. concJiyliatuin 
fragment and disappear. A new form of competition now arises from the climax 
dominants (ride supra \ arising in the community. 

I hope to show in later papers that the decline in the adult population of A. 
ni(/ra is associated with the increasing dominance of these climactics and I believe 
that this is largely a question of competition for food from the more efficient 
stolidobranchs and sponges, while chemical effects from sponges may also be a 
contributors factor I < ioodbody. 1'Hdb). Support for the contention that competi- 
tion for food is important at this stage i> given by the observation that at all stages 
of growth .-/. nit/ni maintains its siphons projecting beyond the rest of the com- 
munity and usually dies when this is no longer possible. This competition for food 
will also affect young .settlers unless they establish themselves in peripheral portions 
of the community. Competition tor .space is of course also of great importance at 
this stage, as almost all available surfaces are now occupied. I lowever, as the climax 
is approached the weight of material in the community increases and sloughing of 
small areas of the community occasionally takes place, exposing bare areas of panel. 
Sloughing of this sort is natural in old sessile communities ( McDougall, 1943; 
I loodbody. l>f>3b) and effectively provides renewed conditions for serai succes- 
sion. Bowever, in the communities under study it was noted that unless the 
bared area mounted to about 30 sq. in. (which was seldom the case) no new 



BTOLOCV OF ASCII) I. \ XIGRA 



131 



TAHI.E II 

The number of new Ascidia nigra appearing each month in all populations, in relation 
to the number of panels exposed. For details see text 



Month 


No. exposed 
panel sides 


No. new 
colonizers 


No. colonizers per 
exposed ijam-1 -idr 


Jan., 1960 


8 


10 


1.25 


Feb. 


8 


2 


0.25 


Mar. 


16 


4 


0.25 


April 


16 


5 


0.31 


May 


16 


3 


0.19 


June 


16 


4 


0.25 


July 


24 


7 


0.29 


Aug. 


24 


8 


0.33 


Sept. 


32 


3 


0.09 


Oct. 


32 


6 


0.18 


Nov. 


40 


78 


1.95 


Dec. 


40 


78 


1.95 


Jan., 1961 


40 


56 


1.40 


Feb. 


40 


28 


0.70 


Mar. 


40 


9 


0.22 


April 


40 


17 


0.42 


May 


32 


11 


0.34 


June 


40 


12 


0.30 


July 


40 


3 


0.07 


Aug. 


40 


10 


0.25 


Sept. 


40 


13 


0.32 


Oct. 


40 


22 


0.55 


Nov. 


40 


38 


0.95 


Dec. 


40 


33 


0.82 


Jan., 1962 


40 


28 


0.70 


Feb. 


40 


9 


0.22 


Mar. 


40 


3 


0.07 



primary colonization occurred and the space was rapidly overgrown by adjacent 
sponges. Thus, we find that in these later stages of serai succession competition 
for both space and food, coupled with possible substances secreted by sponges, all 
combine to inhibit further immigration into the population of A. nujra. 

Examination of Table I shows that in the months of November to January 
there is an increase in the number of new immigrants into the population, particu- 
larly in the first winter, 1960/61. This is best illustrated by expressing the data 
as the number of new immigrants per panel side exposed. In such an analysis the 
newly immersed panels must be excluded and hence only data from panels im- 
mersed for three months or longer are used. Data from population C are not used 
at all as the population suffered damage from an early stage. In May, 1 ( >(1. data 
from population D are excluded as we know that the increased rate of colonization 
there was due to exceptional sloughing of the community. The remaining data are 
analyzed in Table II and Figure 1 and clearly illustrate the increased immigration 
rate in mid-winter. I have shown elsewhere (Goodbody, 1961a) that Ascidia 
nii/ra breeds throughout the year but has a maximum of reproductive activity and 
larval settlement in the winter months, and it seems tolerably certain that the in- 
crease in immigration into the population in November to January is the result of 



132 



IVAN - 'I. BODY 



a massive increase in tin- number of larvae available, with a consequent increase 
in the number of ascidians successfully colonizing in the populations. 

It will be noted that population K differs in two respects from other popula- 
tions. First the initial coloni/ation by SO ascidians in the first two months \vas 
small, and second there was a large additional colonization of 114 animals in No- 
vember January, 1960/61. The panels on which this population developed were 
first immersed in June. 1 ( '6(), and the small initial settlement is in keeping with 
earlier findings ((ioodbodv. 1'^da) that the intensity of larval settlement is minimal 
at this time of the year. The high level of secondary colonization is due to the com- 
bined effects of the mid-winter abundance of larvae and an early retreat and frag- 
mentation of tlu' fiidciiinuiii community. It is usual in these communities for the 
/ >!(lciiiiniiii to be replaced by sponges immediately but, for reasons which cannot at 
present be explained, the / lidcin mini associated with population E retreated rather 
suddenly in November, 1 () (>0. and was not immediately replaced by sponge. As it 
fragmented and retreated it left fresh surfaces available for colonization by A. nigra 
which established itself before the I tidcin mini grew back or was replaced. This 
exceptional circumstance might be considered seriously to bias the figures given in 
Table II and Figure 1. so that it is necessary to point out that the total picture is un- 
affected by the data from population E. If we re-analyze the data in Table II for 
.November and December, 1 ( >6(). and January, 1961, excluding the data for popula- 
tion E, we arrive at the following figures for the number of colonizers per exposed 
panel side: November, 1.19; December. 1.19; January, 0.7. These figures still in- 



2-Ch 




05- 



J FMAMJ JASONDJFMAMJJ ASONDJ FM 
I960 1961 1962 

'KK 1. C'lilonixation l>y . l.v< /'<//</ ni</ni in eaeh month, expressed as the numliiT <>f iu'\v 
individuals appearing per panel sid< exposed. For fnrilu-r details M-e text and Table II. 



BIOLOGY OF ASCIDIA NIGR \ 133 

dicate a higher rate of immigration in the mid-winter months than at other times 
of year. 

In conclusion the data presented in this paper demonstrate beyond doubt that 
Ascidia nigra is a primary colonizer in the sessile community in Jamaica. Settle- 
ment occurs on clean surfaces at a time when barnacles and filamentous algae are 
also colonizing. Subsequently a rapid development of the colonial ascidian, 
Didcniniun conchy liatum, overgrows the panel surface, succeeding the barnacl<-~ 
and algae as the dominant organism in the community. Very small specimens of 
A. nigra are smothered by this growth (Goodbody, 1963a) which also inhibits any 
further settlement of the solitary species. Individuals of A. nii/ra of more than 
about two weeks old can survive the overgrowth and continue to grow in association 
with the Didciiinitin, so that at this stage it is a competitor for space and not for 
food which controls further immigration into the population. Later in the develop- 
ment of the community the Didciiinitiii is replaced by sponges, lamellibranchs and 
stolidobranch ascidians, all of which may effectively compete for food with A. nigra, 
Thus, in the climax community inter-specific competition for food and space is re- 
sponsible for preventing further colonization by A. nigra. A few specimens of A. 
nigra, however, succeed in colonizing at all stages of serai succession. This mav 
occur either when sloughing of the community provides new surfaces for coloniza- 
tion or when larvae settle in peripheral positions in the community where competi- 
tion for food is less intense. 

SUMMARY 

1. Artificial panels were used to allow six populations of Ascidia nigra to de- 
velop and grow naturally. The six populations started life at two-month intervals 
over a period of a year. 

2. With the exception of a population starting life in June, all populations had 
dense colonization in the first two months after immersion of the panels, but few 
new colonizers in subsequent months. 

3. The paucity of new colonizers after initial colonization is due to competition 
from other sessile organisms, first for space and later for food. 

4. In the months November to December there is a marked increase in the num- 
ber of new colonizers, irrespective of the age of the population. This is due to a 
large increase in the number of larvae available for settlement. 

LITERATURE CITED 

GOODBODY, I., 1961a. Continuous breeding in three species of tropical ascidians. Proc. Zool. 

Soc. London, 136: 403-409. 
GOODBODY, I., 1961b. Inhibition of development of a marine sessile community, \\itnrc, 190: 

282-283. 
GOODBODY, I., 1962. The biology of Ascidia ni</ni (Savigny). I. Survival and mortality in an 

adult population. Bio!. Bull., 122: 40-51. 
GOODBODY, I., 1963a. The biology of Ascidia nnjra (Savigny). II. The development and 

survival of young ascidians. Binl. Bull.. 124: 31-44. 
GOODBODY, I., 1963b. Population studies on a tropical ascidian. /'roc. XI 'I. Intermit. Coni/r. 

Zoo!., 1: 113. 
GOODBODY, I., 1963c. The development of a tropical marine sessile community. Bull. Ecol. 

Soc. Aincr., 44: 92. 
McDoucALL, K. D., 1943. Sessile marine invertebrates at Beaufort, North Carolina. Ecol. 

Monogr., 13: 321-374. 



STUDIES ON SPERMATHECAL FILLING IN AEDES 
AKGYPTI (LINNAEUS). I. DESCRIPTION 1 

JACK COLVARD JONES AND RONALD E. WHEELER 2 

/ V/ 1 !/'-/;;/!';// of fiiitonioloiiy. (.'nri'crsity of Maryland, College 1'tirk, Maryland 

In a study on the female reproductive system of C'ule.r pipiens, Kulagin (1901) 
illustrated for the first time a flask-shaped sac opening into the vagina, but he 
mistook it for the accessory gland. Not having seen Kulagin's study, Christophers 
(1923) clearly recognized that the accessory gland of mosquitoes \vas a separate 
structure from the dorsal diverticulum of the vagina and he termed this sac the 
('(teens. Brelje (1924), apparently unaware of Christophers' work, pointed out 
Kulagin's error and described and illustrated the connections between the accessory 
gland, bursa copulatrix, spermathecae, and vagina in MocJilony.v, Cttle.r, Citliseia, 
and Acdes. He termed the dorsal diverticulum of the vagina the bursa copulatrix. 
Brelje also discovered that the male mosquito deposited seminal material into this 
sac. Christophers ( 1 ( ">0, p. 67 ( ' ) stated in his monumental work on Acdes acgypti 
that "the coccus ... is a relatively small structure with the characters of a mucus 
gland," thus repeating Kulagin's confusion of two very different organs. In 1957 
Burcham rediscovered both the structure and function of the bursa in Aedcs and 
subsequently located Brelje's paper. Unfortunately Burcham's thesis was not 
published. Not having seen the earlier work, Hodapp ct a!. (1960) again redis- 
covered the structure and function of the bursa of Acdes. Some of this curious 
history was brought to light by Curtin and Jones (1961). 

It has been known for a long time that female mosquitoes store sperm within 
from one to three spherical organs called spermathecae (Dufour, 1851), but it was 
not until 1957 that Hurcham first explored some of the problems of how the sperm 
reach the thecae. He stated that a few sperm reach the storage organs within 
one minute after coitus, and he noted that few to numerous sperm were present 
within them in two minutes. He further remarked (p. 80) that the number 
". . . steadily increased up to about five minutes after coitus" and that "the 
bursa copulatrix was essentially emptied within five minutes after mating." 
Schwartz ( 1 ( ">1 i found that sperm reached the thecae of A. acgvpti between the 
40th and 15(>th seconds after coitus. Spielman (1964), working with the same 
species, reported that sperm do not reach the thecae during the first 30 to 35 
^crouds after coitus but fill the organs in a period between 40 and 300 seconds. 

It is the purpose of this paper to IM\V a more detailed description of spermathecal 
filling in A. uc</ypti (Bangkok strain) than is currently available. A subsequent 
paper will deal with some of the physiological aspects of filling. 

1 This research was sponsored by \.1.H. Oant GM-06021 and by N.I.H. Development 
Award No. K-3-< i.\!-21,52 ( ^ to the first author. scientific article' number A 1180, contribution 
number .Vol of the Maryland Agricultural Experiment Station. 

'hi. Wheeler's address is I !i, ,1, >^ical Research Laboratory, Ortho Division, California 
Chemical Company, Lucas and Ortho Way, Richmond, California. 

L34 



SPERMATHECAL FILLING IN AEDES 135 

MATERIALS AND METHODS 

The mosquitoes were reared in an insectary at 26 to 29 C. with a relative 
humidity ranging from 70% to 80%. The eggs were hatched in freshly boiled tap 
water that had reached room temperature and approximately 100 larvae were 
pipetted into a stender dish containing 250 ml. of water and a small pellet of 
Purina dog chow. When the larvae pupated, the pupae were sexed by placing them 
laterally on an ice cube and examining the pronounced differences in their external 
genitalia. The pupae were pooled in a beaker of water according to sex and 
placed in a one-cubic-foot screened cage so that the sexes would be completely 
separated at emergence. Adults had continuous access to 5% sugar water and 
the majority of the females were not offered a blood meal. Most of the studies 
were made on 3- to 7-day-old virgins. In the great majority of the experiments, 
the mosquitoes were forced to copulate by the technique of McDaniel and Horsfall 
(1957). Wheeler's modification (1962) of this technique proved essential to 
many of the observations required. Adults were generally anesthetized with 
nitrogen just before use. A fast-drying, non-toxic adhesive (Dekadhese) was 
applied to the head of Ward's #1 or #2 insect pin and gently but firmly pressed to 
the dorsal surface of the thorax. A series of males and females were thus arranged. 
The female-bearing pin was inserted into a cork glued to a microscope slide and the 
preparation placed under a dissecting stereo-microscope. The male-bearing pin 
was inserted into an adjustable holder allowing for gross and fine vertical move- 
ments. The male was moved up and down until his terminalium came into contact 
with that of the female at an angle of about 90. 

Dissections were made by grasping the thorax of the mosquito with sharpened 
Dumont #5 microforceps and the terminalium was placed into a small drop of 
buffered Drosophila saline (Ephrussi and Beadle, 1936). One finely sharpened 
needle was used initially to extract the organs to be studied. Further dissections, if 
needed, were made using two needles. Some extractions were made by using 
a second pair of microforceps to pull out the desired organs. Further experimental 
details are given in the text. 

RESULTS 
1. Efficiency of spcnnathccal filling after forced-copulation 

Virgins of the Bangkok strain force-copulated from 4 to 221 seconds and 
averaged 31.3 seconds with a standard error of 1.6 seconds (132 observations). 
In 18 cases which were allowed only one to five seconds of coital contact, only one 
was inseminated. In 8 cases which were allowed 9 to 10 seconds of coital contact, 
five (62^r) were inseminated. In 23 cases which were allowed 15 seconds coital 
contact, 21 (91.3%) were inseminated. 

Out of 737 cases of forced-copulation, the bursae in 8 females were observed 
to have what appeared to be only male accessory gland material and no visible 
spermatozoa. Three of these cases could be accounted for because the male 
used was found to be sterile and had no sperm cells available. Nevertheless, this 
agametic male copulated readily with 6 females in rapid succession. In two 
other cases, a male's freshly isolated terminalium had been used to copulate intact 
females. But three of the cases could not be accounted for, which presumably 



136 



JACK COLVAK1) JONES . \D RONALD E. WHEELER 



indicates that on rare occasions a normal male- can ejaculate only accessory gland 
material. 

Both sperm and male accessor) inland material were present in all hnrsae of 41 
(82%) of 50 female.s which were force copulated and in all hut one, sperm reached 
the spermathecae. < )f these. 41. 5' , had sperm in all three thecae, 48.8% had 
sperm in two thecae. and ( '.7' , had sperm solely in the large median theca. 

During the first 10 minuter alter forced-mating, sperm moved about within the 
thecae of all females examined i 28 cases; 44 thecae with sperm). In those females 
which were dissected 30 minutes to 6 hours after forced-mating, sperm moved in 

TABLE I 

The extent a ml decree of spermathecal filling and moti/ity of stored sperm in Acdes aegypti 
after different periods of unrestrained muting. Ten females used for each period 







Law spermatheca 


Lateral spermatheca 


Lateral spermatheca 


Bursa 


Time 


Mr, ,11 












degree 
























coitus 


tliecal 
filling 


No. 


Degree 


No. with 
motile 


No. 


Degree 


No. with 
motile 


No. 


Degree 


No. with 
motile 


No. 


Condition 










sperm 






sperm 






sperm 






3 hrs. 


2.70 + 


10 


4 + 


3 


10 


3 + 


3 


10 


1 + 


3 


10 


Distended 


6hrs. 


2.6.? + 


10 


4 + 


1 


8 


3 + 


2 


9 


1 + 


2 


10 


Distended 












2 


2 + 


2 


1 


2 + 


1 






24 hrs. 


2.70 + 


10 


4 + 


8 


10 


3 + 


8 


10 


1 + 


10 


10 


1'artially 


























distended* 


48 hrs. 


2.57 + 


9 


4 + 


10 


10 


3 + 


10 


8 


1 + 


8 


3 


Partillly 






1 


3 + 


















distended*' 1 


72 hrs. 


2.70 + 


10 


4 + 


10 


9 


3 + 


10 


6 


1 + 


8 


10 


Empty 












1 


4 + 




2 


2 + 









* Some sperm present in 50' , of Imrsae; undulations of sperm seen in only one hursa. 
** A few sperm seen in one bursa. 

, to 62$ of the thecae containing them (20 females; 56 thecae with sperm). 
T \\entv-four hours after forced-mating, sperm were active in 80' V' of the thecae 
containing them (5 females; 10 thecae with sperm). 

2. Efficiency <>}' spermathecal lillni</ with cage-copulated niostinitoes 

Using Spiclman's technique (1 ( 'M), individual virgin females of the Bangkok 
strain were introduced into a 4 :< () in. lantern chimney containing a number of 
nnmated males, in order to obtain information on the time required for normal 
mating to begin, and to determine the time of coital contact under Iree mating 
conditions. The chimncv was shaken to stimulate flight and copulation. In 5 
cases, 13 to 32 seconds elapsed before copulation occurred (mean of I'). 4 seconds). 
The mating time under these conditions ranged from ( '.5 to \(> seconds, with a mean 
of 13. J seconds. Ten seconds of coital contact under fret- mating conditions usually 
resulted in insemination. The mean coital time of 13.2 seconds is in close agree- 



SPERMATHECAL FILLING IX AEDES 



137 



merit with Spielman's value of 13.7 seconds lor the Johns Hopkin> strain of A. 
acgypti. Using larger cages, Roth (1948) and ISurcham (1957) obtained a mean 
coital time value of about 16 seconds. 

Three-day-old unmated males and females, 10 of each, were placed in each 
of five one-cubic-foot cages, and allowed to copulate freely for 3, 6, 24, 48, and 72 
hours. The females were dissected and the condition of the bursae, the extent 
and degree of filling of the three spermathecae, and the motility of the stored sperm 
were noted. The degree of filling was qualitatively judged as follows : 4 +, 
numerous sperm ; 3 +, many sperm ; 2 +, few to many sperm ; 1 +, very few sperm ; 
and 0, no sperm detectable. The data for the five groups are summarized in 
Table I. Of the 50 females examined, 92% had sperm in all three thecae and 8% 

TABLE II 

Copulatory behavior and potency of a single six-day-old male Aedes aegypti 
when offered 12 virgin fenales in a 20-minute period 



Virgin female number 
in order of presenta- 
tion to the male 


Seconds of coitus 


Notes 


1 


10.2 


Female escaped after coitus 


2 


13.6 


Bursa full; 2 thecae with sperm 


3 


14.2 


Bursa partially filled; 2 thecae with sperm 


4 


22.4 


Bursa partially filled ; 1 theca with sperm 


5 


10.8 


Female dislodged the male; female not inseminated 


6 


68.0 


\ ninseminated 


7 


13.8 


Female dislodged male; female not inseminated 


8 


25.8 


Uninseminated 


9 





Male repeatedly attempted to clasp female's 






terminaliurn but aedeagus did not erect 


10 


22.8 


Uninseminated 


11 





Male repeatedly erected aedeagus but did not establish 






good genital contact; Uninseminated 


12 





Male erected aedeagus but only feebly clasped female's 






cerci and did not establish good genital contact 



had sperm only in two thecae. Numerous sperm were in the large median theca 
in 98% of the cases, 94% had many sperm in one lateral theca. and 84 c /c had a few 
sperm in the other lateral theca. One to 6 hours after free mating, sperm were 
actively moving in 26.7% to 33.3% of the thecae containing them (30 females; 72 
thecae with sperm). After 24 hours, 86.7% of the thecae contained active sperm 
(10 females ; 30 thecae with sperm ) . After 48 and 72 hours, sperm were active in all 
of the thecae containing them (20 females; 56 thecae with sperm). 

It is evident that data from forced-mating of virgins are significantly different 
from data from unrestrained or free mating between virgins. Thus, coitus lasts 
about twice as long with forced-mating, and the extent of insemination and 
thecal filling is more variable with forced than with unrestrained matings. 
Furthermore, sperm within the thecae become active more rapidly following forced- 
mating. The reasons and significance of such differences are not clear. 



138 JACK COLVARD JONES \NTJ RONALD K. WHEELER 

3. Potency of indk'idinil nnilcs and spermathecal filling 

Each of live previously miniated males which were 6 days old were offered 
4 to 12 virgin female- in succession within a 10- to 20-minute period, using the 
forced-copulation technique. These males successfully copulated with 4 to 9 females 
and inseminated 3 to 5 of these. Data on one male which attempted to copulate 
with 12 females in a 20-minute period are shown in Table II. This individual 
was able to establish good genital contact for 10 to 68 seconds with 9 females, at 
least three of which he inseminated. \Yhen this male was subsequently dissected, 
his accessory glands appeared essentially like those of a once-mated male, but his 
seminal vesicles were shrunken and possessed only a few spermatozoa. As il- 
lustrated by the data in Table II, copulation can occur without insemination and 
even prolonged coitus (as with female #6) does not necessarily result in insemi- 
nation. Erection of the male's aedeagus was not necessarily followed by copulation 
(as with females 11 and 12, Table II). In an earlier study (Jones, 1961), it 
was shown that when virgin males are allowed to copulate freely with a great 
excess of females in a cage for one hour, they inseminate about five females and 
their seminal vesicles are usually completely devoid of sperm and their accessory 
glands are greatly reduced in diameter and have little secretory material. This is 
in striking contrast to the repetitively force-mated males which only rarely get rid 
of all the seminal vesicle sperm and apparently ejaculate much less accessory gland 
material into the females. Conceivably, this difference in the amount of accessory 
gland material in the ejaculate could account for some of the differences already 
noted in the last section. 

Two unmated males were presented to 6 virgins each and allowed to force- 
copulate with each one for 15 seconds, and the females were then dissected after 
23 to 69 seconds. The first male inseminated 4 of the 6 females. The single, 
large, median spermatheca in all 4 of the 6 females had few to numerous sperma- 
tozoa; three of the females additionally had one of the lateral thecae with few to 
numerous spermatozoa. The second male inseminated 5 of the 6 females. Very 
few to numerous spermatozoa were found in the median theca in all 5 cases. 
Four of the females additionally had a few sperm in one lateral theca. In all of 
the above cases, the sperm in the thecae were inactive. 

4. Xnuihcr of spei'iinilozou in the reproductive s\stcin 

Some preliminary estimates on numbers of spermatozoa were made on different 
portions of the male and female reproductive systems after forced-mating, using 
squashed whole mounts stained with aceto-lacto-orcein after Carnoy fixation. 
Sperm heads took the stain strongly and these were counted at a magnification ot 
970 >' with the aid of an ocular grid. The inherent counting errors are considered 
large because the sperm often failed to spread evenly and this failure was especially 
evident with spermathecal squashes. 

V- shown in Table III, the terminal testicular chamber of one unmated male 
had approximately 700 spermatozoa. The terminal testicular chamber of three 
repetitively force-mated males had from 333 to 120'' spermatozoa (mean of 741.3). 
The sperm duct (vas deferens plus vas effereus i of one unmated male had 370 
spermatozoa. The seminal vesicles of three nnmated males had from 3700 to 6309 



SPERMATHECAL FILLING IX AKDES 



139 



sperm (mean of 5132.3), while the seminal vesicles of two repetitively force-mated 
males had from 485 to 1374 sperm (mean of 929.5). 

The bursae of 6 females which were freshly inseminated by a single highly 
potent male (he force-copulated in rapid succession with 7 females) were dissected 
within one to two minutes after each ejaculation. This one male ejaculated 254 to 
2655 spermatozoa, and progressively fewer sperm were released with each succeeding 
forced-copulation (Table III). In sum, this male delivered 6126 sperm to 6 females 
and he did not inseminate the seventh female. 

TABLE III 

Numbers of spermatozoa in aceto-lacto-orcein squashes of different tissues of Aedes aegypti 



Tissue 


No. cases 


Estimated numbers of spermatozoa 


Terminal chamber of testis of unmated male 


1 


700 


Terminal chamber of testes of repetitively, 






force- mated males 


3 


333; 682; 1209 (mean, 741.3) 


Sperm duct of unmated male 


1 


370 


Seminal vesicles of unmated males 


3 


3700; 5388; 6309 (mean, 5132.2) 


Seminal vesicles of repetitively force-mated 






males 


2 


485; 1374 


Bursa 1 to 2 minutes after: 






First ejaculation 


3 


1142; 1744; 2655 (mean, 1847) 


Second ejaculation 


1 


554 (?) 


Third ejaculation 


1 


1248 


Fourth ejaculation 


1 


937 


Fifth ejaculation 


1 


478 


Sixth ejaculation 


1 


254 


Bursa 1 to If hours after first ejaculation 


3 


377; 584; 1142 (mean 701.0) 


Bursa 1 to 1 hours after second ejaculation 


1 


119 


Spermathecae 






Large 


4 


1 H ; 319; 325; 600 (mean, 346.3) 


Small 


4 


68; 200; 250; 500 (mean, 254.5) 


Thecae from 6 females after first ejaculation 






and 1-1 1 hrs. after copulation 


10 


73-400 (mean 225.5) 



The spermathecae of four females were dissected about one hour after forced- 
copulation with previously unmated males. Counts of spermatozoa in the large 
median theca varied from 141 to 600 (mean of 346.3), and counts of spermatozoa 
in one of the lateral thecae varied from 68 to 500 (mean of 254.5). Other counts 
made on spermathecae of unspecified size ranged from 73 to 400, with a mean of 
around 226. 

The bursae of three females were dissected one to 1^ hours after forced-copulation 
with fresh unmated males. The number of spermatozoa that remained in the 
bursa after thecal filling had occurred was estimated to vary from 377 to 1142 
(mean of 701) (Table III). These counts are considered to be much more reliable 
than direct spermathecal counts and suggest that of 1847 sperm (Table III) deposited 
in the bursa, about 1146 (62%) can reach the thecae. This, in turn, suggests that 
about 660 sperm reach the large theca and about 486 reach one of the lateral thecae. 
If these suggestions are correct, then the direct thecal counts of sperm are in error 
by a factor of about 2. 



140 



.1 U"K COLVARD JONES \VI> K<>\ \l.l> K. WHEELER 



From these various estimations llic following suggestions can be made. (1) 
The miniated male has about 5000 sperm available within his seminal vesicles. 
After rapid repetitive forced -mating, the male can ejaculate ahout S_" ', of the 
sperm within his vesicles. ( _' ) The male ejaculates progressively fewer spermatozoa 
into each successive female, i 3 ) Sixty-two per cent of the sperm initially deposited 
in the bursa reach the thecae. and oS' ', remain in this sac immediately after thecal 



filling. 



Cr 




FIGUKK 1. Sagittal section of Acdcx nc</ypti female immediately after ejaculation of the 
male, showing llie seventh (VI It and eighth (VIII) abdominal segments, cerci (CK), upper 
genital lip (UGL), bursal orifii-c (BO), accessory gland duct (AGD), spermathecal eminence 
(SI-".), dorsal valve (DV), ventral vaginal valve (W), and spennathecae (S) of the female. 
Note the spermatozoa (SI*) in packets in the dorso-anterior portion of the bursa and the large 
amount of finely granular male accessory gland secretion. The phallotreme of the male is shoun 
at IT. 

5. 1 lie composition <>j the ejaculate 

The normal ejaculate consists of a relatively small amount of spermatozoa and 
a much larger amount of an acidophilic holocrine secretion of the male's accessory 
glands. I "rcsumablv, the spermatozoa are contained in a small volume ot fluid 
within the seminal vesicles, and it is assumed that some' of tin's seminal fluid is added 
to the ejaculate. When the ejaculate is seen under the dissecting microscope, it 
appears whitish ; when viewed with a compound microscope, it has a greyish yellow 
cast. 



SPERMATHECAL FILLING IN . \KDES 



141 



The male accessory gland secretion includes a clear to finely granular material, 
round to ovoid granules of at least three different sizes, a few free nuclei, and even 
some intact, round to ovoid accessory gland cells of various sizes with large granular 
inclusions. If the accessory glands are ruptured in an open drop of saline, the 
exuding material does not usually vacuolate and the cells, free nuclei, and granular 
inclusions do not ordinarily lyse, although the cells may become swollen. Tin 
accessory gland secretion in an open drop of saline forms a dense, viscous, sticky 
mass which will rapidly clog a micropipette. However, if the saline mount is 
quickly covered with a layer of immersion oil before the glands are ruptured, 
then the exudate can be generally drawn into a micropipette without clogging. 





B 



FIGURE 2. Sagittal section of Acdcs acyypti female during spermathecal filling. In A i> 
shown the upper genital lip (UGL), the swollen bursa (BO, the vestibule (V), the sperma- 
thecal duct (SD), and the dorsal vaginal valve (DV). B is the same section, at greater 
magnification, showing the sperm (SP) assembled in packets on the ventral floor of the bur>a. 
and sperm making the U-turn into the vestibule. Note the position of the accessory gland duct 
(AGD), one spermathecal duct (SD), the posterior vaginal valve (PV), dorsal vaginal valve 
(DV) and ventral valve (VV). The transverse muscles of the spermathecal eminence are 
shown at TM. 



6. Sonic changes in the ejaculate "^ 



the bursa 



At the very moment of deposition, the locomoting packets of sperm are ejected 
dorsally above the accessory gland secretion (Fig. 1). Tn sagittal sections, the 
freshly inseminated bursa measures about 300 to 350 /A in length and varies from 
78 to 100^ in depth. The bursal orifice measures from about IS to 25 /JL. The 
spermatozoa measure about 250 /A (Christophers, I960). \Yhile most of the sperm 
rapidly spread to the edges of the bursa. .some of them become trapped in the 
granular portion of the accessory inland secretion. As Spielman (1964) has 
pointed out, many sperm rapidly assemble on the ventral wall of the bursa facing 



142 



JACK COLVAKI) JONES \VD RONALD E. WHEELER 



the orifice ( l r ig. 2\\, SI' ). Those sperm at the 1)liiul anterior end of the bursa tend 
to remain quite active lor about 17 minutes in oil-covered drops of saline, after 
\vhich they tend to become noticeably less visible and less active. Those sperm at or 
near the bursal orifice tend to he especially active. 

Thirty seconds after ejaculation, the bursal wall in sagittal sections measured 
-.2 to 3.3 p.. Two to three minutes after insemination, the bursal wall was greatly 
swollen (7.5 to !_' // thick ), hyaline, and the cells had large colorless vacuoles. The 
bursal wall was swollen and vacuolated for at least one hour after insemination. 
In fresh unstained whole-mounts of freshly inseminated bursae, we have seen what 
appeared to be a very delicate membrane surrounding the seminal mass, but no 
such membrane was visible in any of the histological sections. 





3. Unstained saline whole-mount dissection of the bursa of Acdcs ac</ypti about 10 
minutes after insemination, showing the fully vacuolated ejaculate. Note the swollen wall (W) 
in A and, at greater magnification (B), the large vacuoles and granules within the ejaculate. 

The largest vacuole in I', is about 20 microns in diameter. 

Three to live minutes after insemination, large vacnoles hrst appear within the 
granular portion of the ejaculate and they steadily increase in number until the 
bursal contents become filled with vacuoles within a granular matrix (Fig. 3). 
The large vacnoles measure about 20 /j. in diameter. These vacuoles are very 
clear in whole mounts but may be indistinct in sectioned material. The ejaculate 
may be fully vacuolated within about 10 minutes at 27 C. At 3(> to 37 C., 
the bursal contents fully vacuolate in about one minute. The completely isolated, 
freshly inseminated bursa will fully vacuolate in a drop of saline covered with a 
laver of immersion oil. When a male's accessory glands were crushed in the vicinity 
of the freshly dissected virgin bursa, the glandular exudate did not vacuolate. Tl 
the female is hosed with carbon dioxide for 5 minutes three seconds after coitus, 
the seminal material within the bursa does not vacuolate. 



SPERMATHECAL FILLING IN AEDES 143 

In many preparations in which the bursal contents we.'- vacuolating or had 
already fully vacuolated, the sperm were not visible through the intact wall of the 
bursa, but when the bursa was opened, sperm were found, r.ursal sperm retain 
a highly variable amount of undulatory activity for about 6 hours and sometimes 
for as long as 24 hours after insemination. In a number of cases, however, the 
sperm within the bursa were mostly inactive within about 75 minutes after 
ejaculation. 

Bursae rilled with vacuolated seminal material were observed up to 6 hours 
after insemination. Twenty-four hours after insemination, the bursae were partially 
distended; the wall was no longer thickened and vacuolar, and the contents of th<- 
sac were finely granular, had a yellowish brown cast, and were devoid of any 
vacuoles. 

The bursa was never observed to contract in saline or oil-covered saline 
preparations of either uninseminated or inseminated A. aegypti. Histological 
sections showed the bursa to be completely devoid of muscles. 

7. Structure of the spermathecae and tlicir ducts 

The median spermatheca of A. aegypti measures about 100 /x. in diameter, and 
each lateral theca measures approximately 75 p, in diameter. The thecae are 
completely devoid of muscle and were never observed to contract in any type 
of preparation examined. When the spermathecae of virgins are opened under a 
deep layer of oil, no bubble escapes. When the thecae are crushed in a drop of 
xylene containing sudan black, a colorless halo of fluid appears immediately around 
them. This colorless watery fluid within the thecae did not react with phenol red, 
neutral red, sudan III, British Drug House Indicator, or with Hydrion papers. 

Although the spermathecal ducts are covered by a single layer of evenly spaced 
circular muscles ( Curtin and Jones, 1961), we have never seen these ducts 
contract in saline or oil-covered saline whole-mounts. The spermathecal ducts, 
when stretched out in saline, measure about 265 p. in length, and the clear lumen of 
these varies from about 2 to 3.5 p.. 

8. Speed of sperm transfer 

Twenty-two females \vere used to study how rapidly bursal sperm could reach 
the thecae after forced-mating. The females copulated with males for 9 to 54 
seconds and were immersed in liquid chloroform 2 to 45 seconds thereafter. Three 
of the females did not become inseminated, although the males had copulated with 
them for 9 to 25 seconds. Eight of the females were killed 2 to 24 seconds after 
coitus and in three of them (37.5%), sperm had reached the thecae. In the 
first of these three cases, the female (which had copulated for 53. 8 seconds 
and was killed in 18 seconds) had only a very few sperm in two thecae. In the 
second case, the female (which had copulated for 28 seconds and was killed in 20 
seconds) had many sperm in the large theca and a few sperm in one lateral theca. 
In the third case, the female (which had copulated for 15 seconds and was killed 
in 24 seconds) had only a few sperm in the large theca. In the five other cases, 
however, sperm were found only in the bursa and none reached the thecae. Eight 
out of the 22 females were immersed in chloroform 30 seconds after coitus and were 



144 | \CR (Oi \ \RD JONE5 \l) RONALD E. WHEELER 

then dissected. In onl\ one of thes< females had sperm reached the large median 
theca. The last three females were killed 40 to 45 seconds after forced-mating, and 
in all three case's mam sperm had ascended to the thecae, and in two of the females 
sperm were in two thecac. Thus, following- uninterrupted forced-coitus, while a 
few sperm apparently are capahle of reaching the thecae of a few females as early as 
IS seconds after copulation, in most cases the sperm begin to till the thecae 
between 30 and 45 seconds. 

That highly variable results can be obtained is shown by the following data. 
Coitus of 7 pairs was permitted for exactly 15 seconds, after which the females 
were killed at 10-second intervals from 30 to 90 seconds after forced-mating, by 
exposing" them to very strong ether fumes. Only a few sperm reached two thecae 
after 30, 40 and 50 seconds. Many to numerous sperm were found in two thecae 
after 60, 70, SO and <>() seconds. 

In spite of the variability of the data, it is evident that spermathecal filling 
in A. ucf/yf'ti is a rapid event, and we are much inclined to agree with Burcham 
(1957) and the data presented by Spielman (1*^)4) that no transfer occurs after 
the first 5 minutes following coitus. 

9. Histological observations on the reproductive tract oj inseminated females before, 
dnrini/ and following spermathecal filling 

Spielman (l l ^>4) stated (p. 341) that ". . . 5 minutes after coitus, sperm 
were scarce in the anterior portion of the . . . btirsa'" and that one hour after 
coitus "the hursa was filled with coarse material and the remaining sperm were 
compressed into the posterior end and into the upper atrium." Our observations 
are not in agreement with these statements. The bursae of 22 mosquitoes were 
dissected 23 minutes to 6 hours after forced-copulation and in all cases many to 
numerous sperm were still present in the distended bursae. Dr. Spielman has 
kindly permitted us to examine the sections which he prepared for his studies on 
spermathecal filling after free mating. Our observations on his material follow. 

Sections made 30 seconds following coitus showed that the bursa was dis- 
tended with ejaculate, the majority of the sperm being antero-dorsally located, 
but at least one dense packet of sperm was seen on the ventral wall of the bursa 
at its orifice. Xo sperm were observed in either the atrium, vestibule, thecal 
ducts, spcrmathecae. or common oviduct. The ventral tuft just inside the female's 
ventral genital lip slanted dorsally into the bursal orifice but did not block this 
opening. In some sections, the posterior valve of the vagina was pressed against 
the surface of the dorsal valve, but in other sections a gap of variable dimensions 
was seen between these two vaginal valves. The ventral valve in some sections 
was pressed against the dorsal valve. 

Thirty-five seconds after coitus, sections showed some male accessory gland 
material within the lumen ot the upper vagina, but no sperm were detected. Numer- 
ous sperm were still in the dorsal portion of the hursa and at its blind anterior 
end. Many sperm in the bursa were seen in ventrally-directed arcs. Dense 
packets of sperm were seen making a sharp I '-turn from the' bursal orifice into 
the spermathecal vestibule- (see Fig. 2). The heads of these sperm were in contact 
with the intima ol the spermatliccal ducts. No sperm were visible in the upper 
vagina, thecae or common oviduct. The ventral tuft slanted upward into the 



SPERMATHECAL FILLING IN AEDES 145 

bursal orifice, blocking the center of it about half way. The spermathecal eminence 
appeared to be elevated and the vestibular opening seemed shifted into a position 
dorsal to the ventral tuft. The posterior vaginal valve in some sections was 
pressed against the dorsal valve. 

Sections made 43 to 60 seconds after insemination of the bursa showed sperm, 
and, in one case, male accessory gland material, free in the lumen of the upper 
vagina (Fig. 2). Many sperm were flattened against the surface of the dorsal 
vaginal valve. No sperm were visible in the lower vagina or in the common 
oviduct. The bursa was distended, and many sperm were ventrally aligned in 
dense packets at the bursal orifice. The median ventral tuft slanted halfway 
across the bursal orifice. The spermathecal eminence still appeared elevated. 
Many sperm were seen making the U-turn into the vestibular opening (Fig. 2). 
Sperm were observed inside two spermathecal ducts, and a few sperm were seen 
inside the spermathecae. The posterior vaginal valve in some sections touched the 
dorsal valve. 

Ten minutes after insemination, sections showed sperm still present in the 
upper vagina, mostly flattened against the dorsal valve. No sperm were visible 
in the lower vagina or the common oviduct. Numerous sperm were still present 
in the bursa and many of them were aligned on the ventral wall at the orifice. 
The ventral tuft appeared to completely block the vestibule in one series of sec- 
tions made at 10 minutes. 

In sections made one hour after coitus, sperm were still within the upper 
vagina, free in its lumen, and against the dorsal valve ; and for the first time, 
sperm were seen in the lower vagina and in the lumen of the common oviduct. 
Numerous sperm were still observable throughout the distended bursa. The 
vestibule appeared fully open to the bursal orifice. The ventral tuft did not block 
the vestibule. Sperm were still detectable within the spermathecal ducts and 
within two thecae. Sperm were especially dense at the entrance to each of two 
thecae. 

According to Spielman (1964, p. 341), the common oviduct contains ". . . 
masses of agglutinated immobilized sperm" one hour after coitus. Dissections 
made on the Bangkok strain after forced-copulation showed a few undulating 
sperm within the common oviduct at 57 to 69 minutes in three females, but no 
sperm were visible in the oviducts of two other females. Two hours after forced- 
copulation, one female had undulating sperm in her common oviduct as far as the 
ampullae, but no sperm were seen in the oviduct of another female dissected at 
the same time. No sperm were visible in the common oviduct of one female 
dissected 6 hours after forced-copulation. 

Our observations indicate that in both freely-mated and force-mated A. aegypti 
many sperm remain within the bursa following spermathecal filling, and that most 
of them are not concentrated at the bursal orifice. It is of considerable interest 
that the female has no anatomical device that could account for the fact that those 
sperm which are aligned at the open bursal orifice no longer attempt to reach 
the spermathecal vestibule. Even sperm which are free within the lumen of the 
upper vagina are no longer oriented towards the vestibule after spermathecal 
filling. 

Numerous dissections were made on force-mated and freely-mated specimens 



146 JACK COLVARD JONES \XD ROXAI.D E. WHEELER 

before, during, and following spermathecal filling, and in not one case were sperm 
ever detected within the spermathecal ducts. It is difficult to reconcile this with 
the presence of a few sperm in these ducts in sectioned material made one hour after 
free-mating. It is possible that during dissection the sperm within the ducts quickly 
entered the spermathecae. 

10. The beliavior of spcnnatoza 

Observations were made on the activity of spermatozoa within various portions 
of the unmated male's intact reproductive system in oil-covered saline whole- 
mounts. The spermatozoa often exhibited intense whirling or spiralling activity 
within the posterior testicular chamber. They were either inactive or showed a 
highly variable degree of activity within the sperm ducts, and sometimes undulated 
within the seminal vesicles. Sperm retained activity within the testes for 54 

TABLE IV 

Changes in motillty of sperm with time in thecae isolated from force- and freely-mated Aedes 
aegypti one hour after insemination. Ten females used for each of the two groups 





% Thecae with moving spermatozoa 


Time after isolation of thecae 






Force-copulated* 


Freely mated** 


less than 10 mins. 


46.4 


33.3 


1 hour 


85.7 


58.3 


2 hours 


64.3 


8.3 


3 hours 


42.9 





4 hours 


10.7 





5 hours 


3.6 






* Twenty-eight thecae contained sperm. 

* Twelve thecae contained sperm. 



to 270 minutes and undulated in some seminal vesicles for 60 to 348 minutes. 
Sperm within the sperm ducts, if active at all, were generally active for less than 
38 minutes and for not more than 196 minutes. 

The spermathecae of force-mated and freely-mated mosquitoes were isolated one 
hour after copulation in a drop of saline and the preparation covered with a layer 
of immersion oil so that the activity of the sperm within the thecae could be noted 
at hourly intervals. As shown in Table IV, sperm were generally more active and 
remained active for a longer period in the force-mated than in the cage-mated 
group. While no activity was observed three hours after isolation of the thecae 
from the cage-mated mosquitoes, sperm were actively moving in 42.9% of the 
thecae of the force-mated females at this time. 

The following types of activity were observed in spermatozoa released from 
seminal vesicles into a drop of saline covered with a layer of immersion oil : 
(1) very rapid locomotion with the short, sharp, thin, stiff, needle-like head piece 
tilting up and down as the long thin tail made rapid, large wave undulations. These 
explosive progressive locomotory movements occurred either in a generally straight 
line in any direction, or the sperm would circle about briefly. In saline drops 



SPERMATHECAL FILLING IN AEDES 147 

which were not covered with a layer of oil, locomotions were only rarely observed. 

(2) Very rapid and intense coiling or lashing motions of the tail were observed, 
especially when the sperm occurred in clusters and when the head was at an inter- 
face. Many times the tails of clustered sperm whirled or lashed synchronously. 

(3) Slow, smooth, regular, large wave undulations of the tail were observed when 
sperm were congregated at the saline/oil interface. (4) Irregular undulations or 
oscillations of highly variable amplitudes were observed in sperm which had ceased 
locomoting or which had stopped the smooth regular undulations. Different por- 
tions of the tail were capable of undulating at very different rates and with differ- 
ent amplitudes. The waves moved away from the head piece. 

Observations were made on the activity and survival time of sperm released 
from the seminal vesicles. Highly variable results were obtained, depending upon 
the technique and the location of the sperm within the preparation. When the 
seminal vesicles were ruptured into an open drop of saline, many sperm which 
reached the edge of the drop quickly lost their motility in one to three minutes. 
Those sperm which did not reach the edge of the drop undulated irregularly and 
lost all activity fairly rapidly. However, those sperm still inside the torn vesicles 
tended to remain active for about four minutes in open saline drops. When the 
vesicles were ruptured in a very small amount of saline that had been covered 
first with a drop of immersion oil, the sperm were often intensely active for two 
to 15 minutes, especially around the surface of the vesicles. Generally, the sperm 
in such preparations lost all activity in three to 87 minutes after release. Those 
sperm which moved out into the layer of oil very quickly ceased moving. When 
the vesicles were opened in a moderate-size drop of saline that had been covered 
with a layer of immersion oil, those sperm which were strongly oriented at the 
saline/oil interface remained active for 16 to 60 minutes, but those which did not 
reach the edge of the drop tended to lose their activity in about one minute. The 
sperm inside the seminal vesicles remained quite active for two to 119 minutes 
(in most cases for about 6 minutes) ; highly variable numbers undulated feebly 
for 182 to 328 minutes. Sperm on the outside of the vesicles tended to have their 
heads oriented to the vesicles' surface and were often very intensely active for about 
four minutes. 

In coverslipped saline mounts in which air bubbles had been trapped, the 
sperm head was frequently strongly oriented to the saline/air interface. 

To study whether chemotaxis might be involved in sperm migration, seminal 
vesicles and bursal sperm were released in the vicinity of intact male accessory 
glands, male accessory gland exudate, and onto freshly excised vaginal tissues. 
In many cases, the tip of the sperm head was strongly oriented to all of these 
tissues, but sperm did not specifically congregate around them. The head of 
seminal vesicle and bursal sperm did not become specifically oriented to fat body, 
testes, somatic muscles, bursa, female accessory gland, or its duct, spermathecal 
ducts, intact or crushed spermathecae, common oviduct, or even to a freshly laid egg. 

To determine whether the sperm head would be oriented in or against the 
direction of a moving stream, the intact seminal vesicles were dissected into a drop 
of saline on a glass slide next to a long rectangular coverslip supported on two 
sides by capillary glass rods. The thin space between the coverslip and slide 
was filled with saline. The seminal vesicles were cut open so that the sperm 



148 JACK COLVARD JONES AND RONALD E. WHEELER 

poured out into the saline just under the edge of the coverslip and a strong current 
was produced by withdrawing saline at the other end. In all such preparations, 
the sperm heads were precisely aligned in the direction of the flowing stream of 
saline. Dead sperm were not precisely aligned. This type of rheotaxis of live 
Aedes sperm is exactly the opposite of that of bull or human sperm which are 
known to orient the head piece against the direction of a moving stream (see, e.g., 
Rothschild. 1962). 

Thus, the head of the spermatozoon of A. uc</yf>ti becomes oriented toward 
certain tissues and becomes aligned in the direction of a flowing stream, and the 
tail piece is capable of propelling the cell rapidly. 



We are indebted to Dr. Andrew Spielman of Harvard University for allowing 
us to examine his histological sections and for many stimulating discussions. Our 
Figures 1 and 2 were taken from material which Dr. Spielman loaned us. We are 
most grateful to Mr. Kenneth W. Ludlam for his help with many of the experi- 
ments. Useful suggestions concerning the manuscript were made by Drs. Norman 
T. Davis, Arden O. Lea, P. T. M. Lum and A. Glenn Richards, and by Elizabeth 
D. Jones. 

SUMMARY 

1. With the forced-copulation technique, the Bangkok strain of Aedes aegypti 
can ejaculate within the first five seconds of coitus but usually does so within 10 
to 15 seconds. The male force-copulates for 31.3 seconds, and 82% of the females 
become inseminated. In 90% of them, spermatozoa reach two of the three thecae. 
With naturally-mated mosquitoes copulation is significantly shorter in duration, 
all of the females become inseminated and in 92 % of them spermatozoa reach all 
three thecae. 

2. The terminal chamber of the testis of an unmated and repetitively force- 
copulated male has about 700 spermatozoa. Each sperm duct has about 370 sperm. 
The seminal vesicles of unmated males have about 5000 spermatozoa, while the 
vesicles of repetitively force-mated males have about 930. 

3. With rapid repetitive force-copulation, the male ejaculates progressively 
fewer spermatozoa into the bursa of each successive female. Sperm counts made 
on 6 ejaculates from one male varied from 254 to 2655. 

4. Counts on spermatozoa remaining in the bursa after spermathecal filling 
indicate that 62% of them leave the bursa, and suggest that about 660 sperm reach 
the large theca and 486 fill one of the lateral thecae. 

5. Most of the sperm deposited in the bursa quickly spread to the edges of 
the sac, and many become aligned on its ventral wall. The wall of the bursa 
greatly swells two to three minutes after insemination. Shortly thereafter the 
accessory gland secretion within the ejaculate begins to vacuolate and may be fully 
vacuolated within 10 minutes and remains vacuolated for at least 6 hours. 

6. With forced-copulation, a few sperm may be capable of reaching the thecae 
within 18 seconds but in most cases sperm begin to reach the thecae between 
30 and 45 seconds after coitus. Complete thecal filling can occur in 90 seconds 
and probably is terminated within the first five minutes or less after coitus. Fol- 



SPERMATHECAL FILLING IN AEDES 149 

lowing spermathecal filling many active sperm remain in the bursa for some time. 
Following spermathecal filling those sperm at the bursal orifice no longer make 
the U-turn towards the open spermathecal vestibule. 

7. Spermatozoa within the isolated but intact male reproductive system may 
remain active for five to six hours in oil-covered saline whole-mounts. Sperma- 
tozoa released from seminal vesicles in oil-covered saline drops exhibit four types 
of movement: (a) brief, rapid, explosive, progressive locomotion, (b) rapid 
synchronous coiling when the cells are in dense clusters and the head is at certain 
interfaces, (c) smooth undulations in situ, and (d) irregular undulations or 
oscillations. The heads of sperm of Aedes become oriented in the direction of a 
moving stream. 

8. Sperm released from the seminal vesicles may become strongly oriented 
toward the male accessory glands and its exudate, and to freshly excised vaginal 
tissues, but they do not specifically congregate about these tissues in oil-covered 
saline whole-mounts. Seminal vesicle sperm do not become oriented to freshly 
excised fat body, testes, somatic muscles, bursa, female accessory gland or its 
duct, spermathecae or their ducts, ovary, common oviduct, or a freshly laid egg. 

CONCLUSIONS 

Many sperm deposited in the bursa of female Aedes aegypti (Linnaeus) rapidly 
locomote around the male accessory gland secretion of the ejaculate and assemble 
on the ventral floor of the sac at its orifice where they undergo rapid and violent 
synchronous coiling movements. The strong orientation of the sperm head to 
certain interfaces presumably guides the long, thread-like contracting cells over a 
U-shaped route directly into the vestibule where they first contact the opening of the 
spermathecal ducts. Bundles of sperm swiftly ascend the ducts, presumably only 
in female fluids, and simultaneously reach two or three thecae. Shortly after 
sperm begin to fill the already fluid-filled thecae, the bursal wall swells and pre- 
sumably secretes material into the ejaculate. After this the accessory gland se- 
cretion of the ejaculate begins to vacuolate, and a short time after this, the still 
active sperm at the open bursal orifice stop moving into the vestibule. 

LITERATURE CITED 

BRELJE, R. VON DER, 1924. Die Anhangsorgane des weiblichen Geschlechtsganges der Stech- 

miicken. Zool. Ans., 61 : 71-80. 
BURCHAM, E. G., 1957. Some characteristics and relations of mating and oviposition of Aedes 

aegypti (Linnaeus) (Diptera: Culicidae). Ph.D. Thesis, Ohio State Univ., 130 pp. 
CHRISTOPHERS, S. R., 1923. The structure and development of the female genital organs and 

hypopygium of the mosquito, hid. J. Mcd. Res., 10: 698-720. 
CHRISTOPHERS, S. R., 1960. Aedes aegypti. The Yellow Fever Mosquito. Its Life History, 

Bionomics and Structure. Cambridge Univ. Press, 739 pp. 
CURTIN, T. J., AND J. C. JONES, 1961. The mechanism of ovulation and oviposition in Aedes 

aegypti. Ann. Ent. Soc. Amer., 54: 298-313. 
DUFOUR, L., 1851. Recherches anatomique et physiologique sur les Dipteres. Mem. Acad. Sci. 

Paris, 11:205-210. 
EPHRUSSI, B., AND G. W. BEADLE, 1936. A technique for transplantation for Drosophila. Amer. 

Nat. ,70: 218-225. 
HODAPP, C. J., P. H. SCHWARTZ AND J. C. JONES, 1960. Some observations on the anatomy 

of the female reproductive system of Aedes aegypti (L.). Anat. Rec., 137: 364-365. 



150 JACK COLVARD JONES AXD RONALD E. WHEELER 

JONES, J. C, 1961. Observations on sexually depleted male Acdes aegypti (L.) Amcr. Zool, 

1 : 362. 
KULAGIN, N., 1901. Der Ban der \\ciblichen Geschlcchtsorgane bei Citlcx und Anopheles. 

Zcitschr. wiss. Zool, 69: 578-597. 
McDANiEL, I. N., AND W. R. HORSFALL, 1957. Induced copulation of aedine mosquitoes. 

Science, 12$: 745. 
ROTH, L. M., 1948. A study of mosquito behavior. An experimental laboratory study of the 

sexual behavior of Acdes aegypti (L.). Amcr. Mid. Nat., 40: 265-352. 
ROTHSCHILD, L., 1962. Sperm movement. Problems and observations. In: Spermatozoan 

Motility (ed. by D. W. Bishop). A.A.A.S. Publ. no. 72: 13-29. 
SCHWARTZ, P. H., 1961. Behavior of spermatozoa in Acdes aegypti (L.). M.S. Thesis, Univ. 

of Maryland, 45 pp. 

SPIELMAN, A., 1964. The mechanics of copulation of Aedes aegypti. Biol. Bull., 127: 324-344. 
WHEELER, R. E., 1962. A simple apparatus for forced copulation of mosquitoes. Mosq. News, 

22 : 402-403. 



NEW SPECIES OF ACOEL TURBELLARIANS FROM 
THE PACIFIC COAST 

EUGENE N. KOZLOFF 

Lewis and Clark College, Portland, Oregon, and Friday Harbor Laboratories, 

University of Washington 

The acoel turbellarians have been accorded very little attention in North 
America. Only a few species have been described, and some of these will have to 
be investigated more completely before their taxonomic position can be estab- 
lished with any degree of certainty. On the Pacific coast, where acoels are 
abundant in bottom sediments, on algae, and in other situations, apparently only 
two species have been named. One of these is PolycJwcrus cannelensis Costello 
and Costello (1938), from central California; the other is Childia groenlandica 
(Levinsen), an acoel which has a wide distribution in North Atlantic waters and 
which has recently been reported from San Francisco Bay, California (Hyman, 
1959). 

During the summers of 1961 and 1962, and the autumn of 1964, several species 
of acoels were taken at various localities on San Juan Island, in the San Juan 
Archipelago, Washington. Mature individuals of three of these were recovered 
in sufficiently large numbers to permit a thorough study of their morphology 
and description as new species. Additional material of one of these acoels was 
found at Charleston, Coos County, Oregon, during the summer of 1964. 

The acoels are a difficult group with which to work. Most of them are small, 
and certain of their syncytial structures are not sharply delimited. Many pub- 
lished descriptions are incomplete and poorly illustrated. There are even some 
rather detailed accounts which do not focus sharply on pertinent details, and 
from which one cannot form a clear picture of the morphology of the acoels con- 
cerned. In deciding the taxonomic position of the new species described here, I 
have relied upon the summary of the genera and higher taxa of acoels given by 
Westblad (1948). 



I wish to express my appreciation to Dr. Robert L. Fernald, Director of tin- 
Friday Harbor Laboratories, for many courtesies which facilitated my work. 

METHODS 

The acoels described in this contribution were for the most part obtained by 
taking up masses of green algae (Uha and Enter onwrpha) and washing them by 
agitation in a pail of sea water. A thin layer of sediment (consisting largely of 
muddy sand from the substrate) obtained in this way was then distributed in large 
culture dishes, in water about 2 or 3 cm. deep. Samples of sediment sucked up with 
a large pipette and examined with a dissecting microscope often contained acoels, 

151 



152 



Krr.KNK N. KOZLOFF 



^oCf *^ Sft i *j f ** 

^ 4t- n^ -^ ^'-^ . ft 

f.VS** 1 * 1 **!** 





7 





Plate I Parotoccllx lulcola 



All figures were prepared with the aid of a camera lucida, but in the case of specimens 
drawn from life (Kiv 1 S), most details were sketched in free-hand. Figures 6 to 11 are based 



NEW ACOELS FROM THE PACIFIC COAST 153 

together with rhabdocoels, alloeocoels, copepods, amphipods, and other small 
organisms. 

My descriptions are based entirely on sexually mature specimens examined 
or fixed soon after collection. (Worms which are immature or which are not 
well nourished are unreliable.) The acoels were studied extensively in life, 
in both transmitted and reflected light. Gentle compression of the worms under 
a coverglass was usually necessary to make certain structures clearly visible. Ad- 
dition of a small amount of a solution of magnesium chloride (approximately 
isotonic with sea water) to the drop of water in which the worms were swimming 
was generally helpful in narcotizing them in an extended condition without obvi- 
ously affecting their appearance. 

Stained whole-mount preparations were less useful than living acoels for 
morphological studies. However, a few whole-mounts of worms fixed in Bouin's 
fluid and stained with alum hematoxylin or borax carmine were prepared for 
permanent records. 

The morphology of the acoels described in this paper was worked out largely 
by study of transverse, sagittal, and frontal serial sections (6 /* or 8 ft). The 
worms were fixed, usually after being narcotized, in Bouin's fluid or in a mixture 
of 90 ml. of a saturated aqueous solution of mercuric chloride with 10 ml. of 
formalin and 5 ml. of acetic acid, and then embedded in paraffin. Iron hematoxylin 
was used routinely for staining ; sometimes the preparations were counterstained 
with eosin, orange G, or fast green FCF. A few series were stained with Harris' 
alum hematoxylin and eosin. 

DESCRIPTIONS OF SPECIES 
Parotocelis Iittcola gen. nov., sp. nov. 

Most of my material of this acoel was taken in shallow pools at the margins 
of a body of water known locally on San Juan Island as Argyle Lagoon (Lat. 48 

on sections (6 /u) of specimens fixed in Bouin's fluid and stained with iron hematoxylin ; certain 
details were supplied from adjacent sections in the same series. 

Abbreviations for all figures : b, brain ; bs, seminal bursa ; ec, ectocytium ; en, endocytium ; 
fg, frontal glands ; g, glands surrounding copulatory organs ; ga, genital atrium ; gp, genital 
pore ; Id, lipid droplets ; m, mouth ; mp, parenchyma! muscles ; ms, subepicytial muscles ; n, nozzle 
of seminal bursa ; ooc, oocyte ; oog, oogonium ; p, penis ; pg, granule-filled masses at tip of penis ; 
s, statocyst ; sd, sperm duct ; sv, seminal vesicle ; t, testis ; v, vagina. 

FIGURE 1. Specimen in contact with substrate; dorsal view. 

FIGURE 2. Rhabdites at left margin of body. 

FIGURE 3. Mature sperm. 

FIGURE 4. Anterior end (specimen slightly compressed under coverglass) ; dorsal view. 

FIGURE 5. Posterior end (specimen slightly compressed under coverglass) ; dorsal view. 
The anterior portion of the vagina, in which the lumen is obliterated by a syncytium containing 
refractile granules, obscures part of the seminal vesicle beneath it. 

FIGURE 6. Frontal section. One of the oocytes is undergoing a maturation division. 

FIGURE 7. Median sagittal section. 

FIGURE 8. Epicytium, epicytial glands, subepicytial musculature, and portion of testis in 
transverse section just anterior to mouth. 

FIGURE 9. Transverse section just anterior to mouth. 

FIGURE 10. Transverse section through seminal bursa of same specimen. 

FIGURE 11. Transverse section through penis, seminal vesicle, and anterior portion of vagina 
of same specimen. 



154 EUGENE X. KOZLOFI'" 

31.3' N.; Long. 123 0.6' \V.i.' The worms are usually abundant in muddy 
sand supporting a growth of I'.ntcroinorpha. P. hiteola has also been collected in 
small numbers at Friday Harbor, among Ulva and Enteromorpha growing on a 
substrate of gravel mixed with muddy sand, at tide levels of about to +2 ft. 

\Yhen gliding actively, the length of P. lit t cola is typically about two and a half 
times the width (Fig. 1). The greatest width is usually near the end of the first 
one fifth of the body. The anterior end is broadly rounded; posteriorly, the 
body tapers rather gradually to a nearly acute tip. The largest specimens are 
about 700 p. in length and 230 ju. in width, but they may become extended temporarily 
to a maximum length of about 840 /*, and a width of about 180 //,. In worms 
which are in tight contact with the substrate, the thickness in the mid-dorsal region 
is usually about one-half the greatest width, so that the body appears definitely 
flattened. When swimming free, the worms become nearly cylindrical. 

The statocyst (Fig. 4) lies about midway between the ventral surface and 
the dorsal surface, near the end of the first one-tenth of the body. In larger 
specimens, the diameter of the statocyst is about 20 p, and that of the statolith is 
about 12 fj.. Viewed on edge, the statolith is nearly hemispherical; the convex 
surface is uppermost. 

In reflected light, the coloration of the body as a whole, excluding the digestive 
endocytium, is whitish, tinged faintly with orange ; the orange color becomes more 
pronounced anterior to the statocyst. In most specimens which contain ingested 
diatoms, the endocytium is green, although in an occasional individual this region 
is brown or yellowish brown. In the area just behind the statocyst, there are small 
masses of a material which appears white in reflected light. Three discontinuous 
longitudinal streaks of this material extend for some distance posteriorly. 

In strong transmitted light, the body is more or less translucent. In the 
endocytium, freshly-ingested diatoms with their characteristic olive-green, vellow- 
brown, or yellow-green color may be seen, together with those which have turned 
green or bluish green. Chlorophylls diffusing out of the diatoms undergoing digestion 
appear to be responsible for the green or blue-green color typically observed in this 
general area. In the ectocytium anterior to the statocyst, there is a crescentic 
band of yellowish-orange pigment ; similar pigment is often found in small areas 
in the posterior part of the body, around the copulatory organs. Lipid droplets 
are scattered throughout the body. The larger of these, which may attain a 
diameter of about 20 /*, arc yellow or orange ; the smaller ones may be nearly 
colorless, yellow, orange, or bluish green, or some mixture of these colors. The 
lipid droplets apparently concentrate pigments which diffuse out of the diatoms. 
The material which is white in reflected light is seen to consist of granules or rods 
of a refractile substance. These granules form small aggregates (Fig. 4) within 
the ectocytium in the dorsal part of the body just behind the statocyst, and dorsal 
and dorsolateral to the endocytium. Under low magnification, the aggregates 

1 Argyle Lagoon is not natural. It was formed after an accumulation of gravel from a 
hillside excavation became deposited, together with sand and debris, on the seaward side of a 
small inlet of North Bay. At high tide, water may enter the lagoon through its narrow connec- 
tion with the inlet. At low tide, some water drains out of the lagoon. However, the water level 
in the lagoon does not vary a great deal, and most of the time it is higher than that of North Bay 
and the inlet. 



NEW ACOELS FROM THE PACIFIC COAST 155 

appear nearly black ; under higher magnification, they appear brown. However, 
the individual bodies in the aggregates are nearly colorless. 

The body is entirely covered by cilia about 10 p. long. Scattered over the body 
surface there are stiff, cilia-like bristles (Fig. 4) up to about 24 ^ long. These 
are probably sensory in function. The bundles of epicytial rhabdites (Fig. 2) 
are rather evenly distributed on both the dorsal and the ventral surfaces. They 
fall into poorly-defined short rows (Fig. 4). 

The mouth is located on the ventral surface, slightly anterior to the middle of 
the body. The pore is kept closed most of the time, but its position may be estab- 
lished by finding a characteristic convergence of rows of cilia. The mouth is 
capable of great distention during ingestion of food and during elimination of 
undigested material, such as diatom frustules. 

The principal elements of the nervous system in this small acoel are not clear 
in any of my preparations. Close to the anterior end of the body, in front of the 
statocyst, a ring-like concentration of nerve tissue encircles the so-called ''frontal 
organ" (the confluence of ducts of the frontal glands approaching the pore through 
which their secretion is discharged). However, the nerve tissue and ectocytium 
are so closely bound together that I have not established how many ganglia con- 
tribute to the brain mass, and I have not been able to trace any important nerves 
leading away from it. 

Much of the anterior quarter of the body is occupied by the frontal glands 
and their secretion (Figs. 6, 7). In sections, the secretion is conspicuous as a 
pale yellowish material which is not appreciably stained by either hematoxylin 
or acid counterstains such as eosin, orange G, and fast green. The pore through 
which the secretion is discharged is circular and is located at the anterior tip of 
the body. The epicytium is supplied with numerous small glands (Fig. 8) within 
which the bundles of rhabdites are formed. The rhabdites are destroyed by the two 
fixatives which I used. 

The subepicytial musculature (Fig. 8) consists of an outer layer of circular 
muscles and an inner layer of longitudinal muscles. In the parenchyma, there are 
scattered longitudinal and dorsoventral muscle fibers ; dorsoventral muscles travers- 
ing the endocytium near the mouth are particularly conspicuous (Figs. 6, 7). The 
musculature associated with the copulatory organs will be described subsequently. 

In the region of the mouth, the endocytium, into which food is ingested, 
occupies most of the body mass mesial to the ovaries and testes (Fig. 7). The 
endocytium extends anteriorly as far as the frontal gland cells and posteriorly 
as far as the anterior edge of the seminal vesicle. The ectocytial layer, which 
is very thin at the level of the mouth, becomes slightly more prominent anteriorly, 
and is very conspicuous in the posterior quarter of the body. Dorsal, lateral, and 
posterior to the copulatory organs, the vacuoles in the ectocytium reach a very 
large size (Figs. 1, 5, 6, 7). 

The testis and ovary on each side of the body are closely apposed for most of 
their length. In the region just behind the frontal glands, the testes are lateral 
and dorsal to the string of small oogonia lying near the ventral epicytium (Figs. 
6, 7). As the oogonia enlarge into oocytes, and therefore occupy progressively 
more of the body mass as they migrate posteriorly (Fig. 9), the testes become 
gradually restricted to a narrow zone lateral to the oocytes. 

From the posterior end of each testis, a delicate duct carries sperm through 



156 EUGENE N. KOZLOFF 

the parenchyma to the seminal vesicle ( Figs. 5, 10). The two sperm ducts enter 
the seminal vesicle at rather widely separated points on its anterolateral surfaces. 
The seminal vesicle (Figs. 5. <>, 7, 11 ) lias a thin muscular wall, and in mature 
specimens invariably contains inactive sperm. Within the seminal vesicle, on its 
posteroventral side, there is a cluster of granule-filled masses which surround a 
small cavity continuous with the lumen of the penis. In life, the cluster resembles 
a group of cells (Fig. 5). but the boundaries of the individual masses are de- 
stroyed by fixation, and I have not been able to distinguish nuclei among the 
granules, which are refractile and are stained by hematoxylin. The penis appears 
to be of a type which is everted during copulation, and presumably when this 
takes place at least some of the granules at its tip are discharged. 

The mature sperm (Fig. 3) of P. luteola are about 150 p. long. Behind the 
appreciably thickened anterior portion (slightly over one-third of the total length), 
the tail of the sperm narrows gradually to a very fine tip. 

The vagina, supplied externally with an outer layer of longitudinal muscles 
and an inner layer of circular muscles, extends at first almost directly dorsally 
away from the genital atrium. This lower portion of the vagina has a distinct 
lumen (Figs. 6, 7) ; the lumen becomes gradually more extensive, and crescentic 
in outline as it is viewed from the dorsal side in living specimens (Fig. 5). The 
surface of the syncytial wall of the vagina next to the lumen is covered with small 
granules which appear to be of the same type as those associated with the tip of 
the penis. Some of these granules are noted within the tissue of the vagina and 
also within the lumen. 

As the vagina arches anterodorsally over the seminal vesicle, the lumen 
becomes obliterated by a syncytial mass continuous with the syncytium of the rest 
of the vagina, and the layer of circular muscles becomes more pronounced. In 
life, the syncytium contains refractile crystal-like granules (Fig. 5) ; in specimens 
which have been fixed and sectioned, the granules are not preserved, and the 
syncytium is conspicuously vacuolated (Figs. 7, 11). Finally, the musculature 
disappears, and the vagina passes insensibly into a syncytium distinct from the 
digestive endocytium and within which the seminal bursa develops (Figs. 5, 7, 10). 
In some living specimens, as well as in sectioned preparations, sperm are observed 
in two or more spaces which may be connected or apparently separate (Fig. 7). 
Extending ventrally or posteroventrally from the bursa is a heavily cuticularized 
nozzle invested by what appears to be a fibrous tissue (Figs. 7, 10). The sperm 
in the seminal bursa usually exhibit considerable activity. 

How the sperm reach the syncytium, within which the rather poorly-defined 
seminal bursa develops, is not clear. I have not been able to distinguish a continu- 
ous clear passage through the syncytium constituting that portion of the vagina 
which lies above the seminal receptacle. However, sperm have been noted in the 
lower portion of the vagina. It is possible that when insemination is effected, 
the sperm entering the vagina are simply forced through the syncytium to the region 
where the bursa is formed. 

In the parenchyma around the seminal vesicle and anterior region of the vagina, 
there are a number of cellular elements which appear to be gland cells. The 
exact distribution of these glands and the pathways by which their secretion or 
secretions are delivered to other organs have not been worked out. 



NEW ACOELS FROM THE PACIFIC COAST 157 

The holotype specimen, in the form of a set of serial sagittal sections, has been 
deposited in the United States National Museum (USNM No. 32902). 

The nature of the brain of P. lutcola, and the fact that its vagina enters the 
genital atrium behind the copulatory complex, indicate that it belongs in the family 
Otocelididae. Westblad (1948) established this family to include a single genus, 
Otocelis, which had previously been referred to the Convolutidae by most students 
concerned with this general group of acoels. 

Westblad (1946) recognized only two species of Otocelis: 0. rubropunctata 
(Schmidt) and 0. giilhuarcnsis Westblad. Ax (1959) pointed out that the acoel 
believed by Westblad to be 0. rubropunctata is quite distinct from the worm de- 
scribed by Schmidt and later studied in detail by von Graff. The true 0. rubropunc- 
tata, which has a single genital pore, is not definitely known to occur outside the 
Mediterranean Sea and Black Sea. Westblad's 0. "rubropunctata," from Scandi- 
navian localities, has separate male and female pores. Ax proposed that it be 
called 0. westbladi. 

Two other acoels have rather recently been added to the genus Octocelis. 
0. dichona Marcus (1954) is distinctive in having the genital pore located at 
the posterior end of the body. 0. sachalinensis Ivanov (1952) is probably more 
nearly similar to 0. rubropunctata than to any other species of Otocelis. How- 
ever, it lacks eyes, and the organization of the penis is very much like that in 
P. lutcola. Certain of Ivanov's figures suggest that the tip of the penis of 0. 
sachalinensis has masses of granules similar to those associated with the penis of 
P. lutcola, although Ivanov did not mention any such masses in the text. 

In the acoel I have described, the most distinctive feature, not shared by any 
of the other known species of Otocelis, is the peculiar nature of the anterior part of 
the vagina, where the lumen appears to be obliterated. It is primarily on the 
basis of this characteristic of P. luteola that I propose a new genus. I am fully 
aware that 0. sachalinensis and P. luteola are similar in a number of respects, but 
the relationship of P. luteola to the genotype (O. rubropunctata} or to the other 
species of Otocelis is probably considerably more remote. 

Raphido phallus actuosus gen. nov., sp. nov. 

This acoel is moderately common in the small inlet of North Bay with which 
Argyle Lagoon communicates. Washings of Ulva detached from substrates of 
muddy sand or gravel at tide levels ranging from about --1 to +4 ft. often contain 
some R. actuosus. 

When extended and gliding actively on a firm substrate (Fig. 12), the length 
of R. actuosus is equal to about four times the width. Anteriorly, the body is 
rounded ; posteriorly, it tapers only slightly. The largest specimens, in a normal 
state of extension, are about SSO ^ long and 220 /j. wide. When the worms are 
in tight contact with the substrate, the ventral surface is flattened, but the thickness 
in the mid-dorsal region may nearly equal the width. When swimming free of 
the substrate, R. actuosus becomes almost cylindrical. 

Of the three acoels described in this paper, R. actuosus is the most active, and 
it is also the most fragile. When the animal is swimming in contact with the 
substrate, its movements are jerky, and the posterior part of the body is often 
twitched back and forth, as if it were being irritated. Addition of a very little 



158 EUGENE X. KOZLOFF 

isotonic magnesium chloride may cause it to disintegrate, and when it is under 
slight pressure from a coverglass it is less likely to maintain its integrity than the 
other two species. 

The statocyst is located near the end of the first one-eighth of the body. In 
larger specimens, its diameter is about 20 /*. The diameter of the statolith is 
about 14 p.. The shape of this structure is approximately hemispherical, and 
the convex surface is uppermost. 

Viewed with reflected light, the body (except for the digestive endocytium) 
is whitish. The endocytium is typically brownish yellow, but it may be greenish 
yellow, or partly of this color. Deposits of bright white material are usually 
very conspicuous in the dorsal part of the body around the statocyst, and in a 
broken streak extending for most of the length of the body along the midline. 

In strong transmitted light, the diatoms taken into the digestive endocytium 
are observed to change from a yellow-brown or olive color to greenish yellow and 
brownish yellow. The pigments diffuse out of the frustules and color the 
endocytium as a whole. Small lipid droplets are abundant in the ectocytial 
parenchyma, especially in the anterior half of the body. Most of these are yellow 
in color, and occur in clusters of various shapes ; some clusters contain a large 
number of droplets, and are confluent with other clusters. The bright white 
deposits noted around the statocyst and along the midline when the worms are 
studied in reflected light are aggregates of small refractile granules which indi- 
vidually are pale yellowish green in color. The aggregates, however, appear 
blackish or brownish. They lie within the ectocytial parenchyma in the dorsal 
part of the body. 

The body is entirely covered by cilia about 8 or 9 p. long. Scattered over the 
body surface are stiff, cilia-like bristles. Most of these are approximately 15 p. 
long, but in the caudal region some of the bristles may reach a length of nearly 
30 p. 

The bundles of rhabdites of R. actnosiis are conspicuous because they are 
closely spaced and are arranged in definite rows (Fig. 13). A particular row 
of rhabdites is never very long, however, and eventually merges with another 
row or terminates as two other rows converge. 

The mouth is located on the ventral surface just posterior to the middle of the 
body. It is capable of rapid distention during the ingestion of food and elimination 
of undigestible residues (largely diatom frustules). 

The brain appears to consist of four major ganglia, one anterolateral and one 
posterolateral to the statocyst on either side. Heavy commissures connect the 
ganglia in front of and behind the statocyst, so that the latter is almost completely 
enclosed within nerve tissue. My preparations do not clearly show the nerve 
trunks which originate from these ganglia. 

The frontal glands and their accumulated secretion occupy a considerable part 
of the body mass in the vicinity of the statocyst (Fig. 17). The secretion appears 
in sectioned specimens as a pale yellowish material. The circular pore through 
which the secretion is discharged is located at the anterior end of the body, but 
is directed slightly downward. The epicytium is supplied with many small glands 
(Fig. 18) within which the bundles of rhabdites are formed. 

The subepicytial musculature (Fig. 18) consists of an outer layer of circular 
muscles and an inner layer of longitudinal muscles. Parenchyma! muscles are also 



NEW ACOELS FROM THE PACIFIC COAST 159 

present ; these are most conspicuous in the anterior part of the body, in the region 
occupied by the frontal glands. 

The digestive endocytitim reaches anteriorly to the frontal glands and pos- 
teriorly nearly to the back edge of the seminal vesicle (Fig. 17). The ectocytial 
layer is rather thin in most regions of the body. It becomes appreciably thicker 
near the anterior end, but is most extensively developed in the posterior part of 
the body, where vacuoles within it are conspicuous lateral, dorsal, and posterior 
to the copulatory organs (Figs. 12, 15. 16, 17). 

The testes and ovaries are approximately one-half the length of the body, 
and anteriorly they reach nearly to the level of the statocyst. In front of the 
mouth, the testes form a considerable part of the body mass. They are located 
above the string of enlarging oogonia, and their distribution is lateral and dorsal 
to the endocytium (Figs. 19, 20). For about one-half of their length, the testes 
of the right and left sides are confluent dorsally. However, as the endocytium 
becomes limited to the dorsal part of the body and the oocytes beneath it become 
very large, the testes become distinctly separate, and finally they occupy only a 
very small portion of the body mass as seen in transverse sections. 

The genital pore (Figs. 15, 17) is located on the ventral surface near the 
beginning of the last quarter of the body. In living specimens, the genital atrium 
contains a number of more or less ovoid masses of granular material (Fig. 15). 
The source of these is not known. In sections of fixed specimens, the secretion 
within the genital atrium has the appearance of a vacuolated coagulum (Fig. 17) ; 
small granules which are stained by iron hematoxylin are scattered through this. 

The seminal vesicle (Figs. 15, 16, 17, 21) is located posterodorsal to the 
genital atrium. The sperm ducts leading from the testes enter it at rather widely 
separated points on its anterolateral surface. The wall of the seminal vesicle is 
thin but muscular. The penis, as seen in sagittal sections (Fig. 17), arches from 
the genital atrium through the upper part of the seminal vesicle, and its lumen 
communicates with the cavity of the seminal vesicle at the rear. The penis is 
invested externally by an outer layer of longitudinal muscles and an inner layer of 
circular muscles, and its lumen is provided with a number of separate and very 
delicate cuticular rods. Presumably, the penis is everted during copulation. The 
cavity of the seminal vesicle is filled with inactive sperm. It is crescentic in out- 
line when viewed from above, but it extends farther anteriorly near the ventral 
side of the seminal vesicle than near the dorsal side. Between the sperm mass and 
the penis there is a large accumulation of granules which are conspicuous in living 
specimens (Fig. 15) and which are stained strongly by hematoxylin (Figs. 16, 
17, 21). 

The mature sperm (Fig. 14) are about 110 \> long, and their structure is very 
interesting. Behind the thickened anterior region, a number of delicate cilia-like 
projections extend away from the sperm. The basal portions of these projections 
are appreciably thicker than the slightly longer outer portions. The posterior 
region of the sperm narrows gradually to a very fine tip. 

The vagina (Fig. 17) extends anterodorsally from the genital atrium. It 
is short, and a part of it is ciliated. Where the lumen terminates, there are glands 
which produce the elongate clusters of granules often noted anterior to the genital 
pore in living specimens (Fig. 15). When worms of this species are compressed, 
the clusters of granules may actually protrude from the genital pore. In sectioned 



160 



EUGENE X. KOZLOFF 



.- 




21 



J'late II Raf>liidopha!lus actuosns 

All figures were prepared with the aid of a camera lucida, but in the case of specimens 
drawn from life (Figs. 12-15), most details were sketched in free-hand. Figures 16-21 are 
based on sections (6 /*) of specimens fixed in Bouin's fluid and stained with iron hematoxylin 



NEW ACOELS FROM THE PACIFIC COAST 161 

preparations, only traces of the clusters can be detected in the anteriormost part 
of the wall of the vagina, and a brownish-yellow coagulum is noted in the lumen 
of the vagina near them. Between the glandular cap of the vagina and the seminal 
bursa, there is a syncytial mass within which the nuclei are rather close together, 
and through which sperm are probably forced into the bursa at the time of 
insemination. 

The holotype specimen, in the form of a set of serial frontal sections, has been 
deposited in the United States National Museum (USNM No. 32903). It was 
collected in the small inlet of North Bay which communicates with Argyle Lagoon, 
San Juan Island, Washington. 

This acoel can be referred to the family Convolutidae, and may be rather closely 
related to certain of the diverse species of Convoluta. The penis lies within the 
seminal vesicle in much the same manner as that of C. divae Marcus (1950), C. 
norvcglca Westblad (1946), and C. flavibacillwm Jensen (see Westblad, 1946), but 
the presence of numerous cuticularized rods within the penis is distinctive. I base 
the genus Raphidophallus largely on this combination of characters. 

Diatoinoi'ora amoena gen. nov., sp. nov. 

This relatively large species is usually found in washings of Ulva growing on 
muddy sand or gravel in the small inlet of North Bay which communicates with 
Argyle Lagoon. Samples collected at tide levels ranging from 1 to +4 ft. generally 
contain D. anioena; as a rule, it is more abundant than R. actnosus. I have also 
found it in washings of Ulva and Enteromorpha taken from muddy sand in South 
Slough at Charleston, Oregon (Lat. 43 20.4' N. ; Long. 124 19.5' W.) at tide 
levels of about +2 to +5 ft. 

When extended and gliding in contact with a firm substrate (Fig. 22), the body 
is about four times as long as wide. The largest specimens are about 1200 p, by 
300 fj,. The body is rounded anteriorly and tapers slightly toward the posterior end. 
Although the body is usually widest near the middle, the width remains almost 
constant in the second and third quarters. The greatest thickness, just behind the 
middle of the body, is nearly equal to the width. When the worms are gliding on a 
firm substrate, the ventral surface is flattened ; when swimming free, the body 
becomes nearly cylindrical. 

(some preparations were counter stained with orange G) ; certain details were supplied from 
adjacent sections in the same series. For abbreviations, see legend for Figures 1-11. 

FIGURE 12. Specimen in contact with substrate ; dorsal view. 

FIGURE 13. Rhabdites near left margin of body at the level of the statocyst. 

FIGURE 14. Mature sperm. 

FIGURE 15. Posterior end (specimen slightly compressed under a coverglass) ; ventral view. 
The masses of granular material shown next to the genital pore lie within the genital atrium. 

FIGURE 16. Frontal section. In the anterior part of the body, the section shows the region 
slightly ventral to the level of the statocyst and pore of the frontal glands ; one of the oocytes is 
undergoing a maturation division. 

FIGURE 17. Median sagittal section. 

FIGURE 18. Epicytium, epicytial glands, subepicytial musculature, and portion of testis in 
transverse section just anterior to mouth. 

FIGURE 19. Transverse section just anterior to mouth. 

FIGURE 20. Transverse section through nozzle of seminal bursa of same specimen. 

FIGURE 21. Transverse section through seminal vesicle and penis of same specimen. 



162 



EUGENE N. KOZLOFF 



* ' ' N 

g ' ' 

'^ . *> - : ' > 



:/ ?.; 

-< 



; a 



r - 



, i >*" i 



; 



r l -- : ...-, 



g .^ J 

!v . 1 : '-' ':' "."'..' ; 



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27 



I'lute III- Diatomovora unwcna 



All figures were prejiared with the aid of i camera lucida, but in the case of specimens 
drawn from life (Figs. 22, 23, 26), most details were sketched in free-hand. Figures 24, 25, 



NEW ACOELS FROM THE PACIFIC COAST 163 

The statocyst lies near the end of the first one-tenth of the body. In larger 
individuals, its diameter is about 25 /JL. The diameter of the hemispherical statolith 
is about 17 /A. 

As viewed with reflected light, the body (excluding the digestive endocytium) is 
whitish, tinged with yellow; the yellow color, concentrated in lipid droplets, is 
prominent in the anterior quarter, and sometimes also near the posterior end. Near 
the dorsal surface, there are generally three discontinuous streaks of bright white 
material ; these streaks originate behind the statocyst and diverge as they extend 
posteriorly. Some specimens have only one conspicuous streak along the midline ; 
in others, the lateral streaks are distinct and the median streak is diffuse. The 
digestive endocytium is usually green, although sometimes it is greenish yellow. 

In strong transmitted light, the ingested diatoms within the endocytium may be 
observed to turn from yellowish brown or yellowish green to a bluish green color. 
The endocytium as a whole is usually pale bluish green. Lipid droplets up to about 
15 ju. in diameter are scattered through the parenchyma, especially in the anterior 
quarter of the body. Some of these are quite yellow, and contribute to the yellowish 
cast noted in specimens examined in reflected light. The discontinuous streaks 
which appear white in reflected light are composed of greenish refractile granules 
(the larger of which are sculptured disks reaching a diameter of about 7 ju.) con- 
centrated in the ectocytium dorsal and dorsolateral to the endocytium. Under low 
magnification, the aggregates of these granules may appear blackish or brownish. 

The body is covered by cilia about 10 /* long. The bundles of rhabdites resemble 
those of R. actuosus; they are closely spaced and are arranged in rather definite 
rows. Most of the rows are short and merge with other rows or simply terminate 
as the neighboring rows on either side converge toward one another. 

The mouth is located on the ventral surface, just anterior to the middle of the 
body. It is capable of being considerably distended during the ingestion of food, 
which consists largely of diatoms and eggs of copepods, and during elimination of 
diatom frustules and other indigestible residues. 

The nervous system has not been studied in detail. The brain appears to 
consist of four major ganglia and their commissures. There are two ganglia on 
either side of the statocyst one anterolateral and the other posterolateral. These 

27-30 are based on sections (6 /a) of specimens fixed in Bouin's fluid and stained with iron 
hematoxylin and orange G (or eosin) ; in most cases, some details have been supplied from 
adjacent sections in the same series. For abbreviations, see legend for Figures 1-11. 

FIGURE 22. Mature specimen (slightly compressed under a coverglass) ; dorsal view. 

FIGURE 23. Posterior end (specimen slightly compressed under a coverglass) ; ventral 
view. The globules on the topographic right side of the penis are within the lumen of the vagina. 

FIGURE 24. Frontal section. Most details have been omitted, and the position of the vagina 
and seminal bursa, which lie some distance below the level of the structures traced, are 
rendered diagrammatically. 

FIGURE 25. Median sagittal section. The vagina and seminal bursa were drawn from a 
number of sections considerably to the left of the section traced. 

FIGURE 26. Mature sperm. 

FIGURE 27. Epicytium, epicytial glands, subepicytial musculature, and portion of testis in 
transverse section just anterior to mouth. 

FIGURE 28. Transverse section just anterior to mouth. 

FIGURE 29. Transverse section through anterior nozzle of seminal bursa of same specimen. 

FIGURE 30. Transverse section through region of genital pore and genital atrium of same 
specimen. 



164 EUGENE N. KOZLOFF 

are fused almost completely, and communicate with their counterparts on the other 
side by thick commissures. 

The frontal glands and their secretion (Figs. 24, 25) occupy much of the 
anterior fifth of the body. The pore through which the secretion is discharged is 
located at the anterior end of the body and is directed slightly downward. The 
glands within which the bundles of rhabdites are formed may be recognized within 
the epicytium (Fig. 27). 

The subepicytial musculature (Fig. 27) consists of an outer layer of circular 
muscles and an inner layer of longitudinal muscles. Parenchymal muscles are also 
present. These are rather abundant in the region of the frontal glands (Fig. 25) 
and in the region of the copulatory organs (Fig. 30). However, parenchymal 
muscles having a nearly dorsoventral orientation are also prominent lateral to 
the endocytium near the mouth (Fig. 28). 

In the region of the mouth, much of the body mass mesial to the testes and 
ovaries is occupied by the endocytial parenchyma into which food is ingested. 
The endocytium extends forward to the frontal glands (Fig. 25). Posteriorly 
it reaches beyond the anterior margin of the seminal vesicle, although it gradually 
becomes restricted to the dorsal part of the body mass, above the enlarging oocytes, 
seminal bursa, and seminal vesicle. The ectocytial parenchyma is rather thin where 
the endocytium is most extensive. It becomes appreciably better developed near 
the anterior end, and is very prominent in the posterior part of the body. Dorsal, 
lateral, and posterior to the copulatory organs, the ectocytium is characterized by 
large vacuoles (Figs. 22, 23, 24, 25, 30). 

The testes are distinctly separate. In the region just behind the frontal glands, 
the testis on either side of the endocytium occupies nearly a third of the body 
mass. Farther posteriorly, however, the oogonia which are ventral to each testis 
become progressively larger, and the testes become gradually more restricted 
(Figs. 28, 29). 

The genital pore (Figs. 23, 25, 30) is located on the ventral surface near the 
beginning of the last one-sixth of the body. The genital atrium has a ciliated 
epithelium and is externally lightly muscularized. The penis has a heavy external 
musculature consisting of an outer layer of longitudinal fibers and an inner layer 
of circular fibers. Its lumen is filled with globules which consist of small granules. 
In some preparations, these granules are stained distinctly by hematoxylin ; as 
a rule, however, hematoxylin seems to be removed from them by routine deslaining, 
and they become readily colored by eosin and orange G. 

The penis leaves the left side of the genital atrium and follows a nearly circular 
ascending path through the seminal vesicle. The cavity of the seminal vesicle is 
filled with inactive sperm and globules of the type observed within the lumen of 
the penis. The delicate sperm ducts passing posteromedially from the testes enter 
the seminal vesicle on its dorsolateral surfaces. 

The mature sperm (Fig. 26) are approximately 250 ^ long. From the thick- 
ened anterior portion, the body becomes gradually narrowed to a very fine tip. A 
conspicuous undulating membrane spirals around the anterior two-thirds of the 
sperm. Undulations of the more slender posterior region appear to be continuous 
with those of the undulating membrane, and perhaps the latter extends almost the 
entire length of the sperm. 



NEW ACOELS FROM THE PACIFIC COAST 165 

The vagina (Figs. 24, 25) has a thick tunic of circular muscles, and leaves 
the genital atrium ventral to the seminal vesicle. It is directed at first slightly to 
the right, then bends toward the midline on its course to the seminal bursa. For 
most of the length of the vagina, the lumen is distinct, and this may contain a 
number of globules of the type noted within the seminal vesicle and penis. Just 
before the vagina reaches the bursa, the lumen is obliterated by a syncytial mass con- 
tinuous with the syncytial wall of the vagina. In life, this mass often contains 
clusters of refractile granules, and is very similar to that which obliterates the 
lumen of the anterior part of the vagina in P. luteola. In fixed and sectioned 
specimens, it is vacuolated and the granules are not preserved. The seminal bursa 
is invested by a heavy coat of fibrous elements ; external to this, there are some 
widely-spaced muscles. 

The bursa has two cuticularized nozzles (Fig. 25) ; one of these is directed 
almost anteriorly, and the other is usually directed dorsally or anterodorsally. The 
bursa generally contains active sperm, but in some specimens it is filled with the 
granule-bearing syncytium which usually characterizes the anterior portion of the 
vagina. It appears likely that unless the bursa is distended by sperm forced into 
it at the time of insemination, it tends to collapse and thus appears to envelop the 
syncytial mass just behind it. 

Gland cells are extensively developed in the parenchyma around the copulatory 
organs, but I have not worked out the pathways by which the secretion or secretions 
of these glands are delivered to other structures. 

The holotype specimen, in the form of a set of serial sagittal sections, has been 
deposited in the United States National Museum (USNM No. 32904). It was 
collected in the inlet of North Bay which communicates with Argyle Lagoon, San 
Juan Island, Washington. 

Like the preceding species, this acoel belongs in the Convolutidae. Its long and 
highly muscular penis is somewhat similar to that of Aphanostoma macrospirijeriiiii 
Westblad (1946) and A. rhomboides (Jensen) (see Westblad, 1946). However, 
neither of these species has a seminal bursa. When a bursa is present in members 
of the genus Aphanostoma, it does not have a cuticularized nozzle, although West- 
blad has questioned the importance of this characteristic in setting Aphanostoma 
apart from Convolnta. The heavily mtiscularized vagina of D. amoena and the 
nature of its seminal bursa, which has two nozzles and a thick wall consisting of 
fibrous elements surrounded by muscles, are characteristics which have persuaded 
me to propose a new genus. 

SUMMARY 

Acoels belonging to three new genera are described. Parotocelis luteola is 
referred to the family Otocelididae. Raphido phallus actuosus and Diatomovora 
amoena are placed in the family Convolutidae. All of these acoels have been 
collected intertidally on San Juan Island, Washington, on substrates of muddy sand 
and gravel supporting growths of Ulva and Enteromorpha. D. amoena has also 
been found at Charleston, Coos County, Oregon. 

LITERATURE CITED 

Ax, P., 1959. Zur Systematik, Okologie und Tiergeographie der Turbellarienfauna in den 
ponto-kaspischen Brackwassermeeren. Zoo/. Jahrb., Abt. f. System., Okol. Geogr. 
Tiers, 87: 43-184. 



166 EUGEXK X. KOZL01-T 



, H. M., AND D. P. CosTF.i.i.o, 1 ( '38. A n f\v species of i'olycliacnts from the Pacific 

coast. . Inn. .M,i</. Nat. Hist., ser. 11,1: 148-155. 
HVMAN, L., 1 ( '5 ( ). Some Turbellaria from the coast of California. Aincr. Mits. Xnrit., No. 

1943. 
IVANOV, A., 1952. Beskishechnye turbelliarii (Acoela) iuzhnogo poberezh'ia Sakhalina. Trudy 

Zoologicheskogo Instituta Akadcmii Nauk SSSR, 12: 40-132. 
MARCUS, E., 1950. Turbellaria Brasileiros (8). Bol. Fac. Fil., Cicnc. Lcir. Unir. Sao Paula , 

Zoologia, No. 15: 5-191. 
.\[ARCUS, E., 1954. Turbellaria Brasileiros XI. Papcis Antlsos Depart. Zool., Sccr. Ayric., 

Sao Paulo, 11: 419-489. 
WKSTBLAD, E., 1946. Studien iiber Skandinavische Turbellaria Acoela. IV. Arkir f. Zooloqi, 

38A, No. 1. 
\\'KSTHI.AI), E., 1948. Studien iiber Skandinavische Turbellaria Acoela. V. Arklv f. Zoologi, 

41 A, No. 7. 



THE SEPARATION OF POST-BASICORONAL AREAS FROM THE 

BASICORONAL PLATES IN THE INTERAMBULACRA OF THE 

SAND DOLLAR, ECHINARACHNIUS PARMA (LAMARCK) 1 

PRASERT LOHAVANIJAYA a AND EMERY F. SWAN 
Department of Zoology, University of Neiv Hampshire, Durham, New Hampshire 03824 

One possible arrangement of the coronal plates of the central portion of the oral 
surface of the test of the sand dollar Echinarachnius parma (Lamarck) is shown 
as Figure 1. In this specimen the post-basicoronal interambulacral areas one 
through four are in contact with the basicoronal plates, but in interambulacrum five 
the post-basicoronal area has become separated from its basicoronal plate. Durham 
(1955) has indicated that the geologically younger genera of scutellinid echinoids 
tend to have the interambulacral columns separated from the basicoronal plates, 
whereas in the older genera these columns and plates are in contact. He also 
noted that in the Pacific Coast sand dollar, Dcndraster excentricus (Eschscholtz), 
a member of one of the younger or more advanced genera, very small or young 
individuals had their basicoronal interambulacral plates in full contact with the 
succeeding plates and that as growth proceeds, the second plate of each ambulacral 
column grows faster than the others and eventually separates the second inter- 
ambulacral plate from contact with the basicoronal interambulacral plate. Of all the 
species he studied for variation, Eclrinarachnius parma was found to be the most 
variable in respect to the separation of the interambulacral columns from the 
basicoronal plates. This study has been made with the aim of determining 
whether any pattern can be noted in this variation. With this in mind three 
questions are posed : 

1. How many areas lose contact, and to what extent does this vary among speci- 
mens within and between collections from different localities? 

2. Is there indication that there is any usual sequence among the areas in their 
loss of contact, and does this vary within and between collections from different 
localities ? 

3. Within areas retaining contact, are there differences in the amount of contact 
between first post-basicoronal plates "a" and "b" with the basicoronal plates? 
Is there any regular pattern of distribution of this asymmetry among the areas, and 
does this vary within and between collections from different localities ? 

MATERIALS AND METHODS 

Four series of specimens were collected intertidally, one series from each of 
the following places : Crow Neck, North Trescott, Washington County, Maine 
(44 52' 37" N., 67 07' 38" W.) ; Bailey's Mistake, South Lubec, Washington 

1 The greater part of this work was included in a dissertation submitted by the senior 
author to the University of New Hampshire in partial fulfillment of the requirements for the 
Ph.D. degree. 

- Present address : New England College, Henniker, New Hampshire. 

167 



168 



PRASERT LOHAVANIJAYA AND EMERY F. SWAN 



County. Maine (44 46' 23" N., 67 03' 16" W.) ; Hampton Beach, Rockingham 
County, New Hampshire (42 54' 07" N., 70 48' 40" W.) ; and Hampton Harbor, 
Rockingham County, New Hampshire (42 53' 59" N., 70 49' 07" W.). To 
minimize the possibility of the introduction of variability resulting from possible 
seasonal differences these collections were all made within as short a time period 
as feasible (September 12-15, 1962). Pertinent environmental characteristics of 
these collecting localities have been discussed by Lohavanijaya (1965). 



a 




a 



FIGURE 1. Oral surface of central portion of test of Echinarachnius par ma, showing contact 
or lack of contact between basicoronal interambulacral plates and first post-basicoronal plates. 
Stippled areas are interambulacral and white areas are ambulacral. 

More careful examination of the specimen shown in Figure 1 reveals not only 
that the post-basicoronal interambulacral areas 1, 2, 3, and 4 are in contact with 
their basicoronal plates, but also that the nature of this contact varies. In area 1 
the first post-basicoronal plate "a" is in contact but "b" has lost contact, whereas 
in area 4 the situation is reversed. In areas 2 and 3 both "a" and "b" remain in 
contact, but it looks as though "b" were approaching loss of contact in area 3 while 
in area 2 the degree of contact appears more nearly equal. 

In order to tabulate such variants for the large numbers of specimens studied, 
the following system of symbols has been devised. 

If both plates "a" and "b" of the first post-basicoronal interanilmlacrals are "in contact" 

with the basicoronal to an approximately equal degree, the condition is designated : + + 

If botli plates "a" and "b" are "in contact" but "a" is to a greater degree, the condition 

is designated : + ~ 



VARIATION IN SAND DOLLARS. II. 



169 



If both plates "a" and "b" are "in contact" but "b" is to a greater degree, the condition 

is designated : \- 

If only plate "a" is "in contact," the condition is designated : + Q 

If only plate "b" is "in contact," the condition is designated : O + 

If both plates "a" and "b" are "out of contact," the condition is designated : O O 

Such data were compiled for the five interambulacral areas for a total of 1280 
specimens. There were a few specimens for which these relationships could not 

TABLE I 

The nature of contact between first post-basicoronal interambulacral plates and basicoronal plates for 

the five areas of the oral surface of the test for series of specimens from four localities. 

N = the total numbers of specimens in each series. The numbers in the bulk of the 

table represent the numbers of specimens having each possible type of contact 

or absence of it (00) for each interambulacral area 



Area 


a b 

+ + 


a b 

H 


a b 
+ 


a b 



a b 
+ 


a b 

- + 


N 


BM* 1 


2 


56 


72 


161 


1 


1 


293 


2 


32 


19 


5 


85 


36 


116 




3 


52 


94 


26 


89 


11 


21 




4 


2 








116 


98 


77 




5 


7 


2 


2 


210 


14 


58 




CN* 1 


8 


73 


106 


105 


2 


1 


295 


2 


30 


17 


5 


68 


79 


96 




3 


61 


71 


39 


53 


19 


52 




4 


2 


3 


1 


88 


109 


92 




5 


8 








225 


28 


34 




HH* 1 


11 


149 


76 


68 





7 


311 


2 


50 


67 


7 


60 


22 


105 




3 


72 


115 


21 


46 


2 


55 




4 


7 


3 





65 


70 


166 




5 


34 


10 


2 


89 


41 


135 




HB* 1 


39 


171 


68 


44 


37 


11 


370 


2 


122 


58 


6 


31 


19 


134 




3 


151 


89 


10 


11 


15 


94 




4 


39 


18 


1 


58 


56 


198 




5 


88 


9 





62 


57 


154 





BM Bailey's Mistake; CN Crow Neck; HH Hampton Harbor; HB Hampton Beach. 



be determined, a few that were malformed, injured, or otherwise so abnormal that 
they were not considered typical, and a very few so far from the usual sizes within 
each series that they were considered unusual. The specimens (23) in these cate- 
gories have not been included in this study. The spines and the superficial organic 
material on the oral surface of the test were brushed off thoroughly. Then, in 
order to make the sutures separating the plates more readily visible, water was 
applied to the test. For determination of the nature of the contact between the 
basicoronal plates and the post-basicoronal areas, examination of the specimens 



170 



PRASKRT LOHAVANITAYA AND EMERY F. SWAN 



TAIU.K H 

Comparison of frequency of occurrence of specimens with the "normal," "1st order" and "2nd order" 
deviant arrangements of interanibulacral areas "out of contact" for specimens with 0, 1, 2, 3, 
4 or all areas "out of contact" for Echinarachnius parma from the four localities studied 



Series 


* of 
specimens 


' , t 
collection 


# of 
areas "out 
of contact" 


Normal sequence 


Deviant sequences 


# 


%* 


1st order** 


2nd order** 


# 


% 


$ 


% 


CN 

(15-70 mm.) 


74 
<M 
36 


24.7 
31.1 
12.0 




1 
2 


74 
91 
18 


100.0 
97.8 
50.0 


Not p 
2 
18 


issible 
2.2 
50.0 


Not possible 
Not possible 
0.0 




41 


13.7 


3 


20 


48.8 


15 


36.6 


6 


14.6 




34 


11.4 


4 


13 


38.2 


15 


44.1 


6 


17.6 




21 


7.0 


all 


21 


100.0 


Not possible 


Not possible 



Average number of areas "out of contact" per specimen = 529/299 = 1.77 


BM 
(50-90 mm.) 


68 
48 

45 


22.4 
15.8 
14.9 




1 

2 


68 
39 
29 


100.0 
81.3 
64.4 


Not p 
9 

14 


assible 
18.8 
31.1 


Not possible 
Not possible 
2 4.4 




58 


19.1 


3 


29 


50.0 


19 


32.8 


10 


17.2 




40 


13.2 


4 


18 


45.0 


15 


37.5 


7 


17.5 




44 


14.5 


all 


44 


100.0 


Not possible 


Not possible 


Average number of areas "out of contact" per specimen = 692/303 = 2.28 


HB 
(20-59 mm.) 


241 
56 
21 


65.1 
15.1 
5.7 



1 
2 


241 
33 
9 


100.0 
58.9 
42.9 


Not p 
23 
4 


assible 
41.1 
19.0 


Not possible 
Not possible 
8 38.1 




26 


7.0 


3 


19 


73.1 


4 


15.4 


3 


11.5 




14 


3.8 


4 


11 


78.6 


3 


21.4 





0.0 




12 


3.2 


all 


12 


100.0 


Not possible 


Not possible 



Average number of areas "out of contact" per specimen = 292/370 = 0.79 



HH 


182 


58.7 





182 


100.0 


Not possible 


Not possible 


(40-65 mm.) 


44 


14.2 


1 


24 


54.5 


20 


45.5 


Not possible 




26 


8.4 


2 


2 


7.7 


12 


46.2 


12 


46.2 




18 


5.8 


3 


8 


44.4 


2 


11.1 


8 


44.4 




22 


7.1 


4 


11 


50.0 


4 


18.2 


7 


31.8 




18 


5.8 


111 


18 


100.0 


Not possible 


Not possible . 



Average number of areas "out of contact" per specimen = 328/310 = 1.06 

" These percentages refer to the percentage of specimens with the indicated number of areas 
"out of contact" having the sequence indicated. 

** First order deviants are those presumed to be normal except for last one out of contact. 
Second order deviants are those combinations in which there appears to be aberrance in sequence 
in loss of contact prior to the last area involved. 



VARIATION IN SAND DOLLARS. II. 



171 



with a hand lens was necessary. Each series of specimens was divided into size 
groups at 5-mm. intervals (except where numbers were inadequate). The data 
thus obtained were tabulated. In Table I these data are summarized by locality and 
area of test but without breakdown into size groups. 

TABLE III 

Distribution of numbers of interambiilacral areas "out of contact" among different 
size groups of Echinarachnius parma from four localities 



Series 


Mean diameter 
J (L + W) 


No. of 

specimens 


Number of areas "out of contact" 


Mean 





i 


2 


3 


4 


5 


CN 


15-23.9 


10 


8 


1 


1 


, 








0.30 




27-34.9 


7 


4 


1 


1 


1 





. 


0.86 




35-39.9 


15 


5 


4 


2 


3 





1 


1.47 




40-44.9 


38 


11 


12 


2 


5 


6 


2 


1.71 




45-49.9 


42 


8 


10 


6 


10 


5 


3 


2.07 




50-54.9 


63 


12 


22 


11 


7 


8 


3 


1.78 




55-59.9 


67 


11 


24 


10 


7 


10 


5 


1.94 




60-64.9 


38 


11 


13 


2 


5 


4 


4 


1.76 




65-69.9 


19 


4 


7 


1 


3 


1 


3 


1.95 


BM 


50-54.9 


30 


7 


9 


3 


4 


6 


1 


1.87 




55-59.9 


66 


17 


9 


14 


7 


11 


8 


2.15 




60-64.9 


59 


21 


8 


4 


13 


5 


8 


1.95 




65-69.9 


52 


5 


11 


7 


11 


7 


11 


2.71 




70-74.9 


46 


6 


4 


11 


11 


4 


10 


2.72 




75-79.9 


40 


12 


5 


5 


10 


3 


5 


2.05 




80-84.9 


10 





2 


1 


2 


4 


1 


3.10 


HB 


20-24.9 


19 


18 


1 














0.05 




25-29.9 


24 


22 


2 














0.09 




30-34.9 


51 


39 


6 


3 


1 





2 


0.49 




35-39.9 


56 


37 


11 


3 


2 


1 


2 


0.66 




40-44.9 


91 


59 


12 


4 


9 


5 


2 


0.85 




45-49.9 


81 


46 


16 


5 


8 


3 


3 


0.95 




50-54.9 


38 


16 


7 


4 


6 


2 


3 


1.47 




55-59.9 


10 


4 


1 


2 





3 





1.70 


HH 


40-44.9 


15 


9 


2 


. . 


1 


? 


1 


1.20 




45-49.9 


75 


44 


12 


7 


4 


5 


3 


0.97 




50-54.9 


121 


71 


19 


8 


7 


7 


9 


1.07 




55-59.9 


83 


54 


7 


9 


4 


4 


5 


0.94 




60-64.9 


16 


4 


4 


2 


2 


4 





1.88 



NUMBERS OF AREAS OUT OF CONTACT 

In Table II several kinds of data are tabulated. The first column on the left 
indicates the number of specimens from the locality indicated having 0, 1, 2, 3, 4, 
or all areas "out of contact." In the next column these numbers have been 
converted into percentages of the total number of specimens used in this study 
from each locality. Then under the tabulations for each locality the average number 
of areas "out of contact" per specimen for the collection from the locality has been 



172 



I'KASERT LOHAVANIJAYA AND EMERY F. SWAN 



calculated. The average numbers of 1.77 for Crow Neck, 2.28 for Bailey's Mistake, 
0.79 for Hampton Beach, and 1.06 for Hampton Harbor suggest that the Maine 
localities have populations that are more progressive in this respect than are those 
from the New Hampshire sites. Noting the size ranges from the localities (indi- 
cated on the table under the initials for the name of each locality) and recalling that 
numbers of areas "out of contact" presumably increase as the animals grow, one 
is immediately beset with the question : Are these differences the result of differences 
in environmental induction or selection on the one hand, or are they wholly the 
result of the differences in size-composition among the collections? Table III and 



3.0 - 



2.5 



^2.0 

p 

1/3 
O> 



O 

2 

m 
rt 
3 
< 



1.0 



0.5 




10 20 30 40 50 

Mean diameter 1/2 (L+W) mm. 



60 



70 



80 



FIGURE 2. Relationship of the average number of areas "out of contact" to size 5 (L+W). 

Figure 2 have been assembled to show the mean numbers of areas "out of contact" at 
5-mm. size (mean diameter) intervals for each of the localities. It is obvious that 
much larger collections and smaller si/.e intervals would be needed to give smooth 
curves on the graphs, but it is quite apparent that : 

(1) For comparable mean diameters up to at least 55 mm. the mean number of 
areas "out of contact" is higher for the (.'row Neck collection than for either 
Hampton Beach or Hampton Harbor, and 

(2) In the range of diameters between 55 and 70 mm. there appears possibly to 
be a tendency toward equal numbers of areas "out of contact" for all the localities. 
Thus for mean diameters of 62.5 mm., the mean numbers of areas "out of contact" 
for the collections from Crow Neck, Bailey's Mistake, and Hampton Harbor all 



VARIATION IN SAND DOLLARS. II. 



173 



fall within the range between 1.75 and 2.00. Although none of the specimens 
from Hampton Beach is this large, extrapolation of the plotted values for smaller 
sizes into this range would place the expected value for this locality very close to 
2.00 areas "out of contact." 

APPARENT SEQUENCE OF Loss OF CONTACT IN INTERAMBULACRAL AREAS 

All possible combinations of areas "out of contact" were listed, and for each 
locality the number of specimens having each combination was tallied. Examination 
of these data along with Durham's (1955) Table 3 (p. 108) strongly suggested that 
the usual sequence in which interambulacral areas lose contact is 5, 1, 4, 2, 3. Thus, 
one would expect specimens "out of contact" for a single area to be most frequently 
"out of contact" in area 5. When two areas are "out of contact," areas 5 and 1 
should be the most frequent combination. This would continue, and the whole 
expected sequence would thus be 0^5-^5 & 1^5, 1, & 4^5, 1, 4, & 2^5, 1, 4, 2, & 3 

TABLE IV 
All possible combinations of normal, 1st order deviants, and 2nd order deviants 



Areas "out 
of contact" 


Normal 


1st order deviants 




2nd order deviants 




1 
2 
3 


All 

5 
5 & 1 
5, 1 &4 


Not possible 
1 ; 2 ; 3 ; or 4 

5, 2; 5,3; or 5,4 
5, 1, 2; or 5, 1, 3 


Not possible 
Not possible 
1 '? 1 VI 

1, - , 1, O , 1, 

1, 2, 3; 1, 2, 


4; 2, 3; 2, 4; or 3, 4 
4; 1, 3, 4; 2, 3, 4; 2, 3, 5; 2, 4, 5; 


4 
5 (all) 


5, 1,4&2 
All 


5, 1, 4 &3 
Not possible 


or 3, 4, 5 
1, 2, 3, 4; 1, 

Not possible 


2,3, 5; or 2, 3,4, 5 



( all areas). These combinations are hereafter called members of the normal 
sequence. Durham's (1955) data for his series from Woods Hole, Massachusetts, 
and the data here presented in Table II for Crow Neck, Bailey's Mistake, and 
Hampton Beach support this sequence, or at least the resulting combinations that 
may be obtained through it. Thus, these combinations of areas "out of contact" 
are the most frequently occurring combinations in the collections aforementioned. 
Durham (1955) noted that the small collection (21 specimens) he studied from 
Hampton Harbor exhibited great variation in respect to loss of contact among the 
interambulacral areas. In the present study 310 specimens were examined from 
this locale, and Durham's conclusion is abundantly supported as can readily be 
seen from examination of Table II. 

The variants from these usual combinations may be conveniently divided into 
two categories. Cases where combinations include areas "out of contact" in the 
normal combination, except for the area presumed last to lose contact, are termed 
"first order deviants." Thus, any specimen having one area "out of contact" other 
than area 5 would be a "first order deviant." "First order deviants" with two 
areas "out of contact" must have one of these areas 5, and the other must not be 
area 1. "Second order deviants" are those combinations which do not include the 
presumed penultimate area among those "out of contact." Thus, for specimens 



174 



1'K ASKRT LOU. \\.\XIJAYA AND EMERY F. SWAN 



with two areas "out of contact," a "second order deviant" must not include area 5 
among the areas "out of contact." In Table IV all possible combinations of 
"normal," "first order deviants." and "second order deviants" are indicated. All the 
theoretically possible combinations have actually been observed among the 1282 
specimens dealt with in this section, except 2, 3 and 4. 

Tables V and VI indicate the numbers of each particular deviant found in each 
collection. In Table II the occurrence of "normal," "first order deviant," and 
"second order deviant" combinations are totalled for each collection for each number 
of areas "out of contact." 

TABLE Y 

.\nnihei- f ^f>eci metis of 1st order deviants in each collection 



Areas "out 
of contact" 


1st order 
deviants 


CN 


BM 


HB 


HH 


Total 





Not possible 












1 


1 





3 


15 


7 






2 





3 


1 


6 






3 


1 


3 


1 


5 






4 


1 





6 


2 








2 


9 


23 


20 


54 


2 


5 ^ 2 


11 


6 





4 






5 K 3 


2 


2 


1 


2 






5 & 4 


5 


6 


3 


6 








18 


14 


4 


12 


48 


3 


5, 1 ,V 2 


7 


6 


3 


2 






5,' 1 ,V 3 


8 


13 


1 











15 


19 


4 


2 


40 


4 


5,1,4 & 3 


15 


15 


3 


4 


37 


5 (all) 


\:>t possible 










_ 














179 



Examination of these tables reveals that for the collections from Crow Neck and 
Bailey's Mistake, Maine, and Hampton Beach, New Hampshire, it is almost a 
generalization that for each number of areas "out of contact" there are more speci- 
mens with "normal" arrangements than with either first or second order deviant 
arrangements. The unique exception to this statement occurs among the specimens 
from Crow Neck with four areas "out of contact." Among these 38.2% have the 
"normal" arrangement and 44.1% have the only possible first order deviant arrange- 
ment that is, areas 5, 1, 4, and 3 "out of contact." There are, however, a few 
other situations (numbers of areas "out of contact" for given localities) where the 
sum of the first and second order deviants exceeds the number of "normal" individ- 
uals. But for the exception noted above, however, in no case does the number 



VARIATION IN SAND DOLLARS. II. 



175 



of individuals with any specific deviant even approach the number of "normal" 
individuals in these localities. 

For the collection from Hampton Harbor the situation is quite different. 
Although there is still a majority of these specimens with one area "out of contact" 
having the normal area 5 "out of contact," the percentage of these is much lower 
than found for the Maine localities and somewhat less than at Hampton Beach. 
In the group with two areas "out of contact" there is a total of only 26 specimens. 



TABLE VI 

Number of specimens of 2nd order deviants in each collection 



Areas "out 
of contact" 


2nd order 
deviants 


CN 


BM 


HB 


HH 


Total 





Not possible 












1 


Not possible 












2 


1 &2 








1 


1 






1 &3 











2 






1 &4 





2 


5 


3 






2 &3 








1 


2 






2 &4 








1 


3 






3 &4 











1 











2 


8 


12 


22 


3 


1,2 &3 





1 





1 






1, 2 &4 





2 


1 


1 






1, 3 &4 





1 





1 






2, 3 &4 


















2, 3 & 5 


3 


4 





3 






2, 4 &5 


2 





2 


2 






3, 4 & 5 


1 


2 














6 


10 


3 


8 


27 


4 


1, 2, 3 &4 


1 








4 






1, 2, 3 & 5 


1 


5 





2 






2, 3, 4 & 5 


4 


2 





1 








6 


7 





7 


20 


5 (all) 


Not possible 

























69 



Of these only two (7.7%} have the "normal" arrangement. While not much 
significance can be attached to these small numbers, it is interesting to note that 
the first order deviant arrangements of areas 5 and 4, and 5 and 2 and the 
second order deviants 1 and 4, and 2 and 4 "out of contact" all exceed the "normal" 
arrangement. These conditions suggest a tendency for areas 4 and 2 to lose contact 
ahead of sequence. Durham's (1955) data for this locality also indicate the 
tendency for area 4 to precede area 1. Second order deviants for specimens with 
three or four areas "out of contact" are also exceptionally high in this collection. 



176 



PRASERT LOHAVANIJAYA AND EMERY F. SWAN 



Here again we suffer from small numbers, but the tendency could be readily ex- 
plained on the basis of deviant sequence's early in their development. 

Why the sequence of loss of contact among the interambulacral areas is so 
unusual at Hampton Harbor is a difficult question to approach. It seems in- 
comprehensible that the Hampton Harbor population is genetically isolated from 
those of Hampton Beach hardly a mile distant by interconnecting water. However, 
there still exists the possibility that even from a common gene pool and common 
reservoir of larvae, there could be a selective difference of survival among genotypes 

TABLE VII 

Nu tubers and percentages of specimens, asymmetrical around the interambulacral radii, having 

"b" (H and -\-(>) and having more contact with "b" than 
(0+ and h) according to area of test and locality of collection 



Area 


Series 


i and + 


+and - + 


No. 


% 


No. 


% 


1 


CN 


179 


98.4 


3 


1.6 




BM 


128 


98.5 


2 


1.5 




HB 


239 


83.3 


48 


16.7 




HH 


225 


97.0 


7 


3.0 


2 


CN 


22 


11.2 


175 


88.8 




BM 


24 


13.6 


152 


86.4 




HB 


64 


29.5 


153 


70.5 




HH 


74 


36.8 


127 


63.2 


3 


CN 


110 


60.8 


71 


39.2 




BM 


120 


78.9 


32 


21.1 




HB 


99 


47.6 


109 


52.4 




HH 


136 


70.5 


57 


29.5 


4 


CN 


4 


2.0 


201 


98.0 




BM 





0.0 


175 


100.0 




HB 


19 


7.0 


254 


93.0 




HH 


3 


1.3 


236 


98.7 


5 


(A 





0.0 


62 


100.0 




BM 


4 


5.3 


72 


94.7 




1115 


9 


4.1 


211 


95.9 




HH 


12 


6.4 


176 


93.6 



after metamorphosis. The other likely explanation is that the differences in 
environmental factors between these proximate localities may affect genetically-like 
organisms in such manner that they develop differently. Regardless of whether 
these differences are genetic or environmentally induced, there still remains the 
question as to what environmental factors might be involved. A somewhat similar 
problem concerning variation in the heart-urchin, Echlnocardhim cordatmn Pennant, 
it occurs in British and nearby waters lias been carefully studied by Nichols 
( \'H>2), who suggests functional advantages for the variants he studied and favors 
the explanation of differences between populations as resulting from differential 
selection. 



VARIATION IX SAND DOLLARS. II. 177 

ASYMMKTRY WITHIN I NTERAMBULACRAL AREAS 

Differences in the amount of contact between first post-basicoronal plates 
"a" and "b" with the basicoronal plates cause deviations from the symmetrical 
arrangements of plates on the two sides of the radius running through the middle 
of the area in question. 

Table I summarizes the number of individuals having the various types of 
contact, or lack of it, between the first post-basicoronal plates and the basicoronal 
plates for each of the interambulacra in the specimens collected from Bailey's 
Mistake and Crow Neck, Maine, and Hampton Beach and Hampton Harbor. 
New Hampshire. Inspection of this table indicates that in areas 1, 2, 3, 4, and 
5, plates "b", "a", "b", "a", and "a", respectively, appear to lose contact more 
frequently ahead of the other member of the pair. It can readily be seen from 
Table VII that the degree of preponderance varies among the areas and within 
areas among the collections from different localities. 

DISCUSSION 

The loss of contact between first post-basicoronal interambulacral plates and 
the basicoronal plates varies in respect to number of areas involved, apparent se- 
quence among areas, and in the asymmetry of contact within the areas which 
appear to be in the process of losing contact. The number of areas "out of contact" 
is subject to increase as the individual grows at least initially. Thus, specimens 
with (or populations averaging) more areas ''out of contact" may be thought of 
as being more advanced or progressive. This agrees with Durham's (1955) idea 
that primitive genera and species near the ancestral stock retain contact whereas 
more highly evolved taxa are characterized by increasing loss of contact. Among 
the regular echinoids Jackson (1912) on similar grounds considered the exsert 
condition of ocular plates to be primitive and the insert condition more progressive. 
From the studies of Jackson (1912), Vasseur (1952), and Swan (1958, 1962) it 
appears that for Strongylocentrotus higher salinities and lower temperatures go 
hand in hand with the more progressive development characterized by more ocular 
plates insert. For the tropical TripncHstes, however, Jackson's (1914) data 
suggest the opposite relationship with temperature. E. panna is essentially a boreal 
species, and the higher numbers of areas "out of contact" in the collections from 
Maine, indicating that they are more progressive, might suggest that this species, 
like Strongylocentrotus, attains a more progressive condition in cooler water. 
Much caution should be used, however, in making even tentative conclusions on tin- 
basis of these few data. One cannot determine a trend from two points (the 
New Hampshire series as compared with those from Maine) ; and when the mean 
number of areas "out of contact" is calculated for Durham's (1955, Table 3, p. 
108) series of E. panna from Woods Hole, Massachusetts, the value obtained is 
1.71. At first glance it is apparent that this figure is nearly up to the overall 
average value for Crow Neck, Maine; but when the effect of the size of the 
specimens is considered, conclusions based on comparison of these overall averages 
become obviously questionable. If the specimens Durham (1955) used for his 
Table 3 are the same ones used for Table 2, which were said to range from 50 
to 62 mm. in average diameter, they are in the size range where a convergence 



178 PRASKRT LOHAVANIJAYA AND KMKKY F. SWAN 

in numbers of areas "out of contact" occurs among the collections from Maine and 
N'ew Hampshire and thus indicate 1 little. 

This convergence, as shown in Figure 2. makes one wonder if the loss of contact 
by additional areas may not cease when a certain size or age is reached. As shown 
hy Jackson (1912) and verified by Swan (1958), this appears to be the case for 
ocular plates becoming insert in Strongylocentrotus drocbachlcnsls. The arrange- 
ment of points (Fig. 2) relating average numbers of areas "out of contact" to 
mean diameter for the sand dollars from Crow Neck certainly appears to suggest 
a curve becoming asymptotic to the base line at some value between 1.75 and 2.00 
areas "out of contact" for diameters above about 45 mm. For diameters of 45 mm. 
and less the "curve" for the population from Hampton Beach appears to be roughly 
parallel to that for Crow Xeck but is displaced toward lower numbers of areas 
"out of contact" at comparable diameters. There is no indication of flattening out 
<>f this curve at mean diameters near 45 mm., and no specimens were available for 
sixes that were appreciably above the diameters where the mean number of areas 
"out of contact" reached 1 .70. The size ranges of the series from Bailey's Mistake 
and from Hampton Harbor are such that they give little help toward answering 
questions, but the great fluctuation shown in the series from Bailey's Mistake in 
regard to numbers of areas "out of contact" intensifies another question suggested 
by the "curve" representing the Crow Neck population. If there is a limit beyond 
which no further areas lose contact, does the value of this limit fluctuate? If so, 
\\liy? These remain as problems for future attack. Before leaving this subject, 
we should be reminded of the fact that Durham's (1955) findings would suggest 
that in sand dollars new plates on the oral side of the test are added up to a 
certain small size, after which no more are added. The variation he notes in the 
numbers of these plates in /:. panua might be related to differences in the time at 
\\liich their addition ceases in different individuals. That the addition of coronal 
plates may cease before regular urchins die or cease growing is indicated by Hsia 
(1948) for two species of Tcnnwplcnnts. No work is known to the present authors 
which indicates whether or not the size or number of plates at which this occurs 
varies within the species from one population to another. Again the temptation 
to make comparisons with better known organisms in other phyla is strong. A 
great many studies have been made on the numbers of vertebrae, fin-rays, and 
other serially repeated structures in fishes; and generally it appears that longer 
developmental periods (i.e., slower growth through the critical stages in develop- 
ment) produce higher counts in meristic structures. Low temperatures, high 
salinities and low oxygen tensions have been shown to retard development and 
produce this effect. Much of the pertinent literature on this subject has been 
discussed and listed by Barlow (1961). That light may also affect the number 
of vertebrae formed appears to be the case in at least some instances (McHugh, 
N54). Perhaps it is no mere coincidence that Strongylocentrotus appears to 
progress further in its attainment of insert ocular plates in colder or more saline 
waters and that Echinarachnius tends to progress toward having more inter- 
ambulacral areas "out of contact" in eastern Maine than in Xew Hampshire. It 
would be interesting to check the numbers of plates on the oral surfaces of the series 
from colder and warmer water to see if those from colder water had a higher average 
number. 



VARIATION' IX SAND DOLLARS. II. 17') 

The sequence of 5, 1, 4, 2. 3 in which interamhulacral areas lose contact is of 
more than a little interest. Although in Strongyloccntrotus the normal sequence in 
which oculars hecome insert is I, V, IV, II with no record of all oculars insert, 
there are many genera of regular urchins where the normal sequence is V, I, IV, 
II, III (Jackson^ 1912). Jackson (1912, 1914), Vasseur (1952), and Swan (1962) 
have all noted that localities differ from one another in respect to the frequency with 
which aherrant variant comhinations of oculars insert occur in various species 
of regular urchins. Thus, the fact that one of the localities here studied (Hampton 
Harbor) is characterized by so many deviant arrangements of areas "out of contact" 
among its sand dollars is not surprising, but at present no explanation can be sug- 
gested. Swan (1962) has noted that certain aberrant variant arrangements of 
ocular plates insert in Strongyloccntrotus are indicative of ''situs inrcrsus." The 
possibility that some of the deviant arrangements of areas "out of contact" in 
Echinarachnius may also indicate such deep-seated reversals of asymmetry should 
be more carefully checked. Initial examination of the first post-basicoronal ambu- 
lacra! plates revealed no deviations from conformity with Loven's (1874) law (cf. 
p. 104, Durham, 1955) that would suggest a reversed pattern. If all specimens or 
any suspected of being reversed were cut frontally or examined with a fluoroscope, it 
should be possible to determine the course traversed by the digestive tract and get 
the best evidence from internal anatomy. 

The pattern of asymmetry around the central axes through the interambulacral 
areas is very strongly marked in areas 1, 4, 5, fairly strongly marked in area 2, 
and rather weakly marked in area 3. It is possible that the deviations from the 
usual arrangements here too might be symptomatic of the more deep-seated 
"situs inversiis." In some respects this study may be considered as an extension 
of Durham's (1955) notable work, which owed a great deal to the earlier thinking of 
numerous workers, of whom Loven (1874) and Jackson (1912) must be singled out 
as especially important. At the same time it is obvious that in the present work 
there are more new avenues of investigation suggested than problems completely 
solved. Workers desirous of making additional studies of variation in irregular 
urchins should, in addition to the approaches used here, become thoroughly ac- 
quainted with the methods of Kongiel (1938), Kermack (1954), Nichols (1959a. 
1959b, 1962) and Kier (1962). 

SUMMARY 

1. The general arrangement of plates on the oral surface of sand dollars is 
discussed. 

2. Variations in this arrangement as they occur in Echinarachnius panua from 
several New England localities are indicated. 

3. As this sand dollar increases in size, there is decreasing contact between the 
post-basicoronal interambulacral areas and the basicoronal plates. 

4. The usual sequence in which this contact is lost among the areas is 5, 1. 4, 2 
and 3, but all possible combinations of areas "out of contact" have been observed, 
except 2, 3 and 4. 

5. The average numbers of areas ''out of contact" for animals of comparable 
sizes vary among localities. 



ISO PRASERT LOHAVANIJAYA AND KMKKV F. SWAN 

<>. The asymmetry of loss of contact \vitliin the interambulacral areas has also 
been found to be highly variable. 

7. The possibility that these variations may be- related to differential environ- 
mental effects upon the rates at which different parts of the growth process occur is 
suggested. 

LITERATURE CITED 

HAKI.OW, G. YV., 1961. Causes and significance of morphological variation in fishes. S\st. Z<>1., 

10: 105-117. 
llrkiiAM, J. \V., 1955. Classification of Clypeasteroid Echinoids. U. of Calif. Pub!. Gcol. Sci., 

31: 73-198 + frontispiece and plates 3 and 4. 
HSIA, W. P., 1948. On the relations between the number of coronal plates and the diameter 

of the test in three species of sea urchins. Contr. lust. Zool. Natn. Acad. Pcipiin/, 

4:25-30. 
JACKSON, R. T., 1912. Phylogeny of the Echini, with a revision of Palaeozoic species. Man. 

Boston Soc. Xat. Hist., 7, 491 pp. + 76 plates. 
JACKSON, R. T., 1914. Studies of Jamaica Echini. /'<//>. Tortitfias Lab. (Pub!. No. 1S2 Cam. 

lust.}, 5: 139-162. 
KERMACK, K. A., 1954. A biometrical study of Micrastcr coranguinum and M. ( Isomicrastcr} 

scnoncnsis. Phil Trans. Roy. Soc.' London, 237B: 375-428 + plates 24-26. 
KIEU, P. M., 1962. Revision of the cassiduloid echinoids. Smithson. Misc. Coll., 144: 1-262. 
KOXGIEL, R., 1938. Rozwazania nad zmiennoscia jezowcow. (Considerations on the variability 

of Echinoidea. ) Annalcs Soc. gcol. Polofinc, 13: 194250. (In Polish with French 

summary and legends for figures and tables. English translation OTS 60-21506 

available from the Office of Technical Services, U. S. Department of Commerce, 

Washington 25, D. C.) 
LOHAVANIJAYA, P., 1965. Variation in linear dimensions, test weight, and ambulacral pores in 

the sand dollar Echinaracliniiis panua (Lamarck). Diol. Bull., 128: 401-414. 
LOVEN, S., 1874. fitudes sur les fichinoidees. Kon</l. Srcnska J'ctcnsk. Akad. Hand!., Stock- 
holm, licit.' scries, 11. 91 pp. + 53 plates. 
Mi HUGH, J. L., 1954. The influence of light on the number of vertebrae in the grunion, 

Lcurcstlics tennis. Copeia, 1954: 23-25. 
NICHOLS, D., 1959a. Changes in the chalk heart-urchin Micrastcr interpreted in relation to 

living forms. Phil. Trans. Roy. Soc. London, 242B: 347-437 + plate ix. 

NICHOLS, D., 1959b. Mode of life and taxonomy in irregular sea-urchins. Systematics Associa- 
tion Publication, No. 3, pp. 61-80. 7 figs. 
NICHOLS, D., 1962. Differential selection in populations of a heart-urchin. Ibid. Publ. No. 4, 

pp. 105-118, 8 figs. 
.SwAN, E. F., 1958. Growth and variation in sea urchins of York, Maine. J. Mar. Res., 17: 

505-522. 
S\VAN, E. F., 1962. Evidence suggesting the existence of two species of Strongylocentrotus 

(Echinoidea) in the Northwest Atlantic. Camid. J. Zoo!.. 40: 1211-1222. 
VASSEUK, E., 1952. Geographic variation in the Norwegian sea urchins, Strongylocentrotus 

droebachicnsis and 5". pallidns. Evolution, 6: 87-100. 



CHROMOSOMES OF TWO SPECIES OF QUAHOG CLAMS 

AND THEIR HYBRIDS 

R. WINSTON MENZEL AND MARGARET Y. MENZEL 1 

Oceanographic Institute and Department of Bioloi/ical Scienee, 
The Florida State University, Tallahassee. Florida 32306 

Two species of quahogs (clams of the genus Merccnaria, formerly Venus) 
occur along the Atlantic and Gulf coasts of North America. Abbott (1954) char- 
acterizes the two species as follows: The northern quahog, Merccnaria incrccnaria 
(L.), ranges from the Gulf of St. Lawrence to Florida and the Gulf of Mexico. 
It has a characteristic smoothish or glossy area on the exterior center of the valves. 
The interior of the valves is white and commonly has purple stainings. The entire 
lunule is three-fourths as wide as long. Two subspecies are listed: M. in. notata 
Say with external zigzag brown mottlings and M. in. tc.vana Dall, from the northern 
Gulf of Mexico, with large irregular coalescing flat-topped concentric ribs. The 
southern quahog, M. campechiensis (Gmelin), ranges from the Chesapeake Bay to 
Florida, Texas, and Cuba. It has a more obese shell and lacks the smooth central 
area on the exterior of the shells. The entire lunule is usually as wide as long. 
Rarely are there brown mottlings on the exterior of the valves, which are always 
white internally. 

It is often difficult to assign a specimen to either species if a single character 
is considered. Fast-growing specimens of M . mercenaries, less than about 25 mm. 
long, lack the characteristic glossy smooth area on the exterior of the valves. Meas- 
urements of length and weight of the two species, grown under the same conditions, 
have shown the small M . uicrccnaria to have heavier shells than M. campechiensis 
of the same length. ( Hherwise typical individuals of M. cainpechicnsis occur with 
internal purple shell stainings and with the brown mottlings of the subspecies M. in. 
notata. Often the lunule of 717. campechiensis is only three-fourths as wide as long. 

The two species hybridize readily in the laboratory (Loosanoff, 1954). This 
paper reports chromosome numbers and behavior in the two species and their 
hybrids at meiosis and early embryonic mitoses. 

MATERIALS AND METHODS 

Live specimens of M. mercenaria were secured from Connecticut, New York, 
Delaware, Virginia, North Carolina, South Carolina and the east coast of Florida. 
The southern quahog was obtained from North Carolina, the east coast of Florida 
and several localities along the Gulf coast of Florida from Tampa Hay northward. 
These clams have been used in several ways: for growth experiments (Menzel, 
1961a, 1962); for clam farming observations (Men/el. l'MI>; Menzel and Sims, 
1962) ; and as brood clams for observations on hybrids (Menzel, 1964). In addi- 

1 Contribution No. 207 from Oceanographic Institute, The Florida State University, 
Tallahassee, Florida. 

181 



182 R. WINSTON MENZEL AX I) MARGARET Y. MENZEL 






/ 
V 



* 
A 



I 




t 

I * S 
V l .v 



Jjf 

^ t 



FIGURES 1-5. 



. 



4 -. ;- 3 



* f* 

/ 






CLAM CHROMOSOMES 183 

tion, laboratory-reared hybrids have been available from the Biological Labora- 
tory, Bureau of Commercial Fisheries, Alilford, Connecticut, and from the marine 
laboratory of the Oceanographic Institute. 

Crosses have been made in our lal (oratory using as brood clams hybrids grown 
to sexual maturity here and the two species from localities listed above. These 
crosses include intraspecific crosses, reciprocal crosses of the two species (F/s), 
F 2 's of the hybrids 5 .17. campechiensis X J* .17. inercoiaria and reciprocal back- 
crosses of the latter F t to each species. All of the above combinations have been 
spawned and reared beyond metamorphosis and settling by the techniques of 
Loosanoff and Davis (1963). Observations of meiosis in eggs from F\ hybrids 
reported here were all made on hybrids from crosses between ,17. incrccnaria males 
from Connecticut and .17. campechiensis females from Florida. 

At intervals after spawning, the eggs and embryos were fixed in freshly mixed 
acetic alcohol (three parts absolute ethanol, one part glacial acetic acid) and stored 
in a freezer at 16 to 18 C. Several dozen embryos in a small drop of fixative 
were placed on a slide and air-dried or flamed. Several drops of iron-acetocarmine 
were added and allowed to stain for two minutes. A coverslip was added and excess 
stain removed by blotting. The coverslip was pressed firmly on the slide and 
the preparation was then heated judiciously over an alcohol flame to clear the cyto- 
plasm and further flatten the eggs. Such temporary squashes usually were examined 
at once with a Zeiss microscope equipped with an apochromatic optical system and 
phase contrast accessories, but they could be stored for several days at 2-4 C. 
without severe deterioration. 

Chromosomes prepared in this way were usually well spread but rather lightly 
stained. Substitution of aceto-orcein, propio-orcein, Gomori's chrom-alum-hema- 
toxylin and Feulgen staining did not result in significantly better preparations. 
Phase-contrast illumination of the acetocarmine slides was used routinely to enhance 
contrast and facilitate analysis. Stages of meiosis from metaphase I to telophase 
II and mitotic figures from early cleavage divisions were readily observed by this 
method. Because of the dense cytoplasm and tough egg membranes, the eggs were 
difficult to flatten sufficiently for microphotography. Hence, most of the stages 
described here are illustrated with drawings made with the aid of a camera lucida. 

Preparations from M. campechiensis were consistently better than those from the 
F x hybrid, which were in turn better than those from M . nicrccnaria. 

OBSERVATIONS 
Early embryology 

Eggs fixed from 15 seconds to 5 minutes after spawning contained oocyte nuclei 
at metaphase I regardless of whether sperm suspension had been added. If the 
eggs were not fertilized, the oocyte nuclei remained at metaphase I for 60 minutes 
or longer and then gradually degenerated in situ. In one unfertilized lot of eggs, 

FIGURES 1-5. Photomicrographs of meiotic metaphase I in clam eggs, phase-contrast 
illumination. Some of the bivalents are out of focus in each photograph. 
FIGURE 1. Mcrccnaria campechiensis, same nucleus as Figure 8, X 900. 
FIGURE 2. M. campechiensis, same nucleus as Figure 10, X 900. 
FIGURE 3. Individual bivalents of nucleus shown in Figures 2 and 10, X 1800. 
FIGURE 4. Fi hybrid, X 900. 
FIGURE 5. M. mercenaries, same nucleus as Figure 12, X 1800. 



1S4 



R. WINSTON MENZEL AND M ^RGARET \. MRXZEL 







" 1 " 



Vf 



V 




4' 






v 



i/ 








\fi * 



7 



l ; K,i'KK 6. Mi-tapliHsc of the second cleavage division in a lcrtilize<t egg of Mcrccmind 
cuinpccliicnsis, 38 chromosomes. Acetocarmine staining, X 1800. 

FIGURK 7. Metapliasc of normal first cleavage division, I-\. hybrid, 38 chromosomes. 
Acetocarmine staining and phase-contrast illumination, X 1800. 



though degenerating nictapliasc I configurations were present 20 hours 
after spawning. Jf an effective sperm suspension \vas added, meiosis proceeded 
rapidly, the first polar body appearing in 10 minutes, and nietaphase II in 15 niin- 
nte>. From nietaphase 1 through telophase II the sperm pronucleus was dis- 
cerned as an increasingly diffuse' nucleus lying at some distance from the meiotic 
spindle.^. Fusion of the egg and sperm proiiuclei was not identified with certainty 
but probablv occurred when the chromosome's ol both were in a mid-prophase 



CLAM CHROMOSOMES 185 

condition preceding formation of the first cleavage spindle. The first cleavage 
division of the zygote nucleus ensued as early as 20 minutes and usually within 
30 minutes after spawning. Subsequent cleavage divisions followed rapidly; eggs 
75 minutes after spawning and contact with sperm suspension often contained too 
many dividing nuclei for analysis. 

Systematic comparisons of the timing of development in the various types of 
fertilizations were not made. Preliminary observations suggested that fertilizations 
in which sperm from the F, hybrids were used (backcrosses to both parental 
species and F 2 ) were followed by somewhat delayed development. In one lot of F 2 
embryos fixed on October 27, 1964, first cleavage metaphase and anaphase were 
found in lots fixed 45-75 minutes after sperm contact. Among 18 embryos in which 
chromosomes could be counted. 5 were diploid, 4 triploid (Fig. 16), 7 tetraploid, 
one had a chromosome number (46) between diploid and triploid. and one egg had 
a diploid and a separate haploid nucleus, both at metaphase. Subsequent lots of 
eggs from similar fertilizations did not exhibit polyploidy (Fig. 7). Occasionally 
an early embryo with dividing haploid nuclei was observed in batches of eggs which 
had not been fertilized either because sperm were not added or because the sperm 
were ineffective. A careful comparison of rates of development under controlled 
conditions should be made. 

Chromosomes 

Both M. incrccnaria (Figs. 5, 11, 12) and M. cainpccliicnsis (Figs. 1-3, 8-10) 
have 19 pairs of chromosomes at metaphase I and 38 chromosomes at embryonic 
mitoses (Fig. 6). At metaphase I the chromosome pairs are small and slender and 
the chromatid split can often be discerned. Most of the chiasmata do not terminalize 
until the onset of anaphase. A typical nucleus from M. campechiensis (Fig. 9) 
showed 19 bivalents with 27 unterminalized and 8 nearly or completely terminalized 
chiasmata (1.89 chiasmata per chromosome pair). The metaphase I bivalents of 
M. mercenaria are similar, but in our material tended to be more compact and 
hence less easily analyzed for chiasma frequency and position. The F, hybrid was 
intermediate in this regard, some figures approaching those of M. campechiensis 
in clarity (Figs. 4, 13, 14). 

In the F, hybrid all the chromosomes were paired regularly as 19 homomorphic 
bivalents at metaphase I. The chiasma frequency was not conspicuously different 
from those of the parents. 

Later stages of meiosis proceeded conventionally in the two species and in the 
hybrid. In one batch of eggs from the hybrid, two anaphase I figures showed an 
apparent bridge between the two groups of chromosomes, one of which is 
shown in Figure 15. Since no fragments were found, the bridges probably resulted 
from lagging separation of chiasmata rather than from crossing-over within a 
heterozygous inversion. At anaphase I in both species and the hybrid the chromo- 
somes at one spindle pole were commonly more compact and darkly stained than 
those at the other. In our squashes we were unable to tell whether either the darker 
or lighter group was consistently destined to be included in the first polar body. 

The mitotic chromosomes of the first and second cleavage divisions were rather 
long and very slender (Figs. 6. 7) but tended to become shorter and more compact, 
at least at metaphase, in later divisions. I'.ecause of the rather high chromosome 



186 



R. WINSTON MH\/KL AND MARGARET Y. MENZEL 






8 




/> 



1 



r 



V 






S '. 



16 

8-16. ChroinoMHiir-, in fertili/ed clam 
ra Incida ; all X 900 except Figure 15, X 1125. 



n 



\ 



10 



13 






14 



Drauin.us maik- with tlu 1 aid of a 



CLAM CHROMOSOMES 187 

number and small cells, it was not practicable to count tbe chromosomes of individual 
nuclei after the second cleavage mctapliase. The individual chromosomes exhibited 
a rather wide range of relative lengths and of arm length ratios. Since the meiotic 
bivalents of the hybrid revealed no evidence of structural differences between tin- 
two species, detailed comparisons of mitotic karyotypes were not made. 

DISCUSSION 

The ease with which hybrids between M. nicrccnarla and .17. cainpecliicnsis 
can be made experimentally and the existence in nature of forms which can be 
interpreted as intermediate suggest that a certain amount of gene flow may occur 
between the two taxa. The homology and regular behavior of the chromosomes of 
the two species revealed at meiosis in the F i hybrid demonstrate that there is no 
gross chromosomal barrier to such gene interchange. 

Ability to exchange genes under experimental conditions does not, of course, 
imply that such interchange actually does occur in nature. Mayr (1963) has re- 
cently reviewed the mechanisms which may serve to keep populations separate 
even though they are sympatric in part of their range and can be successfully 
hybridized under experimental conditions. Porter and Chestnut (I960) suggested 
that in the region of Beaufort, North Carolina, where 717. incrccnaria is confined to 
inland bays and inlets and J\I. cawipechiensis to outer shallow neritic waters, the 
two populations may be effectively separated by differential tolerance to salinity. 
The preliminary observations of F._, and back-crosses suggest also that embryos of 
the species may have an advantage in rate of development over the hybrid offspring 
under certain conditions. 

Regardless of whether and to what degree hybridization and gene exchange 
between J17. mercenaria and 717. campechiensis occur in nature, results so far suggest 
that their hybrids could furnish an important source of variation for the selection 
of improved strains of clams for commercial production, especially for regions in 
which the commercially less desirable 717. campechiensis is naturally better adapted. 



The authors gratefully acknowledge initial financial support from the Graduate 
Research Council, Florida State University, and the assistance of Mrs. Clare Shier 
in preliminary cytological study. Numerous individuals are thanked for kindly 
furnishing brood clams. 

SUMMARY 

Chromosome numbers of n -- 19. 2n -= 38 are reported for Mercenaria mer- 
cenaria, M. campechiensis and their F l hybrids. Meiosis is normal in the hybrids 

FIGURES 8-10. Meiotic metaphase I in Mcrccmiria cainpcchicnsis. 19 bivalents, mostly with 
one or two unterminalized chiasmata. Figure 8 is the same nucleus as Figure 1 ; Figure 10, 
the same as Figures 2 and 3. 

FIGURES 11, 12. Meiotic metaphase I in M crcciuiria incrccnaria, 19 bivalents. Figure 12 is 
the same nucleus as Figure 5. 

FIGURES 13, 14. Meiotic metaphase I in the Fi hybrid, 19 bivalents, the chiasma frequency 
not conspicuously different from that of the parents. 

FIGURE 15. Anaphase I in the Fi hybrid showing one bridge or (more likely) pseudobridge. 

FIGURE 16. Metaphase of an aberrant triploid first cleavage division, F- hybrid, 57 unusually 
short chromosomes. 



188 R. WINSTON MF.XZEL i XI) M \ROARET Y. MENZEL 

and yields no evidence of chromosoiiu- nonhomology or structural rearrangements 
between the two parents. Hie hybrids produce functional eggs and sperm which 
result in normal fertilixation and earl\ embryonic divisions in reciprocal backcrosses 
and at least some Iv.'s. Xo gross chromosomal barrier to gene exchange appears 
to exist between the two species. 

LITERATURE CITED 

ABBOTT, R. T., 1 1 '54. American Seashells. 541 pp. D. Van Nostrand Co., Inc., New York. 
Loos AX OFF, Y. I... I n 54. Xew advances in the study of bivalve larvae. Aiuer. Scientist, 42: 

607-624. 
LOOSAXOKF, Y'. L., AMI IL C. DAVIS, 1963. Rearing of bivalve mollusks. In: Advances in 

Marine Biology, I, pp. 1-136. Academic Press, Inc. (London) Ltd. 

MAYR, E., 1963. Animal Species and Evolution. 797 pp. Harvard University Press, Cam- 
bridge, Mass. 
MKXZEL, R. W., l%la. Seasonal growth of the northern quahog, Merccnar/d nierceuarid, and 

the southern quahog, M. cdinpccliicnsis. in Alligator Harbor, Florida. Proc. Nat!. 

Shellfish. Assoc., 52: 37-46. 
MENZEL, R. \\'., l ( 'nlb. Shrlllish mariculture. Proc. (ailf and Caribb. Fish. Inst., pp. 195-199. 

14th Annual Session. 
MKXZEL, R. W., 1 ( '62. Seasonal growth of northern and southern quahogs, Mcrcciidrin 

incrct'iiitrid and M. cinnpccliit'iisis, and their hybrids in Florida. Proc. Xdtl. Shellfish. 

Assoc., 53: 111-119. 
MKXZEL, R. W., 1964. Observations of quahog clams and their hybrids. Amcr. Ziml., 4(3) 

[Abstract]. 
MKX/KL, R. \\'., AMI 11. W. SIMS, 1962. Experimental fanning of hard clams, Mcrccuana 

iiicrccnurid, in Florida. Proc. Nail. Slid! fish. Assoc., 53: 103-109. 
PORTER, H. J., AND A. F. CHESTNUT, 1960. The offshore clam fishery in North Carolina. 

Proc. Nut!. Shellfish. . Issoc., 51: 67-73. 



STUDIES ON TH K ( )( >PLASMIC SEGREGATION IN THE EGG OF Tl 1 K 

FISH, ORYZIAS LATIPES. III. ANALYSIS OF THE MOVEMENT 

OF OIL DROPLETS DURING THE PROCESS OF 

( )OPLASMIC SEGREGATION 

YOSHI T. SAKAI 1 

Department of Bioloiiy, Tokyo Metropolitan Unircrxlty, Tokyo, Jupun 

Ooplasmic segregation, which generally occurs following fertilization in fish 
eggs, leads to the formation of the blastodisc. Studies of this movement have heen 
done by Spek (1933; Corregotnis, Saliuo, etc.), Roosen-Runge (1938; Brachy- 
danio), Lewis (1949; Brack yd an io ), and Costello (1948; Nerds'), etc. 

Although much attention has heen paid to the protoplasmic movement in the 
yolk or endoplasmic region, observations on the movement in the cortical proto- 
plasmic layer (cortex) have been restricted to the eggs of Brachydanio (Roosen- 
Runge, 1938) and of (,'astcrostcits (Thomopoulos, 1953), neither of which have oil 
droplets in the cortex. In Orysias eggs, the protoplasm and yolk are well separated 
before fertilization and oil droplets are dispersed in the former. On fertilization, the 
oil droplets move toward the vegetal pole at a speed which can be measured 
accurately. 

The present paper deals with analysis of the pattern of the movement of oil 
droplets, both natural and injected, during the formation of the blastodisc in Oryzias 
eggs. 

PART I. MOVEMENT OF NATURAL OIL DROPLETS 

METHODS 

After fertilization in Oryzias. evenly dispersed oil droplets in the unfertilized egg 
cortex migrate toward the vegetal pole, fusing with each other, and finally assemble 
around the vegetal pole, turning the egg upside down within the chorion by buoy- 
ancy. Therefore, to prevent the rotation of the egg during observation, the egg must 
be placed with the animal pole down from the beginning, and fertilized ; it is photo- 
graphed simultaneously from the animal, vegetal and lateral sides at intervals of two 
minutes. The photographs of the egg are magnified 100-fold and superimposed to 
trace the movement of the oil droplets. 

In the polar views, the distance along the curved surface bet ween the oil droplets 
and the animal or vegetal pole is calculated from the tracings of the photographs. 
In the side view, the equator of the egg is taken as a reference line and the distance 
of the oil droplets from the line is calculated. Since the tracings of the moving 
oil droplets are almost parallel to the longitude of the egg. sidewise shifts of the 
droplet are neglected ( Fig. 1 ) . 

1 Present address : Dcpartnu-nt of Zoology, University of California, Berkeley 4, Calif. 

189 



190 



YOSHI T. SAKAI 
AP 




l-'li.l KK 1. 



Tracings of moving oil droplets during ooplasniic segregation. 
AP : animal pole; VP : vegetal pole. 



In the calculations, the egg is considered as a sphere. Since the egg is not 
strictly a sphere hut rather an ellipsoid of revolution, the error coming from the 
approximation must he determined. In Figure 2, S is a sector of a sphere and E is 
that of an ellipsoid of revolution, a and b being the major and the minor axes of the 




IMI.I KK 2. Procedure to obtain the- actual distance or the angle from an apparent distance 
on the surface of sphere and of ellipsoid of revolution. S: a sector of a sphere; E: a sector of 
.HI ellipsoid of revolution; A : position of an oil droplet; a: major axis of ellipsoid of revolution; 
h: minor axis of the ellipsoid of revolution; \: apparent distance of an oil droplet from the 
reference lin< (equator); y: actual distance on the: sphere; z: actual distance of x of the 
ellipsoid of revolution ; : the angle at the center of the sphere embracing y. 



OOPLASMIC SEGREGATION IN FISH EGG 



191 



latter. "A" represents the position of an oil droplet under observation and x is its 
apparent distance from the reference line, as expressed in an angle at the center 
embracing x ; y is an arc of the circle and z is a section of the ellipsoid, which are the 
actual distances along the curved surfaces corresponding to the apparent distance x 
of the sphere and the ellipsoid of revolution, respectively. In other words, the aim 
of the calculation is to obtain either y or z from x. 




-90 



TIME AFTER FERTILIZATION (MIN) 



FIGURE 3. Time courses of the migration of oil droplets in terms of (22 C.). Ordinate : 
the value of Q, taking an equator as ; abscissa : time after fertilization in minutes. AP : animal 
pole; VP: vegetal pole; EQ : equator; arrow: fusion of oil droplets. Alphabetic designation of 
the curves is for use in Figure 4. 

In the Orysias eggs used for the present measurements, the deviation of the 
ellipsoid from the sphere, a b/a, is not larger than 0.08. For simplification of 
the calculation, a b/a is taken as 0.10. On the basis of these figures, the deviation 
of z from y, z y/z, turns out to be less than 0.03, which means that the error 
involved in the approximation as a sphere is negligible. The measurement lor the 
region above \/3/2 b (60 in 6) is supplemented by the measurements in the polar 
views. 

Since an egg can be treated as a sphere, distances through which the oil droplets 
move can best be expressed in 8 because 9 is independent of individual fluctuation 
in the egg size. 



192 



YOSHI T. SAKAI 



K' -i I.TS 

Figure 3 compares the time course of the migration in H of oil droplets initially 
located at different regions of the egg. \\'itliin 2-4 minutes after fertilization, almost 
all the oil droplets shift transiently toward the animal pole to some extent (see the 
left end of Fig. 3). This shift is rather difficult to discern unless one is aware of this 
phenomenon beforehand. During this period, a decrease in the egg volume takes 
place in Oryzias as the result of the breakdown of cortical alveoli (T. Yamamoto, 
1940). However, since the distance is expressed in #. the decrease in the volume 
does not affect the measurement as long as the shrinkage occurs uniformly. Corre- 
spondingly to this stage when the animal region of the Oryzias egg is seemingly 



2.0 



1.0 



en 
O 



(T 







B 



o D 
^E 



-3 F 



20 



30 



40 



50 



60 



TIME AFTER FERTILIZATION (MIN) 

FIGURE 4. The ratio between the distance in ft of the reference oil droplet ( R ) from the 
base line (60) and the distance of a given oil droplet (A, B, C, D, E and F) from the 
line (60) at specified moments (a/'r, h/r, c/r, cl/r, e/r and f/r). Ordinate : ratios a/r, h/r, 
c/r, d/r, e/r and f/r; abscissa: lime after fcrtili/ation. 

contracting. Roosen-Runge ('1'MSj describes, in I'rarliydanio eggs, an increase of the 
egg diameter and flattening of the animal pole region, as observed from the upper 
side. .According to the present writer's observations of Hniclivdiniio eggs made at 
this stage from the side, the egg flattens and so does the blastodisc region. Whether 
or not this flattening of the blastndisc nrion means a contraction at the animal 
region cannot be said at present. 

In Oryzias, the oil droplets remain stationary thereafter for about 20 minutes, 
alter which the movement of the oil droplets is resumed and they move toward the 
vegetal pole. This movement is particularly striking on the animal half. 

< )n the other hand, the vegetal oil droplets, originally located within 30' in H 
from the vegetal pole or beyond 60 on the ordinate of Figure 3, continue to move 
toward the animal side' even alter the transient shift is over. Consequently, all the 1 



OOPLASMIC SEGREGATION IN FISH EGG 193 

droplets are assembled at the latitude of about 60 as a ring. It must be mentioned 
that the ring eventually reaches the vegetal pole after several hours, by the morula 
stage (omitted from Fig. 3). 

If the migration speeds of the oil droplets starting from various levels of the 
egg are compared at various moments after fertilization, it can be said that the 
higher the curve, the steeper the inclination, which means that the closer an oil 
droplet is to the animal pole, the faster it moves. 

Next, the latitude of -60 to which the oil droplets gather is taken as a base 
line, and the positions of seven droplets, A, B, C, D, K, F and R (Fig. 3) from the 
new base line are read in 9 (a, b, c, d, e, f and r) for 20, 30, 40, 50 and 60 minutes 
after fertilization. In Figure 4, the ratios a r, b/r, c/r, d/r, e/r and f/r at the 
specified moments are plotted. As is clear, the ratios for respective droplets remain 
almost unchanged during the migration. This means that these droplets approach 
the base line at a speed proportional to the original distance of the oil droplet from 
the base line, theoretically reaching the base line simultaneously, which is more or 
less what is observed. 

The tracings of the oil droplets look as though the droplets might be attached 
to a stretched rubber membrane and carried to\varcl 60 in by the snapping of 
the membrane, as the result of breaking at the two poles. 

As is well known, the oil droplets frequently fuse with each other during the 
migration. When this happens, the speed of the fused droplet comes close to that of 
the slower or larger partner (see Fig. 3). 

DISCUSSION 

Transient shift of oil droplets toward the animal pole immediately jollowiny 
activation 

From the previous study by the author (Sakai, 1961 ), the unfertilized egg of 
Oryzias, deprived of the chorion, is flattened when observed from the side. On 
fertilization or activation, the egg is further flattened in the region where the 
cortical response is taking place. This flattening (decrease of the tension at the 
surface) spreads from the activated point with the wave of breakdown of cortical 
alveoli. After 2-4 minutes, when the cortical change is almost completed, the egg 
begins to bulge again from the activated point (Sakai, 1961). 

In the fertilized egg enclosed within the chorion, while the tension at the surface 
is decreasing near the animal pole, the egg cortex on the vegetal side must still 
be adhering to the chorion because the cortical response has not yet taken place 
there. By the time the egg cortex at the vegetal pole detaches itself from the 
chorion with decreased tension, tension near the animal pole should have already 
begun to increase. If so, the egg cortex on the vegetal side must be pulled toward 
the animal pole and the transient shift of oil droplets toward the animal pole, 
mentioned in connection with Figure 3, becomes understandable. 

Stationary phase 

Within about 20 minutes after the completion of the cortical response, the naked 
egg bulges higher than before fertilization. This 20-minute period coincides with 
that of the stationary phase, so that it seems that oil droplets do not begin to move 



1"4 YOST1I i SAKA1 

until the tension at the surface reaches a certain level. Similarly, if a part of the 
yolk is sucked out from the egg about 15-20 minutes after fertilization, when oil 
droplets would begin to migrate under natural conditions, the egg is flattened and 
the droplets in the treated egg do not migrate until the egg rounds up again. In 
such eggs having lost a part of the \olk. the formation of the hlastodisc tends to he 
retarded and so does the accumulation of oil droplets at the vegetal pole. The 
recovery of the egg shape (recovery of the level of tension), therefore, seems 
to IK- essential for the initiation of the migration of oil droplets. These observations 
are of interest in connection with the fact that, in Brachydanio eggs, a protoplasmic 
movement inside tin- \olk and a counterstream in the protoplasmic coat begin only 
after the egg becomes exactly round (Roosen-Rtmge, 1938). However, no ex- 
planation is available concerning the manner in which a higher membrane tension 
and the bipolar segregation of the protoplasmic components are connected with 
one another. 

PART II. MOVKM KNT OF INJECTED OIL DROPLETS FOLLOWING ACTIVATION 

After analyzing the movement of the natural oil droplets, it is of interest to see 
how a droplet of oil foreign to the egg will move when it is introduced into the 
egg by injection. 

METHODS 

Salad oil (as a neutral oil; acid value (A.V.) - 0.22), liver oil (as an acidic 
oil; A.V. 0.52), and mineral oil (as a non-polar oil) were used as substances 
to be injected. 

To distinguish the injected oil droplet from the natural ones of the eggs, the 
oil to be injected was previously stained with Sudan III. By using a micro- 
manipulator, an oil droplet of a size similar to that of natural ones (20-70 p. in 
diameter) was injected into the cortical protoplasmic layer (cortex) of the un- 
fertilized eggs, either centrifuged or non-centrifuged, and of the fertilized eggs at the 
one-, two-, and 8-cell stages. When oil droplets are injected into the fertilized 
eggs, the chorion is previously removed by using the hatching enzyme of Oryzias 
(Sakai. l ( '0l ). Since the cortex of the unfertilized eggs is very thin, the tip of tin- 
injection needle sometimes misses it. If the oil happens to he injected into the yolk, 
the oil moves freely by gravity. If the oil is injected at too shallow a layer, the oil 
is apt to be squeezed out of the egg surface into the perivitelline space after the 
alveolar breakdown. As a result, successful injection can easily be determined. 

For measuring the movement of the injected oil droplet, the same procedure 
is applied as that which was used in I 'art I. 

RESULTS \\i> ( INCLUSION 

Hehai'ior <>\ the injected oil droplel 

The oil of Oryzins eggs is a kind of neutral oil because it is stained deeply with 
Sudan III and Sudan black, and also stained a pink color with Nile blue 
sulfate at about pH 7.0. Such a stainabilitv is the same as that of oil droplets of 
Onchdrynchus eggs (K. Yamamoto, 1 ( >58). 



OOPLASMIC SEGREGATION IN FISH EGG 



195 



To test another kind of neutral oil, salad oil is used. After the injection of salad 
oil, the eggs are fertilized or activated by pricking. Although some eggs are 
activated by the injection procedure itself, the behavior of the injected oil is much 
the same as that in the eggs activated after a successful injection in an inactivated 
condition. 

When injected at the equatorial region, the injected oil. on pricking, generally 
migrates toward the vegetal pole, fusing with the natural oil during the movement 
(Fig. 5). In fertilizable but slightly under-ripe eggs, merging of the natural oil 
droplets among themselves rarely occurs but even under such a circumstance, the 
injected oil droplets move to the pole side by side with the natural ones. Occa- 
sionally, the injected oil fails to move, probably owing to imperfection of injecting 
technique, in spite of the successful migration of natural oil droplets lying closer 
to the animal pole than the injected one. In such cases, natural droplets situated 




a 



FIGURE 5. Movement of oil droplet injected at the equatorial region of the unfertilized egg 
(side view). The egg is placed upside down to avoid the rotation of the egg caused by as- 
sembling oil droplets, (a) 20 minutes after fertilization; (b) about 40 minutes after fertilization; 
(c) one hour after fertilization. Injected oil has fused with the natural oil. 

on the animal half overtake the injected oil and pass it closely around its circum- 
ference. However, if the oil droplet migrates at all, it always migrates toward the 
vegetal pole, and never toward the animal pole. Quantitatively, too, the behavior 
of the injected oil corresponds well to that of the natural droplets as shown in Figure 
6. The frequency of the migration is less when the oil is injected close to the 
animal pole than when injected at the equator. 

To clarify whether or not the behavior of the injected oil varies with its prop- 
erties, similar experiments were repeated by using liver oil, mineral oil and the 
oil of Oryzias eggs. These experiments give substantially the same results as that 
of the salad oil. Judging from the fact that the injected oil of Oryzias itself some- 
times fails to move, it is most likely that the failure is not due to the properties 
of the oil but to disturbance caused by the injection technique. 

Relationship between movement of protoplasm and oil droplets 

From the foregoing results, the oil droplets migrate irrespective of the nature of 
the oil itself. However, this still leaves the possibility open that the migration of the 
oil droplets is somehow coupled with the movement of the protoplasm. To make 
sure of this point, the following two conditions were tested : ( 1 ) weakly centrifuged 



YOSHI T. SAKAI 

eggs (100-200 g for about three minutes), with the natural oil droplets shifted to 
one side but leaving the protoplasm undisturbed. (2) strongly centrifuged eggs 
(900-1800 g for 10 minutes), with both oil and protoplasm localized at tin- 
opposite sides. 

In non-injected eggs, shifting of the natural oil by weak centrifugation does not 
interfere with ooplasmie segregation, regardless of the abnormal localization of 




-90 



TIME AFTER FERTILIZATION (M IN) 



Fna'ki-; 6. Time courses of tlu- migration of inji-cti'd oil droplets in comparison \vitli the 
natural droplets. ( )rdinate : the value of ft, taking tin- equator as (I; abscissa: time after 
fertilization in minutes: closed circle: injected oil droplets: open circles: natural oil droplets; 
AI': animal pole; VI': vcjictal pole; KO : equator; arro\\ : fusion of oil droplets. 

the droplets after centrituging. In centrifuged eggs, the injection is made where 
the natural oil droplets arc no longer found, although protoplasm and cortical 
alveoli are still promt in the egg cortex. In spite of the absence of the natural oil 
droplets around the injected oil, it can migrate toward the vegetal pole all by itself. 
' >n the other hand, by strong centrifugation, the protoplasm of the unfertili/ed 
egg can be shifted in the animal region and the oil is massed at the vegetal pole bv 
.orienting the egg by a capillary tube-. As the result, a blastodisc is formed at the 



OOPLASMIC SEGREGATION IN FISH EGG 197 

centrifugal animal side where the protoplasm has already collected. The oil is 
injected at the equatorial region where little protoplasm is found. Under these 
conditions, the injected oil is fixed at the injected point and never migrates toward 
the vegetal pole within the observation period of three hours. 

To further confirm the idea that the migration of oil droplets is caused by the 
movement of the protoplasm, the oil is injected near the equator of the egg at the 
one-, two- and 8-cell stages in which the protoplasmic segregation has almost 
been completed. The injected oil droplets never migrate toward the vegetal pole. 
The relationship between oil and protoplasm is also pointed out by the following 
results. 

When an egg is forced into a capillary, both the migration of oil droplets and 
the formation of the blastodisc are much delayed. Furthermore, if more than one 
protoplasmic accumulation is induced by polyspermy or strong prickings, such 
protoplasmic accumulations are always accompanied by the migration of oil droplets 
toward the opposite side of each accumulation (Sakai, 1964a). Further, the 
experiments on partial activation indicate that the oil migration does take place 
only in the activated half (Sakai, 1964b). 

If the migration of the protoplasm has a leading role over the movement of 
natural oil droplets, the elimination of the oil droplets is expected to have no 
influence on the movement of the remaining protoplasm. In the eggs weakly 
centrifuged at 100-200 y for about 5 minutes just after the fertilization, the cortex 
^protrudes where the oil is forced to gather. Such a mass of oil can be sucked out 
with a micropipette. As in Nereis egg fragments observed by Costello (1940), yet 
the migration of the protoplasm can still occur and form the blastodisc. 

On the other hand, careful observation reveals that the protoplasmic movement 
always precedes that of the oil droplets, that is, by the end of the stationary phase, 
a small amount of protoplasm has already begun to accumulate at the animal pole, 
slightly flattening the yolk surface under it. 

Considering the above-mentioned results in connection with this observation, 
it will be concluded that the migration of oil droplets is a consequence of the 
movement of protoplasm toward the animal pole. 



The author is indebted to Professor K. Dan for his invaluable advice. The 
author's thanks are also due to Dr. M. Yoneda for his kind help in the calculations. 

SUMMARY 

The movement of oil droplets in Oryzias eggs, natural and artificially injected, 
was analyzed during ooplasmic segregation, (a) During 2-4 minutes alter fertili- 
zation, natural oil droplets are shifted transiently toward the animal pole, followed 
by a stationary phase of about 20 minutes. After this phase, all of the oil droplets 
coming either from the animal or from the vegetal side assemble at about 60 
below the equator as a ring and later reach the vegetal pole. The migration is 
faster in droplets coming greater distances than in those coming shorter distances, 
(b) The pattern of the migration of injected oil droplets is the same as that of the 
natural ones, irrespective of their nature. The migration is possible in weakly 
centrifuged eggs in which the protoplasm remains undisturbed in the cortex. 



198 YOSHI T. SAKAI 

However, injected oil droplet. s no longer move after shifting of the protoplasm 
1>\ strong centrifugation or after the completion of ooplasmic segregation. 

LITERATURE CITED 

COSTKU.II, 1). I'., 1'MO. Tlu- iVrtili/ahility of nucleated and non-nucleated fragments of cen- 

trifugcd \ereis eggs. ./. .I/or/ 1 /;., 66: 99-114. 
COSTKI.I.O, I). I'., l')4S. Ooplasiiiic MUD Cation in relation to differentiation. Ann. Xcw York 

Acad. Sri., 49: 663-683. 

LEWIS, \\'. H., 1949. Superficial gel layers of cells and eggs and their role in early develop- 
ment. Ann. I nst. liiuloiiiti. Me.rico, 20: 1-14. 
Roi>sK.\-Rr.\i;K. E. C., 1938. On the early development bipolar differentiation and cleavage 

of the zebratish, Hrtieliyddiiio rerio. Riol. Bull., 75: 119-133. 
SAKAI, Y. T., 1961. Method for removal of chorion and fertilization of the naked egg in 

Oryzius hi fifes. Embryologia, 5: 357-368. 
SAKAI, V. T., l l '64a. Studies on the ooplasmic segregation in the egg of the fish, Oryzlas Infixes. 

I. Ooplasmic segregation in egg fragments. Einlvynlin/ui. 8: 129-134. 
SAKAI. Y. T., 19641). Studies on the ooplasmic segregation in the egg of the fish, Oryzius 

latipcs. II. Ooplasmic segregation of the partially activated egg. Embryologia, 8: 

135-145. 
Si'KK, T., 1933. Die bipolare Differenzierung des Protoplasmas des Teleosteer-Eies und ihre 

luitstehnng. I'rotophtsma, 18: 497-545. 
Tiio.Moi'ouLOS, A., 1 ( )53. Sur 1'oeuf de 1'Epinoche (Gasterosteus ticnlctitiis L.). Hull. Sac. 

Zool. France, 78: 142-149. 
YAMAMOTO, K., 1958. N'itellogenesis in fish eggs (in Japanese). S\mposia Cell Chan., 8: 

119-131. 
YAMAMOTO, T., 1940. The change in volume of the fish egg at fertilization. Proc. /;;;/>. Acud. 

(Tokyo), 16: 482-485. 



GROWTH AND SURVIVAL OF POSTLARVAL PENAEUS 

AZTECUS UNDER CONTROLLED CONDITIONS OF 

TEMPERATURE AND SALINITY * 

ZOULA P. ZEIX-ELDIX AXD DAVID V. ALDRICH 
Bureau oj Commercial Fisheries, Galveston, Texas 

Temperature and salinity may be considered among the most important abiotic 
factors influencing the growth and survival of much of the estuarine fauna. They 
are of particular significance to those organisms that spend certain portions of their 
life cycle in the open sea where both factors are relatively stable, and other 
portions in the estuarine areas where both temperature and salinity may change 
drastically. Although temperature is generally thought to overshadow salinity in 
its effects on migratory organisms, salinity, probably through its osmotic effects, also 
plays a part in limiting some organisms to specific environments. 

Several investigators have attemped to evaluate the importance of temperature 
and salinity to penaeid shrimp of the Gulf of Mexico, but ecological questions con- 
cerning these factors remain unanswered. Although field studies have dealt with 
the relationship of shrimp to salinity, the conclusions reached have differed widely 
enough to warrant further investigation. The interpretation of observations on 
salinity and shrimp abundance in nature is made difficult by changes in other 
environmental factors, some of which frequently vary with salinity. Such factors 
include temperature, light, substrate, food supply, cover and pollution. For this 
reason, controlled-environment studies in the laboratory were employed in the 
present work. 

In an earlier study, Zein-Eldin (1963) determined that under conditions of 
constant temperature and somewhat restricted food supply, grooved Pcnacits post- 
larvae - survived and grew over a wide range of salinity (2-40/e). However, it has 
been suggested that in other migratory Crustacea, notably in the European shrimp, 
Crangon crangon (Broekema, 1941), as well as in juvenile and adult brown and 
pink shrimp, Penaens aztccus and P. diioranun, respectively (\\illiains, i960), 
temperature can influence tolerance to salinity. Thus, further studies were designed 
to test the combined effects of temperature and salinity on the survival and growth 
of postlarval brown shrimp. 

MATERIALS AND METHODS 

The work was of two types, 24-hour survival studies and a 28-day growth 
experiment. For all work, postlarval brown shrimp of approximately 12 mm. 
rostrum-telson length were seined from the Gulf of Mexico surf at the entrance to 
Galveston Bay. The animals were held in the laboratory in aerated water of ap- 

1 Contribution No. 205, Bureau of Commercial Fisheries Hi<)1<>ical Laboratory, Galveston, 
Texas. 

2 As denned by Renfro (1964). 

199 



200 ZOULA P. XKIX-ELDIN AND DAVID V. ALDRICH 

proximately 25',, and 25 ('. for at least 24 hours prior to use. Few mortalities 
occurred during this preliminary holding period. 

The fir.M ohjirti\-c was to ohtain a rapid, crude estimate of postlarval tolerance 
to salinity and temperature in order to provide guidelines for the more detailed and 
sensitive growth study to follow. Accordingly, we selected short-term survival as a 
rough index suited to our needs. 

To determine tin- short-term tolerance of brown shrimp to salinity and tem- 
perature, we exposed groups of experimental shrimp to different levels of the two 
factors for 24 hours. The test levels were chosen to include and extend above and 
below the ranges of salinity and temperature at which large numbers of postlarvae 
have been observed in nature (Bearden, 1'Xil ; Williams. 1955; Baxter, un- 
published ). 

Temperature control of 0.5 C. was maintained by B.( ).D.-type incubators. 
Salinity changes were effected by replacing portions of water in the test containers 
with equal volumes of either distilled water or evaporation-concentrated sea water. 
Salinity was determined by hydrometer and reported to the nearest part per 
thousand. Four series of 24-hour survival experiments were carried out with 
groups of 5 to 30 animals as described below. Series 1, 2 and 4 had an initial salinity 
of 2425',, and an initial temperature of 24 to 2(> C.. matching conditions of 
the holding aquaria. Initial salinity in Series 3 was 40'u, equal to the unusually 
high level at which animals for that series were collected. Following introduction 
of the shrimp into the vigorously aerated experimental containers, stepwise changes 
in both temperature and salinity (0 to 8 steps, depending on the magnitude of change 
involved) were made over a 10- to 12-hour period, to reach the desired conditions. 
The attainment of these conditions marked the beginning of the 24-hour test period. 
At the end of that time, the beakers were removed from the incubators and the 
live and dead postlarvae counted. Failure to show either spontaneous or probe- 
induced activity upon return to room temperature was considered indicative of 
death. No food was provided during the experiment. 

Previous observations of postlarvae in the laboratory had indicated that failure 
to keep them under water mechanically could lead to considerable mortality, due 
to their jumping activity. This type of loss was avoided during the first two series 
by restraining each group of animals in a one-liter beaker whose mouth was filled 
with a stemless funnel 4 inches in diameter. When the 1 beaker was tilled with water, 
all air space .accessible to the shrimp was eliminated, thereby preventing the animals 
from escaping the vessel or adhering to its drv surfaces. Aeration was provided by 
means of an air stone attached to , :i (; -inch ( ).l). Tygon tubing lifted through the hole 
in the funnel. 

In the first two series, we employed only live shrimp per group, hoping thus to 
reduce cannibalism. Although losses caused by escape from the water were success- 
fully avoided, test results indicated that cannibalism occurred at intermediate and 
higher temperatures where the number ot survivors plus the number of dead 
animals remaining per group frequently totaled less than the original number of 
shrimp (Table I ). In such cases, we attempted to discriminate between deaths due 
to cannibalism and mortalities attributable to salimt\ and temperature bv arbitrarilv 
assuming that salinity-temperature combinations causing the rapid death of some 
experimental shrimp were sufficiently severe to inhibit feeding, including cannibal- 



GROWTH AND SURVIVAL OF P. AZTECUS 



201 



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202 



ZOULA I'. ZEIN-KI.DIX \.\l) DAVID V. M.DRHll 



ism. among the- survivors, High rates of penaeid activity, including movement, 
feeding, and molting, have heen observed at temperatures of 25 and 32 C'. in this 
laboratory. Such activities favor cannibalism among shrimp under relatively 
crowded experimental conditions. ( >n this hasis, we counted as mortalities only 
those dead animals visibly present at the end of each 24-hour experimental period. 

To test this assumption. \ve required data unaffected by cannibalism. These 
were obtained in Series 3 by confining each animal within a 11-inch length of 14-mm. 
I'vrex tubing, both ends of which were covered with cotton gauze held in place with 
small rubber bands. While permitting contact between the animal and its experi- 
mental aquatic environment, this procedure prevented "jump-out" losses and made 
physical contact between shrimp impossible. Ten postlarvae thus isolated were 
placed in each test beaker and the experimental conditions established as before. 
The survival results agreed well with those of the two earlier series as interpreted 
above, tending to substantiate our assumption regarding the effect of cannibalism. 

TABU: 1 1 

Schedule nf salinity and temperature changes [initial salinity and temper/it lire 

26%o and 23 C., respectively"] 



Klapsed 
time 
(hr.) 


1 v-iu-d salinity (%o) 


1 >rsired temperature ( C.) 


2 


5 


15 


25 


35 


1 1 


18 


25 


30 


Actual salinity 


Actual temperature 


2 


20 


20 


20 


25 


25 


23 


23 


23 


26 


8 


15 


15 


15 


25 


30 


19 


22 


24 


30 


24 


10 


1(1 


15 


25 


35 


17 


19 


25 


33 


36 


5 


5 


15 


25 


35 


12 


18 


25 


33 


48 


2 


5 


15 


25 


35 


11 


18 


25 


32 



In a fourth series, further survival data were sought at temperature-salinity com- 
binations which seemed to be near the extremes of postlarval tolerance, as 
suggested by results of the three previous series (Table I). In this series the 
importance of acclimation was also estimated. Fach set of experimental conditions 
was duplicated in two two-liter beakers, one for shrimp acclimated as usual, the other 
for animals which were transferred directly from the holding tank to the extreme 
salinity and temperature levels to be tested. Thirty individually confined postlarvae 
were held in each beaker, and 24-hour survival determined as before. 

For the growth study. 46 liters of brackish water and 100 animals were placed 
in each of twenty 15-gal. aquaria. Filtration, aeration, and confinement were 
accomplished as previously described (Zein-Eldin, 1963). .Five aquaria were 
placed in each of tour constant-temperature rooms. The experimental temperatures 
were changed from the initial 23 C. to 11. 18, 25". or 32 C. Water tem- 
perature, although 0.5 C. in a given aquarium, varied as much as 1 C. among 
aquaria in a single room. The initial salinity of 23',, was simultaneously adjusted 
stepwise with temperature, to final levels of 2',,. 5',,, 15',,, 25',,, or 35',, (Table 
J I ) . Fach lank was continuously illuminated bv two 40-w. fluorescent lamps. 



GROWTH AND SURVIVAL OF P. AZTECUS 



203 



Postlarvae were fed live brine shrimp (Arteuiia} nauplii throughout the growth 
experiment. The nauplii in a 0.1-ml. sample of brine shrimp in water were counted 
to estimate number per unit volume, and the volume of food recorded at each 
feeding of the postlarvae. Artcmia nauplii were filtered and washed with distilled 
water before their addition to the tanks, in order to avoid increases in experi- 
mental salinity levels. Live brine shrimp nauplii were present in excess in all 
aquaria during the first 24 days of the experiment. During the last four days at 
32 C., however, the shrimp had grown to such a size that maintaining an excess 
food supply became almost impossible, even though 400,000 to 500,000 nauplii per 
tank (a minimum of 9000 to 10,000 per experimental animal) were supplied per day. 

TABLE III 
Cumulative inorluli-ty [only observed deaths arc included'] 



Elapsed Temperature 
time (C.) 
(days) Salinity (% ) 


2 


32 

5 15 


25 35 


2 


25 
5 15 


25 35 


2 5 


18 
15 


25 35 


2 5 


11 
15 25 35 


1 


5 


1 














1 2 











000 


2 


41 


25 16 


2 











2 2 








4 2 





3 


44 


25 16 


6 











13 3 








77 2 





4 


45 


25 17 


7 





I) 





17 3 








93 5 





5 


45 


25 17 


7 











19 3 








97 12 





6 


45 


25 17 


7 











19 3 


(J 





97 12 





7 


45 


25 17 


7 











19 3 








97 22 





8 


45 


25 17 


7 











19 3 








97 25 


I) 


9 


45 


25 17 


7 











19 3 





(1 


97 32 





10 


45 


25 17 


7 











19 3 








97 37 


(I 


11 


45 


25 17 


7 











24 3 








97 41 





12 


45 


25 17 


7 











26 4 








97 45 





13 


45 


25 17 


7 











27 4 








97 50 





14 


45 


25 17 


7 





{) I) 





27 4 








97 55 





17 


45 


25 17 


7 











29 4 








97 60 


(1 


18 


45 


25 18 


7 





1) 





31 4 





I) 


97 60 





19 


45 


25 18 


7 











45 4 








97 60 


000 


20 


46 


25 18 


7 


23 








57 4 








97 63 





21 


46 


25 18 


7 


39 


I) 





58 5 








97 63 





22 


46 


25 18 


7 


50 








58 5 








97 63 





24 


46 


25 18 


7 


55 








58 5 








97 66 





28 


46 


25 18 


7 2 


55 








58 5 








97 67 


004 


No. of animals removed 
























for measurement 


25 


30 35 


50 50 


40 


50 50 


50 50 


36 50 


50 


50 50 


30 


50 50 50 


No. escaped 


2 








4 


4 3 





1 2 


2 


2 








Observed survivors 


15 


22 25 


29 47 





43 43 


50 49 


41 


46 


48 49 





49 46 43 


Unobserved deaths 


12 


23 22 


14 1 


1 


3 4 


1 


5 2 


2 


1 


3 3 


1 4 3 


Per cent survival 


21 


31 38 


58 94 





93 91 


100 98 


85 


96 


100 98 





98 92 86 



At approximately 5-day intervals, 10 animals were removed from each aquarium. 
These included both the largest and smallest specimens, and eight collected at 
random. The animals were individually measured to the nearest 0.5 mm., blotted 
dry, weighed to the nearest 0.1 mg. with a Mettler HIS analytical balance, and pre- 
served. At the termination of the experiment, all remaining animals were similarly 
treated. The final per cent survival was determined by comparing the total number 
of shrimp remaining at the close of the experiment to the number that theoretically 
should have been present (original number less those that had been removed for 
sampling and a few that had escaped ) . The unobserved deaths recorded in Table 
III were animals not accounted for either as survivors, observed deaths, or those 
sampled for measurement. 

On the assumption that an individual Artemia nauplius weighs an average of 
7.1 f^g. (D. Godwin, unpublished), we estimated conversion efficiency by compar- 
ing the calculated wet weight of the brine shrimp that were fed with the weight 



204 



ZOU \ I 1 . /I.IX-EI.DIX AN'D DAVID V. ALDRICII 



gain of the- surviving pcnacids. Determinations were made only for those tempera- 
ture' and salinity combinations at which survival was S5'/V or greater. 

Although the design of the experiment was similar to that of Costlow, Bookhout 
and Monroe (l l ><>0, l''(>2) in studies of larval crah survival, we did not use the 
-tatiMical methods which they employed. The fitted-surface method of Box and 
Youle ( 1 ( '55 i has proved valuable in industrial applications of physical and chemical 
interactions \\hose principles are sufficiently well defined to permit relatively safe 



40, 



Shrimp 

per 

Series Group 
X = I 5 
0=2 5 

El = 3 10 
O=4 30 




40 45 



IMCCKK 1. IVr cent survival of /'. uztccus postlarvac after 24 honi> at 
indicated K-vrK <>t salinity and temperature. 

extrapolation trom a limited number of experimental observations. However, 
the complex nature of biological responses to temperature and salinity renders such 
extrapolation extremely speculative. In the present study, we have tested a group 
of temperature-salinity combinations which represents a relatively large range of 
levels for each factor. ( )ur interpretations of the results exclude extrapolation. 



l\l Si I. IS VND DISCUSSION 



Short-term s 



The excellent survival of postlarvae for periods of 24 hours under most of the 
experimental conditions suggested a broad /.one of short-term tolerance to both 



GROWTH AND SURVIVAL OF P. AZTECUS 



205 



salinity and temperature (Table I). The animals were quite euryhaline, especially 
at 25 and about 30 C, although a marked reduction in tolerance to salinity 
levels below 10',, was demonstrated at 7 and 15 C. (Fig. 1). A general 
reduction in survival near 35 C., regardless of salinity, suggests a strong tempera- 
ture effect. The absolute limiting ( maximum j temperature for P. aztccits is 
probably only slightly above 35 C. 



35r- 



30 



o 

o 



9 

k- 



25 



20 



- 



v 

I ' 5 



10 



21 








1 1 1 1 


'////,- 80-100% Survival 


for 24 Hours 
' 28 Days 


I 1 


1 



10 



30 



35 



40 



15 20 25 

Salinity (%o) 

FIGURE 2. Long- and short-term survival of P. azteciis at indicated levels of salinity and 
temperature. Numbers indicate 28-day survival in per cent. 

The effect of acclimation in extending ranges of postlarval tolerance is clearly 
shown in the results of the fourth series. In each of the four combinations of 
temperature and salinity, gradually changed conditions permitted better survival 
than did sudden changes (Table I). This effect was considerably more marked 
at 10 than at 35 C. 

28-Day sitri'k'ol 

The survival of postlarvae in the 2X-day experiment further confirmed this 
wide zone of tolerance to salinity and temperature. Although the per cent survival 
for 28 days was somewhat lower than that observed for only 24 hours, in most 
cases the results were much the same (Fig. 2). There is some suggestion of 
greater long-term survival for animals at low temperature and low salinity than 



200 



ZOTI.A 1'. XKIX ELDIN \D DAVID V. ALDRICH 



600 n 






D 






A 
A 



>32 C 




* 






a 






* 




100- 


. 




- 


a 

X 







* 




' 


o 
A 






o-2%o 






g 0-5 % 




* 10- 

4 

= 5H 

7> 


a X - 1 5 %o 
-25% 


l 


i i 1 1 1 


) 




600- 






5 

> 


} 


25C. 


- 


i 




100- 


8 

o 

s 


s 


- 


X 




- 


2 M 

o ; 


18 C. 





8 

A 




. 


/ 













A o 




10- 

-< 
5- 
C 


1 

, 4--t--t r I 5 !) 


i 

.0 


) 5 10 15 20 25 3 



Time (days) 

FlGURI .\ (irii\vtli nl yoiin.L: /'. </:;ViY/i.v at iinlirak'<l levels ,,)' tempi-nitim 1 ami salinity. 



GROWTH AND SURVIVAL OF P. AZTECUS 



207 



for those exposed only 24 hours. This apparently paradoxical situation is probably 
related to the longer acclimation period employed in the 28-day study. 

Survival was markedly reduced at the highest temperature (32C. ) at all 
salinities tested except 35% . Much of the accountable mortality at this tem- 
perature occurred during the first four days (Table III ) and \vas presumably the 
result of the immediate stress caused by the changes in environment. However, 
the stress of salinity acclimation would not seem to explain the poorer survival 
observed at 25 f / C c (very near the initial salinity ) than at 35 c / ( ,. The relatively large 
numbers of unobserved deaths occurring at 32 C. and 2'/ f ., $'/< f , IS'/ic and 
25%c (Table III) suggest two other possible causes of mortality the experi- 
mental temperature per sc, and increased cannibalism associated with high tem- 
peratures (as noted above in Series 1 and 2 of the 24-hour studies). It is possible 
that at 32 C. the one-month period of exposure in the growth experiment elicited 
long-term temperature effects which could not be manifested in the relatively short 
duration of the 24-hour studies. 

TABLE IV 

Increase in mean length (nun.'] of P. azteciis surviving 28 days 
at indicated levels of temperature and salinity 





Salinity (% ) 


Temperature 




( C.) 














2 


5 


15 


25 


35 


32 


2.5.4 


28.9 


19.0 


32.0 


25.8 


25 





21.9 


24.4 


22.3 


22.6 


18 





6.3 


6.5 


7.4 


7.6 


11 








0.5 


0.5 


0.4 



Mortalities occurring at other temperatures were limited to the lowest salinitie.s. 
with stress due to reduced salinity and temperature being sufficient to kill all the 
animals at 2% and 11 C. in only 5 days (Table III). The mortalities observed at 
5 c /co and 11 C., as well as those at 2% and 18 C., probably reflect the cumulative 
effects of stress, since deaths occurred continuously throughout the course of the 
experiment. At 25 C. and 2%-c, however, all observed deaths occurred during a 
four-day period late in the experiment. Although the initial cause of this mortality 
is not known, later deaths (on the 21st through the 24th day) were probably 
due to fouling of water, since brine shrimp were also dying. Furthermore, an 
earlier experiment (Zein-Eldin, 1963) had indicated that P. ac teens postlarvae 
survive well under these conditions. 

Grou'f/i 

Differences in rate of growth were more closely related to temperature than to 
salinity (Fig. 3). The relative effects of the two factors may IK- readily determined 
by comparing the magnitude of growth differences associated with variation in 
salinity (columns) with that due to variation in temperature (rows) (Table IV). 
Differences in mean length between temperature groups were detectable as early as 
the first sampling period (5 days) and increased in magnitude during the experi- 



208 



zori.A p. XKIX Ki.nix \xn DAVID v. ALDRICH 



mental period (Fig. 4). Both length and weight increased much more rapidly 
at 32" and 25 C. than at lower temperatures. The maximum increase in size was 
>1 'served at 32 and 25',,, conditions under which one animal grew to 50 mm. and 
(| (>2 mg., a more than four-fold increase in length and a weight increase of 150-fold 
(Tahle V). The great variation in size noted earlier (Zein-Eldin, 1963) was also 
observed in this experiment, with differences in length between smallest and largest 



45 i- 



32 C. 




-.1 1 1 


1 1 



10 

5 10 15 20 25 30 

Time (days) 

IMGCRK 4. (innvtli of younn" /'. aztecus at various temperatures (salinity: 25',, ). 



animals ranging from 13 to 25.5 mm. in the various salinities at 25 and 32 C. 
n'able V). Almost no growth was detected at 11 C., although survival was 
good at salinities of 15',, and above. \Vith the exception of 32 C. and correspond- 
ing salinity levels of 25',', and below, where mortality more than offset the rapid 
growth rate (Tables IV and VI), the gain in total weight of the survivors was 
comparable within a temperature. However, this gain increased approximately 
10-fold within each level of salinity between II and IS" C., and only slightly less 
between 18 and 25 C. (Table VI). 



GROWTH AND SURVIVAL OF P. AZTECUS 



209 



TABU-: V 

Mean size mid grind h rate of growth-experiment survivors, including 10 anninls 

sampled a I 28 days and shown in Table I'll. S /'.:< range gircH in parentheses. 

Initial ice/ght and length were 6.1 nig. and 12. 1 nun., respectively 



Temperature 
and salinity 


Number of 
survivors 


Weight (mg.) 


Length (mm.) 


Increase in length per 
day (mm. /day) 


32 C. 










i' it 


15 


340.6 (157.7-735.1) 


34.8 (27.5-47.5) 


0.81 (0.55-1.26) 


>' 


22 


447.6 (241.0-667.6) 


39.6 (33.0-46.0) 


0.98 (0.75-1.21) 


15& 


25 


240.2 ( 35.2-542.0) 


31.0 (17.0-42.5) 


0.68 (0.18-1.09) 


25%c 


29 


610.8 (309.0-961.9) 


43.1 (36.0-50.0) 


1.11 (0.85-1.35) 


35 %c 


47 


423.7 (164.7-753.6) 


38.3 (28.5-46.5) 


0.94 (0.59-1.23) 


25 C. 










5%o 


43 


274.0 (115.3-482.0) 


33.9 (25.0-41.5) 


0.77 (0.46-1.05) 


15%o 


43 


375.3 (163.8-681.1) 


37.4 (29.0-46.5) 


0.90 (0.60-1.23) 


25% 


50 


313.8 (101.4-538.0) 


35.2 (24.0-44.0) 


0.82 (0.42-1.14) 


35 %o 


49 


291.0 (108.0-605.7) 


34.4 (24.5-43.0) 


0.80 (0.44-1.10) 


18 C. 










5% 


41 


33.9 (18.0- 56.2) 


18.3 (14.0-21.5) 


0.20 (0.08-0.34) 


15%o 


46 


43.7 (15.3- 77.7) 


19.1 (14.5-23.0) 


0.25 (0.09-0.39) 


25% 


48 


52.8 (15.0-101.4) 


20.1 (14.0-25.0) 


0.29 (0.08-0.46) 


35% 


49 


35.2 (17.7- 62.7) 


18.3 (14.5-22.0) 


0.22 (0.09-0.35) 


11 C. 










15& 


49 


8.7 (5.2-13.1) 


12.6 (11.0-14.0) 


0.03 (0-0.08) 


25%o 


46 


8.8 (6.4-10.8) 


12.7 (11.5-14.0) 


0.02 (0-0.08) 


35% 


43 


7.6 (5.5- 9.7) 


12.4 (11.0-13.0, 


0.01 (0-0.08) 



Growth rate based only upon the steepest portions of the growth curves (i.e., 
between the 10th and 28th days) approached a value of 1.4 mm. per day at 32 
C. and 1.1 mm. per day at 25 C., as against the lower values of 1.1 mm. per day 
at 32 and 0.8 mm. per day at 25 C. over the entire experimental period (Fig. 4, 
Table VII). Although the mean growth rates reported here (Table V) for both 
25 and 32 C. exceed the maximum of 0.56 mm. per day which Pearson (1939) 
reported for laboratory-held postlarvae of P. brasilicnsis (probably P. aztccus*), 
and the maximum of 1.35 mm. per day far exceeds his value, these rates do not 

TABU- VI 

Increase in total weight (g.) of P. azteciis surviving 28 days at indicated levels 

of temperature and salinity. Food conversion efficiency (%) indicated 

in parentheses where survival was 85% or greater 



Temperature 



Salinity (% ) 



(C.) 


2 


5 


15 


25 


35 


32 


4.7 


9.6 


5.8 


17.5 


19.6 (37) 


25 





11.5 (43) 


15.9 (53) 


15.4 (46) 


14.0 (43) 


18 





1.2 (32) 


1.7 (33) 


2.2 (40) 


1.6 (34) 


11 








0.1 (12) 


0.1 (14) 


0.1 (5) 



210 ZOULA P. /KIN' ELDIN AXD DAVID V. ALDRICH 

approach that of postlarval white shrini]), /'. sctijenis, which grew an average of 2.1 
nun. per clay in pond experiments conducted hy Johnson and Fielding (1956). 
A similarly low rate of growth for aquarium-held Mctapcnaciis inastcrsii, which 
ranged in carapace length from 1. to 7.0 mm., has been reported by Dall (195Si. 
Laboratory animals grew only 10 mm. in total length per month at 24 to 28 C., 
as against a natural growth rate of 20 to 30 mm. per month. 

Growth of our laboratory-held P. aztccits postlarvae likewise only approached 
that of slightly larger animals in the field. Yiosca (1920) estimated a growth 
rate of approximately 25 mm. per month for P. sctijenis in the length range 30 
to 150 mm., while Gunter ( r>50) observed a rate of 25 to 40 mm. per month for the 
same species growing from 2S to 100 mm. Extrapolation of Lindner and Ander- 
son's (1956) growth curve for white shrimp gives a rate of 1.50 mm. per day for 
shrimp between 20 and (>5 mm. in length. \Yilliams (1955) determined a rate 
of 1.7 mm. per day for /'. nztccits growing from 37 to 102 mm., a rate also 
estimated by St. Amant, Corkum and Broom (1963) for 51- to 125-mm. specimens 
of this species. It must be noted, however, that these estimates were for shrimp 
at the upper end of the size ranges encountered in the studies described here. 

Although the studies of conversion efficiency were necessarily crude, the 
resulting data indicated that the most efficient utilization of food occurred at 25 
C. (Table VI). Because of the high mortality, no efficiencies could be calculated 
for shrimp held at 32 C. in salinities of 25%c and lower. The conversion values 
should be considered maximal at the highest temperatures since some cannibalism 
probably occurred. However, efficiencies for the animals held at 11 C. are 
probably low since it was apparent that much of the food provided was not eaten. 
At this low temperature, the postlarvae were generally inactive, resting most of 
the time on the bottom. 

Johnson and Fielding (195h), studying P. sctifcnts in aquaria during August 
(temperature not stated), found a mean food-conversion efficiency of 19% for 
juveniles (mean weight 0.9 to 2.1 g.) held one week at 18.5^V. as against an 
efficiency of 24% for juveniles (mean weight 0.7 to 2.1 g. ) held one week at 34%c. 
All animals were fed at a rate of 10% of the initial body weight per day, a rate less 
than that provided in our experiments. The lower efficiencies determined by 
Johnson and Fielding may be due, in part, to the larger size of the animals held, 
since it has been shown in fishes ( Kinne, 19(>0 ) that conversion efficiency decreases 
with increasing size. Furthermore, Johnson and Fielding obtained the maximum 
efficiency of 50% from a group of animals of 0.7 g. mean weight held at 34%o. 
This value compares favorably with those reported here for brown shrimp of 
mean weight 0.3 g. and less, and would indicate that rapidly growing young 
shrimp require 2 to 4 g. of utilizable food to produce 1 g. of tissue. 

The decrease in growth rate of animals held at 32 C. and 15'/r is unexplainable. 
This group of animals consumed less food during the latter days of the experi- 
ment, even though excess food was present. If this decreased growth represented 
a long-term effect of the combined stresses of lowered salinity and increased tem- 
perature, it is strange that such a decrease did not occur among groups held at even 
IOVUT salinities at this temperature. Interpretation of the combined effects of 
lower salinity with 32 C. on growth was complicated hv the high rates of mor- 
tality among these Croups. 



GROWTH AND SURVIVAL OF P. AZTECUS 



211 



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212 XOl'l. A P. ZEIN ELDIN ^ND DAVID V. AI.DRICH 

Biological and ecological implications 

Commercially important Xorth American shrimp of the genus PCIKICHS spawn 
at sea. As shown for /'. sc/il'rnis, the larvae develop in the open sea, migrate 
into the estuarine areas as postlarvae. remain in the less saline estuaries until they 
approach maturity, and then return to the sea (Weymouth, Lindner and Ander- 
son, 1 ( <33; Rurkenroad, 1 ( >34; Pearson, 1939). Various studies in the field have 
suggested that postlarval and juvenile J'cinicns are associated with low salinities 
characteristic of the estuary, and that postlarvae require the lowest salinity for 
growth and survival ( ( lunter, 1 ( H5, 1950; Pearse and Gunter, 1957). Lindner 
and Anderson ( 1 ( >50) concluded, however, that size of white shrimp (juveniles and 
suhadults ) seemed more closely related to locale than to salinity. Gunter, Christmas 
and Killehrew (1 ( >04) have recently presented additional field data indicating 
differences in the natural di.strihutions, with respect to salinity, of the three com- 
mercial species, P. aztccus, I', dnorannn, and P. sctijcnts. In so doing, these 
authors have made certain assumptions. For example (p. 1S4): "If salinity 
meant nothing to these animals they would he evenly distributed relatively over 
the whole range, if food were availahle. The general food hahits of shrimp are 
still largely unknown, hut all indications are they are omnivorous feeders, and shrimp 
do find food over the full salinity range up to pure sea water, although the food 
douhtless changes with si/e." The fact that shrimp do apparently eat a variety of 
food does not, however, indicate that all such foods are of comparable nutritive 
value (Williams, 195 ( >; Zein-Eldin, 1903). Furthermore, there is no evidence that 
food is equally availahle throughout the salinity range occupied by shrimp in 
nature. Onlv in a previous study (Zein-Fldin, 19O3) and in the work reported 
here, has food been equally available to all animals regardless of salinity. The 
24-hour survival experiments, as well as the growth study, indicated that for P. 
aztccits postlarvae, only extreme salinity conditions influence growth and survival. 
Even normal oceanic salinity is not sufficient to interfere with postlarval brown 
shrimp growth and survival when other factors (temperature, food supply, pre- 
dation, oxygen, light, pollution, etc.) are kept relatively constant. In view of our 
results, we suggest that other factors, such as food or cover (which may them- 
selves require relativelv narrow salinitv ranges), are of greater importance than 
salinitv /vr ,sv in determining distribution, growth, and survival of these animals. 

In the present studies, both the survival and the growth data indicated that 
wide ranges of salinity and temperature were well tolerated by postlarval brown 
>hrimp. The combination ot low salinitv and low temperature, however, was not 
favorable, either for survival or growth. That P. aztccns can withstand extreme 
conditions of both factors has been demonstrated in the Held as well, although 
published records have been largelv limited to occurrences of juvenile and adult 
forms (Gunter. I'bO). Hearden (l ( '0l), who found postlarval brown shrimp 
at temperatures as low as f>.5" ( '.. noted a marked decrease in their abundance 
following the sudden cold spell which resulted in this low-temperature value. 
Ken fro and Baxter (unpublished) have reported live postlarvae at 12 C. and 
31.0',, as well as at 2 C. and 30.5',,. supporting our laboratorv evidence that low 
temperatures can be survived when salinities are sufficiently high. Comparable 
data of postlarval occurrence in low-salinity areas are not yet available. Brown 
shrimp ( si/.e not stated ) have also been reported in salinities as low as 0.8%e 



r.RCWTII AND SURVIVAL OK 1'. AZTECUS 



21.S 



(Gunter and Shell, 1958) in Louisiana, while Guntcr and Hall (1963) report a 
34- and a 38-mm. specimen at 0.22/^r in the St. Lucie estuary in Florida. No tem- 
peratures were reported with the latter data, however. It must be noted, neverthe- 
less, that St. Amant, Corkum and Broom (1963) reported maximal spring abund- 
ance of postlarval brown shrimp in Louisiana bays only after water temperatures 
consistently exceeded 20 C. 



35r- 



30 



25 



o 

o 

- 20 

O) 

l_ 
3 
-t~ 
O 

IH 

o> 

15 



10 



23.4 28.9 



19.0 



32.0 



22.6 




0.4 







10 15 20 

Salinity (% ) 



25 



30 



35 



FIGURE 5. Growth and survival of young P. aztccns held 28 days at indicated levels of 
temperature and salinity. Numbers indicate increase in mean length (mm.). Hatched zone 
indicates 80-100% survival. 

The temperature range permitting growth is more limited than the range for 
survival (Fig. 5). Our laboratory studies have demonstrated that growth can 
occur over a wide range of salinity at temperatures of 25 C. and above, and suggest 
that the effect of temperature upon the rate of growth increases rapidly with 
temperature between 11 and 25 C. (Fig. 6). This effect of temperature has 
been confirmed in more recent experiments in which we observed growth at a 
greater number of temperature levels between 15 and 35 C. than tested here. 

The greatest growth differential per 7 C. was observed between the 18 and 
25 C. levels. This difference may well explain the observation of St. Amant, 
Corkum and Broom (1963, p. 25) that "metamorphosis of postlarvae into rapidly 
growing juveniles occurs suddenly after water temperature exceeds 20 C." Above 
25 C., increasing temperature has less effect upon growth. The recent experiments 



214 



ZOULA r. ZEIN KI.DIX AND DAVID V. ALDRICH 



referred to above indicate that grouth is maximal at 30 to 32.5 C. This result, 
coupled with the increased mortality at 32 C. suggests that such a temperature 
Condition, although promoting rapid growth in some individuals, may be above the 
optimum temperature for long-term growth and survival of P. aztecns postlarvae. 
The laboratory evidence suggests that normal winter temperatures render the 
brackish bay systems unfavorable for both survival and growth of brown shrimp 
postlarvae, whereas almost any salinity will provide a favorable environment at 
normal summer temperatures. Thus, the pattern of tolerance to salinity and tem- 
perature observed in the laboratory may explain the seasonal distribution of P. 



l.2 r 




15 20 

Temperature (C.) 



25 



30 



35 



FiGt'RK 6. Effect 'if temperature on the laboratory growth rate of young /'. aztccns (salinity : 
2r',, ; length <>i' experiment : 2X days; initial length of experimental shrimp: 12.1 nun.). 

(tztccits in much the same manner as described by Hroekema (1941) for the 
migratory European shrimp, C rant/on cranyou. Survival of postlarvae within the 
estuary may also be affected by decreases in temperature or salinity. In the 
spring, postlarvae entering bays having relatively low temperatures and salinities 
above ]$'/,<> may be adversely affected by a sudden salinity drop, such as that 
caused by heavy spring rains. Conversely, if the temperatures are intermediate 
(18 C., for example) but salinities low (10'<'r or less), a drop in temperature may 
also decrease survival. Simultaneous decreases in both physical factors tem- 
perature and salinity would be most detrimental to the population in terms of 
both survival and growth. 

Williams (1 ( (>0) bad pre\ iously noted the effects of temperature and salinity 
on juveniles and subadults of I 1 . a:::lccns. Xot onlv did he find that the 96-hour 
survival of 42- to 100-mm. specimens declined with decreasing temperature over 



GROWTH AND SURVIVAL ()! I 1 . AZTElVS 215 

the range 28.8 C. to 8.8 C., but he also determined that survival was most 
markedly reduced at 10/^r (the lowest salinity tested) regardless of the temperature. 
Animals exposed to 8.8 C. showed a greater tendency to lose the ability to 
regulate the osmotic concentration of the serum. It is of interest that juveniles 
were better able to regulate serum concentration than were adults (120 to 150 
mm.) exposed to the same conditions. McFarland and Lee (1963) demonstrated 
that brown shrimp adults were better osmoregulators at higher salinity than at 
lower, with a greater tendency to isosmoticity when the external medium was 
below l8'/ ( (. The latter authors were unable to study animals in salinities below 
5 f /tc to (>'/<( since only one of 12 adults survived 24-hour exposure to this range 
of salinity, despite an acclimation period of almost one week. 

The studies cited above suggest that salinity tolerance may vary not only with 
temperature, but also with size (age) of the shrimp. In demonstrating good 
survival of P. aztecus postlarvae over a broad range of salinity and temperature, the 
findings presented here suggest that postlarvae of this species are better os- 
moregulators than juveniles, which were tested by Williams (I960), or the adults 
tested by McFarland and Lee (1963). Further studies are planned to determine 
the effects of both temperature and salinity upon the osmotic behavior in various 
life-history stages of P. act ecus and I', setifcrus. 

SUMMARY 

1. The combined effects of salinity and temperature upon growth and survival 
of postlarvae of the brown shrimp, Penaciis aztccns. were studied under controlled 
conditions. 

2. Test salinity ranged from 2%o to 40/< c and temperature from 7 to 35 C. 

3. With relatively short periods of acclimation, postlarval brown shrimp 
withstood wide fluctuations in both temperature and salinity for 24 hours. 

4. The range of tolerance to these factors over periods of 28 days was only 
slightly less than that observed for 24 hours. 

5. Postlarvae survived temperatures as low as 11 C. with almost no growth 
for one month in salinities of about lS%c or above. 

6. Growth increased with temperature, with significant growth beginning al 
some temperature above 11 C. but below 18 C. The most marked increase- 
in growth rate occurred in the temperature region between 1 1 and 25 C. 

7. At temperatures below 15 C., young (postlarval) shrimp demonstrated a 
decreased tolerance to low salinity. This reduced tolerance may influence the 
natural distribution and survival of postlarvae, which do not ordinarily enter the 
estuaries in abundance until spring when the temperature has increased to levels 
at which characteristically low estuarine salinities are no longer harmful. 

8. Salinity per sc had little effect on either survival or growth, except at extreme 
temperatures. 

LITERATURE C IT I'D 



BEARDEN, C. M., 1961. Xoto mi pu>tlarvae ol" commercial shrimp ( I't'iuu -n\ ) in South Carolina. 

Cont. Bears Bluff Lab., No. 33, 1-8, Wadmalaw Island, S. C. 
Box, G. E. P., AND P. V. YOULK, 1955. The exploration and exploitation <>f response surfaces: 

An example of the link between the fitted surface and the basic mechanism of tin- 

system. Hitnnctrics, II: 287-323. 



216 zori..\ p. /. MIX -ELD ix AXD DAVID v. AI.DRICH 

BROEKEMA, M. M. M., 1941. ScaMUial movements and the osmotic behaviour of the shrimp, 

Crangon crangon L. Arch. Xccrl. Zool., 6: 1-100. 
I'.i i'K! NROAII, M. D., 1934. The Penaeidea of Louisiana with a discussion of their world 

relationships. Hull. . liner. Mas. A'<;/. Hist.. 68: 61-143. 

COSTI.OW, J. D., JR., C. G. BOOKHOUT AXD R. MONROE, 1960. The effect of salinity and tem- 
perature on larval development of Scsanua cinercitui (Bosc) reared in the laboratorv. 

Biol. Bull., 118: 183-202. 
COSTLOW, J. D., JR., C. ( 1. BOOKHOUT AND R. MOXKOE, 1962. Salinity-temperature effects on 

the larval development of the crab, Panopcus herbstii Milne-Edwards, reared in the 

laboratory. Physiol. Zool., 35: 79-93. 
DAI. i., \Y., 1958. Observations of the biology of the greentail prawn, Metapenaeus inastcrsii 

i llaswell) (Crustacea Decapoda : Penaeidae). .lust. J. Mar. Fresh. Res.. 9: 111-134. 
GCXTER, <;., 145. Studies on marine fishes of Texas. Publ. Inst. Mar. Sci., 1: 1-190. 
GrxTER, ( i., 1950. Seasonal population changes and distributions as related to salinity of certain 

invertebrates of the Texas coast, including the commercial shrimp. Publ. lust. Mar. 

Sci.. 1(2) : 7-51. 
GTXTER, G.. AND G. E. HALL, 1963. Biological investigations of the St. Lucie estuary ( Florida > 

in connection with Lake Okcechobee discharges through the St. Lucic canal. Gulf Res. 

Rep., 1 : 189-307. 
GUXTEK, G., AND W. E. SHELL, JR., 1958. A study of an estuarine area with water-level control 

in the Louisiana marsh. Proc. La. Acad. Sci., 21: 5-34. 
< 1 1 XTER, G., J. Y. CHRISTMAS AXD R. KILLEBREW, 1964. Some relations of salinity to population 

distributions of motile estuarine organisms, with special reference to penaeid shrimp. 

Ecology, 45: 181-185. 
Jonxsox, M. C., AXD J. R. FIELDIXG, 1956. Propagation of the white shrimp, I'cnacus sctifcrus 

( Linn.), in captivity. Tulanc Stud. Zool., 4: 175-190. 
KIXXK, O., 1960. Growth, food intake, and food conversion in a euryplastic fish exposed to 

different temperatures and salinities. Physiol. Zool., 33: 288-317. 

I.INDXKR, M. J., AXD W. W. AXDERSON, 1956. Growth, migrations, spawning and size distribu- 
tion of shrimp, Pcnacus sctifcrus. U. S. Fisli and U'ildl. Scn\, Fish. Bull. 106, 56: 

555-645. 
.Mi FARLAXD, W. N., AXD B. D. LEE, 1963. Osmotic and ionic concentrations of penacidcan 

shrimps of the Texas coast. Bull. Mar. Sci. Gulf Caribb., 13: 391-417. 

PKARSE, A. S., AXD G. GUNTER, 1957. Salinity. /;;: Treatise on Marine Ecology and Paleon- 
tology. Vol. 1, J. W. Hedgpeth, ed. Geological Society of America, Memoir 67, 

129-158, N. Y. 

Pi.Ak.sox, J. C., 1939. The early life histories of some American Penaeidae, chiefly the com- 
mercial shrimp Pcnacus sctifcrus (Linn.). Bull. U. S. Bur. Fisheries, 49(30) : 1-73. 
RKXFRO, \Y. C., 1964. Life history stages of Gulf of Mexico brown shrimp. U. S. Fish ami 

ll'ildl. Scr^.. Or. No. 183, 94-98. 
ST. AMAXT, L. S., K. C. CORKUM AXD J. G. BROOM, 1963. Studies on growth dynamics of the 

brown shrimp, Pcnacus aztccus, in Louisiana Waters. Proc. Gulf Caribb. Fish. Inst.. 

15: 14-26. 

YIOSCA, P., JR., 1920. Report of the biologist. La. Dept. Coiiserv., 4th Bienn. Rep., 120-130. 
\VKYMOUTH, F. W., M. J. LIXDXKR AXD W. \Y. AXDKKSOX, 1933. Preliminary report on the 

life history of the common shrimp Pcuacus sctifcrus (Linn.). Bull. U. S. Bur. 

Fisheries, 48(14) : 1-26. 
\YM.I.IAMS, A. B., 1955. A contribution to the life histories of commercial shrimp {Penaeidae) 

in North Carolina. Bull. Mar. Sci. Gulf Caribb.. 5: 116-146. 
\Yn.i.iA.Ms, A. B., 1959. Spotted and brown shrimp postlarvae (Pcuacus) in North Carolina. 

Bull. Mar. Sci. Gulf Caribb., 9: 281-290. 
WILLIAMS, A. B., 1960. The influence of temperature on osmotic regulation in two species of 

i^tuarine shrimps (Pcnacus). Biol. Bui!., 119: 560-571. 
ZEIN Ki.mx, Z. P., 1963. Effect of salinity on growth of postlarval penaeid shrimp. Bi<>l. 

Bull., 125: 188-1%. 



Vol. 12Q, No. 2 October, 1065 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



TUBE-BUILDING AXD FEEDING IX 
CHAETOPTERID POLYCHAETES 

ROBERT D. BARNES 

Department of Bioloi/y. Gettysburg Cullci/c, licttyslutr,/, 

The tube-building and feeding behavior of the large familiar Chactoptcnts vari- 
opedatus has been known since the studies of Enders (1908, 1909) and MacGinitie 
(1939). Much less was known about the other members of the family Chaetop- 
teridae until 1964. when the Atlantic chaetopterid, Spiochaetopterus ocitlatiis. 
was investigated by the author. This little species, having a pair of long palps 
and inhabiting a straight vertical tube, is a much more typical member of the 
family than is Chaetopterus. The purpose of this study therefore was to investi- 
gate the tube-building and feeding mechanisms of the remaining four chaetopterid 
genera; Tclepsavus, Phyllochactopterus, Ranzanidcs, and Mesochaetopterus. 

The greater part of this study was carried out in Naples, Italy, where repre- 
sentatives of every chaetopterid genus except Mesochaetopterus, occur in the Bay 
of Naples. The author wishes to express his appreciation to the Stazione Zoologica 
at Napoli for the facilities and courtesies extended to him during the three months 
of residence at the laboratory, and also to the National Science Foundation for their 
support of space utilized at the laboratory. The author is indebted to Dr. R. Phil- 
lips Dales of Bedford College, University of London, who provided unpublished 
data from his observations of Mesochaetopterus, and also to Dr. Marion Pettibone 
of the U. S. National Museum for the loan of specimens of Mesochaetopterus. 

MATF.KIALS AND METHODS 

All of the living specimens utilized in this study were collected by dredging 
in water ranging in depth from two to 50 meters. The small size and relatively 
shallow depth of the tubes of these chaetopterids piv\cnU-d any excessive damage 
by the dredge. 

To study feeding and other behavior the worms were transferred from their 
natural tubes into glass capillary tubes. The diameter of the capillary tube was 
critical for the adaptation of the worms to this artificial environment ; but if the fit 
was a good one, the worms lived for as long as two months. The process of trans- 
fer from the natural tube to a glass capillary tube was most easily accomplished 

217 
Copyright 1965, by the Marine Biological Laboratory 



2 IS ROMKK'T I) I'.ARNKS 

by means of a hypodermic syringe \\ith a small needle. The needle was inserted 
into one end of the natural tube and a strong stream of sea water was then in- 
jected. This rapidly forced the worm in an undamaged condition out of the oppo- 
site end of the tube. The needle of the syringe was then inserted into one end of 
the glass capillary tube and the syringe slowly filled. The filling syringe created 
a current of sea water through the tube sufficient to suck the worm into the capil- 
lary tube. 

The capillary tubes were placed within a glass cylinder. A plug of glass wool 
held the tubes in a vertical position against the inner wall of the cylinder, and a 
short piece of large glass tubing penetrated the center of the plug to permit ade- 
quate water circulation. When the worms were not being studied, the entire glass 
cylinder was submerged within a tank of circulating sea water. The worms were 
observed through the cylinder wall by means of a horizontally oriented dissecting 
microscope. The base of the microscope had been removed and the upper optical 
portion was attached in a horizontal position to a ring stand. 

Observations of tube-building were facilitated by confining the worms to very 
short sections of natural tube. The worms would then frequently construct addi- 
tions to the tube. Determination of water current and degree of tube obstruction 
was aided by the use of carmine-stained sea water injected into the tube. A sus- 
pension of carmine particles in sea water was used to study ciliary tracts and feeding. 
Observations of feeding were also aided by using an artificially stained detritus. 
The detritus was prepared by boiling a small amount of cooked pasta in carmine 
and then grinding and suspending it in sea water. 

RESULTS 

Telepsaz'its costantin Claparede 

Tclcpsui'iis costanun Claparede is one of two species known for the genus. It 
is cosmopolitan and in the Western Hemisphere occurs along the Pacific coast of 
North America. This species was collected from the Bay of Naples at depths of 
four to fi\e meters from a bottom of fine sand and silt. 

The tube of Tclcpsavus and the structure of the worm itself are almost identical 
to that of Spiochaetopterus (Fig. 1, P>). Telepsanis secretes an opaque cornified 
annulated tube (Figs. 2 and 3, A ) which is buried vertically in the substratum. 
Only a small part of one end of the tube projects above the surface of the sand. In 
the Bay of Naples the longest dredged tube was 25 cm. but longer tubes are prob- 
ably common. The internal diameter ranged from 1.2 to 1.4 mm. The lower re 
gion of the tube commonly contains a secreted button-like transverse partition (Fig. 
3, E) which is perforated by several openings to allow a water current to pass 
through the tube. 

The total length of TclcpsaTiis costantni averages about S cm. and, as in all 
chaetopterids, the body is divided into three regions. The anterior region ( Fig. 
1, A i contains nine segments, indicated externally only by the presence of the nine 
short lobe-like notopodia ; the anterior nenropodia are lost in rdl chaetopterids. 
Kach notopodium is supplied by a bundle of capillary .setae. The fourth noto- 
podium, in addition, carries a large, heavy, blade-like seta. The anterior end of 
the body (Fig. 3, C) is truncate, the ventral margin projecting beyond the mouth. 



CHAETOPTERIDAE: TUI'.E AND FEEDING 



210 



A small lip flanks the mouth dorsally and lies between the bases of two long heavy 
palps, which may equal or exceed half the body length. 

The entire convex ventral surface of the anterior body region is covered by a 
thick glandular epidermis which secretes the tube. The tube is secreted in half- 
cylinder sections. When an addition to the tube is to be secreted, the worm slowly 



r i , - 



CUTTING 
.-SETA 




PALPS 



ANTENNA'-- ~ 



TELEPSAVUS 



SPIOCHAETOPTERUS 



PHYLLOCHAETOPTERUS 



TEARING 
.- SETA 




PUMPING 
SEGMENTS .. 



^ 



SEGMENTS. 



MESOCHAETOPTERUS 



RANZANIDES 



FIGURE 1. Lateral view of anterior and middle body regions of representatives of each of 
the six chaetopterid genera. Mucous bags are indicated by shaded areas enclosed by dashed 
lines. 

extends almost the entire anterior region of the body out of the tube. Simultane- 
ously, the body is flexed dorsally at the level of the 4th or 5th segment and ap- 
pears very flared and turgid. At the end of the extension movement, the body is 
quickly withdrawn into the tube, leaving behind a delicate half-cylinder of new 
tube. The worm then rotates 180 within the tube and secretes the opposite half- 
cylinder. The length of the addition corresponds to the distance between two 
annulations. 



220 



KOMKKT 1). I5ARNES 



The laying down of a transverse i.artition was never observed in Telepsavus. 
However, since the partitions are identical to those of Spiochaetopterus, they are 
probably constructed in the same manner (Barnes, 1964). In Spiochaetopterus the 
worm assumes a head-downward position in the tube and at the level at which a 
partition is to be placed, the Ik-ad of the worm is slightly flexed dorsally. The 
partition is then laid dmvn during a rapid rotating movement of the anterior end. 




IMC.CKK 2. I'art \ tin- tnl>r of Telepsavus c 

The anterior \entral margin of the head projects through a central opening in 
the partition at the end of the rotation. This single opening is later reduced to 
two to four smaller openings as the secretory .surface of the worm is moved back 
and forth across the partition. "\Yliv the opening is not completely obliterated is 
not understood. 

Partitions are removed exactly as in Spiochaetopterus. The worm assumes a 
head-downward position in the tube. At the level of the jiartition the body is flexed 
dorsally 1S()' J so that the angle of flexure is located at the 4th parapodia. The 
flexed region of the body is now rotated a little to one side, Usually toward the left 



CHAETOPTERIDAE: TUBE AND FEEDING 



221 



side, so that the enlarged blade-like 4th seta is directed downward against the 
periphery of the partition. The seta is now extended, cutting through at the junc- 
tion of the partition and the wall. Then the seta is retracted. Following each 
stroke, the worm rotates slightly in the tube. In less than two minutes the worm 
neatly cuts the partition completely free. The old partition is pushed downward 
and against the tube wall and is gradually incorporated into the wall itself as addi- 
tional reinforcing secretions are laid clown by the worm against the inner surface 
of the tube. 



DORSAL CILIATED GROOVE 



CILIATED 
NOTOPODIAL RINGS 



MUCOUS BAG *'- 








CUPULE 



BLOOD VESSEL 



EJECTION GROOVE - 



_JL -CILIATED GUTTER 



COELOM 



FIGURE 3. Telepsarns custaniui. A. Upper end of tube. B. Dorsal view of three seg- 
ments of posterior body region. C. Dorsal view of anterior end of body showing grooves on 
palps. D. Cross-section of palp. E. Surface view of a tube partition. 

The modified 4th setae are also used to cut open or rupture the tube wall, per- 
mitting the formation of a new extent of tube at this point. Such a new addition 
was always observed at the lower end of the tube and the entrance to the old lower 
section of the tube was sealed over. 

The middle body region commonly contains 31 segments although the number 
is not constant. The parapodia are biramous. The neuropodia (Fig. 1, A) are 
broad, rounded lobes provided with uncinate setae and are used for anchoring the 
body to the tube wall. The notopodia have a foliaceous structure (Fig. 3, F). 
Each notopodium consists of two main divisions, one dorso-medial and one dorso- 
lateral. The dorso-medial division is again divided into two rami. One ramus is 
directed dorso-medially and contacts the corresponding ramus of the opposite noto- 
podium. Together the two opposing rami enclose a large ring-like mid-dorsal 



KOUKKT D. BARNES 

opening. The other ramu> of the dor-M>- medial division is directed ventrally and 
contacts the dorso-lateral division of the- notopodium. This contact likewise en- 
closes a lateral ring-like opening, one on each side. Thus there are three noto- 
podial rings, one dorsal and two lateral, at the level of each segment in the middle 
body region. 

The posterior body region t Fig. -\ B ) varies from a few to many segments. 
The nenropodia of this region are similar to those of the middle body region; the 
notopodia are simple antenna-like projections bearing a seta at the tip. 

The notopodial rings of the middle body region are lined by large membranelles. 
The membranelles beat continually and the beating occurs as several counter- 
clockwise waves mm ing around the ring margin. The beating of the membranelles 
in the notopodial rings of the middle body region drives water through the tube, 
creating the water current upon which the worm depends for respiration, for food, 
and for the elimination of waste. 

Like other chaetopterids, Teli'f>s<.ri us obtains food bv straining the tubal water 
current through a mucous bag. A single mucous bag is employed by Tdcpsai'it* 
costanini. The bag is secreted by the second mid-dorsal notopodial ring (Figs. 
1, A and 3, F) which is slightly heavier than the other mid-dorsal rings of the 
middle body region. The end of the mucous bag is caught by a large ciliated 
cupule located medially just in front of the third dorsal notopodial ring. The cilia 
in the cupule beat backward, and roll the gathered end of the mucous bag into a 
ball. As one end of the bag is rolled up in the cupule. additional mucous film is 
secreted at the opposite end. 

The notopodial ring which secretes the mucous bag is also lined by membranelles 
and all of the water driven through this ring must pass into and through the mucous 
bag. Plankton and tine detritus are strained out and incorporated into the slowly 
enlarging mucous food ball being formed in the cupule. When the food ball has 
reached a certain size, secretion of mucus is halted, and the bag is detached from 
the notopodial ring. Xow the food ball moves out of the cupule and is carried for- 
ward along a mid-dorsal ciliated groove (Fig. 3, F). The flanking ridges of the 
groove bifurcate just in front of the small dorsal lip of the mouth. One ridge of 
the groove passes toward the side of the lip, and the other ridge passes to the oppo- 
site side. Each ridge gradually diminishes. The food ball, on reaching the bifur- 
cation of the ciliated groove, passes over the dorsal lip and into the mouth. 

A small amount of mucus is secreted by the first notopodial ring and is collected 
by a rudimentary cupule located immediately behind the ring. The mucus is never 
elaborated as a distinct bag and appears to be of little importance in feeding. 

As in Spiochaetopterus, the tuo long anterior palps play a minor role in feeding. 
Fach pal]) is provided with a deep ciliated gutter located on the dorsal side (Fig. 3. 
C and D). Small particles which hi come lodged in the gutter are carried down the 
length of the palp by the beating cilia. At the base of the palp the gutter passes 
onto the lateral margins of the mouth. But only rarely in Tclcpsa^'us were parti- 
cles ever observed being carried within the palpal gutter. This was true not only 
for introduced suspensions of carmine particle- and stained detritus but also for 
natural particles. 

The principal function of the palps is the removal of feces and the maintenance 
of an unobstructed tube. Just medial to the ciliated gutter is a small ciliated groove 



CHAETOPTERIDAE : TUBE AND FEEDING 

(Figs. 3, C and D). In contrast to the downward beating cilia of the larger palpal 
gutter, the cilia of the smaller groove beat distally. Any undesirable particles 
which are brought into the tube by the water current must pass over the palps before 
reaching the body proper and the notopodial rings. When such particles contact 
the palps they are quickly swept onto the ejection groove and then transported along 
the groove to the tip of the palp. At the same time the worm moves upward in 
the tube until the palps project out of the tube opening. Material being carried 
along the ejection groove drops from the tip of the palps and falls outside of the 
tube. 

The palps are highly effective in clearing the tube of undesirable material. 
When stained detritus or a suspension of carmine particles was introduced into the 
upper end of the tube, over 90% was quickly ejected by the palps. 

Feces are egested from the posterior anal opening in the form of small pellets. 
The pellets are immediately picked up by the mid-dorsal ciliated groove, which 
runs the entire length of the body and in the middle and anterior body regions also 
functions as the food groove already described. The fecal pellet is carried ante- 
riorly along the groove. In the middle body region the apposing notopodia form- 
ing the mid-dorsal ring separate when the pellet moves through the ring. Their 
separation thus reduces the chance of the pellet being swept out of the groove by 
the opposing beat of the membranelles lining the ring. 

When the fecal pellet reaches the anterior end of the mid-dorsal groove, it does 
not pass over the dorsal lip as does the food ball. Rather, the fecal pellet follows 
either one of the two ridges which separate and swing to either side of the dorsal 
lip. The ridge carries the pellet toward the ejection groove at the base of the palp. 
The fecal pellet is then carried the length of the palp to the tip. Simultaneously, 
the worm moves upward in the tube and projects the palps to the outside so that 
the fecal pellet falls away from the opening of the tube. 

Phyllochaetopterus socialis Claparede 

Phyllochaetopterus socialis Claparede was dredged from a bottom of fine sand 
mixed with silt in about two meters of water. Only small numbers of specimens 
\vere collected. 

This species is a very small chaetopterid, measuring only 15-25 mm. in length 
not including the palps. The structure of Phyllochaetopterus socialis (Fig. 1, C) 
is essentially like that of species of Telepsafus and Spiochaetopterus. It differs 
from the members of the other two genera in only minor respects. In PJi \llo- 
chaetopterus the dorsal lip is very large and the ventral lip is cleft (Fig. 4, D and 
E). There is a pair of small antennae-like structures located directly behind the 
palps. They extend anteriorly only slightly beyond the margin of the head. 
These antennae-like processes occur only in this genus and represent modified 
parapodia, for each contains a single seta. The true palps of Phyllochaetopterus 
are somewhat shorter than those of Spiochaetopterus and Tclcpsai'its, approxi- 
mately equaling the anterior body region in length. The anterior body region 
contains 13 setigerous segments, and the middle body region contains from four to 
ten segments. 

The tube of Phyllochaetopterus socialis tends to be branched and crooked (Fig. 
4, A and C ) . Although part of the tube is always buried, a considerable extent 



224 



R( IBERT D BARNES 



may lie liori/ontally above the surface of tin- Mikstratum. The length of the tube 
ranges from 3.0 to 6.0 cm. and the internal diameter from 0.45 to 0.80 mm. The 
luhe is brown in color and has a tough leathery texture and appearance without 
any external annulations. 

In Phyllochaetopterus socialis one to four worms may inhabit a single tube. 
The individuals may occupy separate branches or the same section of the tube, 
but there is no definite correlation between the number of tube branches and the 
number of occupants. In general there is usually one main extent of tube 
with two openings and any side branches tend to be sealed off from the main sec- 
tion. ^Multiple occupancy of a single tube i> not limited to Phyllochaetopterus so- 






-- DORSAL LIP-. 



-ANTENNA - 



DORSAL CILIATED 
GROOVE 



D 




FIGURE 4. Phyllochaetopterus socialis. A. Three worms within a common tube. B. Sur- 
face view of a partition. C. Part of tube. D. Dorsal view of anterior end of body. E. Ven- 
tral view of anterior end of body. F. A recently constructed section of tube adjoining an older 
section. 



cialis but occurs in some other members of the genus, such as Phyllochaetopterus 
prolifica from the Pacific coast of North America. The condition is believed to 
result from fission of the original builder of the tube. 

Partition:-, are located within the interior of the tube and occur anywhere along 
the tube length. The structure of the partition (Fig. 4, B) is similar to those of 
YV/c/\v</7'//.s- and Spiochaetopterus. A number of times two worms were placed in 
one tube. In one instance one of the worms placed a partition between the two 
occupants. In other cases no partition was constructed and the two worms fre- 
quently passed each other in the tube. 

Phyllochaetopterus secretes its tube in a much less regular fashion than does 
'/\'Icf>str;'its or Spiochaetopterus. The worm lays down small crescent-shaped over- 
lapping -ections of varying size (Fig. 4, F). During the process of secretion, the 
body of the worm is not markedly arched nor projected very far out of the tube. 
Partitions are cut out from the tube in the same manner as in Ti'lcsaTHs and 



CHAETOPTERIDAE: TUBE AND FEEDING 225 

Spiochaetopterus and are probably laid clown in the same way also, but they were 
never observed being secreted. 

As in Tclcf>sonts and Spiochaetopterus, water is driven through the tube of 
Phyllochaetopterus by the beating of the membranelles bordering the rings formed 
by the foliaceous notopodia of the middle body region. 

Phyllochaetopterus utilizes three methods to obtain food. Feeding may involve 
mucous bags as in Spiochaetopterus. A mucous bag is secreted by the middle 
ciliary ring of the more anterior foliaceous parapodia, except for the first (Fig. 1, 
C). Each bag is caught by a cupule located behind the ciliary ring. As many 
as eight mucous bags and rotating food balls were observed being formed at one 
time. 

An alternate method to the use of mucous bags appears to be stimulated by the 
presence of a heavy concentration of food particles in the water passing through the 
tube. Under these conditions the notopodial rings spin out a mucous rope instead 
of bags. The rope picks up and traps particles in the swirling water current 
streaming through the notopodial rings. The rope joins with that of other seg- 
ments to form a continuous twisting strand. Mucus for the rope appears to In- 
supplied not only by the notopodial rings which secrete the mucous bags but also 
by the lateral margins of the mid-dorsal longitudinal ciliated groove. A conspicu- 
ous whitish glandular strip flanks the groove in both the middle and posterior 
body region. The mucous rope extends well into the posterior body region and it 
may well be partly secreted in this region. 

Great quantities of mucus are evident when Phyllochaetopterus is removed from 
its tube. The worm becomes so wrapped up in mucus that it is difficult to handle. 
The source of the mucus may be the glandular strips bordering the dorsal ciliated 
groove. Such large amounts of mucus were not found in any other of the chae- 
topterids studied and none possess the glandular strips. 

The palps also seem to be of some importance in feeding. When large amounts 
of artificial detritus or suspended carmine particles were introduced into the gla^s 
tube, the greater part of this material would be collected by the major groove, or 
ciliated gutter, of the palps and conducted downward to the mouth. Material ap- 
peared to be conveyed as easily when the contact was superficial as when material 
was lodged deeply in the groove. 

Feces are removed by way of the dorsal ciliated groove and the ejection groove 
of the palps. Fecal pellets of specimens in capillary tubes were commonly stuck to 
the rim of the tube opening or even to the underside of a tube partition, where a 
partition had been placed above the worm. The palps of Phyllochaetopterus are 
also used to remove undesirable objects from the tube brought in by the water 
current. But this species is less active than other chaetopterids studied and moves 
rather slowly up and down the tube. 

Ransanides sagittaria ( Claparede i 

Ranzu niilcs sac/ittaria (Claparede), which is known only from the Mediter- 
ranean, is the most abundant chaetopterid in the Bay of Naples. Individuals tend 
to occur together in close associations, and large masses of hundreds of adjacent 
tubes were dredged from fine silt and sand in 10-12 meters of water. The tubes 
are composed of an outer layer of sand grains (Fig. 5, A) separated by an inner 



226 



KOBKRT IX RARNES 



secreted cornified organic lining, probably of similar composition to that of other 
chaetopterid tubes. The length of the tubes ranges from 4.5 to 8.5 cm., with a 
bore of 07-1.0 mm. Although these worms occur in compact masses, there is 
little fusion of adjacent tubes. The tubes are more or less straight, oriented paral- 















PALP 



DORSAL 
CILIATED GROOVE 



DORSAL 
CILIATED GROOVE 



RIDGED 
SURFACE - 



MUCOUS 
BAG 



CUPULE 



FIRST 
PUMPING SEGMENT 




D 



FIGURE 5. Ransanides sn/iitt<n-i<i. A-D. Tube construction. A. Sand grains being brought 
to ventral lip by palps. B. Sand grains lining moved to under (outer) surface of lip. C. Ven- 
tral lip pressing sand grains to rim of tube. D. Worm laying down secreted lining of tube. 
E. Bundle of enlarged 4th notnpodial setae puncturing a tub- partition. F. TuV partition in 
place with five perforations. G. Ventral view of anterior end of body. H. Dorsal view of 
anterior end of body. I. Lateral view of four mMd'e body segments in pumping position. 
J. Dorsal view of section of middle body rcL-ion involved in secreting mucous bag. Mesochae- 
toptcnis tttylori. K. Dorsal view of one .segment of middle body region involved in M-creting 
mucous bag. 



( IIAKTOI'TKKIDAK: TLT.K \XD FEEDING 

lei to each other, and rest vertically in the substratum with one opening of the 
tube at the surface. 

Ranzdiiides is only a little larger than Phyllochaetopterus socialis. The length 
exclusive of the palps, is approximately 2.0 cm. to 2.5 cm. The structure of this 
species (Fig. 1, F) departs considerably from that found in Spiochaetopterus, 
Telepsavus, and Phyllochaetopterus. The palps are long and are similar to those 
of other chaetopterids. The ventral lip (Fig. 5, G and H) is very large and 
flaring. Eye spots are present. The anterior body region consists of 12 setigerous 
segments. The usual single lobate notopodium composes the parapodia. The 
fourth parapodium carries five very heavy setae in addition to a bundle of ordi- 
nary setae. 

The middle and posterior body regions (Figs. 1, F and 5, I and J) are not 
sharply demarcated. All of the parapodia are biramous with the neuropodium unci- 
nate. The first parapodia of the middle body region lie immediately behind the 
12th and last parapodia of the anterior body region and possess a short antenna-like 
notopodium. The second parapodia of this region lie considerably behind the first 
and their notopodia are large and wing-like. The third and remaining para- 
podia are placed at regular intervals and the notopodia have the form of short 
stubby fingers. The length of the notopodia gradually increases posteriorly and 
the notopodia of the posterior body region eventually assume an antenna-like form 
similar to that of other chaetopterids. 

The secreted part of the tube is laid down by the ventral surface of the anterior 
body region as in other chaetopterids. The large flaring ventral lip is responsible 
for molding the outer sand grain layer. In the construction of an addition to the 
tube, the outer sand grain layer is added to the old tube before the inner secreted 
lining. This process begins with the collection of sand grains. The anterior end 
of the worm is projected from the aperture of the tube and may be arched ven- 
trally so that the upper (inner) surface of the ventral lip contacts the substratum. 
Particles of sand adhere to the mucus on the lip surface. Sand particles may also 
be collected by the palps (Fig. 5, A) in the same manner. Adhering particles are 
then conveyed downward to the ventral lip in the major groove of the palp. Not 
infrequently a palp is wiped against the lip. The lip surface is strongly ciliated 
and the sand particles are driven over the edge onto the under (outer) surface 
(Fig. 5, B). Periodically the worm retracts into the tube until the lip fits around 
the rim of the aperture like a collar (Fig. 5, C). In this position the lip acts as a 
mold and the sand grains which have accumulated onto the ventral lip surface are 
added to the end of the tube. At frequent intervals the ventral secretory surface 
of the worm is applied against the inner surface of the sand grains, laying down 
the inner secreted part of the tube and also securing the sand grain layer (Fig. 

5, D). 

Worms living in glass tubes commonly constructed partitions at various points 
within the tube. The partitions of Ranzanidcs do not have the distinct form and 
organization of those of other chaetopterids. The partition is merely a simple sheet 
of secreted material, often oriented obliquely across the tube (Fig. 5, F). Several 
perforations are present to permit the flow of water through the tube. 

In laving down a partition the worm merely bends the anterior end ventrally 
across the tube and secretes a film of material in this position. There is no rota- 



22S K ( (BERT D B \KXES 

lion of the anterior end to form a distinct disc as in otluT chaetopterids. The per- 
forations are ])ro(luce(l by the -4th setae. Following secretion of the ])artition, the 
worm flexes within the tube so that the 1th parapodia are at the level of the flexure. 
The body is twisted slightly i om side and the bundle of 5 heavy setae of the 
right fourth parapodium arc- thrust through the partition and then spread like a fan 
to widen the opening (Fig. 5, E). 

The worm removes the partition by ripping it out. The fourth setae are used 
to tear away the partition at its junction with the tube wall. The body of the 
worm is then pushed through the opening. As in other chaetopterids, the 4th 
setae are also used to rip open the side of the tube wall in order to construct a new 
extent of tube. The old branch of the tube is then sealed off from the addition, 
thus always preserving what is essentially a non-branched straight tube. 

Water is pumped through the tube of Ranzanides by the peristaltic action of the 
segments of the middle body region. Following the longitudinal contraction the 
diameter of a segment at the level of the parapodia is increased until the segment 
fills the tube (Fig. 5, I). Dorsally the two short finger-like notopodia fold over 
the mid-dorsal groove, protecting the groove and forming a dorsal margin of con- 
tact with the tube wall. The segment is then moved posteriorly like a piston driv- 
ing water downward through the tube. The effective stroke occurs in an ante- 
riorly directed wave with one segment moving downward after another. In the 
recovery stroke the diameter of the segment is greatly decreased with a resulting 
greatly lowered water resistance. 

Pumping is more or less continual except when the worm is rapidly moving up 
or down the tube. But the strength and rate of pumping vary greatly and are 
dependent upon the rapidity of the peristaltic waves and the anterior-posterior 
length of the stroke. Although water is commonly driven through the tube to 
the posterior of the worm, reverse pumping was observed on a number of occa- 
-ions, particularly when undesirable material had entered the tube. 

Raii.::ani(lcs is a very active worm. It can move rapidly up the tube, employing 
the anterior notopodia in a somewhat leg-like manner as is true of other chae- 
topterids. Also like other chaetopterids it frequently reverses position within the 
tube. 

Ranrjanuh's employs a mucous bag for feeding. A single bag is utilized and is 
-ecreted by the large wing-like second notopodia of the middle body region (Fig. 
5, J). The bag is caught by the large cupule located halfway between the second 
and third parapodia. 

The palps function as accessory feeding organs, conducting detritus particles 
along the major palpal groove to the mouth. But only small amounts of material 
were observed being obtained in this way. When a suspension of carmine particles 
was injected into a capillary tube containing a worm, the greater part of the sus- 

-ion was ejected back out of the tube. A small amount was collected by the 
major groove of the palps and conducted downward to the mouth, and most of that 
which got past the palps was collected by the mucous bag. 

Rev rse peristalsis, or pumping, of the middle body region is apparently of 
primary importance in ridding the tube of any sudden invasion of undesirable mate- 
rial. In this way the greater part of an introduced carmine suspension was ex- 
pelled. The ejection groove of the palps, however, still functions in removing 



CHAETOPTERIDAE : TUBE AND FEEDING 229 

large undesirable particles which enter the tube. The palps are also important in 
removing fecal waste. Fecal pellets released from the anus travel the dorsal ciliated 
groove along the entire' length of the worm and then are expelled from the tube 
by the palps. 

Mesochaetopterus taylori Potts 

The author did not observe tube-building and feeding behavior in living speci- 
mens of Mesochaetopterus, which can be most easily collected along the west coast 
of North America, but preserved specimens of Mesochaetopterus taylori Potts from 
the coast of Washington were examined. When the structure of this species is 
compared to that of the other chaetopterids studied, a number of deductions regard- 
ing its tube-building and feeding behavior can be made. 

Mesochaetopterus taylori inhabits a very long tube which is oriented vertically 
in the substratum. Although only fragments of tubes were examined by the author, 
the total length of the tube may exceed a meter and numerous collectors have at- 
tested to the difficulty of digging up intact specimens. The tube is composed of an 
outer layer of sand grains adhered to an inner secreted organic lining, but in large 
tubes the sand grain layer is often inconspicuous and the secreted layer has a 
parchment-like texture similar to the tube of Chactoptcrns. Nothing can be stated 
regarding the presence or absence of partitions. 

Species of Mesochaetopterus tend to be somewhat intermediate in size be- 
tween Cliactoptents and the other chaetopterids. The single intact specimen of 
M. tavlori examined by the author had a total length of 25 cm. The anterior 
body region (Fig. 1. E) is essentially like that of other chaetopterids. There are 
nine setigerous segments, with especially well-developed notopodia. The 4th 
notopodia carry a bundle of heavy setae like those of Ranzanides. The palps 
slightly exceed the anterior body region in length and bear both a ciliated gutter 
and an ejection groove. 

The middle body region (Figs. 1, E and 5, K) displays marked resemblances to 
that of Ranzanides. The first pair of parapodia follow immediately behind the 
ninth and last notopodia of the anterior body region. It consists of a finger-like 
notopodium and an uncinate neuropodium. The second and third notopodia are 
aliform and each is followed by a large bivalved cupule. The dorsal surface of 
the body following the first three parapodia of the middle body region is strongly 
concave and transversely ridged. The remaining parapodia of the middle body 
region consist of short finger-like notopodia and uncinate neuropodia. Posteriorly 
the notopodia tend to become antenna-like and these segments may constitute a 
posterior body region as in h'anrjanidcs. A dorsal groove runs the length of the 
body. 

It is possible that Mesochaetopterus taylori constructs its sand grain-secreted 
tube in a similar way to that of K'aiizaiiidcs, although the sand grain layer appears 
to be less important in Mesochaetopterus. The bundle of heavy 4th setae is at 
least employed to open the side of the tube wall. If partitions do exist, then these 
setae may be used to perforate them or remove them. 

The water current passing through the tube is undoubtedly generated by peri- 
staltic contractions of the 4th and remaining segments of the middle body region. 
These segments are virtually identical to the pumping segments of Ranzanides. 



2M) KOMKk r I) BARNES 

The presence of tin- two pair> of aliform notopodia, each followed by a large 
cupule, clearly indicates a feeding mechanism employing two mucous bags (Fig. 
1, E). This conjecture has been con !i lined by the observations of Dales at Friday 
Harbor. Potts ( W14) claimed that when the animal is within its tube, the lateral 
margins of these three segments of the body are arched over toward each other. 
partially enclosing the dorsal surface. \Yithin this enclosed space would lie the 
cupules (the over-arching lateral margins are not included in Figure 1, E in order 
that the mucous bags and cupules can be seen). The transversely ridged dorsal 
surface characteristic of these segments would line the enclosed tubular area, but 
the significance of the ridged surface is difficult to understand. 

The secretion of two mucous bags is not characteristic of all species of Meso- 
chaetopterus. M. viinntns possesses but a single pair of aliform notopodia and 
one cupule. which indicates the formation of only one mucous bag. 

DISCUSSION 

The members of the Chaetopteridae display a relatively uniform structure and 
belmior pattern with regard to tube-building and feeding mechanisms. The only 
atypical member is Chaetopterus variopedatus, which, being the most familiar spe- 
cies, has unfortunately tended to color the conception of the family for many 
zoologists. 

The typical chaetopterid is a small worm inhabiting a more or less straight tube 
oriented \ertically in the substratum. One opening of the tube projects above the 
surface of the sand or mud. A branching tube occurs in species of Phyllochaetop- 
terus and in Mesochaetopterus iitiinttiis, but at least in Phyllochaetopterus socialis 
the branches, even when inhabited by another worm, tend to be sealed off from a 
main section. The tube is always composed of an organic secreted material. The 
secreted material is cornified in Telepsafus and Spiochaetopterus. In Phyllo- 
chaetopterus, Mesochaetopterus, and Chaetopterus it may be leathery or parchment- 
like. In species of two genera, Ranzanidcs and Mesochaetopterus, there is an outer 
layer of sand grains cemented to the secreted part of the tube. In all chaetopterids 
the tube is secreted by the convex ventral surface of the anterior body region. 

A striking feature of the tubes of Spiochaetopterus, Telcpsaviis, Phyllochaetop- 
terus, and Raii:::aiiides is the presence of perforated transverse partitions. In Spio- 
chaetopterns and Tclcpscnus, the partitions are always located near the bottom of 
the tube, and in an earlier paper (1 ( >(4| the author suggested that the partition 
functions to prevent the collapse of the thinner-walled tube in this region. This 
may well be one function ot the partition in these two genera; but it can not be 
the only function, for in Phyllochaetopterus and Ranzanidcs the lower part of the 
tube is not particularly delicate nor are the partitions always limited to this level. 

I'ossibly the chief function of the tube partition is to modify the water pres- 
sure in some way within the tube. The rondition of the tube in Chaelopterus would 
seem to support this conjecture. The tube of Chaetopterus lacks partitions, but the 
two openings of the tube have a much smaller diameter than does the greater part of 

tube lying beneath the surface of the substratum. These differences in the tube 
diameter would reduce the speed ot the water current and perhaps account for the 
absence of partitions in Chaetopterus. If partitions do function in modifving the 



CHAETOPTERIDAE: TUBE AND FEEDING 231 

pressure of the water current, then it is very likely that such partitions will be found 
to occur in the tubes of Mesochaetopterus. 

The fourth notopodia of all chaetopterids carry heavy modified setae. These 
setae are used for cutting open the tube wall to permit the construction of a new 
branch or extent of tube, and they are also used for removing partitions and some- 
times for perforating partitions. In Spiochaetopterus, Tclepsarus and Phyllo- 
chaetopterus, the 4th notopodium carries a single heavy truncate blade-like seta, 
which is adapted for neatly cutting out the more button-like partitions constructed 
bv species of these genera. In other chaetopterids, the 4th notopodium carries a 
bundle of heavy spear-like setae which pierce and tear rather than cut. 

As in most sedentary tubicolous polychaetes, chaetopterids are totally dependent 
upon a current of water passing through the tube. The water current brings in 
oxygen and food and also removes excreted and gaseous waste. The current is 
generated in two ways. In Spiochaetopterus, Tclcpsai'us, and Phyllochaetopterus, 
the current is produced by the beating of cilia (probably membranelles) lining the 
ring-like openings created by the foliaceous notopodia of the middle body region. 
In Chaetopterus, Ranzanides, and Mesochaetopterus, the water current is produced 
by peristaltic contractions of the piston-like segments of the middle body region. 
There are three such segments in Chaetopterus (Fig. 1, D) and they are modified 
somewhat differently from the many pumping segments of Ranzanidcs and 
Mesochaetopterus. 

The primary method of feeding in all chaetopterids is the straining of water 
through a mucous bag, a unique mechanism found in few other animals. The 
secretion of the bag is the function of certain notopodia of the middle body region. 
The number of bags secreted simultaneously varies. A single bag is employed by 
Telepsa-rus, Ranzanidcs, Chaetopterus (Fig. 1, D), and Mesochaetopterus ininutus. 
Two bags are spun by Mesochaetopterus taylori and eight or more by Spiochae- 
topterus and Phyllochactopterus. 

A characteristic feature of the family is a pair of palps that arise from the ante- 
rior dorsal surface just behind the mouth. With the exception of Chaetopterus, 
the palps are heavy and long. In Chactoptcrus the palps are very short and small 
(Fig. 1, D). Each palp is provided with a deep longitudinal groove or gutter, 
lined with downward beating cilia. It is possible that the palp and its ciliated 
gutter represent the ancestral means of obtaining food in the Chaetopteridae. This 
is the method of obtaining food in the Spionidae, a tubicolous family closely related 
to the Chaetopteridae. The evolution of the mucous bag for feeding in the chae- 
topterids permitted the utilization of finer food particles than could be obtained by 
the palps. The palps will play a minor accessory role in feeding in most chae- 
topterids, and in Phyllochactopterus are perhaps as important as the mucous bag. 

The principal function of the chaetopterid palps is that of ejection of fecal pellets 
and unwanted debris which enters the tube with the incoming water current. Fecal 
pellets are carried anteriorly from the anus along a mid-dorsal ciliated groove lo- 
cated to the medial side of the ciliated gutter of the palp. The cilia of the ejection 
groove carry the pellet to the distal end of the palp, which at the time of ejection 
projects from the tube opening at the surface. Similarly, large undesirable parti- 
cles or objects which enter the tube are caught on the palp surface, transferred to 
the election groove, and conveyed back to the exterior. 



ROl'.KKT D. I'.ARNES 

The ejecting function of the chaetopierid palps is correlated with the straight 
vertical tube which these worms inhabit. Although such a tube is open at both 
ends, the lower end is buried in >and and mud. The downward-moving water 
current can leave the tube by passing into the interstitial spaces of the surrounding 
substratum, but large masses of debris or foreign objects, even if they passed 
the worm without interfering with its feeding and pumping behavior, would even- 
tually clog up the lower end of the tube. Feces would also contribute to the 
clogging of the tube and must therefore be removed from the opposite end of the 
tube. The primary function of the palps is therefore to maintain an unobstructed 
tube. 

The situation in Clhtctoplcyus is quite different. The tube is U-shaped and 
provided with both an inhalant and exhalant surface aperture. There is no need 
to move undesired objects back out of the inhalant opening. Unwanted material 
which enters the inhalant opening of the tube is pumped through the tube and out 
the exhalant opening. Significantly, the palps of Clntcloptcrus are greatly reduced 
in size. The major groove is still present but the ejection groove is absent. Fecal 
pellets are also flushed out by the exhalant water current, and the mid-dorsal cili- 
ated groove, which in other chaetopterids extends the length of the body as a 
means of transporting fecal pellets, functions only to carry food balls and extends 
from the cupule to the mouth. The peculiarities of Chactoptcnis are clearly corre- 
lated with the U-shaped structure of its tube. 

The chaetopterids and the spionids probably evolved from some common, an- 
cestral tubicolous polvchaete. having palps as organs for obtaining detritus as food. 
The spionids retained this function of the palps. The chaetopterids, however, shifted 
to a feeding mechanism in which the water current passing through the tube was 
strained through a mucous bag; the palps were employed for maintaining an un- 
obstructed tube. 

The chaetopterids appear to have evolved along three main lines, each of which 
should probably constitute a single genus. One line embraces SpiochaZtopterus, 
Telcrsurus, and riiyllocliuctof'tcnis. in which the water current of the tube is gen- 
erated by the ciliary rings of the foliaceous notopodia. The second line includes 
/\'<ni;::ani(lcs and Mcs<iflniclof>tcni.\\ which have the tubes covered by an outer layer 
of sand grains and pump water through the tube by means of the peristaltic con- 
tractions of a large number of segments of the middle body region. The third line 
is represented by Cliactoptcrus. Mere the tube is U-shaped with two openings to 
the surface, the palps are greatly reduced, and a water current is produced by the 
peristaltic contractions of three speciali/ed segments. Chaetopterus is probably 
more closely allied to the Ranzanides-Mesochaetopterus line than to the Spioclmc- 
topterus-Phyllochaetopterus-Telepsavus group. 

ME MARY 

1. Tube-building and feeding were investigated in members of the chaetopterid 
genera. 'fclcpsin-Hs. Phyllochaetopterus, kauzanidcs and Mesochaetopterus. 

2. With the exception of Chaetopterus, all members of the family construct a 
more or less straight tube oriented vertically in the substratum. The tube con- 
tains one or several transverse perforated partitions. 



CHAETOPTERIDAE: TUHK AXD FEEDING 

3. The tube is secreted by the ventral surface of the anterior body region. The 
enlarged fourth notopoclial setae are used to remove partitions or to tear open tin- 
side of the tube wall in order to construct a new section of tube. 

4. In Tclef>sa"c"its and PhyUocliacioptcnts water is driven through the tube by 
the beating of ciliary membranelles. The membranelles line the ring-like opening- 
formed by the foliaceous notopodia of the middle body region. In Raiizanidcs. 
Mesochaetopterus, and Chaetopterus water is pumped through the tube by the 
piston-like action of segments of the middle body region. 

5. A mucous bag is utilized for feeding in all chaetopterids. The number of 
bags varies from one to many and they are always produced by the middle body 
region. 

6. Except in Chaetopterus, a pair of long palps arise from the dorsal side of the 
head. Each palp carries a large and a small ciliated groove. The large groove, 
in which cilia beat proximally, functions as an accessory feeding device. The 
more important smaller groove, in which cilia beat distally. provides for the ejec- 
tion of fecal pellets and any undesirable material which enters the tube with the 
incoming water current. 

LITERATURE CITED 

BARNES, R. D., 1964. Tube-building and feeding in the chaetopterid polychaete, Spiochaetop- 
terus oculatHS. Biol. Bull, 127: 397-412. 

ENDERS, H. E., 1908. Observations on the formation and enlargement of the tubes of the 
marine annelid, Chaetopterns variopedatns. Proc. Indiana Acad. Sci., 1907 : 12S-135. 

ENDERS, H. E., 1909. A study of the life history and habits of Chaetopterus variopedatits. 
J. Morph., 20 : 479-532. 

MAcGiNixiE, G. E., 1939. The method of feeding of Chactopfcn/s. Biol. Bull, 77: 115-118. 

POTTS, F. A., 1914. Polychaeta from the northeast Pacific. The Chaetopteridae. With an 
account of the phenomenon of asexual reproduction in Phyllochaetopterus and the de- 
scription of two new species of Chaetopteridae from the Atlantic. Zool. Soc. London, 
Proc., 67 : 955-994. 



FACTORS AFFECTING FIREFLY LARVAL LUMINESCENCE 1 

ALBERT D. CARLSON 

Dcpiirtnu'i/t of Biological Sciences, S/<itc University of A Yu r Yi<rk at Stony Brook, 

Stony Brook, AV-rc- York 117W 

The mechanism of control of the adult firefly flash, a burst of light lasting ap- 
proximately 02 second, has been studied intensively (Buck, 1948; McElroy and 
Hastings, l l <55 ; Me Kirov and Seliger, 1961 ; Buck and Case, 1961 ; Case and Buck. 
1963; Buck. Case and Hanson, 1963; Smith, 1963). The larva does not produce 
a flash, but rather a uniform, structureless glow lasting for seconds (Dahlgren. 
1917; Buck, 1948). Histologically its light organs represent a considerably sim- 
pler system because they contain no tracheal end cells, cells which are present in 
the adult light organ and implicated in flash control (Dahlgren, 1917; Snell, 1932; 
Alexander, 1943). 

When an adult firefly is subjected to falling oxygen concentration it remains 
dark for a short period. Then a dull glow spreads over the organ, gains in inten- 
sity, and then slowly declines to extinction. If air is readmitted during this "hy- 
poxic glow" a brilliant pseudoflash is produced, lasting 500 milliseconds or longer 
(Snell, 1932). The tracheal end cell valve theory of pseudoflash control, proposed 
by Snell and reaffirmed by Alexander (1943), implied that adult firefly lumines- 
cence was normally oxygen-limited and that the pseudoflash was independent of 
neural activity. Hastings and Buck (1956) also concluded that central nervou.s 
activity plays no part in the pseudoflash of the adult. Carlson (1961) examined 
the pseudoflash of the adult in more detail and implicated neural activity as well as 
hypoxia. 

In lampyrid larvae Buck (1948) and Hastings and Buck (1956) observed that 
low ambient oxygen induces an hypoxic glow and that subsequently increased oxy- 
gen tension elicits a pseudoflash, which resembles that of the adult. The present 
study of the larval pseudoflash response was initiated to determine in what respect 
the adult and larval pseudoflash differ. It was hoped the differences in turn could 
aid in elucidating the disputed role of the adult tracheal end cell in flash control. 

MATERIALS AND METHODS 

Larvae of the genus 1'holiiris were the- subjects of this study. They were 
collected in the early autumn and stored either in petri dishes on moistened filter 
paper at 4 C. or in dirt-filled dishes at room temperature. The experimental ani- 
mal was secured ventral side up on a narrow glass spatula provided with silver 
stimulating electrodes. The paired stimulating electrodes were usually positioned 
on each side of the ventral nerve cord in the sixth abdominal segment by insertion 

Supported by Grant-in-Aid 31-27 from the Research Foundation of the State Uuiversit\ 
i>t \c\\ York and 1>y a urant from tlu j National Science Foundation. 

'SI 



KIRKFLY LARVAL LUMINESCENCE 



235 



through the intersegmental membrane between the sixth and seventh abdominal 
segments. The spatula was placed in the basal segment of a glass Y-tube which 
was 30 mm. long and 1 cm. in diameter. Oxygen and nitrogen were led into oppo- 
site branches of the Y-tube through paired two-way stopcocks which permitted 
rapid shunting of the oxygen from the animal. Gas mixtures were prepared from 
commercial compressed nitrogen and oxygen metered through two-stage reduction 
valves and calibrated Fishcher-Porter flow 7 meters. Composition was checked by 
gas analysis with a Scholander 0.5-cc. analyzer. Commercial compressed nitrogen, 
referred to hereafter as "nitrogen." was found to contain no more than O.OS/fc oxy- 




FIGURE 1. Stimulated glow and pseudoflash of Photuris larva. In this and all subsequent 
experiments except where noted : Upper trace is photomultiplier output ; middle trace heavy 
line is 21% oxygen and narrow line is nitrogen; marks on narrow segment are time base, 1 
mark per second, reading from left to right. Lower trace : stimulus, 5 volts, 20 msec, dura- 
tion, 10 per second frequency. Electrode pair inserted in 6th abdominal segment in this and 
all subsequent experiments of larval light response. 



gen which was considered to be a negligible amount. A photomultiplier tube ( RCA 
931-A) and dissecting microscope were positioned above the animal and both were 
shielded from stray light by black cloth. 

In preparation for a pseudoflash one valve was rotated 180 to shunt oft the 
oxygen and admit either nitrogen or a nitrogen-oxygen mixture. Rotation of this 
stopcock also opened a signal circuit. At an appropriate time the same valve was 
then rotated back 180 which allowed a higher concentration of oxygen to reach 
the animal suddenly and also closed the signal circuit. The pseudoflash was de- 
tected by a photomultiplier, the output of which was led to one or both channels 
of a Grass Polygraph. In some cases the amount of light recorded in a pseudoflash 
was obtained by integrating the light output with an integrating circuit utilizing 
a Philbrick operational amplifier. 



236 



ALBERT D. CARLSON 



RESI MS 



1. dcncral characteristics <>\ larral //<//// responses 

The light induced in the larval organ by mechanically irritating the animal is 
variable in intensity and duration. An electrically stimulated response which mimics 
the mechanically induced light response is shown in Figure 1. A pseudoflash is 
also shown. The pseudoilashes of the larval and adult forms resemble each other 
more closely than do their respective natural light responses, which confirms 
the observation of I lasting and I'.uck ( 1956). The larval pseudoflash differs from 
the adult pscudoflash, which is illustrated in Figure 2 with a number of spontaneous 
flashes, in being more variable in duration and considerably longer, due to its in- 
creased decay period. Luminescence intensity is uniform over the larval lantern in 
all light responses. 




FIGURE 2. Spontaneous flashes and hypoxic j>lo\v followed by a pseudoflash in a I'liotuns 
adult male. Lower trace equivalent to middle trace in Figure 1. Hypoxic glow begins ap- 
proximately 7 seconds after hypoxic onset; note its relatively low intensity. Note lengthening 
duration of adult spontaneous flash as anoxia proceeds. 

2. Effect oj electrical stimulation and o.\'v</en concentration on larra! light re- 
sponses 

Glow intensity in air is proportional to stimulation frequency up to approxi- 
mately 10 stimuli per second, above which no further increase can be produced. 
Induced glow intensity also varies directly with oxygen concentration between 0% 
and 10'.; oxygen as shown in Figure 3. \Yhereas the adult will produce a glow 
in nitrogen, the larva cannot be induced to glow in this gas after the initial 15 sec- 
onds of perfusion. During continual stimulation glows can be maintained for long 
periods in oxygen concentration as low as 0.25'/o and the glow level responds to 
rapid alternation of oxygen concentration. 

Larvae that have been glowing actively in air can produce pseudoflashes when 
the oxygen tension is manipulated, \on-glo\\-ing larvae must first be mechanically 
or electrically stimulated during the anoxic period before a pseudoflash can be 
produced by admitting oxygen. This .stimulation during anoxia need not elicit 
glowing to be effective in pseudollash production. Larvae left unstimulated for 



FIRKH.Y LARVAL LUMINESCENCE 



237 



periods up to 20 minutes in nitrogen failed to produce a pseudotlash upon re- 
admission of air, but would readily do so if stimulated during the anoxic period. 

Like the glow in air, the maximum intensity of the larval pseudoflash is also a 
function of stimulus frequency. It readies a maximum intensity at about 10 stimuli 




8 12 16 20 24 

OXYGEN CONCENTRATION (%) 



28 



FIGURE 3. Effect of oxygen concentration on the maximum glow intensity attained during 
stimulation in one Pliotiiris larva. Stimulus : 7 volts, 20 msec, duration, 10 per second fre- 
quency. Stimulation applied in various oxygen concentrations until a constant, maximum light 
intensity was attained. Recovery period in air was longer than 60 seconds. Oxygen concen- 
tration randomized during repeated experimental runs. 



per second when the animal is stimulated during a period in nitrogen, as shown in 
Figure 4. The total light output of the pseudoflash was closely related to its maxi- 
mum intensity. If stimulation is continued during readmission of air after hypoxia. 
an after-glow is produced on the falling phase of the pseudoflash, as shown in Fig- 
ure 5. Pseudoflash intensity is proportional to oxygen concentrations at least up 



238 



AU1KRT I). CARLSON 



to 21' i oxygen, as .shown in Figure (>. The pseudoflash shape can be changed 
into a multi-peaked response with rapid alternation of lQ f / ( oxygen and nitrogen; 
see Figure 7. 

3. Effect of liypo.ua duration on pscHdofhish intensity 

\\'itli constant conditions of stimulation, pseudoflash intensity declines as the 
duration of hvpoxia increase^, as shown i" Figure S. T.ong hypoxic durations and 



55 



50 



45 



^40 

c 





o 

35 



>- 
30 



UJ 



25 



o 20 
o 



Ul 

a 



15 



10 



01234 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 

STIMULUS FREQUENCY (stimuli /second) 

l-"i<,ri<K 4. ICtTcct nf stiinuln-. frcinK-iu-y during anoxia on pseudoflash intensity in J'lioturis 
larvai-. I'^arli p lint npivsuiK the mean pM'ii:lollasli intensity of 6 larvae except 2.5 stimuli per 

econd \\liich represents 4 larvae, liar-, indicate 2 standard errors. All intensities are rela 
live to the mean intensity ohlaiiK'd at 15 stimuli per second in the same individual in order to 
eliminate differences in M l ' c niftry "f light-collecting system. Stimulus frequency randomized 
during repealed experimental runs. Stimulus voita.^e constant lor each larva, 20 msec, dura- 
tion. Stimulation applied 5 .seconds after hypoxic onset for a total duration of 5 seconds. 
Ifypoxic duration 15 seconds. Recoverj perm.] l)et\\eeii pseudoflashes lasted 45 seconds; nitro- 
.'II used durinu hypoxic period and pseudoflashes induced with 21'- oxygen. 



FIREFLY LARVAL LUMINESCENCE 



239 










FIGURE 5. Effect of electrical stimulation prior to and during pseudoflash in Photnris larva. 
Lower traces stimulus, 4 volts, 40 msec, duration, 10 per second frequency. 



36 r 



32 - 



- 28 - 



24 - 



320 



z 
x 
< 



o 

Q 
3 
UJ 
CO 

Q. 



16 - 



12 - 



8 - 



4 - 




12 
PERCENT OXYGEN 



16 



20 



FIGURE 6. Effect of pseudoflash-inducing oxygen concentration on pseu loflash intensity 
in one Photnris larva. Stimulus 3 vo'.ts, 20 msec, duration, 10 per second frequency. Stimu- 
lation applied 5 seconds after hypoxia onset for a total duration of 5 seconds. Hypoxia dura- 
tion varied from 16 to 23 seconds. Recovery period between pseudoflashes was 1 to 2 minutes. 
Nitrogen used during hypoxic period. Oxygen concentration randomized during repeated ex- 
perimental runs. 



240 



ALBERT D. CARLSON 



their concoinitantly reduced pseudofiashcs did not affect the intensity of immedi- 
ately following pseudoflashes induced after .short hypoxia. 

I )ISCUSSION 

The similarities bet \\ecn the adult and larval pseudoflashes, with respect to 
induction se(|uence. response to electrical stimulation and response to oxygen con- 
centration, surest that both utilize the same basic process. In both developmental 
forms, the pseudoflush appears to be the result of an accumulation of light-producing 
substance and its rapid oxidation by the inrushing oxygen. 









_ I l- 




















FIGURE 7. Effect of rapid alternation of 10% oxygen and nitrogen on the pseudoflash re- 
sponse of Plwtnris larva. Middle trace: heavy line. W< oxygen; narrow line, nitrogen. Lower 
trace: -timuhis, 4 volt-, 4(1 in-ec. duration. 10 per second frequency. 

Then- are a number of differences, however. between the responses ot the two 
forms. 

(I) Adults which are not Hashing immediately prior to anoxia can produce a 
pseudoflash without stimulation during the anoxic period. Non-glowing larvae 
must first be stimulated in some fashion during anoxia before a pseudoflash can be 
elicited. This might suggest that it is hypoxia alone which triggers the hypoxic 
glow in the adult. However, spontaneous neural activity invariably occurs prior 
to onset of the hypoxic glow in the adult, as observed by Carlson (1962). The 
need to stimulate the larva then perhaps reflects a relative lack of spontaneous 
neural activity. 

(2} The adult can maintain a glow in nitrogen for several minutes, indicating 
that oxygen is still available for the light reaction. The larva is apparently com- 
pletely deoxygenated within 15 seconds in nitrogen because stimulation induces 
no glow after that period as anoxia continues However, the anoxic larval photo- 
cytes are still responsive to electrical stimulation because stimulation initiated after 
the first 15 seconds of the anoxic period makes possible a pseudoflash upon re- 
admission of air. This difference in time necessary to flush out the oxygen during 
exposure to anoxic gas may be a reflection of the relatively more complex tracheal 
supply of the adult (Murk, TH<S). If this assumption is correct the observation 



FIRFFLY LARVAL LUM1XES' 



241 



that the larval pseudoflash occupies, on the average, about six times the duration 
required for the adult pseudoflash cannot be explained on the basis of oxygen diffu- 
sion rates to the photogenic tissue. If diffusion rate controlled pseudoflash dura- 
tion one would expect the adult pseudoflash to lie of longer relative duration due to 



> 









14 



i 



D 6 



o 



20 40 60 80 100 120 

HYPOXIC DURATION (seconds) 



140 






ISO 



FIGURE 8. Decline of pseudoflash intensity with increasing hypoxia duration under con- 
stant conditions of stimulation in one Photuris larva. Stimulus : 6 volts, 4 msec, duration 
and 10 per second frequency. Stimulation applied 15 seconds after hypoxia onset for a total 
duration of 2 seconds. Recovery period between pseudoflashes was 60 seconds; nitrogen usrd 
to produce hypoxic period and pseudoflashes induced with 21% oxygen. First point is average 
of 34 measurements; line shows total range of values for that point. 

the evident impediment to oxygen diffusion noted in its resistance to deoxygenation. 
The explanation for the differences in pseudoflash duration between the two forms 
must lie, therefore, at another level in the luminescence process. 

(3) As illustrated in Figure 2, as anoxia proceeds the adult spontaneous flashes 
decline in intensity and increase in duration and then are replaced by a low level 
glow. Further, this shift from flash response to hypoxic glow fails to develop above 
an oxygen concentration of about 2.5%; instead the adult can continue to produce 



242 ALBERT I) CARLSON 

small, spontaneous or electrically driven flashes (Hasting and Buck, 1950). The 
lar\al glow shows no such discontinuity but is simply proportional to oxygen con- 
centrations below 10'. r under uniform stimulus conditions. One might explain 
these differences on the basis that the larval glow and the adult hypoxic glow are 
similar phenomena in that they represent processes which are oxygen-limited. 
Above about 2.0% oxygen, however, another limiting process may be superimposed 
upon the light reaction in the adult which results in a flash response. This non- 
oxygen-limiting process may involve the tracheal end cell or may be due to im- 
portant biochemical differences within the photocytes of the larval and adult forms. 
There is no comparable experimental evidence that the inactivation of light- 
producing .substance by non-luminescent means, which may occur in the larva during 
anoxia, also occurs in the adult. Long anoxic durations, which apparently result in 
a large inactivation of substance by some dark reaction in the larva, do not preju- 
dice the intensity of later pseudoflashes induced with shorter anoxic periods. It 
would appear that this non-luminescent inactivation of light-producing substance 
Iocs not prevent reactivation for use in subsequent flashes. 

SUMMARY 

1. Electrically stimulated light responses and pseudoflashes were studied in 
larval fireflies, Photitris sp. 

2. The larxal pseudoflash is highly variable, but it is considerably longer in dura- 
tion than the pseudoflash produced by the adult. 

3. Larvae which were not previously glowing in air would not produce pseudo- 
Hashes unless stimulated during the anoxic period prior to admission of oxygen. 
Even with stimulation no glow could be produced in nitrogen. Pseudoflash inten- 
sity is proportional to stimulus frequency up to 10 stimuli per second. Light in- 
tensity is dependent on oxygen concentration up to 10% oxygen under uniform 
stimulus conditions. A multipeaked pseudoflash response can be obtained with 
rapid alternation of 10% oxygen and nitrogen. 

4. Pseudoflash inten.sity declines with increasing hypoxic duration under uni- 
form stimulus conditions. 

5. Differences between the larval and adult pseudoflash response are discussed, 
but no difference could be linked directly to the operation of the adult tracheal end 
cell. 

LITERATURE CITKD 

AI.EXAXDKR, KOHERT S., l c '43. Factors controlling firefly luminescence. /. Cell. Camp. Plivsiol., 

22: 51-71. 
PIIVK, JOHN B., 194S. The anatomy and physiology of the li.nlit or.uan in fireflies. .Inn. N. V. 

A cad. Sci.. 49: 397 482. 

II it K, JOHN, AND JAMES F. CASK, 1961. Control of flushing in fireflies. I. The lantern as a 

neurocftYctor or.<;an. />/'/. Hull., 121: 234 256. 

III i i.. JOHN, JAMES F. CASE AND FKAXK F. ll.\xsn\, JR., 1963. Control of flashing in fire- 

flies. III. Peripheral excitation. Biol. Bull., 125: 251-269. 
CARLSON, AI.BKKT I)., 1961. Effects of neural activity on the firefly pseudoflash. />/'(>/. Hull., 

121 : 265-276. 

RLSON, AI.ISEKT D., 1962. Neural activity during hypoxia in adult firefly. />'/<>/. />';///., 123: 
490. 



FIKKFLY LARVAL LUMINESCENCE 243 

CASE, JAMES F., AND JOHN BUCK, 1963. Control of flashing in fireflies. II. Role of central 

nervous system. Biol. Bull., 125: 234-250. 

DAHLGREN, ULRIC, 1917. The production of light by animals. /. Franklin fust., 183: 323-348. 
HASTINGS, J. WOODLAND, AND JOHN BUCK, 1956. The firefly pseudoflash in relation to photo- 
genic control. Biol. Bull., Ill: 103-113. 
McELROY, W. D., AND J. W. HASTINGS, 1955. Biochemistry of firefly luminescence. Pp. 161- 

198. /;/: Luminescence of Biological Systems (Ed. F. H. Johnson), Amer. Assoc. 

Adv. Sci., Washington. 
MrEi.ROY, W. D., AND H. H. SELIGER, 1961. Mechanisms of bioluminescent reactions. Pp. 

219-257. In: Light and Life (Ed. by William D. McElroy and Bentley Glass), The 

Johns Hopkins Press, Baltimore. 
SMITH, DAVID S., 1963. The organization and innervation of the luminescent organ in a firefly, 

Photuris pciuisylrauicd (Coleoptera). /. Cell Biol.. 16: 323-359. 
SNELL, PETER A., 1932. The control of luminescence in the male lampyrid firefly, Phutitris 

pennsylvanica, with special reference to the effect of oxygen tension on flashing. /. 

Cell. Comp. Physinl., 1 : 37-51. 



THE MECHANISM OF THE SHADOW REFLEX IX CIRRIPEDIA. 

11. PHOTORECEPTOR CELL RESPOXSE, SECOND-ORDER 
RKSroXSKS. \XI) MOTOR CELL OUTPUT 1 

<;. K <;\YIU.IAM 

Department ,>f Biology, Reed Colleuc, Portland, Orcuon ''/"Jrf'J. and the Marine 
Biological /.ahomtory. U'onds Hole. Muss. 02543 

In a previous report ((iwilliam, 19(>3), certain electrical events at various 
locations in the nervous system of cirripedes, associated with changes in the light 
level impinging upon a photoreceptor, were described. At that time it was sug- 
gested that the photorece])tor cells have axons that do not synapse until reaching 
the supraesophageal ganglion, and that these cells influenced the activity of the 
second-order neurons by passive electrotonic conduction of a depolarizing poten- 
tial which occurs when the photoreceptor is illuminated. Recent evidence from 
electron microscopv ( Fahrenbach. 19o5 ) supports this suggestion from a structural 
point of view, and observations to be reported here lend functional support. 

Recent papers on the structure of the crustacean nauplius eye (Kauri. 1962; 
Elofsson, l' 1 ')^! indicate that it is made up of three components, and the evidence 
presented here that adult balanid barnacles possess three (paired lateral and single 
median) photoreceptors suggests that they may well be derived from the three- 
parted naupliar eye found in the larvae of Bahiiins (Kauri. 1962). \Yhile it ap- 
pears that the detailed structure of the three "compartments" of the larval medial 
eye does not coincide with that of the presumed separated components of the adult 
photoreceptors, the mere existence of three distinct components indicates a possible 
developmental source of the three adult photoreceptors. 

In addition, further information has been obtained on neural pathways from the 
ocelli to certain of the muscles responsible for the withdrawal-closure response to 
a shadow that is so characteristic of most barnacles. 

A I. \TKKJALS AND METHODS 

Three species of barnacles have been used in this study. Balanus eburneus 
( lould was used for the intracellular recording, and the animals were supplied by 
the Supply Department of the Marine Biological Laboratory, Woods Hole, Mass. 
Other observations were made on specimens of Bahnins tintinnabulum (L.) and 
ttahnius cariosus (Pallas). The former were supplied by Dr. Eric Barham, Xavy 
Klectronics Laboratory. San Diego, California, and Dr. James Case, University ot 
California. Santa I'.arbara. The latter were collected by the author from the north 
central Oregon coast. 

The lateral eyes of I>. clninicns were exposed by splitting the shell along the 
longitudinal ( rostrocarinal ) axis, carefully removing the opercular valves with the 

ipportrd by a tyrant to thr author ((,-l 3l ( h from the National Scienrc Foundation. 
\ critical reading of the manuscript by J)r. Harold 1',,-inic-, U ratrlully acknowledged. 

'I I 



THE SHADOW REFLEX IN CIRRIPEDIA 245 

body of the animal attached, leaving two "half-shells," each of which bears a lateral 
eye and a short length of retinula cell axons. The eye is located at the junction of 
the fused rostrum-rostrolateral and lateral shell plates and is easily visible 
with the naked eye. These "half-shells" were then mounted, inner surface up. 
with soft wax, in the recording chamber. The pigmented mantle over the photo- 
receptor was then dissected away, and the capsule of the tapetum or reflecting layer 
was removed. This permitted direct viewing of the photoreceptor cells, which 
appeared as two orange-yellow areas in each eye, although histological examination 
reveals three cells. 

Xext, the preparation was treated with trypsin (X 300), 80 mg./lOO ml. of sea 
water, for 45 minutes. Following this treatment, penetration with the micro- 
pipettes could be accomplished in many preparations. Attempts to penetrate cells 
without enzymatic pre-treatment were never successful. 

Glass micropipettes filled with 3 .17 KC1. having a resistance of 10-15 megohms 
in sea water, served as electrodes. The amplifier used was an Argonaut "nega- 
tive capacitance electrometer" which fed into a Tektronix type 502 dual beam 
oscilloscope. 

Other recording techniques, the control of light to the preparation, and the 
making of permanent records are described elsewhere (Gwilliam, 1963). Light 
intensity is referred to as "unit intensity" or "intensity one" or a percentage of unit 
intensity achieved with neutral density filters. Unit intensity was approximately 
1,000 foot-candles at the preparation. 

The preparation used for external recording was achieved in the following 
manner: The opercular valves, bearing the body of the barnacle, were dissected 
free from the shell. This was then placed in a wax-lined dish, opercular plates 
down, and a pin thrust through the median junction of the apex of the scuta. 
The body was then extended along the longitudinal axis away from the terga and 
pinned. This exposed the mouth and ventral surface, brought the adductor muscle 
into view, and made dissection of the median photoreceptor, supraesophageal gan- 
glion, and circumesophageal connectives relatively simple. In this position the lat- 
eral photoreceptors are found beneath the body of the animal close to the scutal 
margin just to either side of the mid-line in B. tintiniiabulitin, but would not be in- 
cluded in the preparation in R. cburncns, for in that species the eyes are displaced 
more basally and laterally onto the shell lining (see above). 

The supraesophageal ganglion must be exposed by dissection, which then makes 
it possible to locate and identify the circumesophageal connectives, the antennular 
nerves (which contain the lateral ocellar axons) and the suprasplanchnic nerves. 
The area overlying the adductor muscle may be dissected away, which exposes the 
median ocellus and its nerve, the adductor muscle itself, and the great splanchnic 
nerve, with its adductor muscle motor branch. Further, one can expose the ventral 
ganglion and the cirral nerves to make the latter accessible for recording motor 
output. All this can be done without disrupting the circuit as illustrated in the 
diagram (Fig. 1 ), so that it is possible to record at any one site and remove sensory 
input as desired. Thus, one can record from cirral nerves or adductor muscle 
motor nerves with the rest of the system intact, cast a shadow, observe the effect, 
and then cut either the lateral or median ocellar nerve and again observe the effect 
of a shadow. The same procedure can be followed when recording from the circum- 



246 (l . ]. , ,\\ ILLIAM 

esophageal connectives, 1ml as it is necessary to cut them close to the ventral 
ganglion for recording, it would no longer IK- possible to record responses to shadows 
in cirral nerves or in the adductor motor supply. 

RESULTS 
Structure 

After dis.section as described almve. the terga and the scutal apex would he at 
the bottom of Figure 1. \\ith the- cirri extending from the top. The general body 
surface viewed is morphologically the ventral surface. In most cases the median 
ocellar nerve can be seen through the thin, usually non-pigmented exoskeleton, and 
the "ophthalmic ganglion" of Darwin (the median photoreceptor) can sometimes 
be seen King very close to the adductor muscle, at which point it is attached. It is 
also usuallv possible to see the great splanchnic nerve which originates on the 
dorsal aspect of the ventral ganglion, runs out laterally to the scuta, and gives off 
a motor branch that supplies the adductor muscle. 

The diagram in Figure 1 is based on B. tintiniiabitluin, the same species illus- 
trated by Darwin ( 1S54, PI. XXVII, Fig. 2), hut apart from differences in orienta- 
tion of the two figures, one significant difference should be noted. Darwin as- 
sumcd (but did not actually see) a connection between the lateral ocelli and what 
he called the ophthalmic ganglion (the median photoreceptor of Figure 1 ) which I 
cannot find. It is clear that the lateral ocellar axons enter the supraesophageal 
ganglion independently of the median ocellar nerve, since severing the median ocel- 
lar nerve at the supraesophageal ganglion does not interfere with responses to 
shadows in the rest of the .system as long as the antennular nerve is intact. 

In a previous paper ( ( i\\ illiam, 1963), I stated that the median photoreceptor 
was probably the onlv one present in B. cariosus, and that it was onlv occasionally 
functional as a photoreceptor in B. cbitrncns. I am now convinced that both of 
these statements are in error, for lateral photoreceptors can be demonstrated physi- 
ologically in B. airiosus, if care is taken not to cut too close to the scuta when dis- 
secting the opercular plates free. The small size of B. cbunicits and consequent 
difficulty in dissecting make it likely that previously the median ocellar nerve was 
damaged in many preparations of that species. 

These new observations, and the fact that B. tlntitinabnlnui, B. baluints, B. cre- 
iiatns and B. hdhnioidcs all possess both lateral and median ocelli convinces me that 
all balanids probably conform to the pattern illustrated diagrammatically in Figure 
1. but that the lateral ocelli are better developed and more obvious in some than in 
others. B. churncns and /!. nnipliilritc are similar in having obvious, pigmented 
lateral ocelli in the position described for B. chiimcus by Fales (1928). In B. tin- 
tnnntlntlniii, B. Ixi/iinns and l>. crcnntns they are not so obvious and lie closer to the 
mid-line, just inside the margin of the scuta in the opercular membrane. In B. 
ruriosiis and B. bahnioidcs the lateral ocelli have not been seen, but can be physi- 
ologically demonstrated to occupy a position similar to that in the last three species 
mentioned. 

The structure of the photorn ,-ptors themselves is reported by Fahrenbach 
(1965) for the median ocellus of B. cariosus and, in less detail, for the lateral ocelli 
of /,'. (iiupliitriic which are virtually identical to B. chiinicus. Fales (1928) reports 
two large photoreceptor cells in each lateral eye of B. ebnrncus, but there are in 



THE SHADOW REFLEX IN CIRRIPEDIA 



247 



fact three (based on examination of serial sections of B. eburneus lateral ocelli). 
In both the median and lateral ocelli examined, there is no ommatidial organization, 
and no evidence of a synaptic layer close to the ocellus. The cell bodies ha\e finger- 
like "dendritic"' projections \\h!ch lu-ar the micnnilli, and each soma has a large 
axon that apparently does not synapse until the level of the supraesophageal gan- 
glion. The size of the axons (15-20 /A in diameter i and the nature of the glial 



Cirral nerve 2-6 



Lateral photoreceptor 



Antennular nerve 



Cirral nerve 1 
Circumesophageal nerve 

Supra-esophageal ganglion 



Great splanchnic nerve 



( \ Median photoreceptor 




Scutum 



FIGURE 1. Diagram of the balanid central nervous system, showing the relationship of the 
photoreceptors to it. Based on B. tintiniuilnilum. Details of branching in the antennular nerve 
are schematic and are included simply to indicate that the nerve is mixed. 

sheath around the ocellar nerve suggest a high value for the length constant of the 
axons. 

Electrical activity 

(a) Balaims eburneus: intracellularly recorded responses 

Although direct proof of penetration of photoreceptor cells is lacking, it was 
assumed \vhen a maintained negative potential was recorded. Further, only those 
preparations which showed reversible depolarization when exposed to a light flash 



248 



i , I ,\\ IU.IAM 



\\rre assumed to have been successfully impaled. Such cells could often be held 
for as long as three hours, hut relatively few such preparations were obtained. In 
the limited time available, only ;i total of twelve preparations met the above criteria 
for any significant length of time. 




F 



IMM-RE 2. Intracellular records from the lateral photoreceptor cells of Balanus cbnrncits. 
Inset time calibration applies to \\ through M; voltage calibration applies to all records. In 
tin- figure and all other-,, upward deflection of the second beam indicates "on." A, sustained 
response. B and C, the response to a 0.8-sec. light flash, B at a lower intensity than C. D, 
response to a flash of unit intensity. [:. 1.0% unit intensity. F, 0.1% unit intensity. Photo- 
cell failed to record in I 1 ', and K. G-K, Decreasing time series. L. The membrane potential at 
the close of the time scries. M, the effect of a light flash on the removed electrode. 

In such cells, membrane potentials recorded varied considerably, depending on 
the immediate history of the penetrated cells. Initial membrane potentials recorded 
on penetration while viewing in relatively bright light were on the order of 30-45 
mV, inside negative. After one hour in darkness these approached 60-70 mV. 

The wave-forms of the potentials recorded when the preparation was exposed 
to a (lash of light are shown in Figure 2. This consists of the familiar "on" transi- 
ent, often, but not always, a secondary rise, followed by a maintained level of de- 
polarization. At "off" this drops very close to the original membrane potential 
level ( Fig. 2. A, B, C). Amplitude of the generator potential was graded in dif- 
ferent light intensities (Figure 2, D, E, F), and the transient disappeared at low 
intensities. The wave-form also varied in light flashes of intensity one, but of 



THE SHADOW REFLEX IN CIRRIPEDIA 249 

different duration (Fig. 2, G-K). In this case only the transient remained in 
flashes less than 0.5 second in duration. 

At the highest intensity the transient may overshoot zero potential, but this 
could not be determined with certainty, because of the shifting membrane potential 
dependent on previous exposure to light (cf. Naka, 1961 ; Naka and Eguchi, 1962a ) 
and to the D. C. drift in the amplifier. However, in a dark-adapted preparation, 
the transient seldom exceeded 55 mV, which suggests that overshoot did not occur 
if membrane potentials reached the values of 60-70 mV which were recorded in 
other cells after dark adaptation. 

It will be noted that these intracellular responses are very similar in form to 
the presumed intracellular response from the median eye of B. cariosits as previ- 
ously reported (Gwilliam, 1963, p. 476) and very similar to the simple electro- 
retinograms recorded from barnacle ocellar nerves, if the difference between A. C. 
and D. C. recording is taken into account. That is, the extracellularly recorded 
"mass" response is directly comparable to the single unit intracellularly recorded 
response, both being almost certainly uncomplicated by post-synaptic events. 

Under the conditions of the observations reported here, it seems highly unlikely 
that the "on" transient has its source in other than the impaled retinula cell. The 
photoreceptor consists of three primary receptor cells, supporting cells, and very 
little else. There are no nearby post-synaptic cells to contribute, so the suggestion 
that the "on" transient originates elsewhere (Burkhardt and Autrum, 1960; Burk- 
hardt, 1962) seems to be ruled out in this material. As Ruck (1964) points out, 
the recorded amplitude alone of the transient argues very strongly against its origin 
outside the retinula cell. 

The records are also uncomplicated by anything resembling ordinary spikes. 
This is also true of the ocellar-nerve recorded ERG when the bundle is uncontami- 
nated with other nerve fibers. There is thus no evidence that the retinula cell axons 
conduct ordinary spikes, despite the relatively great distances over which they pre- 
sumably transmit. 

It may be argued that in the illustrated cases the photoreceptor cell axons have 
been damaged in the exposure procedure, and that this could in turn destroy the 
spiking locus. However, if the "on" transient is accepted as an axonal event, 
its presence in these records argues against extensive axonal damage. It might 
also be argued that the light levels used are insufficient to operate the spiking 
mechanism, but the same light levels serve to inhibit firing of cells in the supra- 
esophageal ganglion, proving that they are adequate to operate the normal post- 
synaptic inhibitory mechanism that leads to the shadow reflex upon release. 

The suggestion put forth by Ruck (1964) that the transient may be a regenera- 
tive event has not been adequately tested in this material, but as Ruck himself 
points out, this will not account for sustained transmitter action on post-synaptic 
cells. 

( b) External recording in B. tintinnabulum and B. cariosus 

1 . Lateral vs. median photoreceptor function 

Having established that two morphologically distinct sets of photoreceptors ex- 
isted, I tried to discover if they had different functions. The records reproduced 
in Figure 3 illustrate the results of observations on the two species. Figure 3, A 



250 



G. F. r, WILLIAM 



illustrates a circumesophageal connective recording of the results of a shadow cast 
on a preparation of B. cariosus with both sets of photoreceptors intact. Figure 
3, B was taken from the same preparation after severing the median ocellar nerve, 
and Figure 3, C after severing both antennular nerves. Figure 3, D is from a dif- 
ferent preparation of the same species, the electrode recording from the motor 




FTGFRF. 3. Function of the lateral and median eyes in />'. luriasits (A-K) and 
/>'. tintinnabulum ((J-K). See text for explanation. 

supply to the adductor muscle, with both sets of photoreceptors intact. Figure 3, E 
is from the same preparation with the antennular nerves severed, and in Figure 3, F 
the median nerve has been M-vered as well. 

These records prove the existence of lateral photoreceptors in B. cariosus, estab- 
lish that both sets are capable of mediating the shadow reflex, and suggest that 
there is no difference in function between the lateral and median photoreceptors in 
this particular pathway. 



THE SHADOW REFLEX IN CIRRIPEDIA 



251 



Figure 3, G is a circumesophageal recording from B. tintinnabulum with both 
sets of eyes intact; in H, the antennular nerves have been cut; and in I, the median 
nerve was also severed. Recordings from the adductor motor supply give the same 
results as in B. cariosns. These records, also, indicate that there is no difference 
in function of the two sets of photoreceptors in B. tintinnabulum. 

Figure 3, J and K were obtained from the cut ends of the nerves containing the 
photoreceptor axons. J is a record of the lateral ocellar ERG taken from the cut 
end of the nerve close to the supraesophageal ganglion, while K was recorded from 
the same relative position from the median nerve. Both records were taken at the 
same overall gain and band pass frequency (0.3-2000 cps) and at the same dis- 




FIGURE 4. Series to illustrate different rates of adaptation to multiple stimuli at different 
points in the responding system. Upward deflection of upper beam in A and lower beam in F 
indicates positivity of active electrode. Membrane potential in F (indicated by the initial sepa- 
ration of the two beams) 60 mV. In F, downward deflection of upper beam indicates "off." 

tance above the bathing medium. The difference in amplitude, rise and decay 
times in the two records may reflect the greater distance of transmission of the lat- 
eral ocellar axons (Fig. 3, J) and probably illustrates the decremental nature of 
ocellar axon transmission. 

It should be pointed out at this juncture that the initial deflection of the ERG 
when recording externally from the ocellar nerve at some distance from the photo- 
receptor with a single active electrode is positive in sign rather than negative as 
erroneously reported in Gwilliam (1963). If the record is taken just distal to, or 
from the region of the photoreceptor cells, the sign is reversed. This result then 
accords with other arthropod ocelli in which the ERG is cornea-negative and 
retinula cell axon-positive when recorded extracellularly, as shown by Ruck (1961) 
and others. 



G. F. GWII.LJAM 



2. The response to multiple slun: 

Gwilliam (1963) briefly reported that the response to multiple shadows at 
different points in the photoreceptor-motor output chain showed different rates of 
adaptation. This was investigated more fully in B. tintinnabulum and B. cariosus. 
two species obtained from rather different habitats and showing different behavioral 
reactions to multiple shadows. The similarities and differences between the two 
species are illustrated in Figin 

Figure 4, A illustrates the non-adapting nature of the ERG in B. cariosus, and 
identical records have been obtained from B. tintinnabulum. Figure 4, B is a 
record of a circumesophageal recording from B. tintinnabulum which illustrates that 
at this point (the presumed .second-order neurons) adaptation is very slow, but will 
fail to follow after approximately 30 shadows of the duration and frequency shown. 
A very similar phenomenon can be demonstrated in the circumesophageal connective 
of B. cariosus. Similarly, the motor output to the cirri in B. tintinnabulum adapts 
very slowly (Fig. 4, C), but the cirral output in B. cariosns adapts very rapidly. 
often failing to follow even after a single shadow (Fig. 4, D ) , and seldom persisting 
for more than four shadows. In both species the motor output to the adductor 
muscle fails to follow after 1-4 shadows (Fig. 4, E of B. cariosits). Figure 
4, F is a record of an intracellular response of one of the giant muscle fibers from 
the adductor muscle in B. cariosus and illustrates the effect of a burst of motor 
nerve action potentials on the muscle junctional potentials ( rf. Fig. 4, E) in re- 
sponse to a shadow. 

If one now turns to an intact, feeding animal and presents shadows of the 
same duration and frequency used in the neurophysiological work, the behavior of 
each species corresponds to the pattern seen in the records of Figure 4. 

B. tintinnabulum will respond by withdrawal of the cirri and valve closure (ad- 
ductor muscle contraction) to the first shadow. If shadow-casting is continued, the 
animal very quickly emerges, but continues to withdraw the cirri at each shadow, 
but after one or a few additional shadows fails to close the valves. 

B. cariosus, on the other hand, responds to the first shadow, quickly re-emerges 
and. after one to four additional shadows, proceeds to execute "fishing" activities. 
completely ignoring the changing light level. 

These responses, of course, will occur in this particular way only in the ab- 
sence of any reinforcing stimuli such as mechanical shocks, or tactile stimuli. If 
the shadow is accompanied by a tactile stimulus or a blow to the dish containing 
the animals, they remain closed for much longer periods of time and do not adapt 
to the dual stimulus nearly so quickly. 

While this difference in behavior is difficult to explain with any degree of confi- 
dence, it is interesting to note that the B. cariosus used in this study were collected 
from the outer Oregon coast \\here the wave action may be severe, and the water 
frequently contains much floating and suspended debris. B. tintinnabulum, how- 
ever, was collected from harbor floats and pilings in relatively quiet bays in south- 
ern California. In the two dilTerin- situations, it may be that a shadow is a more 
"urgent" stimulus in quiet water (i.e., more frequently signals the 1 approach of a 
predator), and continued response is of significant value to the species. In more 
turbulent waters, where shado\\s i|iiiit often signal onlv a piece of floating debris. 
the response may be less significant. 



THE SHADOW REFLEX IN CIRRIPEDIA 253 

DISCUSSION 

The information now available on the structure and function of the adult bar- 
nacle photoreceptors and the nervous system permits a resume which represents a 
fairly complete description of, at least, the obvious pathways and events that are 
involved in the shadow reflex. In no case has the response chain, from photo- 
receptor cell membrane depolarization to muscle junctional potentials, been followrd 
completely through in one species, but by combining information from several it is 
possible to reconstruct the probable chain of events. 

It now seems highly probable that all balanid cirripedes possess two distinct sets 
of photoreceptors : a pair of bilaterally symmetrical lateral ocelli and a single me- 
dian photoreceptor "ganglion." That these receptors contain retinula cells with 
typical arthropod rhabdomere microvilli is now established, and the absence of a 
synaptic layer close to the retinula cells is strongly indicated (Fahrenbach, 1965). 

It is generally held that the adult cirripede eye(s) takes its origin from the me- 
dian eye of the nauplius larva (e.g., Doochin, 1951), and the structure of that eye 
in the larva of Balanus suggests the developmental source of the three separated 
photoreceptors found in the adult. While a detailed comparison of the structure 
of adult and larval eyes (Kauri, 1962; Fahrenbach, 1965) reveals considerable 
difference in numbers of sensory cells and their organization, the existence of three 
components in the larva is very suggestive. It should be recognized that many 
larval structures do not develop directly into adult structures but emerge as the 
definitive adult structures following a phase of larval "degeneration" (Bernard and 
Lane, 1962). 

To judge from the location and structure of the two sets of "eyes" in an animal 
like B. eburneus, it would seem that the median photoreceptor receives light most 
easily when the animal has the opercular valves open and the cirri extended. How- 
ever, it must be recognized that an actively "fishing" barnacle probably casts 
shadows on its own median photoreceptor, which suggests the existence of inhibi- 
tory feed-back mechanism to prevent withdrawal reactions during this process. 

In the lateral photoreceptors of B. clntnicns, the location of light-absorbing and 
reflecting pigments over the inner surface of the PR cells makes it apparent that they 
must receive light either parallel to the shell plates and/or from the outside. In an 
animal like B. tintinnabulum this may also be true, but the inner shielding is less 
well developed. Very little can be said about B. cariosus lateral eyes, for the 
structure has not yet been morphologically identified. 

The structural information on the retinula cells provided by Fahrenbach (loc, 
cit.} helps a great deal in explaining the absence of propagated action potentials in 
barnacle retinula cell axons. The large size of these axons ( 15-20 /x diameter) 
contrasts markedly with those of, for example, the cockroach dorsal ocellus which 
averages 0.5 ^ (Ruck, 1964). Also, the inter-axonal space which is filled with 
glial cell membrane is quite large, so that in contrast to the cockroach, the length 
constant of barnacle retinula cell axons is probably quite large. It thus appears 
that a structural basis for long-distance electrotonic conduction is present. 

The function of these photoreceptors seems to be primarily that of initiating the 
shadow reflex, although other functions may also be imagined. Structural consid- 
erations rule out any image-forming capabilities, and it seems evident that the 
"eyes" are relatively simple light-level and transient-photic-event monitors. No 



254 G. F. GWILLIAM 

difference in function of the lateral and median photoreceptors is so far apparent 
with the techniques used in this study, but more subtle functional differences are 
not precluded. 

In a purely speculative manner, one might imagine the sequence of events lead- 
ing to a shadow reflex as occurring in the following way: self-cast shadows on the 
median photoreceptor would cause a certain amount of depolarization in the second 
order neurons at "off." In an immobilized preparation this would be sufficient to 
trigger the reflex, but in a "fishing" animal this would be countered by inhibitory 
neurons (acting on the same second-order cells) activated by the body movements. 
The balance between these two processes and the inhibitory influence of the illumi- 
nated lateral photoreceptors would serve to keep the second-order cell membrane 
potential depressed below the firing level. If, during this process, the added de- 
polarization (release from inhibition) furnished at "off" by the lateral photorecep- 
tors should impinge on the second-order neurons, the firing level would be reached, 
which would operate the withdrawal-closure reflex; this would in turn shut off the 
inhibitory feed-back mechanism and keep the animal contracted until the shadow- 
was removed, or until firing in the second-order cells ceased (a matter of approxi- 
mately 30-60 seconds ) . 

That there are distortion-sensitive sensory cells present in the mantle lining 
close to the body can be demonstrated by stretching the mantle while recording 
from the cut end of the antennular nerve, the same nerve that carries the lateral 
photoreceptor axons. The spikes shown in Figure 10 (page 482) of the previous 
paper (Gwilliam. I'^o ) are almost certainly in this category. Whether or not they 
inhibit the same second-order cells or the motor cells involved in the shadow- reflex 
is not known. Hoyle and Smythe (1963) have been unable to demonstrate 
peripheral inhibition in barnacles, but central inhibition certainly occurs. 

However, the chain of events as demonstrated to date suggests the following 
interpretation : 

(a) The photoreceptor cells generate a sustained depolarizing potential when 
illuminated. 

(b) This potential is transmitted by passive electrotonic conduction ria the 
large retinula cell axons to the supraesophageal ganglion where the axons synapse 
with the second-order neurons. 

(c) The sustained depolarization probably causes the continual release of an 
inhibitory tran>n litter substance from the terminations of the retinula cell axons 
which prevents the second-order cells from firing, although inhibition may be ac- 
complished by some other mechanism. 

(d) At "off," the inhibition is released and the second-order cells begin to fire. 

(e) The second-order cells synapse either directly or through other interneurons 
in the ventral ganglion with motor neurons. At this level the post-synaptic event 
is excitatory. 

(f) At this level, rather than the previous one, the phenomenon of synaptic 
failure (seen as a failure of motor neurons to respond to multiple stimuli) probably 
occurs. This "tendency to failure" varies in different species and in different motor 
cells of the same species. 

It thus appears that the barnacle retinula cell behaves in a very similar fashion 
to the retinula cell in the insect dorsal ocellus i Ruck. 1962, for summary), the 



THE SHADOW REFLEX IN CIRRIPEDIA 255 

striking difference being the greater distance between the retinula cell and the first 
synapse. This has apparently been compensated for in the barnacle by the morpho- 
logical specializations referred to previously rather than by the development of an 
impulse-propagating mechanism. It seems quite plausible to argue that this mode 
of transmission is fundamental to arthropod photoreceptor cells, and the existence 
of a spiking mechanism (Naka and Eguchi, 1962b) represents a high degree of 
specialization. Washizu (1964), recording intracellular potentials from blowfly 
compound eyes, detected no impulse activity and demonstrated that the "on" 
transient did not overshoot zero potential and was graded. Unequivocal evidence 
of propagated impulse activity in retinula cell axons, on the other hand, is very 
limited. 

SUMMARY 

1. The gross structure of the balanid central nervous system and some of the 
peripheral structures involved in the shadow reflex are described and figured (Fig. 
1 ) . The existence of both paired lateral and single median photoreceptors in sev- 
eral species of barnacles is established, and is probably true for all balanid 
cirripedes. 

2. Intracellular sensory potentials from the lateral ocelli of B. eburneus indicate 
that spiking does not occur in these retinula cells, and that the wave form of the 
response to a light flash is very similar to comparable records from other arthropod 
retinula cells. 

3. No significant difference between the function of the lateral and the median 
ocelli has been shown with the procedures used in this study. 

4. The different rates of adaptation of neurons in the reacting chain have been 
studied. The primary sensory event is non-adapting, the presumed second-order 
neurons adapt very slowly, as does the cirral motor output in B. tintinnabulum. 
The cirral output in B. cariosns, however, adapts rapidly, and so does the adductor 
muscle motor output in both species. This difference in motor output correlates 
very well with the behavior of intact animals. 

5. The probable chain of events leading to the withdrawal-closure response to a 
shadow is summarized. 

LITERATURE CITED 

BERNARD, F. J., AND C. E. LANE, 1962. Early settlement and metamorphosis of the barnarlr 

Balanus amphitrite nivcus. J. Morph., 110: 18-39. 
BURKHARDT, D., 1962. Spectral sensitivity and other response characteristics of single visual 

cells in the arthropod eye. Symp. Soc. E.rp. Bin!., 16: 85-109. 
BURKHARDT, D., AND H. AUTRUM, 1960. Die Belichtungspotentiale einzelner Sehzellen von 

Calliphora erythrocephala Meigen. Z. Naturforsch., 15b: 612-616. 
DARWIN, C., 1854. A Monograph on the Subclass Cirripedia. The Balanidae (or sessile 

Cirripedes) : the Verruddae, etc., etc., etc. London. The Ray Society. 684 pp. 
DOOCHIN, H. D., 1951. The morphology of Balanus improvisns Darwin and Balanus amphitrite 

nircns Darwin during initial attachment and metamorphosis. Bull. Mar. Sci. Gulf 

and Carib., 1: 15-39. 
ELOFSSON, R., 1963. The nauplius eyes and frontal organs in Decapoda (Crustacea). Sarsia, 

12: 1 68. 
FAHRENBACH, W. H., 1965. The micromorphology of a simple photoreceptor. Zeitschr. ZcH- 

forsch., 66: 233-254. 
FALES, D. E., 1928. The light receptive organs of certain barnacles. Blol. Bull., 54: 534-547 



G. F. GY\ il.l.lAAl 

(iwn.i.iAM, G. P., 1963. Tlu- mechanism idow rcilcx in Cirripedia. I. Electrical ac- 

tivity in the supraesophai;ral ganglion and ocellar nerve. >w/. /?//.. 125: 470-485. 

HOYLE, G., .\\i T. SMYTHS l ( '(o. XetiromiiM-ular physiology of giant fibers of a barnacle, 
Helmuts inthilits Daruin. i ^;;;/>. l>it>chcm. Physiol., 10: 291-314. 

KATRI. T.. 1962. On the I'mnta! tilanieiits and nauplius eye in Balanns. Cnistacenitti. 4: 
133 142. 

NAKA, K'.-l.. 1 ( .'()1. Ixecnrdin.u a\ retinal action potentials from single cells in the insect com- 
pound eye. J. Gen. 1'liysiol., 44: 571-584. 

XAKA, K.-l., AND E. Ecucin. l ( '';2a. l-'.i't'ect of background illumination on the retinal action 
potential. Science, 136: 877-87''. 

NAKA, K.-l., A\I> E. EGUCHI, 1962h. Spike potentials recorded from the insect photoreceptor. 
/. Gen. Physiol., 45: 663 680. 

Ki ' K, I'., 1961. Electrophysiology of the insect dorsal ocellus. I. Origin of the components 
of the electro-rctinogram. /. </V;;. Fliysiol., 44: 605-627. 

Kvi-K. P., 1962. On photoreceptor mechanisms of retinal cells. Biol. Bull., 123: 618-634. 

Rtvk. I'., 1"')4. Krtinal structures and photoreception. shin. Rev. Entoin., 9: 83-102. 

W \sinzu, Y., 1964. Electrical activity of the single retinula cells in the compound eye of the 
l>'o\vfly Ciilliplior,! grythrocephalct Meigen. ('<u;;/>. Biochcm. Pliysiol., 12: 369-387. 



OBSERVATIONS ON THE NUTRITION OF 
MONOGENETIC TREMATODES 

D. W. HALTON ' AND J. B. JENNINGS 
J >i'ptirtmcnt of Zoology, The (Jni-rcrsity of Leeds, England 

Relatively little information is available regarding the general pattern of nutri- 
tion in the Trematoda Monogenea, but there are indications that the two sub-orders 
of this class of parasitic flatworms differ considerably as regards the nature of their 
diet. The Monopisthocotylea so far investigated are reported to feed on the epi- 
dermal tissues and associated secretions of the host organism, whilst the Polyopis- 
thocotylea appear to be largely sanguinivorous and take in little host tissue other 
than blood (Goto, 1895; Heath, 1902; Folda, 1928; Gallien, 1934; Sproston, 
1945; Llewellyn, 1954; Jennings, 1956, 1959; Uspenskaya, 1962; Kearn, 1963). 

Other differences between the two sub-orders, concerned with nutrition, are seen 
in the cellular structure of the digestive organs. Thus, in the Monopisthocotylea 
the intestine is lined by a continuous and unpigmented gastrodermis ; but in the 
Polyopisthocotylea the gastrodermis is typically discontinuous and consists of colum- 
nar cells, containing varying amounts of brownish or black pigment, interspersed 
with areas devoid of cells and consisting only of thin basement membrane (Baer 
and Euzet, 1961). In a number of species the pigment has been identified as 
hematin, a degradation product of hemoglobin (Llewellyn, 1954; Jennings, 1959). 

These differences in gastrodermal structure within the Monogenea are pre- 
sumably related to the differences in diet and they may reflect, also, further differ- 
ences in the site and course of the digestive process. In the present investigation, 
therefore, the relationships between diet, gut structure and digestion in the Mo- 
nogenea have been studied, as part of a comparative survey of nutrition within this 
class of Trematoda. 

MATERIALS AND METHODS 

The following species of Monogenea, listed systematically with details of their 
hosts and parasitic locations, have been examined : 

MONOPISTHOCOTYLEA 

Callcot\le kroyeri Diesing. Cloaca of the thorn-back skate, Raid clavata and 
the starry ray, Raid radiata. 

Entobdclla hippoglossi Miiller. Skin and general body surface of the halibut, 
Hippoglossus hippoglossus. 

Udonclla calit/oniin Johnston. Egg sacs of copepods (Caligus sp. ) found on 
the head and in the buccal cavity of the cod, Gadns callarias. 

1 Present address : Department of Zoology, The Queen's University, Belfast, N. Ireland. 

257 



D. W. IIAI.TMX \M) J. B. JENNINGS 

POLYOPISTHOCOTYLEA 

Polystoma intcgcrriiiiiuii Fruhlich. Urinary bladder of the common frog, 
Rana ft inporaria. 

/liplozoon parado.nts Nordinann. Gills of the minnow, Pho.viiuis pho.vinus. 

Discocolvlc sat/ittdtit i.enckart. Gills of the trout, Sahno trutta. 

Diclidophora ;//<T/</;/<// Kiihn. Gills of the whiting, Gadus nicrlant/ns. 

Octodactylus [\tlinaia Leuckart. Gills of the ling, Molva niolra. 

ricctanocot\le (/iinnin/i Beneden & Hesse. Gills of the grey gurnard, Trigla 
gurnardus. 

To determine the food of each species, and to study the structure of the gut, 
.specimens were fixed in Bouin, Susa, or I0 c /c formalin immediately after removal 
from the host, and serial sections cut at 5 /JL after impregnation and embedding in 
polyester wax (m.pt. 37 C.) or paraffin wax (m.pt. 56 C.). For identification 
of intestinal contents sections were examined by one or other of the following 
methods : 

1. The alcian blue method for mucins (Steedman, 1950). 

2. The periodic acid-Schiff (P.A.S.) method for mucins and carbohydrates. 

3. The mercuric bromphenol blue method for proteins (Mazia. Brewer and 
Alfert, 1953i. 

4. The benzidine method for hemoglobin (Pickworth, 1934). 

5. The application of various solubility and bleaching tests for hematin (sum- 
marized by Jennings, 1959). 

6. The Gmelin test for hematoidin and bile pigments. 

7. The Turnbull's and Prussian blue methods for ferrous and ferric salts. 

8. Various routine histological methods, e.g., hematoxylin and eosin, Mallory's 
trichrome stain, Feulgen's reaction for nuclei, etc. 

To aid identification of the chosen food the host organs were fixed and examined 
by the above methods, for comparison of tissue components with the trematode's 
intestinal contents. Further, where the trematodes had obviously only recently fed, 
they were induced to regurgitate the food, by gentle pressure, and the material so 
obtained examined either fresh or after treatment as a fixed and stained smear. 

In the study of the feeding mechanisms the trematodes were observed alive upon 
their hosts, \\henever this was possible, and others were fixed and sectioned in situ. 
The latter process was facilitated by fixation in warm (40 C.) Bouin, or by plung- 
ing the host organ and attached flat worms into isopentane, cooled to - - 160 C. in 
liquid nitrogen, followed by transfer of the frozen mass into fixative held at - 1 C. 

The site and course of digestion were investigated by isolating recently fed trema- 
todes in aerated salt or fresh water ( Hedon-Fleig saline with added glucose for 
r<>lysf<nini ) and fixing individuals at progressive intervals up to three days, the 
maximum survival time for most .species. The progressive breakdown and absorp- 
tion of the tood was followed in sections prepared and treated as above. Enzyme 
activity in the alimentary system was investigated histochemically, using frozen or 
45 C. paraffin wax sections prepared after fixation at - 1 C. in 10% formalin 
buffered to pi I 7.0. The histocheniical methods employed included the indoxyl 
acetate- method for non-specific eMerases (Holt, 195S), both metal-salt and azo-dye 
method- |".,r alkaline and arid phosplialases ( ( ioniori. 1 ( >52; Bnrsloiie, 1958). the 



NUTRITION OF MONOGENEA 259 

Tween 80 method for lipase (Gomori, 1952) and the L-leucyl-/3-naphthylamide 
method for leucine aminopeptidase (Burstone and Folk, 1956). 

OBSERVATIONS 
MONOPISTHOCOTYLEA 

1. Calicotyle kroyeri 

Calicotylc kroyeri feeds exclusively on epidermal cells and mucoid secretions 
derived from the lining of the skate cloaca. In many instances the gut lumen of 
specimens fixed immediately after removal from the host contained mucus, staining 
strongly with alcian blue and P.A.S., together with numerous large cells 10-12 p. in 
diameter and containing prominent nuclei (Fig. 1). These cells are identical with 
the epidermal cells in situ on the cloacal wall or lying free in the mucoid material 
coating the walls of the cloacal chamber. 

The mouth in C. kroyeri is anterior and ventral, and surrounded by a poorly 
defined oral sucker. The anterior lip of the sucker contains unicellular glands whose 
secretions are used for adhesion to the cloacal wall, and the posterior portion bears 
a tongue-like valve which on contraction cuts ofT the cavity of the sucker from the 
rest of the alimentary system (Fig. 2). The pharynx is highly muscular and de- 
void of gland cells, and is used to suck in the semifluid mucus and desquamated 
epidermal cells which are always present in the cloaca. The cloacal wall and its 
epidermis are always intact and undamaged, even when many specimens of Calico- 
tyle are present, and it appears that the pharynx never removes living epidermal 
cells or breaches the epidermis. 

The pharynx leads via a short esophagus into the intestine, which consists of two 
simple unbranched ceca. The esophagus is surrounded by many acidophllic gland 
cells (Fig. 2) which open into its lumen, but the function of their secretion remains 
unknown. 

The intestinal ceca are lined by a single-layered continuous gastrodermis, made 
up of columnar cells 16-18 /A tall and 6-8 //, wide, with granular cytoplasm and basal 
vesicular nuclei (Fig. 1). The cells go through a secretory cycle in which a small 
vacuole appears basally and then increases in size as it moves to the distal portion 
of the cytoplasm. The vacuoles eventually pass out into the gut lumen where they 
may remain as visible and discrete structures for varying periods before they finally 
disappear. 

The entire gastrodermis consistently shows a strongly positive reaction for non- 
specific esterases, apart from the vacuoles whose finely granular contents remain 
unstained (Fig. 3). 

The mucoid and cellular elements of the food are progressively homogenized as 
they lie in the gut lumen, demonstrating the occurrence of extracellular digestion. 
The enzymes responsible for this originate, presumably, in the esophageal glands 
and from the vacuoles released by the gastrodermis. No inclusions were seen in 
the gastrodermal cells, apart from the vacuoles, but the intense esterase activity seen 
in the cytoplasm suggests the occurrence of some intracellular digestion following 
absorption of partially digested material from the gut lumen. 

Non-specific esterases are found also in the cuticle, notably that of the anterior 
ventral region and that lining the oral sucker, and may be used by the trematode 



260 



D. W. HA1 T< IN AND .1. B. JENNINGS 




g.c. 



* 













FlGi RE. 1. (.'alicufylc hri'iyct-i. 'l'ran^\-ci->c ->^riimi of an intestinal arum .shoxvin.L; tlie 
uolated continuous sja-Orodrrnii- and, in tin- Innicn, rn-cntly in.^c^tcd lins) cpidi-rinal cells. 
Il<.-inato\ylin, rosin and alcian blur. Scale: 1 cm. 20 /,. 



NUTRITION' OF MONOGENEA 261 

in some form of extracorporeal digestion to accelerate the sloughing-off of spent 
cells from the cloacal epidermis. 

2. Entobdclla hippoglossi 

Examination of the intestinal contents of E. hippoglossi fixed immediately after 
removal from the host showed that in this species, as in Calicotylc, the food consists 
entirely of host epidermis and mucus, and no traces were found of ingested dermal 
tissue components such as chromatophores or blood cells. 

The ventral subterminal mouth leads directly into the pharynx, which is consid- 
erably modified from the usual trematode type to form a muscular-glandular feed- 
ing organ (Fig. 5). The anterior portion of this organ is entirely muscular, with 
flexible lips, and can be protruded through the mouth for application to the host 
epidermis. The posterior portion is glandular and consists of 40-50 large acido- 
philic gland cells, separated from each other by muscle fibers. Each cell communi- 
cates individually with the lumen of the anterior part of the feeding organ by a 
fine duct, and each duct opens distally at the apex of a large papilla (Fig. 4). 

The gland cells give no reaction with the indoxyl acetate method for non-specific 
esterases, but fresh frozen sections applied to thin films of solidified 2/o aqueous 
gelatine cause liquefaction and cavitation in the area covered by the glandular por- 
tion of the feeding organ, indicating the presence of a proteolytic enzyme. The 
feeding organ of E. soleae is reported to produce a similar gelatine-splitting protease 
(Kearn, 1963), and it seems likely, therefore, that protease production in the 
pharynx is characteristic of the entobdellid trematodes as a group. 

It was not possible to observe E. hippoglossi in the act of feeding, but the pres- 
ence on the host skin of circular lesions of the approximate diameter of the feeding 
organ indicates that the proteolytic secretions are used to erode and dissolve epi- 
dermal tissue prior to ingestion. This is supported by the fact that relatively feu- 
intact epidermal cells are found in the intestinal contents, even when the gut is full 
and the trematode obviously only recently fed. Generally the gut contents are quite 
homogeneous, acidophilic and stain only lightly with alcian blue or P.A.S., in 
marked contrast to the situation seen in Calicotylc. 

The feeding organ leads posteriorly into the intestine via a short esophagus, into 
which open the ducts of numerous unicellular glands lying in the parenchyma of the 
anterior portion of the body. These esophageal gland cells are intensely basophilic 
but the function of their secretion could not be detected. 

FIGURE 2. Calicotylc kroycri. Longitudinal section through the anterior region, o. g., 
oesophageal glands; p., pharynx; v, tongue-like valve which can close off the pharynx from 
the oral sucker. Mallory. Scale 1 cm. = 200 /j.. 

FIGURE 3. Calicotyle krdyeri. Horizontal longitudinal section of the anterior region, 
showing the pharynx (p.) and portions of the two intestinal ceca. The gastrodermis in each 
cecum shows intense non-specific estcrase activity. Holt indoxyl acetate method. Scale : 
1 cm. = SOn. 

FIGURE 4. Entobdclla hippoglossi. Transverse section through the posterior glandular 
region of the feeding organ, showing the gland cells (g. c.) and papillae. Mallory. Scale: 
1 cm. = 75 /j.. 

FIGURE 5. Entobdclla hippoglossi. Longitudinal section through the anterior region show- 
ing the muscular-glandular feeding organ, g. r., glandular region ; m. r., muscular region. 
Hematoxylin and eosin. Scale : 1 cm. = 125 /JL. 



262 1). \v. 1 1 ALTON AND .1 IJ. JKNNINtiS 

The intestine is divided into two ceca which re-unite posteriorly by means of a 
commissure and give off over their entire length branched diverticula. It is lined 
throughout by a continuous gastrodermis consisting of uniform flattened cells, 
12-15 p long and 5-7 p. tall, with finely granular cytoplasm and basal vesicular 
nuclei. Gland cells are absent and no enzyme activity could be demonstrated. The 
only variation obsenable in the gastrodermis is in the height of the constituent cells, 
and this is related to the amount of food present in the lumen, the cells becoming 
even more flattened as the intestinal walls stretch to accommodate newly ingested 
material. 

The amount of material in the gut lumen decreases with time, after feeding, 
but without noticeable change in consistency from the relatively homogeneous con- 
dition in \\hich the food is ingested. This fact, together with the absence of gland 
cells from the gastrodermis, suggests that the bulk of digestion in E. hippoglossi is 
effected by the secretions poured on to the food from the glands of the feeding 
organ before and during ingestion, aided perhaps by the secretions of the esophageal 
glands. The gastrodermis would thus appear to be entirely absorptive in function 
and to play little or no part in the production of the digestive juices. 

3. Udonclla caligontin 

Udonclla caligontin lives attached to the egg sacs of copepods (C aligns sp.) 
which in turn are ectoparasitic in the buccal cavity and on the head region of cod, 
halibut and ling. 

The only recognizable material found amongst the gut contents of Udonclla 
was a mucoid substance staining lightly with alcian blue and P.A.S., and often the 
intestine contained only a finely granular acidophilic digest. Nothing can be seen 
to suggest that Udonclla feeds on the copepod tissues or body fluids, and it is con- 
cluded that the trematode ingests mucus, and perhaps sloughed-off epidermal cells, 
from the fish skin or mucous membrane adjacent to the copepod's point of 
attachment. 

The mouth in Udonclla is anterior and ventral, and leads directly into the large 
muscular pharynx. This can be protruded slightly through the mouth but is not 
armed or equipped with glandular elements so that it is unlikely that it penetrates 
host tissues. Feeding, therefore, is probably a case of merely sucking in the mate- 
rial lying on the fish epidermis. 

The intestine in Udonella, in contrast to that in most other Monopisthocotylea, 
is undivided and extend^ almost to the posterior end of the body as a simple sac, 
reminiscent of the sac-like gut of many rhabdocoel Turbellaria. It is lined by a 
flattened and continuous gastrodermis similar to that found in Entobdclla. 

Digestion appears to be entirely intraluminar, judging from the appearance of 
the gut contents, and nothing was seen to indicate intracellular digestion, as the 
gastrodermis shows no trace of esterase activity. 

POLYOPISTHOCOTYIJ .A 
1. Polvsloina integerrimum 

Polystonia integerrimum is sanguinivorous, feeding on blood drawn from the 
capillaries of the frog urinary bladder, and no host tissues other than blood were 
found in the gut content s. 



NUTRITION OF MONOGENEA 263 

The ventral mouth is encircled by an oral sphincter and leads into the cavity 
of the oral sucker. This is lined by cuticle and surrounded by numerous unicellular 
glands which open via long branched ducts over the external surface of the sucker 
and also into the oral cavity. The glands produce a granular proteinaceous secre- 
tion which stains strongly with the Mallory and Mazia methods, but gives no re- 
action for esterases or phosphatases. The distribution of the ducts conveying the 
secretion to the exterior indicates that it is probably used in adhesion. 

The oral cavity is linked with the large muscular and bulbous pharynx by means 
of a short cuticle-lined buccal tube, into w r hich the anterior portion of the pharynx 
projects. The wall of the pharynx contains, in addition to muscular elements, a 
number of large cells w r ith prominent nuclei and nucleoli, and a series of small 
vacuoles of unknown function ranged along the inner and outer surfaces at regular 
intervals (Fig. 6). 

The pharynx leads via the esophagus into a bifid intestine whose ceca run the 
length of the body and give off branches which in turn repeatedly subdivide and 
anastomose. The esophagus is a short muscular tube surrounded by numerous 
unicellular glands arranged in t\vo distinct zones. The cells of the inner zone, 
immediately around the esophagus, are smaller and produce a granular secretion 
giving an intensely positive reaction for alkaline phosphatase, while the outer 
larger cells produce a more coarsely granular and strongly acidophilic secretion 
quite free of phosphatases (Fig. 7). Fresh frozen sections and aqueous extracts 
of the esophageal region rapidly cause cavitation in gelatine films, indicating the 
production of a proteolytic enzyme by these esophageal glands, but it was impossible 
to determine which type of gland cell was responsible. 

Both types of gland cell discharge through long unbranched ducts which enter 
the pharynx at its posterior end and run forward between the cuticular lining and 
the underlying musculature to open finally into the anterior end of the pharynx 
lumen (Figs. 6 and 7). 

During feeding the oral sucker is flattened and flared against the bladder wall, 
and contraction of radial muscles within the sucker draws up a plug of bladder 
tissue whose tip reaches the anterior end of the pharynx. The plug is held secure 
by the constricting grip of the oral sphincter around its base while the sucking action 
of the pharynx, aided no doubt by proteolytic secretions from the esophageal glands, 
ruptures capillaries and draws blood into the intestine. Bladder tissue is never 
ingested, however, and very little damage is caused to the bladder epithelium. 

The structure of the gastrodermis in Polystoina has been described elsewhere 
(Jennings, 1959). In brief, the gastrodermis is a single-layered discontinuous 
structure made up of columnar cells 16-18 /A tall and 8-10 ^ wide, with basal 
vesicular nuclei and cytoplasm containing varying amounts of the pigment hematin, 
interspersed with areas devoid of cells and w r here only a thin basement membrane 
separates the gut lumen from underlying body tissues (Fig. 8). The hematin is 
contained within spherical vesicles up to 8 ^ in diameter and the number of these 
increases with age, so that a mature cell is loaded with pigment and the nucleus 
obscured. When this condition is reached the vesicles are extruded into the gut 
lumen or, more commonly, the entire cell disintegrates either in situ or after being 
shed from the gastrodermis. The vesicles themselves persist intact for some time, 
but eventually rupture to discharge their contained hematin. New, younger cells 



264 



D. \Y. HALTON VND J. B. JKXXIXGS 












w 



Pi. ' 



\ 



) 








I CGI ! 6 Polystoma inteyerrimum. [.oii^itudiiial Motion of tin. 1 anterior region, y. c., 
cell- posiciior to the ])liar\u\ whose duels run forward between llie inner eutionlar lining 
and musculature of the pharynx and open in its anterior portion; L, intestine, lined by a dis- 



NUTRITION OF MONOGENKA 265 

grow up to replace the spent cells and fill in the gaps in the gastrodermis, and thus 
the latter structure is in a state of constant degeneration and renewal. 

Digestion in Polystoma occurs by a combination of extracellular and intra- 
cellular processes. Erythrocytes entering the intestine are immediately hemolyzed 
and within three hours of ingestion their nuclei have also disintegrated. The freed 
nuclear material mixes with the other gut contents and causes the whole mass to 
stain lightly with Feulgen, but this reaction eventually disappears as digestion 
progresses. 

The intraluminar phase of digestion is accompanied by absorption of semi- 
digested substances by the smaller younger cells of the gastrodermis, and their 
cytoplasm becomes swollen with spherical aggregations of material showing the 
same staining reactions as that remaining in the gut lumen. These cells show in- 
tense alkaline phosphatase activity along their distal margins and this is obviously 
concerned with the process of absorption. The enzyme is best visualized by the 
azo-dye method since the black cobalt-sulphide end product of the calcium-salt 
technique may be masked by any hematin present. 

Absorption from the gut lumen continues until no stainable material remains. 
This situation is reached 2448 hours after a meal, depending upon the amount of 
blood ingested. Digestion is completed intracellularly in the vesicles within which 
material is aggregated as it is absorbed from the lumen, but of the enzymes con- 
cerned in the process, only non-specific esterases could be demonstrated histo- 
chemically. These are localized within the vesicles and cannot be demonstrated 
in the cytoplasm of the gastrodermal cells. 

As intracellular digestion proceeds, stainable material disappears from the vesi- 
cles and is replaced by granules of hematin resulting from degradation of the hemo- 
globin content of the meal. The hematin remains within the cell and thus the 
amount seen in a mature cell about to be shed from the gastrodermis probably repre- 
sents an accumulation from the digestion of several meals. 

Extrusion of hematin vesicles or the shedding of intact spent cells occurs 2448 
hours after a meal and consequently there is at this time a marked increase in the 
amount of hematin lying free in the gut lumen. Many of the freed vesicles, prior 
to rupturing, still show traces of esterase activity and this confirms a suggestion 
made in an earlier account (Jennings, 1959) that enzymes concerned primarily with 
intracellular digestion may remain in the hematin vesicles and be eventually trans- 
ported to the gut lumen where they are released when the vesicles rupture. Due 
to the ramifications of the gut in Polystoma there is never complete evacuation be- 
tween meals, and it is likely that these enzymes of intracellular origin will still be 

continuous pigmented gastrodermis and containing hematin granules mixed with heavily staining 
hemolyzed erythrocytes ; o. s., oral sucker containing material regurgitated from the intestine ; 
vi., vitellaria. Mallory. Scale : 1 cm. = 75 /*. 

FIGURE 7. Polystoma integcrrimum. Longitudinal section of the anterior region, showing 
intense alkaline phosphatase activity in the inner zone of gland cells associated with the pharynx. 
Abbreviations as in Figure 6. Gomori azo-dye method. Scale : 1 cm. = 75 /j.. 

FIGURE 8. Polystoma integerrimum. Transverse section through the gastrodermis, show- 
ing the discontinuous structure and the intracellular aggregations of hematin. Mallory. Scale : 
1 cm. = 20 /i. 

FIGURE 9. Diplosoon paradoxum. Longitudinal section of an individual fixed in situ on 
the host gill. g. f., gill filament ; pi., plug of gill tissue drawn up and held by the buccal sucker. 
P.A.S. Scale: 1 cm. = 250 M. 



266 D. \Y. MAI |M\ AND .1. H. JENNINGS 

present in the lumen when the next meal is taken, and thus contribute to the intra- 
luminar digestive phase. 

No specific source of the intraluminar digestive enzymes was located, other than 
the lu matin vesicles, and it seems likely, therefore, that the proteolytic secretions of 
the esophageal glands will play .in important part in extracellular digestion, entering 
the intestine with the fond and initiating hemolysis and nuclear breakdown. 

The principal endprodnci of hemoglobin digestion in Polystoma is hematin but 
a small proportion of the hemoglobin is converted to hematoidin, an iron-free, acid- 
soluble crystalline substance closely related to the bile pigments. Hematoidin crys- 
tals are only rarely found in histological preparations of Polystoma, however, due 
to their solubility in the .standard fixatives, but can be seen in fresh squash prepara- 
tions of the gastrodermis in about 10% of the cells. 



2. Diplosoon pt 

Diplozoon /'tirado.vum feeds predominantly on blood, but small amounts of gill 
tissue, epithelial cells and mucus are also found amongst the gut contents. 

The adult Diplozoon consists of two individuals united in permanent copulation, 
with organic fusion of their bodies midway along the long axis, so that the com- 
posite individual is X-shaped. Each individual retains a terminal ventral mouth 
opening into a buccal cavity which bears laterally a pair of buccal suckers. An oval, 
muscular pharynx, devoid of glandular elements, protrudes slightly into the buccal 
cavity and leads backwards into the intestine. This extends posteriorly in each 
individual as a single much-branched cecum, and where the bodies of the two indi- 
viduals fuse, the two ceca unite by a median canal, so that the two intestines are 
confluent. 

ni[>!o.::oon lives attached to the gills of the host minnow by the clamps of the two 
opisthaptors and during feeding one or both of the anterior ends attaches itself to a 
gill filament by means of the buccal suckers. The grip is aided by adhesive secre- 
tions produced by clusters of gland cells around the buccal cavity which open on to 
the anterior body surface. The buccal suckers draw up a plug of gill tissue (Fig. 
9), in much the same manner as the oral sucker in Polystoma draws up a plug of 
bladder tissue. The plug extends through the buccal cavity to the pharynx, which 
is protruded slightly and applied to the tip. Prolonged suction bursts the super- 
ficial blood capillaries, and blood, together with a small amount of gill tissue, enters 
the intestine. There is no evidence indicating the use of histolytic secretions to 
effect rupture of the gill capillaries, and no serious damage is caused to the gill fila- 
ments by the feeding activities of the trematode. 

The gastrodermis resembles that of Polystoma in that it- is a discontinuous and 
decidtu us structure whose individual cells contain the characteristic hematin-laden 
vesicles. The cells are interspersed with areas of basement membrane either devoid 
of cells or covered by thin, extremely flattened and unpigmented young cells. 

The course of digestion follows closely that observed in Polysloma, hemolysis 
of the er\ throcytes occurring during or very soon after ingestion and being followed 
by partial intraluminar digestion. Soluble substances are absorbed by the gastro- 
deimis and digestion sul>~<-i piently completed intracellularly. with the production 
of hematin as a \isible insoluble endproducl. As in I'olys/o'ini the cells actively 
absorbing materials from the gut lumen show intense alkaline phosphatase activity 



NUTRITION OF MONOGENEA 267 

distally and this decreases as the cell ages and reduces its digestive functions. No 
esterase reaction could be demonstrated in the gastrodermis, but the entire nervous 
system shows intense cholinesterase activity and this provides a simple but effective 
means of demonstrating the system in toto (Halton and Jennings, 1964). 

The vitelline glands of the reproductive system are in intimate contact with the 
intestine for most of its length and show at all times positive reactions for alkaline 
phosphatase, lipase and aminopeptidase, indicating metabolic activity possibly con- 
cerned with absorption and utilization of food materials from the gastrodermis. 

In a few instances the intestine of newly fed Diplozoon contained, in addition 
to the hemolyzed blood, a number of reddish needle-shaped crystals 1 50-200 /j, in 
length. These were water-soluble but could be fixed in absolute ethyl or methyl 
alcohol, when they stained strongly with the benzidine technique for hemoglobin. 
The crystals gradually disappeared in the living animal as digestion progressed, 
and they probably resulted from crystallization of hemoglobin released from 
hemolyzed erythrocytes and concentrated by absorption of water from the gut con- 
tents during the early stages of digestion. 

3. Discocotyle sagittata 

Discocotyle sagittata appears to feed exclusively on blood drawn from the super- 
ficial capillaries of the trout gills. 

The mouth is anterior and ventral, and opens into a buccal cavity possessing 
laterally a pair of very large bilobed buccal suckers. The buccal cavity opens 
posteriorly into a small muscular non-glandular pharynx. 

It was not possible to observe Discocotyle in the act of feeding, but judging from 
the similarities in structure and habit it is likely that the breaching of the host capil- 
laries and the ingestion of blood are effected in the same manner as in Diplozooii. 
The buccal suckers are larger and more powerful than in the latter species, how- 
ever, while the pharynx is relatively smaller, so that the suckers probably play a 
greater part in creating the necessary suction. No evidence of the production or 
use of proteolytic secretions could be found, and no significant amount of damage 
is caused to the gill filaments by the feeding activities of the trematode. 

Neither gill tissues nor mucus were observed in the gut contents of the speci- 
mens examined. 

The pharynx opens directly into the bifid intestine whose ceca extend to the 
posterior end of the body and give off numerous lateral branches which in turn sub- 
divide and ramify between the vitellaria and other organs. 

The gastrodermis resembles that of Polystoma and Diplozoon, and is made up 
of large hematin-laden cells, 18-20 ^ long and 4-6 ^ tall, which are interspersed 
with smaller, flattened non-pigmented cells and areas completely devoid of cellular 
elements. 

The appearance of the gastrodermis indicated that digestion in Discocotvle fol- 
lows much the same course as in Polystoma and Diplozoon, and this was con- 
firmed from histological examination of individuals fixed at progressive intervals 
after removal from the host. Hemolysis and intraluminar digestion are accom- 
panied by active absorption of the products by the gastrodermis, with subsequent 
completion of digestion and production of hematin within intracellular vesicles. 
Absorption is particularly noticeable in the smaller non-pigmented cells, and both 
these and the larger cells show intense distal alkaline phosphatase activity. 



-<>> s D. W. HALT ND J. B. JENNINGS 

Mematin is eliminate'! from thi gastrodermis by extrusion of the vesicles or by 
ilie sloughing oil" of intact spent < 

It was not possible to de nonstrate ibe presence <if proteolytic enzymes in the 
gastrodermis, bv histocliei: nethods, but as in Diplozoon the vitellaria give 

strong positive reactions for lipase and aminopeptidase. 



4. Diclidophora 

Diclidophora incrlautji lecds chieflv upon blood but small amounts of gill tissue 
and mucus are also ingested. 

The mouth is ventral and subterminal, and opens into a typical buccal cavity 
with lateral paired buccal suckers. The pharynx is spherical, muscular and devoid 
of glandular elements, and feeding is effected by suction of the host tissue. 

A long esophagus links the pharynx with the bifid intestine whose ceca give off 
lateral much-branched diverticula. The latter are enveloped by the numerous 
\itellaria of the reproductive system. 

The gastrodermis in Diclidophora, as in the other Polyopisthocotylea already de- 
scribed. is a discontinuous and deciduous structure whose cells contain varying 
amounts of hematin and show the characteristic distal zone of alkaline phosphatase 
activity. The cells are much smaller than in the other genera investigated, how- 
ever, and even when fully mature and loaded with hematin are only 6-8 ft long and 
3-4 p tall. 

Digestion in Diclidophora is effected by a combination of extra- and intra- 
cellular processes and follows much the same course as in Polystoma, except that 
hematin appears to be the sole endproduct of hemoglobin degradation and no 
traces of hematoidin were found. A small amount of non-specific esterase activity 
can usually be demonstrated in the gut contents of specimens fixed soon after feed- 
ing, but this does not increase in amount with time and appears, in fact, to be de- 
rived from the gill tissue ingested along with mucus as the subsidiary component 
of the diet. In control sections of whiting gill approximately 10% of the epi- 
thelial cells showed non-specific esterase activity and it is likely that the activity 
seen in the Diclidophora gut contents originates in these cells. 

The gastrodermal cells show no enzyme activity, other than alkaline phosphatase, 
that could be detected by the techniques used, but the vitellaria, as in the other 
genera studied, give positive reactions for lipase and aminopeptidase. 

5. Octodactylus paluiala 

Ocioditctylns palmata feeds predominantly on blood drawn from the host gill 
.ipillaries but as in Diclidophora and Diplozoon, this diet is supplemented by gill 
tissue and mucus. 

terminal mouth opens into a buccal cavity which possesses a pair of large 
lateral buccal suckers, dill tissue is drawn up through the mouth, and suction by 
the bulbous and highly muscular pharynx ruptures the capillaries and draws blood 
into th line. The pharynx is devoid of gland cells and its action in procuring 

the ears to be entirely mechanical. 

Tin gul , of recently fed Octodactylus generally include mucus and gill 

lie in somewhat larger quantities than are found in Diclidophora and Diplosoon, 
but no appreciable damage to the gill filaments of the host was observed. 



NUTRITION OF MONOGENEA 26 

The intestine is of the- usual polyopisthocotylean type, being In' lid with the coca 
of considerable length and giving off many branched lateral diverticula. 

The gastrodermis differs somewhat from that of the other genera examined in 
that only relatively few areas are completely devoid of cells at any one time, and 
these are usually restricted to the walls of the two main ceca. The cells are small, 
as in Diclidophora, and range from 3-8 /L in height and 6-8 /i in width. The great 
majority of the cells contain hematin but the pigment granules are generally all 
confined within a single large vesicle, 3-6 /JL in diameter, rather than distributed 
amongst four or five smaller vesicles as, for example, in Polystoina. The larger- 
sized vesicles often fill the entire cell and displace the nucleus to one side away 
from its normal basal position. In fixed preparations the vesicles appear as solid 
masses of hematin, but in fresh squashes the individual pigment granules are free 
and exhibit constant Brownian movement within the confines of the vesicle. 

Digestion in Octodactylus follows the pattern observed in the other polyopis- 
thocotyleans studied. Hemolysis is completed very soon after ingestion and then 
intraluminar digestion is accompanied by absorption and the completion of digestion 
intracellularly. The gastrodermal cells show a distal zone of high alkaline phos- 
phatase activity which is particularly intensified during absorption. 

The hematin resulting from intracellular degradation of hemoglobin accumulates 
within the vesicles until eventually the distal margins of the individual cells break 
down and the hematin is discharged into the gut lumen. During this process, and 
while the cell is recovering, the cell becomes crescent- or cup-shaped and the dis- 
organized distal margin shows only diffuse alkaline phosphatase activity. Cells in 
this condition may continue to absorb material from the lumen, however, and often 
show a single small secondary hematin vesicle basally. This increases in size as 
the cell recovers from expulsion of the primary vesicle and moves distally, almost 
fills the cell, and is eventually expelled. 

The gut contents in Octodactylus often show non-specific esterase activity but, 
as in Diclidophora, there is every indication that this originates in the gill tissue 
and not in the gastrodermis. No other enzymatic activity could be demonstrated 
histochemically in the intestine, but again lipase and aminopeptidase were abundant 
in the vitellaria. 

6. Plcctanocotylc guniardi 

None of the specimens of Plcctanocotylc gnrnardi available for examination was 
recently fed, but since the gastrodermal cells contain at all times large amounts of 
hematin it is concluded that blood forms the dominant, if not the sole, component 
of the diet. No traces of gill tissue or mucus were found, but this could con- 
ceivably be due to the progress of digestion since the previous meal. 

The alimentary system resembles that of Diclidophora or Diplozoou, with paired 
buccal suckers, a muscular pharynx and a bifid, much-branched intestine. The 
gastrodermis is of the typical discontinuous and deciduous type, with somewhat 
sickle-shaped pigmented cells interspersed with naked areas devoid of cells. 

DISCUSSION 

These observations on the nutrition of a number of monogenetic trematodes con- 
firm indications available from previous accounts that there is a fundamental differ- 
ence between the Monopisthocotylea and Polyopisthocotylea as regards the domi- 



270 I). \v. HAI.TOX AND J. I 1 .. JENNINGS 

nant components of the diet. The three monopisthocotyleans studied, from quite 
different parasitic locations, all feed on the host's epidermis and epidermal secre- 
tions, and similar feeding habits have been described in Entobdclla squcnnata 
(Heath, 1902), Mcgalocotylc nntrc/iiiata (Folda, 1928), Lcptocotylc minor and 
Acanthocotyle sp. (Llewellyn. l ( '54i, Entohdclla solcac, Capsala inartinicri, Tro- 
chopus sp. and slcantliocotylc sp. ( Kearn, 1963). Thus, this type of diet would 
appear to be a characteristic feature of the Monopisthocotylea. Uspenskaya 
i 1 () 62), however, .states that in four other species (Dactylogyrus Testator, D. so- 
licliis, Anchylodiscoides pcinisilitri and Tetraonchus inoncntcron) varying amounts 
of blood are found in the intestinal contents, together with gill tissue and mucus, 
but the latter substance's predominate. 

In the Polyopisthocotylea, in marked contrast, blood forms the major, and some- 
times the only, component of the diet. Of the species examined in the present 
study, Polystount integerrimum and possibly Discocotylc sagittata feed entirely upon 
the host's blood, while Diplozoon paradoxum, Diclidophora mcrlangi and Octo- 
dactyltts palmata supplement the blood diet with varying quantities of gill tissue 
and mucus. Ingestion of blood, or the presence of an intestinal pigment which is 
presumably hematin and hence indicative of a blood diet, has also been reported in 
Hc.\'acol\lc sp., Onchocotyle sp., Octocotyle sp. and Microcotyle sp. (Goto, 1895) ; 
Axine spp., and Diclidophora spp. (Goto, 1895; Llewellyn, 1954) ; the larval and 
neotenic adult stages, as well as the normal adult stage, of Polystoma integerrimum 
(Gallien, 1934; Llewellyn, 1954); Kuhnia scombri (Sproston, 1945; Llewellyn. 
1954); Hc.vabotJi riitm appendiculata and Anthocotyle mciiitcci (Llewellyn, 1954) 
and Pricca c \bhtni and Protomicrocotyle caran.r (L^spenskaya, 1962). 

It is reasonable to suppose that the earliest Monogenea lived ectocommensally 
upon fish in much the same sort of way as modern Tennocephalida live on crus- 
tacean and other hosts. The fish epidermis and its mucoid secretions would form 
a readily available and rapidly replenished source of food to the flatworm, once the 
association was established, and by utilizing this the primitive Monogenea would 
become truly ectoparasitic. On this view the modern monopisthocotylean Mo- 
nogenea, living as a rule upon the external surface of the host, retain ancestral feed- 
ing habits and the only modification found is the evolution in groups such as the 
entobdellid species of a specific feeding mechanism involving the use of histolytic 
"salivary" secretions. Even species such as Calicotylc which have sought the shel- 
ter of the host's cloaca, and are apparently on the way to becoming endoparasitic, 
still use the original type of food. 

The polyopisthocotylean Monogenea, on the other hand, are predominantly gill 
parasites, having migrated into the branchial chamber of their piscine hosts, and 
they have departed considerably from the supposedly primitive feeding habits. The 
highly vasculari/ed gill I'da.ments offer an extremely nutritious and, again, readily 
available food in the form of blood, and the basic monogenean feeding mechanism 
of a suctorial pharynx is capable of obtaining this food without any modification 
other than slight elaboration of the oral and buccal sucker system. Thus, the dif- 
ferences in diet between the Monopisthocotylea and Polyopisthocotylea have not 
affected the feeding mechanism, and the anterior region of the alimentary system 
remains remarkably constant in structure throughout the Monogenea and, indeed. 
the l)i^enea. This unitormitv contrast- sharply with the situation in the Turbel- 



NUTRITION OF MONOGENEA 271 

laria, where considerable diversification in the form of the pharynx is linked with 
the utilization of a wide variety of prey, ranging from Protozoa and many other 
invertebrates to tunicates (Jennings, 1957). 

The differences in diet within the Monogenea do, however, have considerable 
effect upon the cellular structure of the gastrodermis. In the Monopisthocotylea 
digestion of the food creates no particular problem as regards the elimination of 
unwanted endproducts, even though the process is completed intracellularly. In 
the Polyopisthocotylea, however, the diet of blood and the retention of the intra- 
cellular digestive phase result in the intracellular production of hematin. The 
elimination of this insoluble substance is achieved at the expense of the continuity 
of the intestinal lining, and produces the discontinuous or deciduous gastrodermis 
characteristic of the sub-order. This involves wastage of cellular materials and thus 
the Polyopisthocotylea appear to be incompletely adapted to a blood diet. A more 
complete adaptation would be the extracellular formation of hematin, or the degra- 
dation of hemoglobin along some other pathway which allows the unwanted iron 
to be eliminated in a soluble form. The Trematoda are in fact capable of evolving 
such digestive processes, and these are seen in certain sanguinivorous Digenea such 
as Hacmatolocchus and Haplomctra (Halton, unpublished work). 

A compensating factor arising from the disintegration of gastrodermal cells in 
the Polyopisthocotylea is that intracellular enzymes are released to mingle with the 
gut contents and initiate breakdown of the next meal. Unfortunately it has proved 
impossible to localize the source of other digestive enzymes in either the Mono- 
pisthocotylea or the Polyopisthocotylea with the techniques at present available. It 
seems likely that the secretions of esophageal glands, poured on to the food during 
ingestion, play an important part in the extracellular phase of digestion, but this 
cannot be conclusively demonstrated. Certainly, however, the gastrodermis in the 
Monogenea has not evolved to the point of specialization of cellular function, for it 
shows no signs whatsoever of differentiation into glandular and absorptive or 
phagocytic components. In this respect it differs radically from the gastrodermis 
of other members of the phylum Platyhelminthes, such as the triclad Turbellaria, 
and of other acoelomates, such as the Rhynchocoela, where well differentiated gland 
cells occur and where there is separation of secretory and absorptive or phagocytic 
functions (Jennings, 1962a; 1962b). 

SUMMARY 

1. A comparative study has been made of the food, feeding mechanism, gut 
structure and digestive processes in representatives of the two sub-orders of the 
Trematoda Monogenea. 

2. The two sub-orders differ fundamentally as regards the dominant compo- 
nents of the diet, the Monopisthocotylea feeding on the epidermis and associated 
mucoid secretions of the host while the Polyopisthocotylea feed primarily upon the 
host's blood. In some instances the Polyopisthocotylea supplement the diet with 
small amounts of host tissue and mucus. 

3. The feeding mechanism in both groups consists basically of a muscular 
pharynx, and ingestion is the result of muscular suction, aided in some cases by 
histolytic secretions produced in pharyngeal or esophageal glands and used to erode 
the host tissues. 



D. \Y. HALT* 'X . l> J- I'. JENNINGS 

4. The two sub-orders differ considerably with regard to the structure of the 
gasirodennis, that of the MonOj being a continuous cellular structure as in 
mosl other animals while in the Polyopisthocotylea it is a discontinuous and decidu- 
ous structure \\hose cells cont; varying amounts of the pigment hematin. 

5. Digestion in hoth the Monopisthocotylea and Polyopisthocotylea is effected 
by a combination of extra- and intracellular processes, but in the Polyopisthocotylea 
intracellular degradation of hemoglobin results in the accumulation within the Castro- 
dermal cells of insoluble hematin, and the elimination of this substance results in 
the deciduous gastrodermi> characteristic of the sub-order. 

LITERATURE CITED 

I'.AI-K. J. (',., AND L. Ei ,\ i. 1961. Traite de Zoologie. Anatomie-Systematique Biologic. Tome 

IV. Edit. Pierre- 1'. < .rasse, Masson et Cie, Paris; 944 pp. 
DONE, M. S.. 1 n 58. I listochemical demonstration of acid phosphatase with naphtho] 

AS-phosph /. Nat. Cancer InsL, 21: 523-53''. 

BURSTOXK. M. S.. A \D J. E. FOLK, 1956. Histochemical demonstration of aminopeptidase. /. 

1 1 isloclieui. Cylocheiii., 4: 217-226. 

FOLDA, F., 1'L'S. Mei/a!i>f,>t\'le iiiiin/inata, a new genus of ectoparasitic trematodes from tin- 
Rock Fish. Publ. Puget Sound Mar. (Biol) Slat., 6: 195-206. 
GALLIE.X. I... 1 ( '34. Rcchcrchcs experimentales sur le (limori)liisme evolntif ct la biologie de 

J'ulysloiiia iiileiierriiiiiiiu Frohl. Tra-r. Sta. Zool. iriinercit.v. 12: 1-182. 
GOMORI, (i.. 1 ( >52. Microscopic Histochemistry. Univ. of Chicago Press, Chicago. 
GOTO,- S., 18 n 5. Studies on the ectoparasitic tremafodcs of Japan. /. Coll. Sci. Tokyo. 8: 

1-273. 

HALTON, D. \Y., AND J. B. JENXIXC.S, 1964. Demonstration of the nervous system in the mono- 
genetic trematode Diplozoon ^iinidu.riiiii Nordmann by the indoxyl acetate method for 

esterases. Nature, 202: 510-511. 
HEATH, H., 1902. The anatomy of Efibdclhi sqiiauuila sp. nov. Proc. Calif. .Ian!. Sci. 

(Zool.}, 3: 109-136. 
MOLT, S. J., 1958. Studies in enzyme histochemistry. Proc. Ro\. Soc. London, Scr. 11, 148: 

465-532. 
1 1 S.MXGS, J. B., 1956. A technique for the detection of Polystunnt intci/crriiiuiin in the common 

frog (Rana temporaria) . J. Helminth., 30: 119-120. 
IKXM.XGS, J. B., 1957. Studies on feeding, digestion and food storage in free-living flatworm^ 

(Platylu-Iinintlu-s: Turlx-llaria). Biol. Bull. 112: 63-80. 
II.VXIXGS, (. I!.. 1 () 5'^. Studies on digestion in the moiiogenetic trematode folysto/ua integer- 

rimum. J. Helminth., 33: 197-204. 
IEXXIXGS, J. B., l%2a. A histochemical study of digestion and digestive enzymes in the rhyn- 

chocoelan Linens ruber (O. F. Miillcr). Biol. Bull., 122: 63-72. 
IKXXIXT.S, |. H., I ( >o2l>. Inirther studies on feeding and digestion in triclad Tnrbellaria. 

Biol. Bull, 123: 571 581. 
KKAKX, G. C., 1963. Feeding in some mcmogenean skin parasites: lintobdclht sol cue on Solea 

solca and Acunlli, sp. on R'aia chn'nlti. J. Mar. Biol. .Issue., 43: 749-76''. 

LI.KXVKU.VX, J., 1954. Observations mi the food and gut pigment of the Polyopisthocotylea 

(Trematoda : \\\ I, J'tinisitolot/y, 44 : 428-437. 

Af.\/iA, D., P. A. |'>KI-\\IK \\n M. \MII;I, 1953. The eytochemical staining and measurements 

of protein with mercuric hrompheiiol hlue. Biul. Bull., 104: 57-67. 
Pic K\\ OKTII, F. A., l'J34. A iirv, method of study of the brain capillaries and its application to 

the regional localisation of mental disorder. ./. ./;;/., 69: 62-71. 
OSTIIX, X. (i., 1 ( '45. 'I 11- Kuhnia n.g. (Trematoda: Monogenea). An examination 

of thr value of some specific characters, including factors of relative growth. Pi 

si/olof/y. 36: 1/6 1 
STEEDMAN, H. F., 1 ( '5(). A.lcian Blue sii.S. A ne\\ stain for mucin. Ounrt. J. Mier. Sei., 91: 

477-47". 

' 5KAYA, A. \ ., I ( 'n2. pitanii mouogei ut icheskikh sosal'schikov (Nutrition of the 
nogenoidea). D nauk. S.SS.R., 142: 5-12. 



DIGESTIVE ENZYMES OF THE CRYSTALLINE STYLE OF 

STROMBUS GIGAS LINNE. 1 I. CELLULASE AND 

SOME OTHER CARBOHYDRASES 

SHIRO HORIUCHI 2 AND CHARLES E. LANE 

Institute of Marine Science, Unircrsity of Miami, Miami, Florida 33149 

The crystalline style is a flexible hyaline rod composed of mucoprotein gel. 
Among marine molluscs it occurs in most lamellibranchs and in a few gastropods. 
The digestive enzymes of the crystalline style are set free into the gut lumen by 
dissolution of the style as it is rotated against a cuticular gastric shield by the 
cilia of the style sac epithelium. Yonge (1932) pointed out that Strombus gigas, 
feeding on fine filamentous algae, was the largest of herbivorous gastropods. 
Yonge's observations were later confirmed by Robertson (1961) and by Randall 
(1964), who showed that this conch feeds unselectively on delicate macroscopic 
algae, on unicellular algae, and on algal detritus. Microscopic examination of the 
stomach contents of Strom bus gigas Linne from the Miami area has confirmed the 
ability of this animal to digest cell walls of filamentous green algae and to dissolve 
algal cytoplasm. These changes were particularly marked in green algae adherent 
to the surface of the head of the crystalline style. 

A cellulase enzyme system would facilitate use of algal cellulose by 6". gigas. 
Cellulolytic activity has been demonstrated in the crystalline style of the clams, 
Mya and Mactra (Lavine, 1946), an oyster, street and a mussel, Mytilits (Newell, 
1953), an African bivalve, Caclatnni and a marine snail, Melanoidcs (Fish, 1955), 
the wood-boring pelecypods, Bankia (Nair, 1955, 1957) and Teredo (Greenfield 
and Lane, 1953), and the lamellibranchs, Cardhtm and Sc orbicular ia (Stone and 
Morton, 1958). During feeding experiments Dean (1958) observed that algal 
cells of Cryptomonas were destroyed while this alga was swimming near the style 
of an oyster, Crassostrea. In this paper the cellulase activity of the crystalline 
style of S. gigas will be described. Subsequent papers in this series will examine 
other enzymes of the crystalline style. 

MATERIALS AND METHODS 

Specimens of the queen conch, S. gigas, which is native to Southeast Florida and 
the West Indies, were collected from shallow water adjacent to Virginia Key, 
Miami, Florida. They were maintained in sea water pens on the laboratory 
grounds until they were used. The style of S. gigas is large ; one of the largest in 
the present series of samples measured 18.5 cm. long, 0.55 cm. wide, and weighed 

1 Contribution No. 630 from The Marine Laboratory, Institute of Marine Science, Uni- 
versity of Miami. 

2 Postdoctoral Fellow of The Heart Institute of The National Institutes of Health 
under Grant No. HE-5489. Present Address : Biological Laboratory, Sophia University, 
Tokyo, Japan. 

273 



SHIRO HORIUCHI AND CHARLES E. LANE 

2.100 gin. The average moisture content was 82.4% and N averaged 8.19% by a 
micro-Kjeldahl method. Broken algal fragments and other detritus are ofte i em- 
bedded in the substance of the portion of the style (ca. 0.5 cm. in length) protruding 
from the style sac into the lumen of the gut. 

The animals were separated from the shell and the style removed quickly. The 
exposed head of the stvle was cut off and discarded. The surface of the re- 
mainder of the style was scraped with a sharp knife to remove adherent debris. It 
was then washed by shaking several times in sterile sea water. The styles were 
then homogenized in a sterile \Yaring Blendor with sterile 0.66 M phosphate buffer 
ipll n.55 i made isotonic with sea water by the addition of NaCl. This medium 
was O.h20 .17 XaCl, the same as that used earlier for the extraction of cellulase 
in Teredo ( ( Ireenlield and Lane, 1953). The viscous homogenate was stored at 
4 C. for 12 hours, then centrifugal at 20,200 g at 4 C. for 30 minutes. The small 
residue was discarded. The supernatant solution was lyophilized, and the result- 
ing powder was stored at - 10 C. There was no significant loss of cellulolytic 
activity during three months of storage. The powder was dissolved in citric acid- 
sodium phosphate buffer at room temperature and centrifuged briefly at low speed. 
The supernatant solution was filtered through a Millipore membrane. The sterility 
of the resulting enzyme solution was established by broth culture with Difco Tryptic 
Soy Broth. Cultures were negative for growth at both 24 and 48 hours at 30 C. 
Control experiments employed the sterile enzyme solution that had been boiled for 
10 minutes. 

Cellulolytic activity was estimated both by the formation of reducing sugar and 
\iscosimetrically. Reducing sugar was estimated by the methods of Somogyi (1928, 
1952) as modified by Nelson (1944). Optical density was measured at 500 m/u. 
with either the Beckman Model DU spectrophotometer or Coleman Model 6A 
spectrophotometer. Samples of sodium carboxymethyl cellulose (CMC) from Her- 
cules Powder Company, of different degrees of polymerization (D.P.) were used 
as substrates in the cellulase assay. Purified sodium alginate (Fisher Scientific 
Company), and carrageenan (Marine Colloids, Inc.) were also employed. In test- 
ing for cellobiase. glucose was estimated by the Glucostat Special reagent (Worth- 
ington Biochemical Cor]).). The pH of the reaction mixture was determined with 
a Beckman glass electrode before and after incubation. Digestive activity was ex- 
pressed as //g of reducing sugar liberated per milligram of dry style extract per 
hour. 

Changes in substrate viscosity were measured in Ostwald viscosimeters. 
Three ml. of substrate solution (CMC to final concentration O.S' < } were mixed in 
the viscosimeter with 3.0 ml. of Mcllvaine's citric acid-sodium phosphate buffer 
(pH 6.75). After thermal equilibrium in the 35 C. water bath had been achieved, 
the time required to empty the capillary was noted. One-tenth ml. of enzyme 
solution was added and mixed in a stream of air bubbles. The time required to 
empty the capillary was measured at intervals from the time of mixing. 

RESULTS 

Filtration through Millipore membranes does not affect the activity of style en- 
zyme solutions as judged by two different criteria (Tables 1 and II I. The yields 
of reducing -oigar from CMC 70 of three different degrees of polymerization in- 



ENZYMES OF THE CRYSTALLINE STYLE 



275 



TABLE I 

Hydrolytic activity of crystalline style extract before and after bacterial filtration* 



Substrate 


Unfiltered enzyme 
solution 


Mi Hi pore-filtered 
enzyme solution 


Boiled enzyme 
solution 


CMC 70 M 


90.5 9.0 


89.7 10.3 


11.7 1.7 


Phosphoric acid-swollen 








cellulose 


10.3 0.7 


10.9 1.1 


3.8 0.4 


Cellulose powder 
Sodium alginate 
Carrageenan 
Cellobicse 


2.1 0.3 

5.7 0.3 
2.4 0.7 
8.9 0.5 


1.9 0.3 
6.1 0.5 
2.7 0.5 
8.7 0.5 


0.7 0.2 
0.6 0.3 
0.7 0.2 
3.7 0.4 



* Activity expressed as ^tg. reducing sugar/mg. crystalline style powder /hour. 
Each value is the mean and standard deviation of 10-17 determinations. 
Ki'uctants incubated at 35-37 C. at pH 6.75. 

cubated with crystalline style enzyme solutions are shown in Table III. This table 
shows that different degrees of substitution in the CMC substrate had only a slight 
effect on the activity of the enzyme preparation. These results differ somewhat 
from those reported for Liinnoria by Ray (1959). In this form maximum cellulase 
activity was found when CMC 70 High was the substrate. When the cellulase ac- 
tivity of 6". gigas style extract was estimated viscosimetrically, however, the results, 
shown in Figure 1, more nearly resemble those reported by Ray. Whatman No. 1 
filter paper, swollen in 85 % phosphoric acid and suspended at 0.5% concentration 
in phosphate buffer and the same concentration of Whatman cellulose powder, was 
also used as a substrate. These results also appear in Table I. The style enzyme 
solution is less effective on these substrates than on the CMC samples used. Figure 
2 shows the linear relationship between cellulase activity and enzyme concentration 
measured by protein nitrogen. The relationship between pH and cellulolytic activity 
is shown in Figure 3. There is a single peak of activity between pH 6.8 and pH 
7.2. When determined viscosimetrically after 60 minutes incubation the pH opti- 
mum was 6.75. The pH of gut contents of 6". gigas ranged from 6.25 to 6.65. 

TABLE II 
Viscosity of enzyme-substrate* before and alter bacterial filtration 



Substrate 



Incubation time 





15 min. 


30 min. 


60 min. 


90 min. 


120 min. 




f I 67.8 1.4 


54.7 1.2 


44.9 2.0 


39.6 2.2 


36.5 2.4 


CMC 70 M 


1 II 74.2 3.1 


62.9 3.2 


52.1 3.3 


47.4 2.7 


44.5 1.8 


Sodium alginate 


] I 89.0 3.2 
I II 88.1 2.2 


80.9 2.8 
82.2 3.8 


67.7 2.1 
72.0 3.2 


58.2 2.9 
63.9 db 3.2 


52.3 2.8 
56.7 2.7 




/ I 90.9 1.9 


83.3 2.5 


74.7 2.2 


68.4 1.8 


64.3 2.2 


Carrageenan 


\II 90.7 5.8 


86.9 0.1 


79.8 0.6 


74.2 0.4 


70.7 0.1 



* Yisccsity is expressed as time in seconds for capillary emptying. 

I = Unf Itered enzyme solution. 

IT = Millipore-filtered enzyme solution. 



276 



SIIIKi ) IIORIUCH] AND i HARLES E. LANE 



TAHI.I. 111 
activity oj the < rystalh ^trombus i;/i;.v wiili unions substrates 



Substrate 


D.S. 


Viscosity 


fig. glucose/mg. of enzyme In. 


CMC To low 




30 cps. 


31.4 


CMC 70 mol. 


d.77 


7o cps. 


41.1 


CMC 70 hiiili 


0.72 


2100 cps. 


15.0 


CMC" )() hi.Ji 


0.94 


280 cps. 


17.8 


CMC 120 h 




1 .-55 cps. 


7.8 



Suollen filter paper 

( Yllulo-r powder . 0.7 

Incubation at so and pi I 7. -1 lor CMC, pH 7.3 for other substrates. 

The optimum temperature and the temperature of inactivation of the enzyme 
solution were determined. The relationship between the incubation temperature 
and cellulase activity showing an optimum about 40 C. is presented in Figure 4. 
Thermal stability of the enzyme solution was determined by assaying residual ac- 
tivity after heating the enzyme solution to various temperatures for fifteen minutes 



CO 

o 
o 

CO 



CO 

<I 

LU 

cr 
o 

LU 
O 




CONTROL 



CMC 70 LOW 



CMC 70 
MEDIUM 



CMC 70 HIGH 







10 20 30 40 50 
INCUBATION TIME (MINUTES) 



I-'K,I ]<]. 1. Ki'tVrt of crystalline style enzyme on CMC 70 of different viso^ity. Final 
concentration of CMC /o was 0.8$ ; pi I was n.75 and tcinprratuiv S3 C. 



ENZYMES OF THE CRYSTALLINE STYLK 



277 




ct 

QK _L_ 

0.05 0. 10 

ENZYME CONCENTRATION (Mg N) 

FIGURE 2. Cellulase activity of crystalline style extract at various enzyme concentrations. 
Reaction mixtures containing 12 ing. CMC 70 of medium viscosity were incubated at 30 C, 
pH 6.8 for two hours. 



ml 



100 - 



CO 

o 
o 



CO 



cc 

<t 

50 

CO 



o 



LU 
CC 








8 



pH 



FICUUK 3. Cellulase activity of the crystalline style at various pi 1 levels. Suhstrate was 
CMC 70 of medium viscosity ; temperature was 30 C. ; time was two hours. 



278 



SHIRO IIORIUCHI AM) CHARLES E. LANE 



(Fig. 5). Activity was unditnini-Ucl up to 45 C. Between 45 C. and 50 C.. 
cellukilytic activity was markedly reduced and was 90% destroyed at 70 C. 

Cellobiase activity (Table EV) was estimated by incubating cellobiose with 
crystalline style en/.yme solul llucose produced was determined by the spe- 

cific glucose oxidase method \Yorthington Biochemical Corp.). As compared 
with the cellulase activity, the cellobiase acthity is slight. Since the crystalline 
style of -V. gigas appears i deficient in cellobiase, it appears that digestion of 

cellulose by this animal does not necessarily include cellobiose as an intermediate 
(Levinson and Reese. 1"50). 



200 

LU 
CO 

8 



CO 



a: 
% 

CO 



o 
o 



100 








20 30 40 

TEMPERATURE C 



50 



FIGURE 4. Effect of temperature on cellulolytic activity of the crystalline style extract. 
Substrate was CMC 70 medium; pH 6.75; incubation time was two hours. 

The relationship between decreasing viscosity and increasing concentrations of 

reducing sugar during cellulolysis is presented in Figure 6. 

DISCUSSION 

There is general reluctance to attribute cellulase enzyme activity to higher 
ia because symbiotic microorganisms are i ivolved in cellulose breakdown in 
r animals. Indeed, Morton (1952. 1960), Newell (1953) and Barring- 
ton i have emphasi/ed the occurrence of spirochaetes in the crystalline style 
of many bivalves. Some, at least, of the cn/ymatic capability of the style is attrib- 
uted to these symbionts. 



ENZYMES OF THE CRYSTALLINE STYLE 



279 



CE 
LU 
Q_ 



UJ 
CT 



50 



CO 








j_ 



30 



40 50 60 

TEMPERATURE C 



70 



FIGURE 5. Temperature stability of cellulase in the crystalline style. Activity of the 
heated enzyme, assayed after two hours in a reaction mixture containing CMC 70 medium, at 
.15 C., pH 6.8. 

Levinson and Reese (1950) suggested that at least two kinds of enzymes were 
involved in the complete degradation of native cellulose. First, a C l enzyme causes 
a rapid decrease in viscosity by converting native cellulose to linear anhydroglucose 
chains. These are then hydrolyzed to the soluble sugars glucose and cellobiose by 
C x enzymes (Gascoigne and Gascoigne, 1960; Levinson and Reese, 1950). Our 
results strongly suggest that the cellulolytic capability of the crystalline style of 6". 
gigas, together with certain other amylolytic activities, are of molluscan rather than 
bacterial origin. If it be assumed that CMC in solution is a straight-chain mole- 
cule made up of 1 ,4-/3-glucose linkages (Levinson and Reese, 1950), then all the 

TABLE IV 

Cellobiase activity of the crystalline style of Strombus gigas Linne 



pH 



5.7 



6.6 



7.6 



Glucose 



16.3 



16.2 



10.3 



0.5 % cellobiose in Mcllvaine buffer was incubated at 35 C. for three hours. Reaction 
mixtures contained 1.8 mg. of lyophilized style. 



280 



SHIRO IIOR1UCHI AND CHARLES E. LANE 



soluble derivatives of cellulose u>ed in ilii> study were hydrolyzed by a C x enzyme of 
the crystalline style. 1 )igestion of algal cellulose by .V. (jif/as probably includes some 
preliminary microbiological degradation followed by extracellular digestion by style 
i'ii/yines in the .stomach (Evans ;ind Jones, 1962). Digestion of lower molecular 
weight sugars and other partially digested foods is probably completed intracellu- 
larly in phngocytic cell- of the digestive diverticula. 



CO 

o 

CO 

> 




^ 50 - 



CO 

<t 



o 

UJ 
Q 



30 60 90 

INCUBATION TIME (MINUTES) 



120 



IMI.CKK 6. Tin- relationship between decrease in viscosity and formation of reducing sugar 
during cellulolysis by the crystalline style. Reaction mixture contained O.b'/f CMC 70 medium 
viscosity; ]>H 6.75 at 35 C. 

Si'.M M AKY 

The crystalline style was extracted in buffered saline and the extract subse- 
quently lyophilixed. The activity of the resulting enzyme powder was determined 
by measuring the amount of reducing sugar it liberated from various substrates under 
different conditions, and by measuring the decrease in viscosity of these substrates. 
Cellulase activity was proportional to enzyme concentration. The pH optimum was 
between pi I 6.8 and pi I 7.2. ( tptimum temperature for enzyme activity was 40 C. 
P.etween 45 and 50 C'., cellulolytic activity was markedly reduced. C'ellobiasc 
activity of the style extract was slight. Bacteria-free extracts were as active as 
unsterile preparations. Some implications of these observations arc discussed. 

LITERATURE CITED 

KINGTON', K. J. \V., l ( 'f)2. Di{ic-ii\-c nes. hi: Advanci-s in Comparative Physiology 

rmd Biochemistry. O. Lo\\vnstein, Editor. Ac;id<'inic 1'ros, N. Y., Vol. 1, pp. 1-65. 
|)i. \x. I)., 15<S. Xe\v property crystalline Mylr of Cruxxoxtrca riri/iiiica. Science, 

128: S3". 



ENZYMES OF THE CRYSTALLINE STYLE 281 

EVANS, W. A. L., AND E. G. JONES, 1962. Carbohydrases in the alimentary tract of the slug, 
Arion ater L. Coinp. IHuchan. Physiol., 5 : 149-160. 

FISH. G. R., 1955. Digestion and the production of sulphuric acid by Mollusca. Nature, 175: 
733-734. 

GASCOIGNE, J. A., AND M. M. GASCOIGNE, 1960. Biological Degradation of Cellulose. Butter- 
worths, London. 

GREENFIELD, L. J., AND C. E. LANE, 1953. Cellulose digestion in Teredo. J. Biol. Chan., 204: 
669-672. 

LAVINE, T. F., 1946. A study of the enzymatic and other properties of the crystalline style of 
clams : evidence for the presence of a cellulase. /. Cell. Camp. Physiol., 28 : 183-195. 

LEVINSON, H. S., AND E. T. REESE, 1950. Enzymatic hydrolysis of soluble cellulose derivatives 
as measured by changes in viscosity. /. Gen. Physiol., 33: 601-628. 

MORTON, J. E., 1952. The role of the crystalline style. Proc. Malacol. Soc. London, 29 : 85-92. 

MORTON, J. E., 1960. The function of the gut in ciliary feeders. Biol. Rev., 35 : 92-140. 

XAIR, N. D., 1955. Cellulose activity of the crystalline style of the wood-boring pelecypod 
Bankia indica Nair. Current Sci., 24 : 201. 

NAIR, N. B., 1957. Physiology of digestion in Bankia indica; the enzymatic activity of the 
crystalline style. /. Sci. Industr. Res., 16C : 39-41. 

NELSON, N., 1944. A photometric adaptation of the Somogyi method for the determination of 
glucose. /. Biol. Chem., 153 : 375-380. 

NEWELL, B. S., 1953. Cellulolytic activity in the lamellibranch crystalline style. /. Mar. Biol. 
Assoc., 32: 491-495. 

RANDALL, J. E., 1964. Contributions to the biology of the queen conch, Strombus gigas. Bull. 
Mar. Sci. Gulf Carib., 14: 246-294. 

RAY, D. L., 1959. Some properties of cellulase from Limnoria. In: Marine Boring and Foul- 
ing Organisms. D. L. Ray, Editor. University of Washington Press, Seattle, pp. 
372-386. 

ROBERTSON, R., 1961. The feeding of Strombus gigas and related herbivorous marine gastro- 
pods. Nohilac Naturae, 343 : 1-9. 

SOMOGYI, M., 1928. The distribution of sugar in normal human blood. /. Biol. Chem., 78 : 
117-127. 

SOMOGYI, M., 1952. Notes on sugar determination. /. Biol. Chcni., 195: 19-23. 

STONE, B. A., AND J. E. MORTON, 1958. The distribution of cellulases and related enzymes in 
Mollusca. Proc. Malacol. Soc. London, 33: 127-141. 

YONGE, C. M., 1932. On the size attained by the crystalline style in Tridacna and Strombus. 
Proc. Malacol. Soc. London, 20: 44-45. 



T11K HEMOCYTE!, OF RHODNIUS I'ROLIXUS Sl'AL. 

COLVARD JONKS 
I >cp(irtnii'iit at Eni ly, University of Maryland, College Park, Maryland 

Wigglesworth ( l l '. : ' . -Mtied four kinds of hemocytes in Rhodnlns nymphs, 

and in ] ( )55 he identifu additional types. His interest centered on the most 

abundant hemocyte, which will he referred to in the present paper as the plasnuito- 
eyte. Jle ( 195(>a, pa.^e 142) stated that plasmatocytes ". . . show definite signs 
of secretory activity just at the time when hormone of the thoracic gland is being 
produced," and he ( 1^5(>b, page 97) concluded that one of the distinct functions of 
the plasmatocytes is that ". . . in the early stages of moulting they seem to play 
some essential part in the production of the moulting hormone by the thoracic 
glands." Wigglesworth (1956b) demonstrated that the plasmatocytes possess 
mucopolysaccharide inclusions and mentioned that this material was liberated during 
the later stages of moulting. The signs of secretory activity which Wigglesworth 
stated occur in the plasmatocytes of fourth stage nymphs were an average in- 
crease in cell sixe and the sudden appearance of many, clear, non-staining vacuoles 
in the cytoplasm between the third and fourth day after these insects took a blood 
meal. 

The initial purpose of these studies was to examine quantitatively these two cri- 
teria for secretory activity in the circulating plasmatocytes of Rlwdniits in fresh. 
unfixed, unstained coverslipped samples of hemolymph with dark phase contrast 
microscopy. As these studies were being made, it became evident from the form 
and behavior of the different types of hemocytes that a change in the terminology 
of the different cells was needed. 

Hemocytes were examined daily throughout the fourth and fifth stages and in 
adults at 970 to 1400 X. The hemocytes were also studied using supravital meth- 
ods and in fixed and stained smears. Hemolymph was obtained from a severed leg 
or antenna. 

RESULTS 

1. General observations 

The hemolymph of Rhodnius is a clear, pale, straw-yellow, watery fluid. When 
examined in a hanging-drop preparation or in a moist chamber the hemolymph 
does not obviously coagulate or gel in 24 hours, although a very finely granular 
precipitate may form on long standing as the drop partially darkens. In a very 
f<-\v cases, a rare plasmal veil may be observed. In a moist chamber, the hemo- 
lymph slowly darkens and finally appears either pale brown or has an irregular scat- 
tering of dark sooty patches, but the drop never becomes generally dark- brown or 
uniformly black in vitro. None of the hemocytes darken or blacken. 

2. Phase contrast observations and classification 

the classification and terminology suggested by Jones (1962), the various 
type- ocytes of l\'liodniiis can be readily identified in fresh, undiluted, un- 

fixed, unstained hemolymph with a dark phase contrast microscope. 

282 



HEMOCYTES OF RHODMUS 283 

a. Prohemocytes 

The prohemocytes are always small, mostly round to ovoid cells, generally with 
a relatively large, single, centrally-located, round nucleus (Plate I, Figs. 1 and 2). 
Usually the nucleus has extremely fine, dark grey, granular chromatin material 
around a single, slightly excentric round nucleolus. Prohemocytes have a relatively 
small amount of smooth, dark grey, homogeneous, or sometimes finely granular cyto- 
plasm. In some cases, the nucleus may be excentric and ovoid, with a single slight 
indentation; the nucleolus may he irregular in shape, or, in a few cases, absent. 
Prohemocytes can be seen with a few, relatively large, dark grey, round inclusions. 
On a number of occasions, prohemocytes have been seen to degenerate in vitro : the 
small, round, cartwheel-like nucleus is generally ejected, and the cytoplasm rounds 
up into a pale-grey sphere with fine dancing particles within and around it (Plate I, 
Fig. 3 ). On many occasions, a prohemocyte has been seen to undergo unmistakable 
and intense ameboid movements /;/ vitro, when none of the other hemocytes made 
comparable movements. During such ameboid movements, the nucleus was fre- 
quently constricted or otherwise distorted. In older nymphs, the prohemocytes 
measured from 5 to 7 microns in diameter. Mitotic divisions were seen only in 
cells slightly larger than the typical prohemocyte. At metaphase, the chromosomes 
were so tightly packed that they appeared as a single dark-grey bar. The meta- 
phase plate was contained in a clear, hyaline central zone within the cell. Vacuoles 
and granules were usually conspicuous in dividing cells. Prophases could not be 
accurately identified in the preparations examined. 

b. Plasmatocytes 

Plasmatocytes are exceedingly variable in form (Plate I, Figs. 5, 6, 9, 10-15). 
The most common variety is an ovoid cell with a single, large, centrally-located, 
round-to-ovoid nucleus containing a single, round-to-ovoid nucleolus. The cyto- 
plasm contains sharply-outlined round, ovoid, or short rod-shaped, or tear-drop- 
shaped granular inclusions (Plate I. Figs. 5, 6, 14, 15). With dark phase micros- 
copy, the edges of these inclusions are generally sharp black and the enclosed 
granular space bright. 

In unfixed plasmatocytes, the cytoplasm often contains few (t\vo) to many 
(about 32) round or irregular, clear, non-refringent, watery, colorless vacuoles of 
various sizes (Plate I, Figs. 11-13). Some plasmatocytes can be seen with large, 
less sharply-defined, round or spherical, grey inclusions, often having a very pale 
greenish cast. 

Plasmatocytes tend to send out several to many, fine, thread-like pseudopodia 
(Plate I, Figs. 5, 11-13). In some cases, exceedingly thin cytoplasm will spread 
out from the cells and terminate in extremely delicate spikes. Round, spindle, and 
irregular forms of plasmatocytes abound in the hemolymph. Small, medium and 
large varieties of each of the above may be found in a single preparation. 

Although the plasmatocytes have a distinct tendency to spread out and form pe- 
ripheral thread-like pseudopodia in thin wet films, they were very rarely observed 
to perform vigorous ameboid movements comparable to those seen in some pro- 
hemocytes. Although the plasmatocytes are probably capable of ameboid activity 
in vivo, such movements do not critically distinguish plasmatocytes from other types 
of hemocytes. 



JM 



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PLATE I 

I'lra^t appearance <>l Rhodnius hemocytes. 

l*"i(;ri<i'> 1 AMI 2. 'J'ypieal prolienioeyti's. 

FIGURE 3. Lysed proheiih- Tlie nucleus is at the ri.ulil and the Cytoplasmic envelope 

;ind uraiinK^ at the left. 

FlGURK 4. Small spiml!< ytoid with excentric nucleus and without inclusions. 

FlGt'KK 5. Non-vacuolated iila^nialueyte with thread-like p^endi ipodia. 



HEMOCYTES OF RHODNIUS 

The sizes of plasmatocytes throughout the fourth stadium of Rlwdniiis were 
noted but were so highly variable in both unfixed and methanol-fixed films within 
and between individuals that no difference in average sizes could be detected at any 
time during the stage with the methods used. Since many more hemocytes are 
available in the fifth stadium, plasmatocytes were measured every day between the 
first through ninth, eleventh through sixteenth, and on the twentieth days after 
nymphal feeding. Plasmatocytes in unfed fifth stage nymphs and in unfed adult- 
were also measured. In all, 335 plasmatocytes were measured. The minimum 
values for individual widths varied from 4.4 to 8.8 microns and the maximum values 
from 8.8 to 17.6. The minimum values for individual lengths ranged from 7.7 to 
11 and the maximum values from 12.1 to 66 microns. The mean widths of the 
plasmatocytes during the fifth stadium varied from 7.7 to 12.2 and the lengths from 
9.9 to 20.6 microns. The overall mean dimensions of the plasmatocytes for the 
period examined were 9.8 (standard error 0.3 ) by 14.5 (standard error 0.6) microns. 
There was a distinct tendency for the plasmatocytes to decrease in length during 
the fifth stadium while the widths did not change greatly. Measurements were 
made on a series of coded slides, but the writer was unable to accurately distinguish 
between plasmatocyte sizes at any time during the fifth stadium. 

Although Wigglesworth (1955 ) stated that the average size of the plasmatocytes 
increased between the third and fourth days after fourth stage nymphs feed, meas- 
urements made on the cells shown in his Figure 5 A and B show statistically insig- 
nificant differences in mean width, length, circumference and area (Table I). The 
same applies to the plasmatocytes illustrated in his Figure 6 (Table I). But what 
is striking about Wigglesworth's Figures 5 and 6 is that the fixed adherent plas- 
matocytes of fourth stage nvmphs are strikingly and significantly smaller than the 
unfixed circulating forms ( Table I ) . Whether this is clue to fixation or to the 
spreading-out of plasmatocytes in the fresh preparation is not clear. The fixed 
adherent plasmatocytes illustrated in Wigglesworth's Figure 6 average about the 

FIGURE 6. Non-vacuolated plasmatocyte without pseudopodia. 

FIGURES 7 AND 8. Round and ovoid oenocytoids with characteristically excentric nucleus 
and with dark, smooth cytoplasm. 

FIGURE 9. Small plasmatocyte without inclusions, vacuoles, or pseudopodia. 

FIGURE 10. Small plasmatocyte with a few inclusions, vacuoles, and blunt pseudopodia. 

FIGURES 11-13. Typical vacuolated plasmatocytes with obscured nucleus and large watery 
vacuoles and discrete inclusions. 

FIGURES 14-15. Spindle plasmatocytes showing very fine granules (mitochondria?) and 
larger inclusions (mucopolysaccharide?). 

FIGURE 16. Spindle form of granular hemocyte. 

FIGURES 17-19. Typical ovoid granular hemocytes. 

FIGURE 20. Large granular hemocyte with one, short blunt extrusion at upper right. 

FIGURE 21. Round granular hemocyte. 

FIGURES 22-23. Partially lysing granular hemocytes. 

FIGURE 23a. Very small intact granular hemocyte with obscured nucleus. 

FIGURE 24. Lysing granular hemocyte, showing tin- hyaline cytoplasm and round cart- 
wheel-like nucleus. 

FIGURE 25. Fully lysed granular hemocyte. 

FIGURE 26. Large oenocytoid with glassy rod-like inclusions. 

FIGURE 27. Large oenocytoid with finely granular network and one vacuole. 

FIGURES 28-29. Typical large oenocytoids with characteristic excentric nucleus and long 
threadlike filamentous inclusions. 

FIGURE 30. Adipohemocyte with excentric nucleus and various sizes of fat-like droplets. 



286 



JACK O ILVARD JONES 



same as the unfixed circulating forms measured in the present study. It is con- 
cluded from the present observations and from calculations on Wigglesworth's fig- 
ures (Table I), that a change in the sizes of plasmatocytes is not a practical cri- 
terion for their possible secretory activity because of an inherently large individual 
variation. 

In fresh wet films, various inclusions in many plasmatocytes were observed to 
very rapidly turn into clear, colorless vacuoles. Unfixed circulating plasmatocytes 
of 72 fourth stage Rlwdniits were classified as either with or entirely without vacu- 
oles in differential counts of generally 100 hemocytes per insect per group of two 
to five nymphs, two to five- days after ecdysis, and daily after the nymphs fed, for 
a period of 12 days. Each insect was used only once. During the fourth stage the 
mean percentages of plasmatocytes in fresh hemolymph varied from 22.7 to 61.5. 

TABLK I 

t.'til< illations made from plasmatocytes oj jourth .v/</r Rhudnins, us illustrated 
hy Wigglesworttl (1V55), with standard errors 



FisuM- 


No. 

iMf.iMMrnients 


No. 

( ,-ll.x 


Days alter 
nymphal 
feeding 


Mean width 
U) 


Mean length 
(/O 


Mean 
circumference 
(/.) 


Mean area 

<M-) 


5A 


1 

2 


4 


3 


12.40.6 
11.60.4 


29.62.9 
27.82.8 


9 1.8 14.9 
98.817.9 


156.819.8 
157.826.6 


SB 


1 
2 


3 


4 


13.70.9 
13.01.3 


36.02.9 
,U.42.5 


127.020.0 
132.714.2 


186.2 36. 6 
185.630.0 


6A 


1 
2 


7 


3 


8.30.3 

8.2 0.2 


16. 2 1.0 
16.5 1.0 


33.8 1.4 
38.6 1.7 


66.9 5.7 
66. 7 5.7 


6B 


1 

2 


6 


4 


10.1 0.8 

io.4o.9 


16.0 1.1 
16.11.1 


26. 6 2.6 
39.9 2.9 


108.3 17.0 
105'3 6.7 



with an overall mean of 44.2. Of these. 83.8% to 96.8 '/ (mean of 92.3%, standard 
error of 0.8) had few-to-many vacuoles in their cytoplasm. Even in unfed nymphs. 
94.3% of the plasmatocytes encountered in differential hemocyte counts were vacu- 
olated. A few extremely vacuolated plasmatocytes were seen in only four insects 
during the fourth stadium. It i> evident from these findings that vacuolation of 
circulating plasmatocytes cannot be a very useful criterion of changes in their pos- 
sible secretory activity during the fourth stage. It is not clear why there should 
be such a great discrepancy between the present findings and those of Wigglesworth 
( ]''55 i. \Yiggle>worth'.s Figure 5 shows very few vacuoles on the third day and a 
considerable (about 10-fold) increase- in vacuoles on the fourth day in circulating 
plasmatocytes. Hi> Figure (> indicates that fixed adherent plasmatocytes have 
many more vacuoles than the unfixed circulating forms on the fourth day. 

Unfixed circulating plasmaiocytes of fifth stage h'liodniits were sub-divided 
into those with no or very few vacuoles and those with few to many vacuoles in 
differential counts of generaib 200 hcmocvtes per insect per group of four to li\e 
nymphs. Thirty-nine insects were used for the first seven days after ecdysis. a 
Separate group being examined each day. One hundred and one nymphs were 



HEMOCYTES OF RHODNIl'S 287 

used to stud}- changes following the blood meal, generally five insects being studied 
daily from the day the nymphs took a blood meal to the twentieth day thereafter. 

During the first seven days after ecdysis to the fifth stage, plasmatocytes in 
differential hemocyte counts varied from 47.1 to 68.7. and 95.9% to 98.4% of them 
were vacuolated. On the da\ the insects fed and during the next day, differential 
counts of plasmatocytes averaged 44.7 r r to 53.8% , and of these 9(>. 1 ' , to 99.3% were 
vacuolated forms. Between the first and second days after the nymphs took blood, 
however, a spectacular shift in the presence of vacuolated plasmatocytes was re- 
corded. From the second through the twentieth flay, circulating plasmatocytes 
averaged 29.8% to 63.7% (overall mean of 51.5% ). Between the first and second 
days after nymphal feeding, the percentage of plasmatocytes which were classified 
as vacuolated forms dropped from 96% to 40%. and thereafter more or less steadily 
declined to about 8% on the twentieth day ( i.e., before ecdysis to the adult stage). 

\\ hen five newly-ecdysed fifth stage Rhodnius were submerged for one minute 
in water at 55 C, it was observed that most (97.5% ) of the plasmatocytes still 
vacuolated in ritro; but when nymphs which had taken a blood meal were similarlv 
heat-fixed, very few of their plasmatocytes vacuolated in ritro. Fifth stage nymphs 
were heat-fixed on seven representative days after the blood meal and hemocytes 
from three to five insects for each day were examined. In differential counts, the 
plasmatocytes were classified as those with few or no inclusions and those with 
conspicuous round, ovoid or irregular, phase-dark inclusions. Plasmatocytes aver- 
aged 32.7% to 49%, and 82.1% to 93.5% (mean of 89.6%) of them had inclusions. 

Clearly the problem of vacuolation of plasmatocytes in Rhodnius needs further 
study. But, if one assumes that the secretory activity of plasmatocytes is indeed 
correlated with cytoplasmic vacuolation, as \Yigglesworth ( 1955 ) has indicated, 
then the present data collected on unfixed hemolymph could be interpreted to mean 
( 1 ) that the circulating plasmatocytes are highly secretory throughout the entire 
fourth stadium, and during the fasting period after ecdysis to the fifth stage, and 
(2) that there is a remarkable decrease in their secretory activity shortly after fifth 
stage nymphs take a blood meal. Additional studies are clearly needed before such 
interpretations can be accepted. The present data do not support the idea that 
circulating plasmatocytes suddenly become secretory during the time when the 
thoracic gland hormone is being produced in either the fourth or fifth stadia. 

c. Granular hemocytes 

The granular hemocytes of Rhodnius are typically larger and noticeably thicker 
than plasmatocytes in thin films of hemolymph examined with phase microscopy 
(Plate I, Figs. 16-20). However, very small granular hemocytes have been seen 
in fresh hemolymph films (Plate I, Fig. 23a). Freshly withdrawn granular hemo- 
cytes often have a very pale, yellowish-brown cast, are ovoid to spindle in shape, 
and are characteristically filled with numerous, discrete, round, granular inclusions 
of a mostly uniform size from 0.5 to 1 micron. The granules generally tend to 
obscure the relatively small, round, centrally-located nucleus in fresh material. 
With the ordinary bright field microscope, the granules in these unfixed cells appear 
quite vague and could easily be overlooked or mistaken for fine droplets (vacuoles). 

Of the numerous granular hemocytes examined in this study (approximated 
50,000), only 20 were seen with one, short, blunt, or spike-like clear pseudopodium. 



288 JACK COLYARD JONES 

On a few occasions granulocytes have been seen to retract very rapidly their spindle 
ends and round up. I'nlikc aocytes, some of the granular hemocytes have 

been observed to rock hack and n>nh very slightly in situ. Unlike plasmatocytes, 
they were never observed to send out pseudopodia in vitro. 

In unfixed films of her .ymph, the granular hemocytes occur in two verv di>- 
tinct forms: those which rmain intact for long periods in vitro (Plate I. Fig>. 
16-20) and those which suddenly degenerate or lyse generally shortly after with- 
drawal of the hemolymph sample (Plate I, Figs. 22-25). Many granular hemo- 
cytes of various >i/< been watched as they degenerate /';/ vitro. The intact 
cell seems to twist suddenlv. contract or constrict along its longitudinal axis ; and 
the cell may then collapse like a balloon, releasing the round, cartwheel-like nucleus 
and many fine dancing granules (Plate I. Figs. 21-23). Many times, as the cell 
breaks down, the cytoplasmic envelope fragments into two or more spherical hyaline 
masses with some enclosing dancing granules, as well as with granules around the 
masses. The granules suddenly released from the disintegrating granulocyte are 
much more sharply outlined than in the intact cell and may have a very faint green- 
ish cast. The granules and cytoplasmic fragments do not quickly vanish but tend 
to maintain their identity for a considerable time. The cytoplasmic fragments and 
extruded nuclei greatly complicate both differential and total hemocyte counts. 
The lysing or lysed granular hemocytes strikingly resemble cystocytes (but they 
do not lead to coagulation or gelation of the plasma ). 

Submersion of Rhodnius in a water bath at 55 C. for one minute generally did 
not reduce the percentages of granulocytes lysing in vitro, but collection of fresh 
hemolymph into 0.75% Versene largely prevented lysis of these cells. Collec- 
tions of hemocytes in \% to 3% Versene severely damaged the hemocytes: the 
surface of the cells appeared abnormally thickened. 

Widths and lengths of granulocytes were measured in unfixed wet films of 
hemolymph from fifth stage nymphs on eleven representative days after they took 
a blood meal. Of the 130 granular hemocvtes measured, the minimal values for 
individual widths ranged from 4.4 to <->.9 microns and the maximal values from 1 1 
to 16.5; the minimal values for individual lengths ranged from l '. ( > to 13.2 microns 
and the maximal values from 22 to 35.2. The mean dimensions of the granular 
hemocytes were 10.3 (standard error 0.3) by IS. 8 (standard error 0.4) microns. 
There \vas no marked change or trend in the length-width measurements of grannlo- 
cytes during the fifth stage after feeding. 

d. Oenocytoids 

The cells which will be termed the Oenocytoids of Rhodnius occur in two dis- 
tinct forms. In fresh hemolymph examined with phase microscopy, the first 
variety is a relatively small, round, ovoid cell with one or two sharp spindle en<K 
and the cytoplasm is very smooth, dark grey, and homogeneous. The nucleus is 
sharply outlined, round and characteristically excentric (Plate I. Figs. 4. 7 and Si. 
-erond, and most commonly encountered variety of oenocytoid is a very large 
plasi vie-like cell, often occurring as hi/arre variations of the spindle form 

I Plate I. ; 26-29). These large cells are characteri/ed bv having extremeb 

line, long, phase-dark filaments at the spindle ends and b\ a large excentric nucleus, 
often with two nucleoli. The inclusions are sometimes in the form of an irregular 



HEMOCYTES OF RHODNIUS 

finely granular network or appear as delicate, faintly outlined glassy rod: 

have a very faint greenish cast (Plate I, Fig. 26). The large bizarre 

often occur in clusters of two to four, and in some cases appear fused to each c 

On some occasions, irregular nuclei and apparent hinucleate forms have been s< 

Some of these cells have been seen to send out a few filamentous cytoplasmic 

extensions. 

e. Adipohemocytes 

On rare occasions, hemocytes with an excentric nucleus and many brilliant fat- 
like droplets of various sizes have been encountered in the hemolymph (Plate I. 
Fig. 30). These cells should be termed adipohemocytes only when they can be 
clearly distinguished from fat body cells. At various times typical large fat body 
cells can appear in the hemolymph (in some cases, at least, their appearance results 
from accidental dislodgement at the time of sampling). Adipohemocyte-like cells 
have been seen in fixed whole-mounts of thoracic glands. The scarcity and erratic 
occurrence of the small adipohemocytes in the hemolymph make it most imprac- 
tical to include these cells with other hemocytes in most differential counts. Since 
Wigglesworth (1955) has indicated that all gradations between adipohemocytes 
and typical fat body cells may be found in Rhodiiiits, it is useful to place all circu- 
lating cells containing fat droplets in a special category where they may be listed 
separately. 

f. Granulocytophagous cells 

Unmistakable plasmatocytes have been seen engulfing a single intact granulootr 
and/or the nucleus of the lysed form (Plate II, Figs. 31-34). In addition, very 
large, plasmatocyte-like cells, measuring 20 to 35 microns or more in diameter, have 
been observed with two to eight engulfed intact granular hemocytes or their nuclei 
(Plate II, Figs. 35-39). These large phagocytes tend to send out characteristically 
very extensive pseudopodia, which may extend 30 microns and more beyond the 
main portion of the cell (Plate II, Fig. 39). While the very large forms may be 
only a hypertrophied form of plasmatocyte, they are sufficiently distinctive in si/e 
and activity to be listed separately in differential counts, and for convenience will 
be termed granulocytophagous cells. Whether the large granulocytophagous cells 
are, in fact, giant plasmatocytes is by no means certain. 

3. Observations on supravital preparations 

To further characterize the different types of hematocytes, fresh drops of un- 
fixed hemolymph from fifth stage nymphs were collected on slides previously coated 
with a thin, even, dry film of the following dyes: (a) neutral red, (b) phenol red, 
(c) congo red, (d) eosin Y, and (e) Janus green H. Wet coverslip films were 
examined with and without phase contrast. 

Neutral red was picked up by the plasmatocytes and concentrated uithin yellow, 
orange, or red cytoplasmic inclusions. The granular hemocytes and oenocytoids 
did not encorporate neutral red. The nuclei and cytoplasmic fragments from the 
lysing or already lysed granulocytes also did not stain. None of the hemocytes 
encorporated phenol red or congo red. The nuclei of lysing and fully disintegrated 



2 ( )() JACK i i >LVARD JONES 

granular heniocues stained pale rose with eosin Y but the cytoplasm and granules 
did not stain. Other types of hemocytes did not definitely eucorporate the eosin. 
With Janus green B. the granules within intact granular hemocytes quickly became 
a pale but distinct slate blue; those nuclei which were ejected from lysing granulo- 
cytes did not stain. Many vacuolated plasmatocytes had a dull slate-blue cast to 
them but the vacuoles did not stain. In some plasmatocytes brilliant sky-blue inclu- 
sions were visible. Other hemocyte tvpes did not stain siipravitally with Janus 
green B. 






32. 

31 




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- * * ''(I) J 

-. e. 

Vl. -> . -* ,, ': 



t ~*^af i 

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34 



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37 






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33 '. 



PLATE II 

FIGURES .U-^4. Plasmatocytes which lia\'e each (.n.ynlfccl a .sin.iilr granular hemocyte. 
Fici'KKs 35- >V>. I-ar.yi- granulocytophagous cells which have ciigulU-d two or more yranulur 
liemoryti-.- and/or their nuclei Note the very extensive pseudopodia in leisures 37-39. 

4. Observations on ji.\-cd and stained smears 

In thin and thick, air-dried smears of hemolvinph obtained from uulixed or heal- 
lixed Rhodnius and subse(|uently treated with ethanol. methanol or Caruoy prior to 
staining with Giemsa or Wright stain or with Harris hematoxylin and eosin, the 
granular hemocytes generally could not be readily recognixed, and none of the hemo- 
cytes appeared sharply acidophilic. In those granulocytes which were identifiable. 
the round inclusions did not stain but appeared as sharply outlined circular bodies, 
often \\ith a faint yellowish casl against a pale blue or faint grey cytoplasm. 

When the hemolymph was fixed with alcoholic Bouin and stained with eosin 



HEMOCYTES OF RHODNIUS 

azur-II, those granulocytes which could be recognized had faintly pink- 
cytoplasm with colorless granules. The prohemocytes were varying shades 
blue with faint red-purple nuclei. The oenocytoids were basophilic around the 
nucleus but the abrupt spindle ends of these cells were often distinctly eosinophilic 
In some oenocytoids a fine purple-staining meshwork could be seen. 

In general, regardless of the fixative employed, the different types of hemocyte: 
did not stain sharply with vertebrate blood stains and were often very difficult to 
distinguish. Even the best stained smears were notably inferior to the examina- 
tion of fresh unstained cells with phase contrast microscopy. 

It is important to note, however, that Wigglesworth (1955) observed that the 
adherent granular hemocytes of Rhodnins were acidophilic after fixation with Car- 
noy or Bouin and staining with either Alasson trichrome, hematoxylin and eosin, or 
with Prenant's ferric trihematin. In osmium-ethyl gallate preparations, Wiggles- 
worth (personal communication ) observed that the inclusions of the granular hemo- 
cytes appeared as colorless spheres or vesicles. In ordinary stained preparations, 
he found the granular hemocytes to be conspicuous for their lack of granularity. 

DISCUSSION 

The terms used here for three of the hemocytes of Rliudnius present no problem 
relative to the terms used by Wigglesworth (1933, 1955) for the same cells; thus, 
the prohemocytes are equivalent to his proleucocytes, plasmatocytes are the same as 
his phagocytes or amebocytes, and the adipohemocytes are comparable to his adipo- 
leucocytes or lipocytes. A complex problem arises, however, with the changes in 
the names of two clear-cut types of hemocytes: (1 ) the cells which are here termed 
the granular Iicinocytcs of Rhodnins are referred to by Wigglesworth as oenocytoids 
and possibly also as large granular cells, and (2) the cells which are here termed 
oenocytoids are referred to by Wigglesworth as large non-granular spindle cells 
and as non-phagocytic giant hcuioc\tcs | Wigglesworth (personal communication) 
considers these to be variant forms of the plasmatocyte] . 

Wigglesworth (1933) considered the granular hemocytes to be comparable to 
the oenocytoids of Poyarkoff (1910) because (1) when fixed, the cytoplasm ap- 
peared homogeneous and stained with eosin, and (2) they were specifically phago- 
cytized by certain other hemocytes. Nevertheless, the cells here termed granular 
hemucytcs of Rhodniits differ strikingly from the oenocytoids of most other insects 
in that (1) the nucleus in intact cells is centrally located whereas in most oeno- 
cytoids the nucleus is characteristically excentric (Hollande. 1909, 1911 ; Poyarkoff, 
1910; Bogojavlensky, 1932; Rooseboom, 1937; Yeager. 1945; Jones, 1962). (2) 
The granular hemocyte of R/wdniits is never a binucleated cell whereas oenocytoids 
may have two nuclei (Hollande, 1911 ; Bogojavlensky, 1932). (3) The cytoplasm 
of the granular hemocyte is not dense or elaborately textured whereas in most oeno- 
cytoids the cytoplasm is dense and contains canaliculi, threads or crystals (Bogo- 
javlensky, 1932; Yeager, 1945; Nittono, 1960; Jones, 1962). (4) The granular 
hemocytes of Rhodniits stain with eosin in certain preparations (Wigglesworth, 
1933), whereas the oenocytoids of many other insects are generally amphophilic or 
basophilic (Bogojavlensky, 1932; Yeager, 1945; Nittono, 1960; Jones, 1962), 
and at best are faintly eosinophilic (Yeager, 1945). (5) The granular hemocytes 



2 n 2 JACK CO! \ AtfD JONES 

of A'liodiiins are often vcr\ numi i the hemolymph (they can make up 30 f ',', to 

70% of the cells encountered in ; ; ', icnrial counts), whereas the oenocytoids have 
not been reported to rise ev< 10% level (Yeager, 1945; Jones, 1950; Nit- 

tono, 19(>() i. (6) Tlie gra-; ar lieuiocytes of Rhodnius do not pick up neutral red 
and thus differ from the & of several insects which are said to encorporate 

this dve ; oenocytes an -aid to stain supravitally with methylene blue, trypan 

blue, and Bismarck bro i Ilollande. 1914; Poisson, 1924; Bogojavlensky, 1932; 
Koch. 1945; Ocli.se. . (7) The granular hemocytes of Rhodnius do not 

possess the bizarre shapes of those oenocytes of Rhodnius illustrated by YYiggle.s- 
worth ( 1953. his Fi 239 g, h. and ki. nor do granular hemocytes possess the 
spindle-shaped clefts or needle-like crystals which some of the oenocytes have 
1 \\ iggles\\ nrtli's Kig. 239 e. f, h and i ). It is important to mention, however, that 
Wigglesworth (personal communication) believes the granular hemocytes resemble 
certain small oenocytes in stained preparations of Rhodnius (see, for example, his 
Fig. 239 c and d). " 

\Yith phase contrast microscopy, the granular hemocytes of Rhodnius resemble 
the non-phagocytic granulated blood cells of many other insects (Poisson, 1924, see 
his Fig. 21? Bogojavlensky, 1932; Millara. 1947; Jones, 1959 and unpublished i . 
The granular hemocytes of Rliodnins do not closely resemble the oenocytoids of 
Mysia (Ilollande, 1909). Melolontlm (Hollande, 1911), Psylliodes (Hollande, 
1911 i. Galleria (. Metalnikov and Gaschen. 1922). Notonccta (Poisson, 1924). Cal- 
H[>honi (Rooseboom. 1937), Prodenia (Yeager, 1945). Ephcstia (Arnold, 1952), 
Tenebrio (Jones, 1954). Drosophihi (Rizki and Rizki. l t 59 < their "crystal cells"). 
Uninhy.r ( Xittono. 1960). or GalcruccUa (Jones, unpublished). Wigglesworth 
( 1933 i stated clearly that the cells which we term gninuhir hemocytes have no con- 
nection with the oenocytes of RJwdnius. 

The cells which are termed the oenocytoids of Rhodnius ('and which Wiggles- 
worth considers to be a variety of plasmatocyte) have the following characteristics 
in common with the oenocytoids and/or oenocytes of many other insects: ( 1 ) they 
are generally large, thick, often quite bizarrely-shaped cells, (2) they have one or 
sometimes two nuclei with conspicuous nticleoli and a dense, often complexly tex- 
tured cytoplasm. (3) they may occur in discrete clusters, and (4) they are not 
numerous in the circulating hemolymph. They differ from stationary oenocytes 
most conspicuously in not being eosinophilic cells. Whether the cells here identified 
MS oenocytoids are related to or are derived from the stationary oenocytes of Rhod- 
nius is not known. For a long time it has been claimed that oenocytes are capable 
of budding off certain hcmocyte.s ( Koschevnikov, 1900; Kollman, 1908; Hufnagel. 
1918; f'oisson. 1 ( >.24 i hut generally the investigators do not clearly distinguish be- 
\veen small oenocytes. large plasmatocytes, granular hemocytes. or oenocytoids. 

Several of the hcmocvte> of Khadnius might be secretory (Y.//., plasmatocx tes. 

nular hemocytes, and oenocytoids). Various cytological criteria are needed be- 
' can accurately assess the situation. Total and differential hemocvte counts 

KJuxInhts will be presented in subsequent papers. 

/ork was supported by Grant HE 05193 and Award 1 K-3-GM-21,529 

from th National 'Institutes of Health. Bethesda. Maryland. Scientific Article Xo. 
01 no. 3655 of the ^Faryland Agricultural Kxpenment Station. 



HEMOCYTES OF RHODNIUS 

I am indebted to Mrs. Daisy P. Liu for much help with some of the initi; 
vations and for rearing the insects. I am particularly grateful to Sir ' 
worth for reading the initial manuscript and for his stimulating comments. 

SUMMARY 

1. The hemocytes of R/iodniits f^rolLrns have been studied with phase contrast 
microscopy, after supravital staining, and in fixed and stained smears. 

2. With phase contrast microscopy, the following categories of circulating cells 
can be readily identified: (a) non-dividing and mitotically-dividing prohemocytes, 
(b) non-vacuolated and vacuolated plasmatocytes, (c) intact and quickly lysing 
granular hemocytes, (d) oenocytoids with and without special cytoplasmic inclu- 
sions, (e) adipohemocytes, and "fat body cells, and (f ) granulocytophagous cells. 

3. This classification and terminology are compared with those of Wigglesworth. 
It is suggested that the cell which Wigglesworth terms an oenocytoid is more com- 
parable to the granulated blood cells of other insects and may be referred to as a 
granular hemocyte. It is suggested that the cells which Wigglesworth refers to as 
large non-granular spindle cells and nnn-phagocytic giant hemocytes are comparable 
to the oenocytoids of other insects. 

4. Yacuolation of plasmatocytes can be prevented by heat-fixing fed Rhodnius. 
Lysis of granulocytes can be prevented by collecting hemolymph into 0.75 r r 
Versene. 

5. Attempts to correlate an increase in sizes of circulating plasmatocytes with 
secretion of the thoracic gland hormone in fourth and fifth stage nymphs were not 
successful because of the great variability in the sizes of these cells. 

6. Since most circulating plasmatocytes in differential hemocyte counts of un- 
fixed fourth stage nymphs were identified as the vacuolated type, no correlation was 
possible between their vacuolation and the secretion of the thoracic gland hormone. 

7. In unfed fifth stage nymphs, most of the circulating plasmatocytes were classi- 
fied as vacuolated cells. Between the first and second days after the nymphs took 
a blood meal, the percentages of plasmatocytes identified as vacuolated cells abruptly 
decreased and steadily declined during the rest of the stadium. 

LITERATURE CITED 

ARNOLD, J. W., 1952. The haemocytes of the mediterranean flour moth, Ephcstia kuhniclLi Zell. 

(Lepidoptera: Pyralididae). Canad. J. ZooL, 30: 352-364. 
BOGOJAVLENSKY, K. S., 1932. [The formed elements of the blood of insects.] Arch. Rnss. 

Auat.. Hist, ct Einhryiil.. Lcnini/nid. 11: 361-386 (in Russian). 
HOLLANDS, A. C., 1909. Contribution a 1'etude du sang des Coleopteres. Arch. ZooL c.r/vr. cf 

gen. (5 ser.), 2: 271-294. 
HOLLANDS, A. C., 1911. fitude histolngique comparee du sang- des insectes a hemorrhee et des 

insectes sans hemorrhee. Arch. ZooL c.vper. ct ijcn. (5 ser.), 6: 283-323. 
HOLLANDK, A. C., 1914. Les cerodecytes ou "oenocytes" des insectes considered an point do vue 

biochemique. Arch. Anat. Micros., 16: 1-66. 
HUFNAGEL, A., 1918. Recherches histologique sur la metamorphose d'un Lepidoptere (Hyfo- 

noincnta padella L.). Arch Zool. exper. et gen., 57 : 47-202. 
JONES, J. C., 1950. The normal hemocyte picture of the yellow nu'iiKvurm, Tcnchrio inolitor 

Linnaeus. loiva State Coll. J . ScL, 24 : 355-361. 
JONES, J. C., 1954. A study of mealworm hemocytes with phase contrast microscopy. Ann. 

Ent. Soc. Aincr., 47 : 308-315. 



.1 \CK COLVARD JONES 

JUNES, J. C., 1959. A phase contrast .study of the blood-cells in I'mdcnin cridania (Order 

Lepidoptera). Quart. J. Wicr. Sci., 100: 17-23. 

JONES, J. C, 1962. Current concepts ning insect hemucytes. .liner. 7,ool., 2: 209-249. 

KIHH. J., 1945. Die Oenocyten von Drosnpliilii mclanotjaster. Rcr. Snissc Zool., 52: 415-420. 
KOI. i. MAX, M., 1908. Recherches sui les leucocytes et le tissue lyni|)lioidc des invcrtebres. Ann. 

Soc. Nat. Zool, 9 : 1-238. 
ECos< IIEVXIKOV, G. A., 1900. Vcbet den Fettkorper und die Oenocyten der Honigbiene (Apis 

mcllijcra L.). Zool. , in;., 23: 337-353. 
MKTAI.XIKOV. S.. AND H. GASCIIEN, 1922. Immunite cellulaire et luiniorale cliez la chenille. 

Ann. Inst. I'astcur, 36: 231-252. 
MILLARA, P., 1947. Contribution a 1'etude cytologique et physiologique des leucocytes d'insectes. 

Hull. Soc. France et !<cl<n,,uc (Paris), 81: 129-153. 
XITTOXO, V., 1960. Studies on the blood cells in the silkworm, Boinhy.r inori L. Bull. Scricnlt. 

/:.r/\ Stut. ( Tokyo), 16 (4) : 171-266 (in Japanese, with English summary). 
OCHSE, \Y., 1946. Untersuchungen iiber die Oenocyten von Sialis Intaria L. Rcr. Sitisse 

Zool, 53: W-71. 
POISSON, R., 1924. Contribution a 1'etude des Hemipteres aquatiques. Bull. Biol. France ct 

Bchjiquc, 58 : 49-305. 
Pi'VAKKOKF, E., 1910. Recherches histologiques sur la metamorphose d'un Coleoptere (la 

galerque de Tonne). Arch. Aunt. Micros.. 12: 333-474. 
RIZKT, M. T. M.. ANT) R. M. RI/KI, 1959. Functional significance of the crystal cells in the 

larva of Drnsophilii iiiclan/n/astcr. J. Binfhys. Cytol., 5: 235-240. 
ROOSEBOOM, M., 1937. Contribution a 1'etude de la cytologie du sang de certains insectes, avec 

quelques considerations generales. Arch. Nccrl. Zool.. 2: 432-559. 
\\'ir.c,i,Ks\voKTn, V. B., 1933. The physiology of the cuticle and of ecdysis in Rhodniits proli.vnx 

(Triatomidae, Hemiptera) ; with special reference to the function of the oenocytes and 

of the dermal glands. Quart. J. Micr. Sci., 76: 269-318. 
\YIGM.ESWURTH, V. B., 1953. The Principles of Insect Physiology. E. P. Button, New York, 

546 pp. 
YYiGGLESWORTii, Y. B.. 1 U 55. The role of the haeniocytes in the growth and moulting of an 

insect. Kluxhiins proli.vus (Hemiptera). /. E.vp. Biol., 32: 649-663. 
WIGGLES WORTH, V. B., 1956a. The function of the amoebocytes during moulting in Rhodiiius. 

Ann. Sci. Nat. Zool., 18: 139-144. 
\VIGGLESWORTII, V. B., 1956b. The haemocytes and connective tissue formation in an insect, 

Rhodnins proli.vus (Hemiptera). Quart. J. Micr. Sci., 97: 87-98. 
YKAGKR, J. F., 1945. The blood picture of the southern armyworm (Prodcnia cridania). J 

Auric. Rex., 71 : 1-40. 



EVIDENCE FOR TRANSAMINASE ACTIVITY IN THE SLIME MOLD, 
DICTYOSTELIUM DISCOIDEUM RAPER * 

JEROME O. KRIVANEK AND ROBIN C. KRIVANEK 
Department of Zoology, University of South Florida, Tampa, Florida 33620 

As the developmental cycle of the slime mold, Dictyostelium discoidcmn Raper, 
proceeds from the myxamoeba stage to the mature sorocarp stage, there occurs a 
reduction in proteinaceous materials and an increase in polysaccharide carbohydrate, 
as shown by Gregg, Hackney and Krivanek (1954) and Gregg and Bronsweig 
(1956). On the basis of these quantitative studies, Gregg and his associates sug- 
gested that the protein components may serve as precursors not only for energy- 
source intermediates of development, but also for the synthesis of carbohydrate nec- 
essary for stalk formation. Attempting to define the metabolic mechanisms respon- 
sible for this inverse relationship, Krivanek and Krivanek (1959) chromatographi- 
cally analyzed the amino acid components of the slime mold in both hydrolyzed and 
unhydrolyzed tissue. Their findings suggested that deamination may be one 
process relating protein degradation to carbohydrate synthesis. These authors did 
not exclude the possibility, however, that other metabolic mechanisms, e.g., trans- 
amination, may be instrumental in this linkage. 

Utilizing a spectrophotometric technique to observe the change in the charac- 
teristic absorption band of DPNH at 340 m/x, Wright and Anderson (1959) demon- 
strated the occurrence of "aspartic-pyruvic transaminase." However, these au- 
thors, as well as others (Meister, 1950; Aspen and Meister, 1958), have expressed 
the lack of definity of such an analytical technique because of its broad specificity. 

In view of the important role which transaminase activity plays in relating pro- 
tein and carbohydrate metabolism, more precise evidence than that thus far pre- 
sented would seem desirable. It is therefore the intent of this paper to demonstrate 
that specific transaminase activities are indeed operative in the slime mold, Dictyo- 
stclhnn discoidcum. 

MATERIALS AND METHODS 

D. discoidciiin was cultured in the manner described by Bonner (1947), using 
Escherichia coli as the bacterial associate. 

The following two transamination reactions were studied : 

Reaction I : glutamate + pyruvate a-ketoglutarate + 1-alanine 
Reaction II : glutamate + oxaloacetate a-ketoglutarate -f 1-aspartate 

The primary technique employed in studying these reactions was that of progressive 
chromatography as described by Hird and Roswell (1950). 

1 This research supported by Grants G-1908 and G-14422 from the National Science 
Foundation. 

295 



296 J. (). Kkl\. \.\TK AND R. C. KKIVANKK 

I iidi\ idnals in the desired stag* i \eiopment were harvested from the Petri 

j)lates and homogenized in ice-cole >>phate buffer, pi [ 7.4, using an homogenizer 
of the type described by Gregg . 54). The final volume of the homogenate 

was 5 nil. The extent of /ation was determined by microscopic inspec- 

tion t the In miogenatc. 

After centrifugal ion. o1 :omogenate. the supernatant or soluble fraction (S) 

was withdrawn from tl culate centrifugate or insoluble fraction (I). Hoth 

fractions were then madf up to the original volume of 5 ml. 

One-mi, samples oi .raction were then transferred to five separate reaction 

tubes live tubes for each fraction series. To the control tube in each series was 
added 1 ml. of phosphate buller only. To each of the remaining four tubes in each 
series were added ., ml. glutamate and \ ml. oxaloacetate or pyruvate, depending 
upon the reaction under consideration. The concentration of the glutamate, 
oxaloacetate and pyruvate varied and will be discussed under Results. To this 
point all step- were carried out under ice-cold conditions. 

It is conceivable that 1-alanine may be formed from pyruvate. 1-aspartate from 
oxaloacetate, and a-ketoglutarate from glutamate by reactions other than trans- 
amination. Therefore, supplementary controls were utilized to determine whether 
the appearance and disappearance of the appropriate substrates were mutually inter- 
dependent. In this particular control series, only one of the initial reactants, i.e., 
glutamate, was added to the reaction tubes, with subsequent treatment of these 
controls being the same as for the phosphate buffer controls and the experimental 
series. 

Immediately upon the addition of the last substrate, the reaction tubes were put 
into a closed anaerobic environment, consisting of gaseous nitrogen and pyrogallol. 
and the mixtures were allowed to react at a temperature of 37 C. Reaction tubes 
from both soluble and insoluble series were generally removed after 30, 120, 180 
and 240 minutes and processed. Control tubes were processed in the same man- 
ner and for the maximum time interval. 

At the end of each incubation period, to each of the tubes were added two 
volumes of warm ethanol (60-70 C.) to precipitate the proteins. After centrifu- 
gal ion, the supernatant fluid was withdrawn and dried //; raciio at room tempera- 
ture. The residue after evaporation was then resuspendecl in 1 ml. of distilled 
water, and identical fractions from each preparation were spotted on Whatman No. 
1 filter paper for a uni-directional chromatographic separation of the amino acids. 
Among the various solvents used were: propanol-water (80:20), n-butanol-acetic 
acid-water (250:60:250), n-butanol-acetone- water (10:10:5), and n-butanol-ace- 
tone-water (5:4:1 ). Development of the spots was accomplished by means of spray- 

'he chromatograms with a solution of 0.3% ninhydrin in 95% ethanol. Identifi- 
cation of the unknown spots was determined by means of positional comparisons 
-'ecu the unknown spots and spots nf known amino acids applied to the same 

pol determination of the alpha-keto acids (oxaloacetate, alpha-ketoglutarate 
ruvate) was done by .separating them as their 2,4-dinitrophenylhydrazones 
- method described by Block, Durrum and Zweig (1 ( >58). To sam- 
ple- of reaction mixtures, after deproteinization with warm ethanol, was 
added 1 ml. < J.I dinitrophenylhydrazine dissolved in 6 N HC1. After 30 



TRANSAMINATION IN DICTYOSTEl.il M 

minutes' standing at room temperature, the hydrazones were extracted in 
tory funnel with three 7|-ml. washes of a chloroform : ethanol (80:20) solul- 
The hydrazones. now in the latter solution, were then extracted with 7\ ml. of '. 
Na 2 CO 3 . After washing the hydrazone-containing Na 2 CO 3 solution with 5 ml. o 
chloroform-ethanol solution, the Na 2 CO.,, solution was then acidified with 2\ ml 
6 N HC1 in the cold. The resultant acidified Na 2 CO : . solution was further washc-d 
with three portions of the chloroform: ethanol solution totaling 10 ml. The 10 ml. 
of washings were then evaporated under a gentle air stream. 

For a chromatographic separation of the 2,4-dinitrophenylhydrazones, the resi- 
due after evaporation was dissolved in 2 ml. absolute ethanol and spotted on What- 
man No. 1 filter paper in !-/*!. amounts. Identification of the spots was determined 
by preparing samples of known alpha-keto acids, processing them in the same man- 
ner as the experimental series and spotting them on the same paper with the 
experimental s. 

Detection of the hydrazones of the keto acids was accomplished by initially in- 
specting the chromatogram for yellow spots (distinctive of hydrazones), then spray- 
ing the paper with a 2% ethanolic KOH solution which imparts a red-brown color 
to the spots, and/or scanning the paper with a UV light which caused the spots to 
fluoresce. 

Since the paper chromatographic method did not afford a clear separation be- 
tween glutamate and aspartate, a paper electrophoresis procedure was utilized 
(Block, Durrum and Zweig, 1958). This method is specific in its separation of 
aspartate, glutamate, histidine, arginine, lysine, and the monoamino-monocarboxylic 
acids. A phthalate buffer, pH 5.9, was used in a Spinco paper electrophoresis appa- 
ratus usually run at 500 volts, 18 amperes for three hours. 

K'Ksri/rs AND DISCUSSION 
. /. Amino acids 

Figure 1 shows the chromatographic results of Reaction I, i.e., when tissue 
fractions, soluble and insoluble, were incubated in the presence of 1/40 i\I glu- 
tamate and 1/10 J\I pyruvate. It is to be noted that with lengthening periods of 
incubation (A is shortest, D is longest), color intensities of the glutamate spots 
decrease in both soluble and insoluble series. No corresponding spots were evi- 
dent in the control series which were incubated for four hours the maximum time 
for the experimental series. The appearance of alanine in the insoluble series lagged 
behind its appearance in the soluble series. Thus, after two hours' incubation 
alanine was first seen in the former series while only 30 minutes were necessary for 
it to appear in the soluble series. 

The results relative to Reaction II are seen in Figures 2 and 3. In Figure 2, 
the spots of aspartate and glutamate in the soluble series are in close spatial rela- 
tionship to each other, with aspartate trailing glutamate. The chromatographic 
technique did not clearly delineate the two compounds although development of the 
chromatogram with dicyclohexylamine did allow better interpretation than did nin- 
hydrin. It is to be noted that aspartic acid increased in intensity in the soluble 
series. A corresponding decrease in the intensity of glutamate was also evident. 
In the insoluble series, no aspartate was apparent in either the chromatographic or 



298 



T. O. KKIVAXKK AND R. C. KRIVANEK 



INCREASING 
INTENSITY 



o o 

DECREASING 
INTENSITY 



o 



o 







A B C D E 

s s s s s 



INCREASING 
INTENSITY 



DECREASING 
INTENSITY 



o 



A i B i C r D i E i 







GLU ALA 



FIGI RE 1. Exact reproduction of chromatogram showing amino acid results of Reaction I. 
Incubation times: A series, -1 hour; B series, 2 hours; C series, 3 hours; D series, 4 hours; 
K Aeries (phosphate control), 4 hours. "S" denotes soluble fraction, "I" denotes insoluble frac- 
tion, GLU glutamate known; ALA = alanine known. 



INCREASING 

INTENSITY 
x 

o D o 



*l^. 



INCREASING 
INTENSITY 

o o 



o, 



f\ ' ' ' \ 

'-' I / v/ 



' i 



B s C s D s E s A , B i C i D r E i 







ASP GLU ALA 



MI.' iix;n oduction of cbroniato^rani shoxvini; amino acid results of Reaction 

Incubation times same as in iM.uun- 1. ASP aspartate known. 



TRANSAMINATION IN DICTYOSTELIL'M 



299 



GLUTAMIC 



ASPARTIC 



oc-ALANINE 



FIGURE 3. Exact reproduction of electrophoretic pattern showing amino acid results of 
Reaction II. Length of run: 3 hours; voltage: 500; amperage: 18 amps. Veronal buffer of 
pH 8.6. Letter notations same as in Figure 1. 

electrophoretic determinations. It should he mentioned at this point that no de- 
crease in color intensity of glutamate was observed when 1/20 J\I concentration was 
used. When 1/40 ,17 glutamate was used, a perceptible decrease was evident. 
However, this decrease was less profound than in Reaction I. 

In addition to the expected aspartate product of transamination Reaction II. 
an unexpected product alanine was also present in both soluble and insoluble 
series. No alanine was detected in the control series. The appearance of alanine 
could be accounted for by way of oxaloacetic acid being decarboxylated to pyruvic 
acid, with transamination subsequently occurring to form alanine. Such a trans- 
formation could be mediated only through the action of a decarboxvlnse. 

The chromatographic separation of alanine, aspartic acid, and glutamate was 
supplemented by an electrophoretic separation. Using the electrophoretic technique 
previously described for the separation of monoamino-monocarboxylic amino acids, 
complete validation of the chromatographic results was accomplished as shown in 
Figure 3. A decrease in glutamate intensity and increase in alanine and aspartic 
acid intensities were noted. 

B. Kcto-acids 

The keto-acid determinations essentially follow expectation if transamination is. 
in fact, operative in the slime mold. 



W .1. ( ). KRIVANEK AND R. C. KRIVAXKK 

\\hen tissue extracts were incubated in 1/40 .17 glutamatc and I/ 10 .17 oxalo- 
acelic acid, Reaction II, tin resul shown in Figure 4 were achieved. Alpha 

ketoglutarate, one of the end pru i the reaction; is seen to increase in intensitx 

with increasing tinu- of incuh: of the tissue. The decree of color intensity of 

the soluble series reniaiiu ^lier than that in the insoluble series. However, 

oxaloacctate. one of tin ' reactants, did not display any reduction in intensity 

as might have been exp< Since our method did not distinguish between pyru- 

vate and oxaloacetate ! s i^nre 4), pyruvate may have been generated during the 
course of the reaction would again not be unreasonable in view of the previ- 

ouslv stated possibil!i\ that oxaloacetate may be decarboxylated to yield pyruvate 
in the slime mold. 

Various concentrations of oxaloacetate were used in addition to the 1/10 il/ con- 
centration. When a lower concentration (1/20 .17) was used, no spot was evident 
at the 1 oxaloacetate locus chromatographically. In addition, the enzymatic conver- 
sion of this substrate took place rapidly, for alpha-ketoglutarate appeared after only 
15 minutes of incubation. Increasing the concentration of oxaloacetate to 1/5 .!/ 
seemed to have an inhibitory effect on the reaction, as indicated by (1) large spots 
at the oxaloacetic acid locus, (J) extremely small ketoglutarate spots, and (3) no 
alanine or aspartic acid being formed. ( hi the basis of these test concentrations. 
1/10 .17 oxaloacetate was chosen as being the optimal concentration. 

Figure 5 shows the results of incubating homogenized tissue with 1/40 M glu- 
tamic acid and 1/10 .17 pyruvate. Here, as in the case of Reaction II, alpha- 
ketoglutarate increased in color intensity with increasing lengths of incubation time-. 








008 



INCREASING INCREASING 

INTENSITY INTENSITY 



O C? 



B s C s D s E s A t B T Cj D, E,. oC-KETO OXAL PYR MESO 

GLUT ACET 



\. I ; .\,K t ic]ii (idiu-tinn of chromatogram shoxvins koto-arid n-Milts of Reaction II. 

on tiii]i'> sanu- as in Figure 1. a-KKT( )( il.L"!' = a-ketoglutarate known: ()X.\I..\CET 

! kiio\\n; \'\\\ -- pyruvic acid known; MI-'.SO mcsoxalic acid known. 

Meso .u'id known was spotted 1o aid idrnlit'ication of nnknoun spots. Note lori of nn- 

kn< iv noin) -, of -pot application. 



TRANSAMINATION IN DICTYOSTELIUM 301 





oooo o 



INCREASING INCREASING 

INTENSITY INTENSITY 



d tf & o 



o 



A, B. C D. E. A, B T C D, E, OC-KETO PYRUV 



S S S S S I T I I I 



GLUT 



FIGURE 5. Exact reproduction of chromatogram showing keto-acid results of Reaction I. 
Incubation times same as in Figure 1. Abbreviations same as in Figure 4. 

With pyruvate, as with oxaloacetate at this concentration, no progressive diminution 
in color intensity occurred with increasing lengths of incubating times. Similar 
reasoning and possible concentration effects can be applied to this event as were 
applied to the oxaloacetate results. 

Brief mention should be made of two unidentified spots evident only in Reaction 
II experiments. Their possible importance lies in the fact that they occurred only 
in the experimental series and were not evident in the controls. The locus of "spot 
A," when present, was invariably midway between the point of origin (point of ap- 
plication of the test solution on chromatogram) and the alpha-ketoglutarate locus. 
Although every attempt was made to reproduce exact conditions between each run, 
"spot A" was not always detected, occurring more times than not. It appeared 
when using 1/20 M and 1/10 M concentrations of oxaloacetic acid. 

The second unidentified spot, "spot B," was consistently present, its locus being 
at or slightly above the points of application of the experimental samples on the 
chromatogram (see Figure 4). The various concentrations of oxaloacetic acid did 
not affect its appearance. Not only was it present in all test series, but there was 
also a tendency for it to increase in color intensity with increasing incubation times. 
Its absence from the controls and from the point of application of a known oxalo- 
acetic acid solution is to be especially noted. The significance, if any, of unidenti- 
fied "spots A and B" is at present not apparent. 

SUMMARY 

Progressive chromatography and paper electrophoresis techniques have demon- 
strated qualitatively the occurrence of two transaminating systems in the slime mold, 
Dictyostelium discoideinn. These systems are glutamic-aspartic (or glutamic- 



302 j. o. KRIVANEK: AND R. C. KRIVANEK 

oxaloacetic) transaminase and glutamic-alanine (or glutamic-pyruvic) transaminase. 
However, in experiments designed to demonstrate the glutamic-aspartic trans- 
aminase, alanine was also produced, indicating the presence of an oxaloacetic-pyruvic 
decarboxylase. The evidence for transaminases confirms the existence of pathways 
for the conversion of protein to carbohydrate in the slime mold. 

LITERATURE CITED 

ASPEN, A. J., AND A. MEISTER, 1958. Determination of transaminase. In: Methods of Bio- 
chemical Analysis, Vol. VI. Interscience Publishers, New York. Pp. 131-161. 
BLOCK, R. J., E. L. DURRUM AND G. ZWEIG, 1958. A Manual of Paper Chromatography and 

Paper Electrophoresis. 2nd Edition. Academic Press, New York. 
BONNER, J. T., 1947. Evidence for the formation of cell aggregates by chemotaxis in the 

development of slime mold Dictyostclium discoidcum. J. E.rp. Zoo/., 106: 1-26. 
GREGG, J. H., AND R. D. BRONSWEIG, 1956. Biochemical events accompanying stalk formation 

in the slime mold, Dictyostclium discoidcum. J. Cell Comf>. Physiol., 48: 293-300. 
GREGG, J. H., A. L. HACKNEY AND J. O. KRIVANEK, 1954. Nitrogen metabolism of the slime 

mold Dictyostclium discoideum during growth and morphogenesis. Biol. Bull., 107: 

226-235. 
HIRD, F. J. R., AND E. V. ROSVVELL, 1950. Additional transaminations by insoluble particle 

preparations of rat liver. Nature, 166: 517-518. 
KRIVANEK, J. O., AND R. C. KRIVANEK, 1959. Chromatographic analyses of amino acids in the 

developing slime mold, Dictyostelium discoidewn Raper. Biol. Bull., 116: 265-271. 
MEISTER, A., 1950. Reduction of a, 7-diketo and a-ketoacids catalyzed by muscle preparations 

and by crystalline lactic dehydrogenase. /. Biol. Chcm., 184: 117-129. 
WRIGHT, B. E., AND M. L. ANDERSON, 1959. Biochemical differentiation in the slime mold. 

Biochim. Binphys. Acta, 31: 310-322. 



THE OVARY AND ANAL PROCESSES OF "CHARACODON" 

EISENI, A VIVIPAROUS CYPRINODONT 

TELEOST FROM MEXICO 1 

GUILLERMO MENDOZA 

Biology Department, Grinnell College, Grinnell, lozva 

Early classifications of the Mexican fishes of the family Goodeidae, such as those 
of Jordan and Evermann (1896-1900), Meek (1902, 1904), Regan (1906-1908) 
and Hubbs (1924, 1926), were based largely on characteristics concerned with the 
type of jaws, teeth, length of intestine, etc. Actually, many of the species were 
placed in genera now included in entirely different families, such as the Poeciliidae. 
Meek recognized the natural relationships of the Goodeidae, using such criteria as 
(1) viviparity, (2) specialization of the anal fin, and (3) geographic distribution, 
although he continued to base his classification on the older criteria. 

In 1939 Hubbs and Turner revised the taxonomic structure of the goodeids, 
basing the new classification primarily on characteristics of the ovarian structure 
and the trophotaeniae, processes extending from the peri-anal region in the embryo 
and assumed to be used for respiratory and nutritive functions during gestation. 
The authors concluded that the ovarian and trophotaenial characters indicated the 
lines of phyletic relationships better than previously used taxonomic schemes. This 
new classification has been used by workers since 1939. However, De Buen pub- 
lished a key to the family (1942-1943) in which he used the Hubbs-Turner criteria 
to distinguish genera but reverted to the more usual characteristics to distinguish 
species. On the other hand, in his recent key to the fishes of Mexico, Alvarez 
(1950) used the customary taxonomic features but did not refer to the Hubbs- 
Turner criteria. 

Recently there has been some question about the validity and classification of 
"Characodon" eiscni Rutter, synonymized by Hubbs and Turner (1939) with 
Characodon variatus (= Xenotoca variata} of previous classifications (Meek, etc.). 
The need for a careful study of this species was suggested to me by Robert R. 
Miller of the University of Michigan. It was agreed that I would examine the 
ovary and trophotaeniae whereas Dr. Miller would reappraise the taxonomic position 
of the species on the basis of other characters. For various reasons, it has been 
decided that this portion of the study should be published now, to be followed later 
by Dr. Miller's taxonomic analysis. 

In the process of comparing the ovarian and trophotaenial characteristics of 
"Characodon" eiscni and Xenotoca variata, certain discrepancies in structure have 
become apparent to the writer: (1) there are serious differences in the ovarian and 
trophotaenial structures of the species described here, "Characodon" eiseni, and 
those of Xenotoca variata with which it has been synonymized by Hubbs and Turner 

1 This study was supported by Grants No. G16726 and GB2378 of the National Science 
Foundation. 

303 



304 



(iUILLER.MO MKNDOZA 







POSTERIOR 
MEDIAN 

PROCESS 




I mm 



LATERAL 
PROCESSES 

PLATE I 



ANTERIOR 
MEDIAN 
PROCESS 



OVARY AND PROCESSES OF C. EISENI 305 

(1939) ; (2) regardless of the identity of the species, lack of agreement between the 
ovarian and trophotaenial structures calls into question the applicability of the 
Hubbs-Turner criteria in this particular species. 

THE GOODEID OVARY AND TROPHOTAENIAE 

The ovary of the goodeid fishes is a single, hollow, spindle-shaped structure, con- 
tinuous posteriorly with the oviduct which in turn opens to the outside at the genital 
pore immediately behind the anus. The ovary is further divided into two lateral 
halves by a median vertical septum. The nature of the median septum is very 
important in the Hubbs-Turner classification scheme. The septum may be single, 
complex and attached at the mid-dorsal and mid-ventral lines as in Alloophorus 
robustus and Goodca luitpoldii (Plate I, Fig. 1) or it may be divided into dorsal 
and ventral halves as in Xenoophorus captivus and Neoophorus dlad (Turner, 1933 ; 
Hubbs and Turner, 1939). The halves may then be long or short and may be 
rolled in one lateral direction or the other ; other variations occur. A second im- 
portant characteristic of the ovary is the location of the ovigerous tissue. Eggs may 
be found in the walls of the ovary (e.g., Alloophorus robustus (Plate I, Fig. 1) ; in 
some species they may also be found in the septum (Goodca luitpoldii and others). 
In species such as Neotoca bilineata (Plate I, Fig. 2), the median septum is thin 
and bears no eggs ; germ cells are restricted to two lobulated folds that protrude into 
the ovarian lumen from the dorso-lateral walls of the ovary (Turner, 1933; Men- 
doza, 1940). 

In addition to these characteristics, the trophotaeniae were also used by the au- 
thors in the classification of the species. These trophotaeniae usually are exten- 
sions of the peri-anal lips and may occur in one of two basic forms ; they may have 
the form of a small flower or "rosette" as in Goodea luitpoldii (Plate II, Fig. 6), 
Neoophorus diasi and Allotoca dugcsii (Turner, 1937, Hubbs and Turner, 1939), 
or they may have the shape of a ribbon, the number of ribbons varying with the dif- 
ferent species. For example, Characodon lateral-is (Turner, 1937) and Hubbsina 
turneri (Mendoza, 1956) have only two posteriorly directed processes (Plate II, 
Fig. 8) ; Neotoca bilineata (Turner, 1937) and others have three processes in the 
form of a "trident" extending caudad (Plate II. Fig. 7), but Zoogoncticus cuit- 
seocnsis (Plate II, Fig. 11), on the other hand, has 10 to 12 processes (Turner, 
1937). Furthermore, the ribbon-shaped processes may be sheathed, in which case 
the epithelium of the process is separated from the central medulla by a space as in 
Neotoca bilineata and Skiffla Icnnac (Plate III, Figs. 12-13). In non-sheathed 

FIGURES 1-2. Diagrammatic transverse sections of two goodeid ovaries (from Hubbs and 
Turner, 1939) to show the basic structure of the ovary and the location of the ovigerous tissue. 
Eggs are shown in black. 

FIGURE 1. Alloophorus robustus. 

FIGURE 2. Neotoca bilineata. 

FIGURE 3. A section of an immature ovary of "Characodon" ciscni. showing a large number 
of eggs in the anterior region where the median septum is not well formed. 

FIGURE 4. A section of a mature post-partum ovary of "Characodon" ciscni. showing few 
eggs in the ovarian wall and a much-folded median septum. Figures 3 and 4 are tracings from 
photographs ; in these figures the ovarian lumen is stippled. 

FIGURE 5. The trophotaeniae of "Characodon" ciscni. The sheathed nature of the processes 
shows clearly. The drawing is a tracing of a photograph. 



306 



GLJILLKRMO MENDOZA 



8 




PLATE II 

FIGI-RKS 6-11. Representative types of trophotaeniae from different oodi-id Buries. All 
figures except 9 arc taken from Hubbs and Turner (1939). 
FIGURE 6. Goodca hiitpoldii. 
FIGURE 7. Neotoca bilineata. 
FTCURK 8. Characodon latcralis. 



OVARY AND PROCESSES OF C. EISENI 307 

processes found in species such as Alloophorus robustus (Plate III, Fig. 14), 
Zoogoncticus cuitzeocnsis, etc., the epithelium is immediately adjacent to the central 
core; there is no subepithelial space (Turner, 1937; Hubbs and Turner, 1939). 

MATERIALS AND METHODS 

All specimens used in this study, living and preserved, were obtained from 
Robert R. Miller, Curator of Fishes, Museum of Zoology, University of Michigan. 
The material examined came from the Manantial "El Sacristan" at Tepic, Nayarit, 
near the type locality for Rutter's species. The writer expresses his gratitude to 
Dr. Miller for the specimens, for the suggestion that this study be made and for 
valuable suggestions made during the writing of this manuscript. 

The description of the ovary in the present paper is based on a study of approxi- 
mately 75 gonads. Over 40 ovaries from preserved specimens were studied in toto ; 
the others were sectioned, stained by standard techniques and examined microscopi- 
cally. The nature of the median septum is best analyzed in a whole gonad by re- 
moval of the embryos and by examination of the entire organ under a dissecting 
microscope. Analysis of the septal structure solely from microscopic sections would 
be very tedious at best and probably very unreliable. For the study of the proc- 
esses, at least 200 embryos were examined, ranging from neural tube stages to speci- 
mens ready for birth (13-14 mm.) ; observations were made on living, preserved 
and sectioned specimens. Since the processes undergo serious changes just prior 
to birth, it is imperative that description of the processes be based on embryos less 
than maximum size. 

OVARY 
Gross structure 

The ovary is a spindle-shaped organ attached by a strong band of connective 
tissue to the anterior wall of the body cavity. Two median mesenteries further sup- 
port the ovary ; one is a very short membrane to the pigmented roof of the coelom, 
the other is a long mesentery to the posterior section of the gut. 

In a mature female the resting ovary normally measures 2-3 mm. in diameter; 
the length of the combined ovary and oviduct varies from 10-30 mm., depending 
on the size of the female. A gonad with developing embryos varies according to 
the size of the female and the age and number of the contained young. A repre- 
sentative measurement of an ovary of a 55-mm. female with embryos 11-13 mm. 
long is 20 X 10 mm. (length by diameter). 

The ovary is a typical goodeid ovary ; it has a muscular wall, a central lumen 
and a median septum (Plate I, Figs. 3-4). The gonad is divided approximately 
into equal halves by a much-folded longitudinal, median septum This membrane 
is quite variable in its structure for it may be complete, only partially complete or 
fully divided into dorsal and ventral halves. A complete septum is one that ex- 
tends the length of the ovary as a single, continuous sheet. However, the septum 

FIGURE 9. "Characodon" ciscni. This is a semi-diagrammatic, ventral view of the processes 
in Figure 5. 

FIGURE 10. Xenotoca variata. 
FIGURE 11. Zoogoncticus cuitseoensis. 



GUILLEKMO MENDOZA 




process epithelium 
sheath space 




medulla 



12 




0.1 mm 



13 




I'l.ATE III 




14 



FIGURES 12-14. Sections of trophotaeniae of three goodeid species. All drawings are 
tracings from micropr ejections. 

FIGURE 12. Ncntoca bilmcata (11 mm.). Note the delicate epithelium and the generoti> 
sheath space around the medulla. 

FIGUUK 13. Skiffia Icnuae (6 mm.). Sheathed processes similar to those of Neatnc,! 
bilineata. 

FIGURE 14. .!// Chorus ri>t>ustus (10 mm.). The process epithelium is thick; the sheath 
space is absent. 



OVARY AND PROCESSES OF C. EISENI 309 

may be complete but perforated by one or more openings of various sizes, usually 
at the posterior end. If partially complete, the septum is normally intact in the 
anterior region but is divided into dorsal and ventral halves in the posterior region 
of the ovary. All possible gradations occur in the degree of completeness of the 
septum; as little as 25% or as much as 90% of the septum may be intact. If the 
septum is not complete, it is divided into dorsal and ventral components which may 
be approximately equal in size or markedly unequal. Among the 43 dissected 
specimens the following variations in the septum were found : 

TABLE I 

Structure of the median septum 



Number of ovaries 



Condition of median septum 



12 
13 

14 
4 



Complete; intact the full length of the ovary. 

Partially complete; some perforations and partial division into dorsal and 

ventral halves. 

Divided into two complete and equal halves. 
Divided into two complete but unequal halves. 



Despite variations, the total height of the septum is much greater than the diameter 
of the ovary, thereby throwing the septum into many folds. Side extensions or 
branches of this membrane are numerous. 

Ovigerous tissue occurs more often in certain locations but it is also quite varied 
in its distribution. Eggs invariably are found in the anterior half or third of the 
ovary although they may extend throughout most of the gonad in juvenile speci- 
mens. Eggs occur in the ovarian wall and in the septum but more often they are 
found in the anterior, ventral and lateral walls of the ovary. Eggs that occur in 
the septum are confined primarily to the ventral edge but they may occur anywhere 
along the septum (Plate I, Fig. 3). 

Histology 

Histologically, the mature ovary resembles other goodeid ovaries (Turner, 
1937; Hubbs and Turner, 1939; Mendoza, 1940, 1956). In a non-gravid ovary 
both the septum and the internal walls are extensively folded. The stroma of the 
gonad is formed of a delicate network of collagenous, mesenchyme-like connective 
tissue that contains the many eggs and, in mature ovaries, many large blood vessels. 
A large artery and vein follow a path along the mid-dorsal and mid-ventral lines of 
the gonad. embedded in the muscular wall. The internal epithelium is sqviamous 
or low cuboidal ; nuclei are large, rounded and vesicular. The epithelium evidently 
does not attain the elaborate structure found in Neotoca bilineata (Mendoza, 1940). 
A very extensive capillary plexus lies in a sub-epithelial position in the septum and 
in the internal ovarian wall ; the plexus is very conspicuous in the mature ovary 
but poorly developed in the immature gonad. Nests of early oogonia occur in the 
ovarian wall and the septum ; eggs attain a maximum size of 250-300 /*. The fol- 
licle that surrounds each egg is squamous in smaller eggs but columnar to compound 
in eggs of maximum size. A thin vascular connective tissue "theca" surrounds 



310 



GUILLERMO MENDOZA 



each follicle. A spongy or tumescent condition of the ovary occurs only in early 
stages of development ; in advanced stages of gestation the ovarian walls and septa 
are thin and collapsed. The muscular wall of the mature gonad is very thick and 
is formed of smooth muscle and connective tissue. In the ovary proper the muscle 
cells tend to run in a circular manner but there is much random orientation ; actual 
whorls of cells and longitudinally oriented cells occur at random in the muscular 
layer. A heavy layer of connective tissue borders the muscle layer on the external 
and internal surfaces ; connective tissue fibers also occur in the muscle layer. In 
the juvenile ovary the muscular wall is very thin. In the region of the oviduct, the 
smooth muscle cells in the wall are arranged in two orderly layers ; one is longi- 
tudinal, narrow and external in position, the other is circular, wide and internal. 

TROPHOTAENIAE 

There are four basic processes ; one is median in position and anterior to the 
anus ; the other three extend posteriorly ; two are lateral and one is median and 
posterior to the anus (Plate I, Fig. 5). Any one process may be modified, degener- 
ate or completely missing. Any process may be secondarily split, the point of bifur- 
cation occurring at a proximal or distal position along the process. Splitting is 
more likely to take place in one rather than in two or more processes at one time 
and, although splitting may be found in any process, the total number of ribbons 
seldom exceeds six. Sometimes two or even three of the processes arise from 
one common base. 

The anterior process is invariably short ; the posterior median process tends to be 
the longest but the lateral processes approach or may even exceed it in length. At 
the point of maximum development, one or more of the processes extend to the 
caudal fin and often extend beyond the tip. This size relationship is true for embryos 
at all lengths, 6 mm. or 13 mm. The following examples are illustrative of proc- 
esses in embryos 12-13 mm. long. The maximum length recorded for any process 
was 7.5 mm. in a 13-mm. embryo ready for birth. The maximum length is normally 
retained until time for birth although embryos frequently begin to resorb the 
processes even before birth ; some specimens just prior to birth have been observed 
with processes that extend only to the anal fin. Processes normally measure 0.3-0.4 
mm. in typical maximum width although some of 1.0 mm. have been observed in 
exceptional cases. At optimal development, processes appear turgid, smooth, trans- 
lucent ; as birth approaches, they become compact, less translucent and have a 
"furry" appearance. 

TAIH.I. 1 1 
tij anal processes in millimeters 



Specimen 


Anterior median 


Right latt-i.il 


I-ci't lateral 


Posterior median 


1 


1.5 


4.5 


5.0 


4.5 


2 


1.5 


5.5, 6.5 


5.5 


6.0, 5.0 


3 


1.5 


6.5 


6.0 


6.0, 7.0 


1 


2.5 


5.5 


6.0 


5.0 


5 


1.0, 1.0* 


5.0 


4.0, 4.0 


5.0 



|)oi!l)le figures indicate split processes. 



OVARY AND PROCESSES OF C. EISENI 



311 



Processes are unquestionably sheathed; a central medulla is separated by a 
sheath space from the epithelial covering (Plate I, Fig. 5). The sheath is normal for 
specimens up to stages approaching birth ; at this time, the characteristic may be lost. 
The sheath characteristic may be visible even in the proximal peduncle that forms 
the base of the processes. The sheath space may be extensive and continuous or 
broken up into smaller vesicles (Plate IV, Figs. 16-17). The medulla or core 





IS 






16 






PLATE IV 

FIGURE 15. Trophotaeniae of Goodca Initpoldii (8 mm.). This "rosette" type process has 
a large sheath space in small embryos but the space is absent in older embryos. 

FIGURES 16-17. Trophotaeniae from two 8-mm. specimens of "Characodon" eiseni. The 
epithelium shown is thick ; the sheath space is variable in appearance ; it is generous, restricted or 
absent in some regions. All figures are tracings from microprojections. 



312 GUILLERMO MKXDO/A 

normally measures 0.16-0.24 mm. in maximum width although some measurements 
of 0.35 mm. have been noted. The medulla is normally attached to the dorsal 
epithelium although it may attach to any area of the epithelium. On occasions, the 
medulla may even protrude beyond the surface of the process, carrying the process 
epithelium out with it. 

Histology 

Two types of cells form the epithelium of the processes ; one is an extension of 
the gut epithelium, the other is a continuation of the epidermis. Because of the 
origin of the processes, the former is found on the ventral surface of the processes, 
the latter on the dorsal surface. The cells derived from the gut epithelium are 
cuboidal to low columnar and are normally 8-10 /JL high; the nucleus is primarily 
spherical, basal in position, vesicular and 3.5-5.0 ^ in size. The cells show a con- 
spicuous "brush border" that, in the light of modern microscopy, is probably a 
surface covered with microvilli. The position of the nucleus and the stratification 
of the heterogeneous cytoplasm are evidence of a physiologically active cell. The 
basement membrane of these cells is extremely delicate. The fact that mitotic figures 
are seldom seen indicates that the processes probably grow at the base. The 
epithelium derived from the epidermis is very thin, composed of flattened cells 
normally arranged in an irregular double layer and often vacuolated. The transition 
between the two types of cells is abrupt. The large cuboidal cells normally form 
75% or more of the epithelial surface. The most typical appearance of the epi- 
thelium occurs in embryos 10 mm. or less in length ; as time approaches for birth, 
the epithelium and, indeed, the entire process undergo marked changes. 

The medulla is formed of a mass of loose, spongy, connective tissue. Fibrocyte 
nuclei are approximately 10 ^ in length, oval, pale, finely granular and homogeneous 
in appearance. Approaching birth, many phagocytes appear in the tissues. As 
is true for other goodeids, the blood supply to the processes is very rich, forming 
an extensive capillary plexus on the medullary surface. The vascular character 
is a property of the medulla, not of the epithelium. Occasionally, capillaries or 
large vessels protrude beyond the surface of the entire process in the region of the 
"epidermal" epithelium. The medullary connective tissue is continuous with the 
submucosa of the gut and the sub-epidermal connective tissue of the body surface. 

DISCUSSION 

The present description of the ovary and the trophotaeniae of "Characodon" 
ciscni differs from that given for Xcnotoca (variata} by Hubbs and Turner (1939) 
in some respects ; there are two serious differences and other minor ones. In their 
study, the median septum of Xenotoca is described as "entire, attached dorsally and 
\cntrally, much folded" (Hubbs and Turner, 1939, Table II). This property places 
Xcnotoca in the second phyletic line, along with Alloophorus and Chapalichthys. 
However, in the present study only 12 of the 43 ovaries were found to follow this 
description. An additional 13 ovaries had a septum essentially complete but with 
minor or more serious variations, whereas 18 ovaries had a septum divided distinctly 
into dorsal and ventral halves. In this species, therefore, the median septum is 
inconsistent or variable in form and thus is an unreliable criterion for use in classi- 



OVARY AND PROCESSES OF C. EISENI 313 

fication. Using this criterion, "Characodon" eiseni may well be classified in three 
phyletic lines, numbers 2, 6 or 7 (Hubbs and Turner, 1939, Table II). 

No serious discrepancies were found in the description of the location of the 
ovigerous tissue although it appears to the writer that, except for the extreme 
anterior end, eggs seldom occur in the median septum ; they occur mostly in the walls 
of the ovary and especially at the anterior end. This again differs somewhat from 
the revision of Hubbs and Turner where Xenotoca is likened to Alloophorus and the 
latter is described as having ovarian "walls . . . almost devoid of ovigerous tissue" 
(p. 13). 

Hubbs and Turner describe the trophotaeniae of Xenotoca as 6 to 8 in number, 
very long and unsheathed (Plate II, Fig. 10). The processes are said to "arise by 
dichotomous branching from three backwardly projecting trunks, one median and 
two lateral" (p. 25). Having examined more than 200 specimens, the writer 
concludes that there are four basic processes in "Characodon" eiseni, the one 
posterior median process and two lateral processes as described by Hubbs and 
Turner for Xenotoca but with an additional median process anterior to the anus. 
The writer agrees with Turner that there is much secondary splitting (Turner, 
1937). Furthermore, although the writer agrees that there may be as many as 6 to 
8 processes, this is a number seldom attained ; 4 to 6 processes is a much more 
representative number. The slight disagreement in number is a minor matter but 
it is important that a fourth antero-median process be recognized as part of the 
basic set of processes. 

Another serious difference arises in the matter of the presence or absence of the 
sheath around the process. Xenotoca is described as having unsheathed tropho- 
taeniae (Turner, 1937; Hubbs and Turner, 1939), thereby placing it in a category 
with Alloophorus robnstus, Chapalichthys encaustus, and Zoogoncticus cuitseoensis. 
The size of the embryos, the stage of development and the number of specimens 
examined may well affect the conclusions drawn. In embryos of "Characodon" 
eiseni approaching birth, the processes do tend to show an absence of a sheath but 
in younger stages there is no question of the presence of a sheath, although even 
this is variable in degree of formation. Following Hubbs and Turner, the presence 
of the sheath in the processes should place this species with genera such as Skiffia, 
Ollentodon and Neotoca, in the subfamily Girardinichthyinae rather than in the 
Goodeinae. The three genera listed are the only other ones in which the processes 
are stated to be sheathed. While this paper is not intended to include an evolu- 
tionary analysis of the species, it seems to the writer that the arrangement of 
processes in "Characodon" eiseni could easily have arisen from the "trident" 
arrangement present in species such as Neotocoa bilincata, simply by the addition of 
a short median process anterior to the anus. Finally, a minor difference arises 
in regard to the nature of the process epithelium. The writer is not in agreement 
that the ". . . epithelium ... is everywhere simple and of irregular height" 
(Hubbs and Turner, 1939, p. 25). In the younger embryos the epithelium has a 
dual structure, depending on whether it is continuous with the epidermis or the 
gut epithelium. The double nature is very clear ; each of the two types tends to be 
quite regular in its own structure. The irregularity referred to may be true in 
stages just before birth when the entire process undergoes marked changes, 
preceding its resorption at about the time of birth. At this time the epithelium does 



314 GUILI.KRMO MKXDOZA 

become most irregular and is even sloughed off. The writer appreciates the fact 
that this is a minor matter, a detail readily noticed in a descriptive, histological 
study but likely to be missed in a paper of broader scope and concerned primarily 
with overall taxonomic matters. 

In conclusion, the writer points out the overall taxonomic impasse in which 
these newer facts place "Characodon" eiseni. First, the description of the median 
septum of the ovary is not in agreement with that given for Xenotoca variata by 
Hubbs and Turner ( 1939). Second, because of the variability of the structure of the 
median septum, "Characodon" eiseni can be placed in different phyletic lines within 
the family as determined by the nature of the median septum. Hence this criterion 
is unreliable for the classification of this species. Third, the sheathed processes 
found in "Characodon" eiseni are totally different from the solid, non-sheathed 
processes described for Xenotoca variata. Fourth, the presence of sheathed 
processes in this species is inconsistent with the type of median septum described 
here for "Characodon" eiseni or that of Xenotoca variata. In the classification 
scheme devised by Hubbs and Turner, species that have sheathed processes have an 
ovary with a thin, delicate, median septum and ovigerous tissue confined to two 
dorso-lateral folds (e.g., Neotoca bilineata}. Such an ovary has but little in common 
with that described for either "Characodon" eiseni or Xenotoca variata. Thus two 
major criteria (ovarian structure and type of process) are at odds with each 
other and one criterion (ovarian structure) fails to discriminate between two or 
more phyletic lines. 

The facts brought out in this paper raise the serious question whether taxonomic 
criteria based on ovarian and trophotaenial structures can be used successfully 
in the case of this species. At the same time, this paper does not propose to extend 
this conclusion to the entire goodeid family since this study is limited only to one 
species, "Characodon" eiseni. It may well be that a restudy should be made of the 
degree of variation in the structure of the septum in some or most species of the 
family, particularly those species in which the septum is ovigerous. It is not likely 
that a median septum of the Neotoca bilineata type (thin and non-ovigerous) will 
vary much. The criteria set up by Hubbs and Turner in 1939 no doubt will still 
prove to be valuable, even though there may be exceptional forms such as this 
species in which the criteria are not absolutely discriminatory. Lastly, a taxonomic 
analysis of this species using other conventional criteria should help to clarify the 
taxonomic relationships of "Characodon" eiseni and Xenotoca variata. 

SUMMARY 

The ovary and trophotaeniae of "Characodon" eiseni Rutter are described. The 
median septum of the ovary is variable in structure ; the septum may be a single, 
continuous sheet or it may be divided into dorsal and ventral halves. Ovigerous 
tissue is confined primarily to the anterior region of the ovary but mostly to the 
ovarian wall. There are four trophotaeniae (processes), two lateral and two 
median, one anterior and one posterior to the anus. The processes are further 
described as sheathed. The above facts are not in agreement with previously 
published descriptions of Xenotoca variata with which this species has been synony- 
mi/ed. The above facts are further contradictory with each other in assigning 



OVARY AND PROCESSES OF C. EISENI 315 

"Characodon" eiseni to a particular evolutionary line within the family Goodeidae. 
The paper shows that the goodeid taxonomic criteria based on ovarian and tro- 
photaenial structure are not discriminatory when applied to "Characodon" eiseni. 

LITERATURE CITED 

ALVAREZ, J., 1950. Claves para la determination de especies en los peces de las aguas 

continentales Mexicanas. Sec. de Marina, Direction General de Pesca e Industrias 

Conexas, pp. 1-136. 
BUEN, F. de, 1942-1943. Los peces de agua dulce de la familia Goodeidae. Boletin Biol. Lab. 

de la Universidad, Universidad de Puebla, Mexico, pp. 111-148. 
HUBBS, C. L., 1924. Studies of the fishes of the order Cyprinodontes. Parts I-IV. Misc. Publ. 

Mus. Zool., Univ. Michigan, No. 13, pp. 1-31. 
HUBBS, C. L., 1926. Studies of the fishes of the order Cyprinodontes. Part VI. Material for 

a revision of the American Genera and Species. Misc. Publ. Mus. Zool., Univ. 

Michigan, No. 16, pp. 1-87. 
HUBBS, C. L., AND C. L. TURNER, 1939. Studies of the fishes of the order Cyprinodontes. 

Part XVI. A revision of the Goodeidae. Misc. Publ. Mus. Zool., Univ. Michigan, No. 

42, pp. 1-80. 
JORDAN, D. S., 1923. A classification of fishes including families and genera as far as known. 

Leland Stanford Jr. Univ. Publ., (Univ. Ser.}, 3: 77-243. 
JORDAN, D. S., AND B. W. EVERMANN, 1896-1900. The fishes of North and Middle America. 

Bull. U. S. Nat. Mus., 47 (4 parts), pp. 1-3313. 
MEEK, S. E., 1902. A contribution to the ichthyology of Mexico. Field Columbian Mus. Publ., 

No. 65, Zool. Ser., 3: 63-128. 
MEEK, S. E., 1904. The fresh-water fishes of Mexico north of the Isthmus of Tehuantepec. 

Field Columbian Mus., Publ, No. 93, Zool. Ser., 5: 1-252. 
MENDOZA, G., 1937. Structural and vascular changes accompanying the resorption of the 

proctodaeal processes after birth in the embryos of the Goodeidae, a family of viviparous 

fishes. /. Morph., 61 : 95-125. 
MENDOZA, G., 1940. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a 

member of the family Goodeidae. II. The cyclic changes in the ovarian soma during 

gestation. Biol. Bull, 78: 349-365. 
MENDOZA, G., 1956. Adaptations during gestation in the viviparous cyprinodont teleost, 

Hubbsina turneri. J. Morph., 99: 73-96. 

REGAN, C. T., 1906-1908. Pisces. Biologia Centrali-Americana. 
TURNER, C. L., 1933. Viviparity superimposed on ovoviviparity in the Goodeidae, a family of 

cyprinodont teleost fishes of the Mexican Plateau. J. Morph., 55: 207-251. 
TURNER, C. L., 1935. The use of the embryonic rectal processes of the embryos in a revision of 

the classification of the family Goodeidae. Anat. Rec., 64 (Suppl.) : 137-138. 
TURNER, C. L., 1937. The trophotaeniae of the Goodeidae, a family of viviparous cyprinodont 

fishes. /. Morph., 61 : 495-523. 



SYMBIOSIS OF HYDRA AND ALGAE. 

II. EFFECTS OF LIMITED FOOD AND STARVATION ON GROWTH 
OF SYMMIOTIC AND AFOSYMBIOTIC HYDRA 

LEONARD MUSCATINE 1 AND HOWARD M. LENHOFF 

/tit'ision tij Marine Hinlo</y, Scrimps Institution of Oceanography, University of California, 

San Dicf/c. Culifiirnia, and Laboratory for Quantitative Biolouy, 

University of Miami, Coral Gables, Florida 

Very few measurements have been made on the effect of symbiotic algae 
on growth of their various invertebrate hosts. Recently Karakashian (1963) dem- 
onstrated that the algae symbiotic with Paramecium bursaria exert a strong 
influence on the growth of the host. These studies were carried out with sym- 
biotic and aposymbiotic individuals of known genetic and nutritional history 
cultured in a defined medium. Culture techniques (Loomis, 1954; Muscatine and 
Lenhoff, 1965) now permit a similar approach using green hydra, thus affording 
insight into an association of algae and a metazoan. Previous studies on the role 
of algae in green hydra (Goetsch, 1924; Haffner, 1925) were carried out in 
undefined media, and lacked the quantitative precision necessary for critical 
evaluation. 

The present study describes experiments on the growth, survival, and protein 
turnover of hydra with and without algae as a function of exogenous food supply. 
Possible mechanisms of interaction between algae and host are discussed. A 
preliminary note on some of this work has appeared elsewhere (Muscatine, 1961). 

MATERIALS AND METHODS 

All experiments were carried out with Chlorohydra I'iridissima, Carolina 
strain 1960. The culture medium, and methods for maintaining animals in the 
laboratory, sampling individuals for experiments, obtaining algae-free controls and 
conducting growth experiments are described in a previous paper (Muscatine 
and Lenhoff, 1965). 

"Pale green" hydra containing known amounts of algae intermediate between 
green and albino (== algae-free) were obtained in the following manner. Green 
hydra were placed in culture solution containing 0.068 AI glycerine, which causes 
the gradual elimination of algae (Whitney, 1907, 1908). At daily intervals for 
eight days, groups of (en "uniform" (cf. Lenhoff and Bovaird, 1961) hydra were 
removed, rinsed in clean culture solution, and exposed to C 14 O 2 for exactly 24 
hours, using a procedure described by Muscatine and Lenhoff (1963). These 
labeled animals were (hen rinsed in several changes of clean culture solution, and 
placed on a Millipore filler (IIA-47) in a drop of deionix-.-d water. When relaxed, 
the animals were flattened on (he filter by application of suction (cf. Lenhoff. 1959). 

address : Department of Zoology, University of California, Los Angeles, California. 

316 



SYMBIOSIS OF HYDRA AND ALGAE. II. 



317 



The filter was dried, glued to an aluminum planchet and assayed for radioactivity. 
The level of radioactivity of untreated green hydra controls was considered to 
represent the net photosynthetic activity of the normal algal flora. Glycerine- 
treated animals having fewer algae had proportionally less radioactivity. Albinos 
served as controls for animal fixation of C 14 O 2 . Figure 1 shows the radioactivity 
of each group plotted against time grown in glycerinated culture solution. Hydra 
sampled after four and six days of glycerine treatment were judged to contain, 



240 


- 








GREEN CONTROL 




200 

X 

t- 


< i 


- 


100 * 


< 






X 

o 


o: 






H 


> 160 




_ 


O 

80 co 


X 














2 


LJ 


< 


> 


H 
X 


1- 






rn 


^ 






H 


? 120 


- 


- 


60 o 


N, 






r 


CO 






r 


8 80 


- 


- 


40 > 















( 


m 


40 


- 


{ 


20 
m 




ALBINO CONTROL V 






<$ i i 


i i i i 9 o 





12345678 
DURATION OF EXPOSURE TO 0.068 M GLYCERINE (DAYS) 

FIGURE 1. Radioactivity accumulated by green hydra exposed first to 0.068 M glycerine for 
periods up to 8 days, and then to C 14 O 2 for 24 hours. Vertical bars denote twice the standard 
deviation of the mean number of counts per minute. Non-glycerine-treated green controls, in 
18 trials, gave 203.2 32.0 counts per minute per hydranth. Albinos gave less than two counts 
per minute above background. 



from this reference curve, approximately 10-20% and 4-6%, respectively, of their 
usual normal complement of algae. These animals were washed with several 
changes of clean culture solution one hour before experiments. 

To graft the heads (hypostome and tentacles) of green hydra onto the bodies 
(gastric region and below) of albinos, one-day starved stock hydra were bisected 
transversely. Appropriate pieces were threaded on a hair and held together by 
gentle pressure with watchmaker's forceps. Adhesion began within a minute or 
two and grafts were available after 15-30 minutes. The approximate algal 
content of green heads was estimated by first exposing whole intact "uniform" 
green hydra to C 14 O 2 in a standard manner (Muscatine and Lenhofif, 1963), and 



318 



L. MUSCATINE AND H. M. LENHOFF 



then cutting each animal in two just below the hypostome and tentacles. Each 
head and body was then dried separately on a planchet and assayed for radio- 
activity. In five replicates, green heads were found to contain 30.5 2.3% of 
the normal complement of photosynthetically-active algae in an entire animal. 

S 35 -labeled mouse liver (specific activity 1000-2500 counts per minute per 
microgram protein nitrogen) was prepared, administered to hydra and fractionated 
as described by Lenhorf (1961). Radioactive material was assayed, with cor- 
rection for background, with an end window gas flow counter (Nuclear-Chicago 
Clll-B). 



80 



t/j 

x 



Q. 



40 



cr 

oJ 

CD 



(0) 



(b) 



(c) 




DAYS 

FIGURE 2. Semi-logarithmic plot of growth of green (closed circles) and albino (open 
circles) C. viridissima (a) fed daily, (b) fed every second day, and (c) fed every third day. 
Arrows indicate time of feeding. 

RESULTS 
1. The effect uj amount oj joud un yrou'th oj ijrccn and albino C. viridissima 

In a previous paper (Muscatine and Lenhoff, 1965) we reported that green 
and albino C. viridissima grew at nearly identical logarithmic rates when fed 
daily on excess Artcmia nauplii. This is illustrated in Figure 2, curve a. A 
doubling time of about 1.5 days is the maximum growth rate (k mn x) for this 
species under these conditions (Muscatine and Lenhoff, 1965). Growth of 
albinos at k max indicates that algae are not essential for logarithmic growth as 
long as there is ample exogenous food. However, when food was limited, 
growth rates of green hydra always exceeded those of algae-free individuals, as 
shown in curves b and c. Curve b shows that the growth rate of green hydra 
fed every second day deviated only slightly from the rate of animals fed daily. 
Growth of albinos, on the other hand, lagged after the second feeding, and in- 
creased only after a third feeding. Green hydra produced nearly twice the 



SYMBIOSIS OF HYDRA AND ALGAE. II. 310 

number of buds produced by albinos. When the diet of excess Artemia nauplii 
was further limited to a feeding every third day (curve c), growth of green hydra 
dropped off sharply during the first two-day interval without food, but resumed 
a nearly normal rate immediately after the next feeding. Growth of albinos also 
dropped off after two days without food but continued to lag through the second 
feeding without resuming a normal rate. Again, green hydra produced almost 
twice the number of buds produced by albinos. 

Since hydra are normally given excess Artemia larvae at a feeding, there was 
the possibility that in experiments with limited feeding, green hydra had simply 
taken in more food. This was tested by feeding green and albino hydra daily 
with single Artemia nauplii. This regime both controlled and limited the food 
intake. Freshly hatched larvae were fed to individual green and albino hydra 
with a tapered pipette allowing the larvae to leave singly. Since the number of 

TABLE I 

Growth of replicate cultures of green (G) and albino (A) C. viridissima fed daily but only 

on single Artemia nauplii. Numbers in parentheses indicate total number 

of shrimp given to each culture 









No. 


of hydranths on day 


Exp. 


i 


2 


3 


4 


5 


6 


7 


k 






(5) 


(9) 


(15) 


(19) 


(23) 


(30) 




G 


10 


17 


22 


30 


41 


60 


71 


0.277 


G 


10 


18 


25 


31 


41 


57 


71 


0.277 


1 A 


10 


20 


23 


25 


30 


35 


36 


0.121 


A 


10 


17 


19 


22 


27 


37 


39 


0.187 






(2) 


(3) 


(3) 


(4) 


(5) 


(8) 




G 


4 


7 


8 


10 


13 


21 





0.346 


G 


4 


5 


8 


9 


11 


17 





0.277 


G 


4 


6 


8 


9 


11 


17 





0.277 


A 


4 


5 


6 


6 


9 


11 





0.198 


A 


4 


6 


6 


7 


7 


12 





0.210 


A 


4 


5 


6 


7 


10 


12 





0.231 



hydranths in each culture changed as the experiment progressed, a second shrimp 
was given to some individuals in order that cultures would receive the same 
number of shrimp. In this case the additional larvae were fed to maturing buds. 
Table I shows that under these conditions the average growth rate of green hydra 
(0.29) still approached that of well-fed individuals, while the average for albinos 
(0.19) was significantly lower (p < 0.05). 

Some experiments were carried out to determine if a greater capacity for gastro- 
dermal phagocytosis might have accounted for the increased growth of green 
hydra on a limited food supply. Twelve green and 12 albino hydra were each 
fed a small piece of S 35 -labeled mouse liver along with excess Artemia nauplii. 
Fractionation by differential solubilities showed that 80% of the isotope was bound 
in the alcohol, trichloroacetic acid-insoluble liver fraction, i.e., the residual protein 
fraction, and was thus favorable for tracing the course of food protein from the 
gut lumen into phogacytic digestive cells. At hourly intervals during the six 



320 



L. MUSCATINE AND II. M. LKNHOFF 



hours following ingestion, duplicate pairs of green and albino hydra were bisected 
longitudinally, and the gut contents (ingested but not phagocytized) were washed 
out with culture solution onto a planchet. The radioactivity of this material was 
then measured and compared to that remaining in the hydra tissues (phago- 



100 



80 



CO 
LU 



00 
CO 



60 



CO 



40 



20 



o 
o 








_L 



_L 



34 
HOURS 



3. Rate of phagocytosis of S 35 -labeled mouse liver by replicate cultures of green 
(closed circles) and albino (open circles) C. i-iridissiwa. 



cytized). The curve in Figure 3 represents the rate of phagocytosis of sulfur- 
labeled tissue. Phagocytosis proceeded relatively slowly over the first two hours, 
more rapidly during the next two to three hours, and then more slowly after five to 
six hours as the phagocytic capacity of gastrodermis reached a maximum. Both 
green and albino hydra phagocyti/ed 85-95 9^ of the labeled tissue and at similar 



SYMBIOSIS OF HYDRA AND ALGAE. II. 



321 



rates, indicating that the absence of algae did not impair the phagocytic capacity of 
albino C. viridissima. Thus, the difference in growth of green and albino on a 
limited food supply was not simply the result of a quantitative difference in food 
intake. This conclusion is further borne out by starvation experiments. 

2. The effect of starvation on survival of green and albino C. viridissima 

Goetsch (1924) described an experiment in which green and albino hydra 
were placed in the same aquarium with little food and the albinos gradually died 
out. He concluded that albinos live only when well-supplied with food. These 
observations were confirmed by randomly placing 10 green and 10 albino C. 
viridissima in a 10-gallon aquarium containing aged tap water, several Gambusia 
sp., common aquatic plants, and a sparse population of an unidentified ostracod. 
This laboratory "ecosystem" was observed daily but otherwise unattended. After 
three weeks the number of green hydra had at least trebled while no albinos 
could be found. 

TABLE II 

Results of 8 replicate experiments showing Ike mean ( standard deviation of the mean) 

number of green, pale green and albino C. viridissima surviving starvation, 

and the range of survival times 







No. of hydranths on day 


Range of 


Group 


% 




survival 




algae 


















(days) 









2 


4 


6 


8 


10 


12 


14 




Green 


100 


10 


20.51.5 


24.7 1.5 


28.5 1.9 


29.5 4.3 


29.0 2. 4 


32. 5 0.7 


31.00.0 


28-30 


Pale green 


10-20 


10 


20.71.7 


24.2 3.1 


23. 7 3.4 


23.43.7 


23.0 3.6 


21.04.9 


22. 2 3. 2 


24-26 


Pale green 


4-6 


10 


21. Oil. 


25. 5 3.5 


24.5 3. 5 


24.5 3. 5 


21.06.0 


13.04.0 


8.0 6.0 


17-20 


Albino 





10 


18.23.5 


20.63.7 


18.7 4.3 


12.5 4.2 


5.6 3.2 


1.7 


0.6 


10-12 



To obtain quantitative data on starvation, five green hydra were placed in 
30 ml. of culture solution in a Petri dish (100 mm. X 15 mm.). Five albinos 
were similarly treated. Also starved in the same manner were two different 
groups of five "pale green" C. viridissima, one containing 4-6% and the other 
10-20% of the normal algal flora. The animals were illuminated but not fed, 
and the culture medium was changed once daily. The number of hydranths in each 
vessel was recorded daily. The results are shown in Table II. During starvation 
green hydra produced buds for 12 days and survived for nearly four weeks, 
gradually becoming smaller during this time, and finally disintegrating. "Pale 
green" individuals did not appear to change, judging from their relative shades 
of green, until after about 10 days of starvation when the 5% "pale green" group 
seemed noticeably whiter. Albinos produced buds for about 6 days and most 
survived for only 10-12 days. One or two individuals, in subsequent starvation 
experiments, survived for as long as 17 days. Unlike green and pale green 
hydra, most of the albinos disintegrated soon after they discontinued budding. 
This unusually premature event was characterized by crumbling of tentacles and 
body tube until all that remained of each albino was an amorphous accumulation 
of whitish debris. It was frequently difficult to decide when an albino w r as 
"dead." This was arbitrarily taken as the time at which the crumbling of 



322 



L. MUSCATINE AM) II. M. I.KXHUKI< 



tentacles \vas first noticed. Goetsch also observed the disintegration of starving 
albinos in contrast to the gradual diminution of similarly treated green hydra. 

From the data on starvation in Table II, it was possible to estimate the degree 
to which the number of algae influenced survival. In Figure 4 the percentage of 
algae contained initially by the hydra in each group is plotted against (1) the 
average number of hydranths present at 12 days starvation, and (2) the range 
of maximum survival times. In the first curve, 12 days was chosen because it repre- 
sents the time at which no albinos remain, although data from 8-14 days give curves 
of essentially the same character. The shape of the resulting curves is interpreted 
to mean that survival ability of a starved C. viridissima is not appreciably impaired 




20 40 60 80 

% PHOTOSYNTHETICALLY-ACTIVE ALGAE 



100 



FIGURE 4. Survival of starved C. viridissima as a function of the number of algae contained. 
Using data from Table III, the per cent of photosynthetically-active plant material is plotted 
against the number of hydranths present after 12 days' starvation (open circles) and mean 
survival (open squares). 

until the level of its photosynthetically-active plant material drops below 15-20% 
of its normal value. Thus, only about 5-10% of the normal algal flora appears 
necessary for half-maximum survival ability of the starving host. 

3. The effect of starvation on turnover of S 35 -Iabelcd food 

To compare the turnover of protein by starving green and albino C. viridissima, 
we measured the rate at which radioactivity was released into the medium by hydra 
which had previously ingested S 35 -labeled mouse liver. Duplicate groups of 10 
green, 20 albino, and 10 "pale green" (15% algae) hydra were fed sulfur-labeled 
liver and allowed to regurgitate the uneaten portion 6 hours later. Immediately 
after regurgitation 10 of the labeled albinos were decapitated and unlabeled green 
heads of known algal content were grafted onto the labeled albino bodies as 



SYMBIOSIS OF HYDRA AND ALGAE. II. 



323 



described under Methods. Each group of hydra (green, "pale green," albino, and 
graft) was placed in 2 ml. of culture solution in depressions of plastic temperature 
control blocks (Coral Research and Development, Miami, Fla.) maintained at 
22.5 0.25 C. At 24-hour intervals for five days, the culture fluid was removed 
from each group and the animals and vessels were rinsed with 0.5 ml. of culture 
solution per group. The solution and rinsings were combined, dried on planchets 
and assayed for radioactivity. A fresh 2-ml. portion of culture solution was added 
to the hydra. After five days the animals were removed and assayed for radio- 
activity. The sum of the radioactivity of fluid samples is the total S 35 present at 
the beginning of the experiment. Material released is expressed as a per cent of 
this total. In five experiments (Table III) the loss of S 35 by the groups of hydra 
always bore the same relationship, although considerable variation was encoun- 
tered from one experiment to another. Figure 5 shows the rates of loss in one 

TABLE III 

Per cent of 6' 36 lost by groups of green, albino, pale green (10-20% algae) and grafted 
(30.5% algae) C. viridissima during 5 days' starvation 



Expt. 


Green 


Albino 


Pale 


Graft 


1 a 


10.8 


22.8 








b 


14.2 


23.5 








2 a 


19.9 


40.5 





. 


b 


23.2 


33.2 








3 a 


19.1 


89.5 


29.6 


28.2 


b 


29.9 


53.5 


24.4 


31.0 


4 a 


12.0 


24.6 








b 


14.0 


34.8 





. 


c 


11.1 


35.4 








5 a 


35.2 


68.5 


57.3 


48.1 


b 


40.6 


82.1 


46.3 


61.1 



experiment. Invariably albinos lost material to the medium faster than any other 
group. Pale green and grafted hydra which contained, respectively, 15% and 30% 
of the normal complement of algae lost material at a lower rate. Green hydra lost 
material at about half the rate of albinos and retained labeled material about twice as 
long. The relationship between rate of loss of labeled material by a group of hydra 
and its algal content is similar to that illustrated by the curves in Figure 4, where 
relatively few algae have nearly the same effect on the host as does a full complement 
of algae. The ability of the algae in grafted individuals to modify the rate of loss of 
material from the labeled albino body, despite the localization of algae in the un- 
labeled head, exemplifies a "replacement therapy" type of experiment, and suggests 
that the algae in this case might act by releasing something which diffuses through 
a distance. 

That less material appeared in the medium of green and pale green hydra than 
in the medium of albinos implied that the algae either (1) directly affected the 
catabolic activities of the host cells, or (2) accumulated the labeled material after 
it was released by the animal cells. This was investigated in preliminary experi- 
ments in which sulfur-labeled hydra were starved for five days and then homogenized 



324 



L. MUSCATINE AND H. M. LENHOFF 



by ultrasonic vibration. By gentle centrifugation most of the algae were separated 
from the bulk of the animal tissues, but mutual contamination could not be avoided. 
In two trials the resulting algal pellet contained 3.8% and 6.8% of the total radio- 
activity in the entire homogenate, tentatively indicating that the algae did not 
accumulate the isotope but depressed the rate of protein catabolism of the host. 








DAYS 



FIGURE 5. Rate of loss of S^-labeled material by green ( ), grafted ( (I ), pale green (O) 
and albino (O) C. riridissima containing, respectively, 100%, 30%, 20% and 0% photo- 
synthetically-active plant material. 

DISCUSSION 

The results of this study lead to the conclusion that symbiotic algae favorably 
influence the growth, reproduction, survival, and protein turn-over of C. viridissiina. 
The growth rate of green C. viridissiina on a limited food supply was consistently 
greater than that of aposymbiotic controls (Fig. 2; Table I). This difference 
was not the result of a proportionally larger food intake by green hydra (Fig. 3), 
but of some intrinsic factor associated with the presence of algae, since even 
during starvation green hydra produced more buds, survived longer, resisted 
disintegration and displayed a lower turnover of sulfur-labeled protein compared 
to aposymbiotic controls (Tables II, III). These results lend quantitative sup- 
port to the observations of Goetsch (1924), who noted that (1) well-fed albino 
hydra grew as well as well-fed green individuals, either in light or in darkness, 
and (2) when starved in the light, green hydra survived for nearly twice as long 



SYMBIOSIS OF HYDRA AND ALGAE. If. 325 

as aposymbiotic controls. Partially "infected" hydra survived at least as long 
as any of the aposymbiotic controls and appeared "less depressed." Goetsch 
concluded that the algae were not essential when food was abundant but probably 
played some role in augmenting survival when certain stresses, e.g., starvation, 
were imposed on the host. Karakashian (1963) demonstrated a positive influence 
of algae on growth of the ciliate protozoan, Paramecium bursaria, when bacterial 
food was present in low concentration and when cultures were starved but 
illuminated. A positive correlation of mean numbers of algae per paramecium 
with growth rate and survival time was also observed. Our results thus 
parallel these in several respects. 

The adaptive value of symbiosis with algae is particularly evident from the 
behavior of albinos in these experiments. Their low budding rate and tendency 
to disintegrate early during starvation, and poor growth on a limited food 
supply would be disadvantageous for survival in an environment where limited 
food and periods of starvation are frequently encountered (Welch and Loomis, 
1924). These observations perhaps explain why aposymbiotic adult C. viridis- 
sima have not yet been found in natural waters, although algae-free eggs are often 
produced by at least one strain of green and albino C. viridissima (unpublished 
observations). 

Possible mechanisms by which the algae influence growth of the host 

1. Gas exchange and waste uptake 

Geddes (1882) suggested that symbiotic algae augment the well-being of their 
animal hosts in several ways, including (1) by taking up carbon dioxide and pro- 
ducing oxygen during photosynthesis, thus facilitating host respiration, and (2) 
by taking up host excretory wastes, such as ammonia, thereby creating a less toxic 
micro-environmental milieu for the animal. These interactions undoubtedly take 
place to some extent in most associations but as yet there is little direct evidence 
that any of them are essential to the animal (see Droop, 1963). In fact, they ap- 
pear to be non-essential for C. viridissima since individuals without algae grow at 
kmax as long as they are well fed. Similarly, well-fed green and albino C. viridis- 
sima grow at nearly identical rates in darkness where photosynthetic gas exchange 
is again ruled out as an augmenting factor (Goetsch, 1924; our unpublished 
observations). 

2. Utilization of algal metabolic products 

As suggested by Geddes (1882) and others (Keeble, 1908; Boschma, 1925; 
Gohar, 1940, 1948) a host could benefit by digesting its symbiotic algae or utilizing 
their extracellular products. On the basis of the observations in this study, little 
can be said regarding digestion of algae by C. viridissima. Since there is no ap- 
parent decrease in number of algae after two to three weeks' starvation, and since 
10-20% of the normal flora can sustain the starving host, digestion of algae seems 
unlikely but is not ruled out. As noted by Yonge (1944) symbiotic algae probably 
resist digestion since the majority are found in animals which display intracellular 
digestion. 



326 L MUSCATIXK AND II. M. LENHOH 

However, there is evidence that C. viridissima utilizes products of algal metabo- 
lism. Experiments with C 14 O 2 show that 10-20% of the labeled carbon fixed by 
the algae is transferred to the animal where some is incorporated into major 
chemical fractions (nucleic acids, proteins, etc.). The specific activity of C 1 * in 
algae-free green hydra tissues in these experiments was 50-100 times greater than 
that in albino control tissues where some carbon was assimilated solely by hetero- 
trophic fixation (Lenhoff and Zimmerman, 1959; Muscatine and Lenhoff, 1963). 
Similar transfers take place in other coelenterate-algae associations (Muscatine and 
Hand, 1959; Goreau and Goreau, 1960) and are implied to occur in others (Sar- 
gent and Austin, 1949. 1954; Odum and Odum, 1955 ; Burkholder and Burkholder. 
1960). 

The results of this study and demonstration of the utilization of products of 
algal metabolism by host cells lend support to the conclusion that the algae in C. 
viridissima augment growth of the host by nutritional supplementation. Support 
for this view comes also from the observation that adequate food can replace the 
need for symbiotic algae (Goetsch, 1924; Fig. 2, this paper). A similar observa- 
tion was reported by Parker (1926) and Karakashian (1963) for P. bursaria. The 
inability of albino C. viridissima to withstand starvation and the tendency to disinte- 
grate undoubtedly reflects a loss of function by this species. Neither green C. 
viridissima nor the non-symbiotic species, H. littoralis, show this reaction to starva- 
tion, which could be symptomatic of a nutrient deficiency, as a result of a metabolic 
lesion. The growth lags and slow responses of albinos to intermittent feeding (Fig. 
2) may represent the time needed to accumulate essential nutrients from the limited 
food supply. In contrast, green hydra did not exhibit extended growth lags or 
delayed responses to intermittent feeding. Auxiliary metabolites received from 
the algae probably offset any deficiency, though only temporarily, since, as shown 
in starvation experiments (Table II), the algae cannot sustain the animal indefi- 
nitely without some exogenous food. Information on carbon turnover rates by 
the algae, their extracellular products, the growth requirements of the host, and the 
peculiarities of the metabolism of algae-free individuals should bring to light the 
details of mechanisms of host-symbiont interaction in this association. 

Part of this investigation was carried out at the Laboratories of Biochemistry, 
Ho\\ard Hughes Medical Institute, Miami, Florida during the tenure of a Post- 
doctoral Fellowship from the Division of General Medical Sciences, United States 
Public Health Service (1963) to Leonard Muscatine, and an Investigator Award 
of the Howard Hughes Medical Institute to Howard M. Lenhoff. We thank Mr. 
I. Bovaird, Mr. Alfredo Lopez, and Mr. Enrique Nagid for technical assistance. 

NOTE ADDED IN PROOF 

Slobodkin ( 1964) has recently demonstrated that the "ecological efficiency" 
(yield energy /food energy) of experimental populations of C. viridissima is about 
four times higher than that of Hydra littoralis (a non-symbiotic species), but only 
in populations grown in the light. The implication is that photosynthetic carbon 
is available to C. viridissima for energy. Slobodkin, L. B., 1964. Experimental 
populations of Hydrida. /. Ecol. (Sup pi.} , 52 : 131-148. 



SYMBIOSIS OF HYDRA AND ALGAE. 11. 327 

SUMMARY 

1. When fed daily on Artcmia nauplii, green and albino C. viridisshna grew at 
nearly identical logarithmic rates. With limited food, growth of green hydra al- 
ways exceeded that of albinos. This difference was not the result of a quantitative 
difference in food intake. 

2. Green hydra survived starvation for about four weeks, gradually diminishing 
in size. Albinos survived only 10-12 days, succumbing to starvation by relatively 
sudden disintegration. 

3. The relationship between survival ability and algal content was non-linear. 
Animals with 20% of the normal flora survived nearly as well as those with a full 
complement of algae. 

4. Turnover rate of sulfur-labeled protein during starvation showed the rela- 
tionship albino > pale green > green, among the groups tested. The presence of 
symbiotic algae appears to depress the rate of protein catabolism. 

5. It is concluded that symbiotic algae augment growth, budding, and survival 
of C. viridisshna (Carolina strain 1960) by a mechanism which does not appear to 
involve gas exchange or waste removal by the algae. 

6. Evidence is presented in support of the hypothesis that algal metabolic prod- 
ucts augment growth and survival of C. viridissima. 

LITERATURE CITED 

BOSCHMA, H., 1925. The nature of the association between Anthozoa and zooxanthellae. Proc. 

Nat. Acad. Sci., 11:65-67. 
BURKHOLDER, P., AND L. M. BuRKHOLDER, 1960. Photosynthesis in some alcyonacean corals. 

Amcr. J. Bot. 47: 866-872. 
DROOP, M., 1963. Algae and invertebrates in symbiosis. In: Symbiotic Associations. Soc. Gen. 

Microbiol. Symp. no. 13. B. Mosse and P. Nutman, Eds. Cambridge, pp. 171-199. 
GEDDES, P., 1882. The yellow cells of radiolarians and coelenterates. Proc. Roy. Soc. Edin- 
burgh, 11: 377-396. 
GOETSCH, W., 1924. Die Symbiose der Susswasser-Hydroiden und ihre kiinstliche Beeinflussung. 

Zcitschr. Morph. Okol. Tiere, 1: 660-731. 
GOHAR, H. A. F., 1940. Studies on the Xeniidae of the Red Sea. Publ. Mar. Biol. Sta. 

Ghardaqa, 2: 25-118. 
GOHAR, H. A. F., 1948. A description of some biological studies of a new alcyonarian species 

Clariilaria hamra Gohar. Pub. Mar. Biol. Sta. Ghardaqa, 6: 1-33. 
GOREAU, T. F., AND N. I. GOREAU, 1960. Distribution of labeled carbon in reef -building corals 

with and without zooxanthellae. Science, 131: 668-669. 
HAFFNER, K., 1925. Untersuchungen iiber die Symbiose von Dalyellia viridis und Chlorohydra 

Tiridissima mit Chlorellcn. Zeitschr. iviss. ZooL, 126: 1-69. 
KARAKASHIAN, S., 1963. Growth of Paramecitim bursaria as influenced by the presence of 

algal symbionts. Physiol. ZooL, 36: 52-67. 
KEEBLE, F., 1908. The yellow-brown cells of Convoluta paradoxa. Quart. J. Micr. Sci., 52: 

431-479. 

LENHOFF, H. M., 1959. Migration of CMabeled cnidoblasts. Exp. Cell Res., 17: 570-573. 
LENHOFF, H. M., 1961. Digestion of ingested protein by Hydra as studied by radioautography 

and fractionation by differential solubilities. Exp. Cell Res., 23: 335-353. 
LENHOFF, H. M., AND J. BOVAIRD, 1961. A quantitative chemical approach to problems of 

nematocyst distribution and replacement in Hydra. Devel. Biol., 3: 227-240. 
LENHOFF, H. M., AND K. F. ZIMMERMANN, 1959. Biochemical studies of symbiosis in 

Chlorohydra viridisshna. Anat. Rec., 134: 599. 
LOOMIS, W. F., 1954. Environmental factors controlling growth in hydra. /. Exp. ZooL, 126: 

223-234. 



328 L. MUSCAT1NE AND II. M. LENHOFK 

MUSCATINE, L., 1961. Symbiosis in marine and fresh water coelenterates. In: The Biology of 

Hydra, H. M. Lenhoff and W. F. Loomis, Eds., University of Miami Press, Miami, 

Florida, pp. 255-268. 
MUSCATINE, L., AND C. HAND, 1958. Direct evidence for transfer of materials from symbiotic 

algae to the tissues of a coelenterate. Proc. Nat. Acad. Sci., 44: 1259-1263. 
MUSCATINE, L., AND H. M. LENHOFF, 1963. Symbiosis : On the role of algae symbiotic with 

hydra. Science. 142:956-958. 
MTSCATINE, L., AND H. M. LEXIIOKF, 1965. Symbiosis of hydra and algae. I. Effects of some 

environmental cations on growth of svmbiotic and aposymbiotic hydra. Biol. Bull., 

128: 415-424. 
ODUM, H. T., AND E. P. ODUM, 1955. Trophic structures and productivity of a windward coral 

reef community on Eniwetok atoll. Ecol. Monogr., 25: 291-320. 
PARKER, R. C., 1926. Symbiosis in Paramccmm bursaria. J. Exp. ZooL, 46: 1-11. 
SARGENT, M. C., AND T. S. AUSTIN, 1949. Organic productivity of an atoll. Trans. Amcr. 

Gcophys. Union, 30: 245-249. 
SARGENT, M. C, AND T. S. AUSTIN, 1954. Biologic economy of coral reefs. U. S. Geol. Survey 

Prof. Paper 260-E, pp. 299-300. 
WELCH, P. S., AND H. A. LOOMIS, 1924. A limnological study of Hydra oligactis in Douglas 

Lake, Michigan. Trans. Amcr. Micr. Soc., 43: 203-235. 
WHITNEY, D. D., 1908. Further studies on the elimination of the green bodies from the 

endoderm cells of Hydra riridis. Biol. Bull., 15: 241-246. 
VONGE, C. M., 1944. Experimental analysis of the association between invertebrates and 

unicellular algae. Biol. Rev., 19: 68-80. 



THE DEVELOPMENT OF EGGS OF THE SCREW-WORM FLY 

COCHLIOMYIA HOMINIVORAX (COOUEREL) (DIPTERA: 

CALLIPHORIDAE) TO THE BLASTODERM STAGE 

AS SEEN IN WHOLE-MOUNT PREPARATIONS 

JOHN G. RIEMANN 1 
Entomology Research Division, Agric. Res. Serr., USD A, Mission, Texas 

In studying effects of radiation or chemical mutagens on insect germ cells, being 
able to examine the chromosomes of nuclei in freshly deposited eggs would provide 
many advantages. Meiotic division of the oocytes typically occurs after the eggs 
are deposited. Thus, both the meiotic nuclei and the mitotic ones of the cleavages 
or later stage divisions could be studied. In addition, only in freshly laid eggs can 
the chromosomes of sperm be directly examined after treatment that produces domi- 
nant lethal mutations. In spite of these advantages, only a few such studies have 
been made of the nuclei of very young eggs, to ascertain the presence of chromo- 
somal aberrations or abnormal nuclear divisions and death (i.e., Sonnenblick, 1940, 
on Drosophila; Whiting, 1945a, 1945b, von Borstel, 1955, on Habrobracon). 
Neglect of this kind of study has probably been due in part to technical difficulties 
in collecting and preparing enough eggs at the exact stage of development required. 
In addition, nuclei and chromosomes of such eggs, as a rule, are very small and 
thus difficult to study. 

Recently the author and his associates became interested in determining whether 
chromosome aberrations induced in the sperm or oocytes of screw-worms, Cochli- 
omyia hominh'ora.r (Coquerel), could be studied in the young egg, in at least a 
reasonably satisfactory manner. Sectioned material was quickly found unsatisfac- 
tory for this purpose and emphasis was placed on whole-mount preparations. 
Normal development to the stage of blastoderm formation, as followed in the whole- 
mounts, is described herein. Chromosome aberrations actually found in young 
eggs after treatment of screw-worm flies with gamma radiation and the alkylating 
agent, tretamine, w r ere discussed in an earlier paper (LaChance and Riemann, 
1964). 

MATERIALS AND METHODS 

The sexually mature females of the Florida Normal strain used in this study 
usually oviposit readily when offered warm lean meat. Each female will then nor- 
mally produce 200-300 eggs within a period of 15-20 minutes. These eggs, ce- 
mented together in a compact mass, are each about 0.7 mm. long and no wider than 
0.16 mm. Each one is covered with a rather thick chorion which must be removed 
in making a whole-mount preparation. 

1 Present Address : Metabolism and Radiation Research Laboratory, Entomology Research 
Division, State University Station, Fargo, North Dakota 58103. 

329 



330 JOHN (-. K I KM ANN 

To obtain eggs of a fairly uniform age, individual flies were allowed to oviposit 
for one minute in shell vials placed in a water bath heated to 35 C. The flies were 
then discarded and the vials with the deposited eggs, usually 10 to 20 in number, 
were removed from the water bath and held at room temperature (24-26 C.) until 
fixation. 

The whole-mount procedure was essentially that described by von Borstel and 
Linclsley (1959) in their modification of the technique developed by Schmuck and 
Metz (1931). First, the eggs were dechorionated by shaking them gently in \% 
sodium hypochlorite (commercial bleach diluted 1:5 in distilled water) for not more 
than two minutes. By the end of this period most eggs had lost their chorions. 
The eggs were removed from the hypochlorite solution by straining them through 
a cloth. They were then washed briefly in distilled water and placed in rows on 
22-mm. coverslips by means of an artist's brush. Each egg was then gently punc- 
tured (usually at the posterior or blunt end) with a fine needle so that a thin 
stream of ooplasm flowed onto the surface of the coverslip. This puncturing was 
essential for good fixation and the extruded ooplasm served to attach the eggs firmly 
to the coverslips. As soon as possible after puncturing, the coverslip with its at- 
tached eggs was placed in Kahle's fixative. All fluids used in preparing the eggs 
were kept at room temperature. 

It took about 4-5 minutes to handle 10-15 eggs, from the begintning of dechori- 
onation to placing the eggs in the fixative. Fewer eggs could be handled somewhat 
faster. In general, different groups of eggs fixed at comparable times after depo- 
sition were quite similar in stage of development. Also, puncturing one end of the 
egg or the other, or even allowing the nuclei to flow out of the egg, made little 
difference in the timing of the stages of development, since fixation occurred rapidly. 

After fixation, the eggs were stained according to the Feulgen procedure with 
Schiff's solution, as outlined by von Borstel and Lindsley. Because of the eggs' 
rather considerable thickness, the stained and dehydrated specimens were usually 
mounted on another coverslip instead of on a slide. In this way the mounts could 
be easily turned over to permit examination of both sides under high magnification. 
Generally the preparations were allowed to clear for a few days in the mounting 
medium (Diaphane) before they were examined. This was particularly desirable 
for examining cleavage nuclei. An optical system that produced little contrast was 
also found to be highly desirable for examining eggs. 

More than 600 eggs were examined in preparing this description of egg develop- 
ment in the screw-worm. Two series of eggs fixed at 15-minute intervals through 
the first two hours of development and one series fixed at 5-minute intervals from 
the first to the third hour were prepared. Also prepared were numerous other 
series of eggs less than one hour old, particularly less than 25 minutes old, since 
this time interval covered the most significant periods in determining the effects of 
mutagens on germ cells. 

All drawings were made with a camera lucida. However, to save space, some 
nuclei were drawn somewhat closer together than they would have appeared in true 
scale. 

OBSERVATIONS AND DISCUSSIONS 

LaChance and Leverich (1962) demonstrated that the meiotic nuclei of screw- 
worm eggs go through prophase I and metaphase I while the eggs are still in the 



EARLY DEVELOPMENT OF SCREW-WORM FLY 331 

ovaries. By the time the eggs have reached maturity in the 5-day-old females, the 
oocytes are in early anaphase I, in which stage they remain until the eggs are actu- 
ally deposited. The chromosomes (six pairs) of these anaphase nuclei are much 
contracted, and each meiotic figure appears to be merely a single, very small chro- 
inatin mass in which some of the homologous chromosomes probably remain in con- 
tact with each other. Each nucleus is located in the superficial ooplasm at a point 
on the dorsal surface of the egg about one-fifth of the total egg length from the 
anterior end. 

In a few eggs, fixed individually as early as three minutes after deposition, the 
nuclei were still in the condensed early anaphase characteristic of the mature ovarian 
eggs. Nuclei 1-2 minutes older had longer chromosomes and were usually in late 
anaphase or telophase I (Fig. 1). After telophase I, the egg nuclei went at once, 
without any interphase stage, through the second meiotic division, which was com- 
pleted in 7-8 minutes. At the end of telophase II (Fig. 2), all egg nuclei had 
formed a straight line that extended from the site of the original anaphase I nucleus 
to a point somewhat further into the interior of the egg but still near the surface. 
The position of the line of nuclei (and hence the division plane at anaphase I) with 
regard to the long axis of the eggs varied widely from egg to egg. In some eggs 
it extended posteriorly, in others anteriorly, and in still others in various inter- 
mediate positions, although always with one end located further in the ooplasm than 
the other. 

The chromosomes of the meiotic nuclei were extremely small and as a rule indi 
vidual ones could not be recognized, even in the anaphase figures where they were 
usually fairly well separated. 

Immediately after completion of the second division, the terminal nucleus (the 
one farthest inside the egg) swings out of line and moves as the female pronucleus 
to a central position in the egg interior at about the same level on the long axis of 
the egg as the original oocyte. As the female pronucleus moves, it is transformed 
into an interphase nucleus surrounded by a membrane. The other three meiotic 
nuclei remain in their original positions as the polar bodies. However, they quickly 
follow the pronucleus into interphase. This transition starts in the other terminal 
nucleus a little before it does in the two medial ones. 

During the period when the meiotic divisions are taking place, the heads of such 
sperm as may be present (from 1-5), are seen as short Feulgen-positive rods (Figs. 
1 and 2), usually located in the deeper ooplasm of the same general level as the 
oocyte nuclei. Occasionally a more elongate, threadlike sperm head, such as normal 
motile sperm possess, was noted, but usually only the short rods were seen, even 
in the youngest eggs. Sperm tails were presumably present in the eggs but they 
did not stain and could not be detected. 

In near synchrony with the oocyte nuclei, all sperm heads in an egg were trans- 
formed into interphase nuclei. One of these nuclei moved to a position immediately 
adjacent to the female pronucleus to become the male pronucleus. Thus, by the 
end of about 9 minutes, only interphase nuclei were present in the eggs. When first 
formed, these nuclei have a diameter of about 5^ microns which increases to 12 mi- 
crons. Presumably during this 9-minute period cell synthesis takes place, involving 
among other things DNA replication. 

By the end of about 12 minutes, all nuclei, including the polar bodies and any 



332 



JOHN' G. R1F.MANN 



extra sperm nuclei that might he present, have entered into early prophase (Fig. 3). 
By 1415 minutes they have usually reached metaphase with the two pronuclei 
being included in a single unit to complete syngamy. The first cleavage division 
(Fig. 4) quickly follows and succeeding cleavages occur in rapid succession (Fig. 



t 





\ 



2 



s 

s \ 

\ . 



PN 






3 



FIGURE 1. Four-5-minute egg, telophase I. 
FIGURE 2. Seven-8-minute egg, telophase II. 

FIGURE 3. Eleven-12-minute egg, late prophase before syngamy. 
had disappeared from one of the pronuclei. 



The nuclear memhrane 



5). Approximately 5 minutes separate the first few cleavages, but the others occur 
somewhat less rapidly. By the end of one hour the eighth and final internal cleavage 
division is usually in progress. Thus, at 25-26 C. cleavage within the interior of 
the egg occupies a period of about 45 minutes, with the average division cycle lasting 
only about (> minutes. This rate is somewhat faster than the cleavage cycle of about 
9-10 minutes at 20-30 C. reported for Drosofihila (Sonnenblick, 1950) and 10 



KARLY DKYKLOPMKNT OF SCRKW-WORM FLY 



333 



minutes at 20 C. for Calliphora t'icina (R. and D. ) [erythrocephala > 

( A[elander, 1'tfo) and may represent the most rapid rate of mitosis reported for 

multicellular animals ( Mazia, 19(>1 ), although it would seem rather 

other higher Diptera develop as rapidly. 



PB 




CN 



I 



4 



PB 





FIGURE 4. Fifteen-16-minute egg, first cleavage telophase. 
FIGURE 5. Thirty-31 -minute egg, fourth cleavage metaphase. 

Observing cleavage nuclei was rather less satisfactory than observing meiotic 
ones. In part, the difference was due to the obstruction caused by the overlying 
ooplasm even after it had cleared, but in addition the cleavage nuclei appeared to 
stain less deeply. Again, individual chromosomes could not lie distinguished as a 



i . i||\ G. l< I KM ANN 

ruli 1 . Sometimes male and femaU chromosome complements remained somewhat 
separated during the first cle; '.sion, hut more usually they could not be 

recogni/ed after the\ had. , , the first metaphase figure. Viewing cleavage 

divisions \vas made easier 1>\ fact that they all occurred in a plane rather closeK 

parallel to the egg surface. 

The first cleavage ' .;. nail] in the area in which svngamv occurs. \\"itlt 

repeated divisions there is a -.pread of nuclei, and hv the end ot the eighth division 
they are rather evenly ited along the length of the egg. 

The somewhat slo ; \elocity of the later internal cleavages appeared to he 
associated with a ch ' in duration of the different stages of cell division. Very 
careful timing was rei|iiircd to obtain metaphase. anaphase, or telophase figures 
(hiring the first 2 3 cleavage divisions. On the other hand these stages were found 
more often than interphases or prophases in eggs fixed at various later times. All 
the first S cleavages are probably completely synchronous in the intact egg, but in 
the usual stained specimen a slight gradient ot development awav from the site of 
puncturing was noted. 

After the end of the eighth cleavage division in the interior of the egg, most 
nuclei have migrated near the surface of the egg to form the incipient blastoderm or 
blastema by the end of 1 hour and 10 minutes. After the blastema is first formed, 
four other cleavage divisions of most of the nuclei occur. The first of these divi- 
sions takes place shortly alter the nuclei complete their movement to the egg sur- 
face. The last division is usually underwav or completed by the end of the second 
hour. This cycle, lasting about 15 minutes, compares rather closely with the 17- 
minute cycle that Agrell (1963) and Melander (19()3) reported for the same four 
di\i.xions in ('. rvV/m/. After the 12th division a prolonged interphase takes place. 
at the end of which further divisions occur. During the long interphase period, 
cell membranes are formed around the nuclei located in the surface ooplasm. to be- 
come the definitive blastoderm. 

These last four cleavages are not completely synchronous like the first S, but 
instead appear to follow the pattern reported by Agrell ( 1<>(>3) for ('. t'icina. For 
the hrst three divisions there was a rather slight mitotic gradient from the anterior 
end of the egg. and also during the llth division some nuclei near the posterior end 
were observed to divide earlier than those more anterior. During the 12th division 
a definite mitotic gradient proceeding from both ends of the egg was noted (Fig. 6). 

Xot all ol the earlv cleavage nuclei migrate to the surface ooplasm to form the 
blastema. Some remain behind as the so-called yolk nuclei or vitellophags. These 
divide out of synchrony with the blastema nuclei, to form rather massive clusters of 
nuclei ( Fig. 7 \ by the time the 12th division is completed. No evidence was seen 
to indicate that anv ot the blastema nuclei migrate inward to increase the' number of 
yolk nuclei. 

Shortly before the 10th cleavage a cluster of nuclei appear outside of the blas- 

at the posterior end of the egg. These are the .so-called pole cells which pre- 

here as in other species, include' the primordial germ cells. In ('. t'iciini 

i actually only nuclei ) are also set off at about the time of the 10th divi- 

''. according to Soiineublick (1950), the first ones appear at the time 

vision in l^rosopliilti. Xo divisions were detected among the pole cell 

nuclei dii ii riods when the later cleavages occurred. 



EARLY DEVELOPMENT OF SCREW-WORM 

Melander (1963) described in great detail how the "pseudo-chiasmi ,ied 

during the 9-1 3th nuclear divisions in C. I'icina, result in chromosome diminution 
by causing the loss of small chromosome fragments. No attempt was made, to 
study possible chrosome diminution in the screw-worm. However, in tli< bl 



PC 



t 



~* . Jt* 



FIGURE 6. One hour and 59-60-minute egg. The nuclei at either end have gone into 
interphase while those in the central region are still dividing; 400 X. 

FIGURE 7. Two hour and 15-16-minute egg, showing the clumped yolk cells and the 
interphase nuclei of the blastema after the completion of the 12th division; 320 X. 

Explanation of captions: C N, cleavage nuclei; P B, polar bodies; PC, pole cells; P N, 
pronucleus ; S, sperm cell ; Y, yolk cells. 

many anaphases exhibited configurations that appeared to be similar to the pseudo- 
chiasmata described by Melander. Thus, it seems probable that the events in the 
two species are at least somewhat similar. 

As stated earlier, the polar bodies and surplus sperm nuclei of screw-worm egg> 
go into metaphase at the same time as the pronuclei and remain in this stage until 
they eventually disappear. The sperm nuclei can be seen during early cleavages 



336 



[OHN (.. RIEMANN 



I \BU I 

1 iif seuuen<f in tiif develop ..v-7ew/ eggs through blastoderm jonnution* 



Minutt 



i) 1 



l (, 
5-8 

8 l ' 



I. 1 I 



14-16 

29 SI 






Minutes 



Sperm mo\e into are 44-46 

which remains in e.irh meiotic 

anaph.ise I. 59-61 

Meiotic division 1 is completed. 
Meiotic division li i- c ompleted. 74-76 

All nuclei go into interphasu. 1'ro- 

nnclei move l< adjacent position in ')(! ( > 1 

tin- interior of the egg. 
All nuclei pass synchronously into 104-106 

metaphase. Syngamy occurs as 120-122 

the two pronuclei form a single 150 

figure. 

( 't 'inplel ii ni o| clea\ age I . 
Cleavage 1 1 1 in progress or completed, 
4-8 nuclei. 



Stage "I <lr\rlo|,iiirnt 



Cleavage V completed and division 

\ I may lie in progress; S2 nuclei. 
Cleavage \ III generally in progress; 

1 28 dividing nuclei. 
Blastema formed with division IX 

usually in progress. 
Division X in progress. I'ole cell 

nuclei set aside. 

Division XI usually in progress. 
I >ivision XII usually in progress. 
Cell membranes around surface nuclei. 

definitive blastoderm. 



* Oviposition .ii S5 ('., development after one minute at 24-26 C. 

but not during tin- later om-s. The polar bodies remain until the blastema is formed 
but then quicklv undergo dissolution. Very rarely we observed one of the haploid 
polar bodies dividing at the same time as the first cleavage nucleus. 

1. /'olys/n'riny in screw-worm c;/(/s 



It has lon^- been considered a general rule that in insects each egg is penetrated 
by several sperm ( \Yigglesvvorth, 1950). However, Hildreth and Lucchesi (1963) 
tound that, contrary to the observations of earlier workers, the eggs of Drosophila 
melanogaster and /). I'irilis usually recei\'ed only a single sperm. As a result of 
their studies, they raised the question as to how common polyspermy might actually 
be in insects. 

Many screw-worm eggs contained only a .single sperm, but more often than not 
they had two or more sperm. In no egg, however, were more than 5 sperm found 
and this number was quite unusual. Also, an inseminated fly often deposited a few 
eggs that had not been fertilized. The presence of these unfertilized eggs probably 
explains why somewhat les, than 100'; of the eggs from normally inseminated fe- 
males usuall hatch. 



TABLE II 

Distribution annul in u MI in file of 

I nlerlili/cd eggs 
Eggs containing one sperm 
Kggs i ontaining two sperm 
Kggs ( ontaining three sperm 
I '. CO ling lour sperm 

Potal numl >l vgs 

Total number of sperm 



e] 



2 

30 
39 

1 I 
.1 

88 
L62 



EARLY DEVELOPMKXT OF SCREW-WORM FLY 



337 



The distribution of sperm in 88 eggs from 10 different females is shown in Table 
II. All of these eggs were fixed before they were 8 minutes old, when the rodlike 
sperm heads are relatively easy to count. 

Occasionally a sperm nucleus was observed very near one of the polar bodies 
although in no instance had a second zygote actually been formed. However, the 
appearance of an occasional gynandromorph probably indicates that a second zygote 
is sometimes formed. 

3. Development of unfertilised eggs 

Virgin screw-\vorm females oviposit nearly as readily as inseminated ones, but 
their eggs apparently never hatch. However, young eggs in which no sperm could 
be detected often completed both meiotic divisions. To determine how far develop- 
ment advances in unfertilized eggs, whole-mounts of 8-20 eggs were prepared from 
each of 15 virgin flies. One portion of the eggs were fixed at 5^-7 minutes after 

TABLE III 

'L 'he development of eggs from virgin females 





Age at fixation 




5J-7 min. 


14-16 min. 


1 i-2 hrs. 


No. of females 


5 


6 


4 


No. of eggs 


53 


72 


60 


Aborted meiotic division I 


12 






Meiotic division I completed or in progress 


19 






Meiotic division II completed or in progress 


22 






Pronucleus not formed or did not migrate to interior of^egg 




35 


33 


Normal migration of pronucleus 




33 


24 


Cleavage I completed or in progress 




4 


3 


Cleavage II completed or in progress 











deposition, another portion at 14-16 minutes, and a third at \\-2 hours. To elimi- 
nate any possibility that dechorionation and other steps involved in the handling of 
the eggs might initiate development, these operations were limited to not more than 
5 minutes immediately prior to fixation. 

Results of observations on the stained preparations are shown in Table III. 
They demonstrate that fertilization is not strictly necessary for initiation of develop- 
ment. However, it is also evident that the presence of sperm in the eggs has some 
influence even on meiotic divisions, for nearly one-third of the nuclei fixed at 5^-7 
minutes had failed to complete the first division. It was also obvious that some of 
the other nuclei could not have gone through the second division. Even in eggs in 
which the two divisions were completed, the pronuclei had often failed to migrate 
into the interior of the egg. Thus, in over half the eggs fixed at 14 minutes or later, 
clumps of chromatin were found only at the site normally occupied by the three 
polar bodies. In some eggs, each of these clumps was observed to be composed of 
four smaller clumps representing all of the meiotic nuclei. Observations on other 
unfertilized eggs fixed at 8-9 minutes demonstrated that when meiosis was com- 



338 JOHN G. RIEMANN 

pleted, the oocyte nuclei went through the usual interphase stage before returning 
to metaphase. 

Scoring of the older unfertilized eggs was easier because development always 
stopped at a stage at which all nuclei were in metaphase. Apparently no nuclei had 
disappeared by the time the oldest preparations were fixed. 

The author wishes to express his appreciation to Dr. Leo LaChance, who sug- 
gested this study and helped in many ways during its progress. He also wishes to 
thank Miss Ann Leverich and Miss Sarah Bruns for their technical assistance. 

SUMMARY 

1. Development of screw-worm eggs from the first meiotic division to blasto- 
derm formation was studied from whole-mount preparations. Both meiotic divi- 
sions were completed by 7-8 minutes. Syngamy at 1415 minutes was quickly 
followed by the first cleavage division. The first 8 cleavages took place within the 
interior of the eggs, and the last of these occurred approximately one hour after egg 
deposition. After the 8th division, most cleavage nuclei moved near the egg sur- 
face to form the blastema by the end of about 1 hour and 10 minutes. This move- 
ment was followed by four more divisions of the blastema nuclei. The last of these 
divisions was underway, or had been completed, by the end of the second hour. 
There was then a prolonged interphase period, during which cell membranes formed 
around the blastema nuclei to become the definitive blastoderm. 

2. The first 8 cleavages were synchronous, but for the last four divisions an 
anterior-posterior gradient was evident. During the llth division, and especially 
during the 12th division, there was also an accompanying mitotic gradient proceed- 
ing from the posterior end of the egg. 

3. After the 8th cleavage some nuclei remained behind to form the yolk nuclei 
or vitellophags. Also, the pole cells were set aside after the 9th division but prior 
to the 10th. 

4. A low order of polyspermy was found in most eggs, but many of them re- 
ceived only a single sperm. Most unfertilized eggs completed at least the first 
meiotic division, but very few of them achieved the first cleavage division and none 
developed further than this stage. 

LITERATURE CITED 

AC-.KELL, IVAR, 1963. Mitotic gradients in the early insect embryo. Arkiv. Zool, 15: 143-148. 
HILDRETH, P. E., AND J. C. LuccHESi, 1963. Fertilization in DrosnphiJa. Dcrcl. BioL, 6: 

262-278. 
LACHANCE, L. E., AND A. P. LEVERICH, 1962. Radiosensilivity of developing reproductive cells 

in female CocliUoinyia hominivorax. Genetics, 47: 721-735. 
LACHANCE, L. E., AND J. G. RIEMANN, 1964. Cytogenetic investigations on radiation and 

chemically induced dominant lethal mutations in oocytes and sperm of the screw-worm 

fly. Mutation Res., 1: 318-333. 
MAZIA, DANIEL, 1961. Mitosis and the physiology of cell division. In: The Cell, Vol. Ill, pp. 

77-440. Edited by J. Brachet and A. E. Mirsky. Academic Press, New York and 

London. 
MELANDER, YNGVE, 1963. Chromatid tension and fragmentation during the development of 

Calliphora crythroccphala Mcig. (Diptera). Ilercditas, 49: 91-106. 



EARLY DEVELOPMENT OF SCREW-WORM FLY 339 

SCHMUCK, M. L., AND C. W. METZ, 1931. A method for the study of chromosomes in entire 

insect eggs. Science, 74: 600-601. 
SONNENBLICK, B. P., 1940. Cytology and development of the embryos of X-rayed adult 

Drosophila mclanogastcr. Proc. Nat. Acad. Sci., 26: 373-381. 
SONNENBLICK, B. P., 1950. The early embryology of Drosophila mclanogaster. In: Biology of 

Drosophila, Chapter 2, pp. 62-167. Edited by M. Demerec. John Wiley and Sons, 

New York. 
VON BORSTEL, R. C., 1955. Differential response of meiotic stages in Habrobracon eggs to 

nitrogen mustard. Genetics, 40: 107-116. 
VON BORSTEL, R. C., AND D. L. LINDSLEY, 1959. Insect embryo techniques. Stain Tech.. 34: 

23-26. 
WHITING, A. R., 1945a. Effects of X-rays on hatchability and on chromosomes of Habrobracon 

eggs treated in first meiotic prophase and metaphase. Amcr. Naturalist, 79: 193-227. 
WHITING, A. R., 1945b. Dominant lethality and correlated chromosome effects in Habrobracon 

eggs X-rayed in diplotene and in late metaphase I. Biol. Bull., 89: 61-71. 
WIGGLESWORTH, V. B., 1950. The Principles of Insect Physiology. Fourth ed. Dutton, New 

York. 



THE RELATIONSHIP OF SALINITY TO LARVAL SURVIVAL 

AND DEVELOPMENT IN NASSARIUS 

OBSOLETUS (GASTROPODA) 1 - 2 

RUDOLF S. SCHELTEMA 
Woniis Unit- OiV(/;i</</n//>/nV Institution, Woods Hole, Massachusetts 0-543 

Not until recently has the relationship of salinity to the survival and develop- 
ment of marine larvae been seriously examined through laboratory observations. 
The earliest studies upon mollusks were confined almost exclusively to the em- 
bryonic and very early shelled pelagic stages of pelecypods. Thus, Seno, Hori 
and Kusakabe (1926), Amemiya (1926), and Rao (1951) determined the effect 
of reduced salinities upon the early development of, respectively : Ostrea gigas and 
Crassostrca virginica: Ostrea angulata and Ostrea cdulis ; and Ostrea madrasensis. 
Because the early larvae of Crassostrca virginica survived salinities far lower than 
those found in the habitat of the adult, Clark (1935) concluded that the effect of 
salinity was unimportant in determining the mortality of oyster spat in Malpeque 
Bay, P.E.I., Canada. Turner and George (1955) showed experimentally that the 
larvae of Venus mercenaria would not swim past a salinity discontinuity of 20 and 
15 parts per thousand (%c~). The very interesting results of Haskin ( 1964) 
reveal a direct relationship between salinity and swimming activity of oyster 
larvae (Crassostrca virginica}. Haskin also demonstrated that light intensity 
and its spectral composition modify the response elicited from late "eyed" oyster 
larvae. Wells (1961) has compared the "salinity death points" of the adults and 
early larvae of two species of gastropods, TJiais floridana and Cerithium floridanuui. 
In the former, little difference between the "salinity death point" of the adult 
and larva was found, whereas in the latter species the "salinity death point" of the 
larva was at a higher salinity than of the adult. Not until the investigations of 
Davis (1958) upon Crassostrea virginica and Venus mercenaria and of Davis and 
Ansell (1962) on Ostrea edit I is have observations on the effect of salinity over the 
entire pelagic period of larval molluscan development been made. Stickney (1964) 
in addition has recently cultured Mya arenaria larvae and noted their response to 
salinity. No laboratory experiments on the relationship between salinity and 
growth in estuarine prosobranch gastropod veliger larvae have been published. 
Field studies on the effect of salinity upon survival and development of larval 
mollusks deal largely with commercially important species (Nelson and Perkins, 
1930; Carriker, 1951; Korringa, 1952; Kunkle, 1957; Haskin, 1964; etc.) and no 
attempt to summarize this work is made here. 

1 Contribution No. 1621 from the Woods Hole Oceanographic Institution, Woods Hole. 
Massachusetts. 

2 This research was supported in part by grants 17883 and GB-2207 from the National 
Science Foundation. I wish to thank my assistant. Mr. (Jonlon Enk, for his help during the 
conduct of sonic of the experiments described here. 

340 



RELATIONSHIP OF SALINITY TO LARVAL SURVIVAL 341 

A common species which inhabits the intertidal flats of estuaries along the east 
coast of North America from Chaleur Bay in the Gulf of St. Lawrence to northern 
Florida is the mud snail or basket shell, Nassarius obsoletus Say. The ecology, 
certain aspects of which have recently been reviewed (Scheltema, 1964), is rather 
well known and the pelagic larval development and early post-larval life history have 
been described (Scheltema, 1962a). The lower limits of salinity at which the 
adults of N. obsoletus are naturally found range between 15/cc and 20%o. 

Though much is known about this ubiquitous species, nothing has yet been 
reported on the effect of salinity upon survival and growth of the veliger larvae. 
I report here upon the results of some experiments with the larvae of N. obsoletus 
which (1 ) demonstrate the low r er limit at which salinity becomes lethal to both 
the larvae and adults, and (2) show the effect of reduced salinity on growth be- 
tween the time of emergence from the egg capsule to the completion of larval 
development. 

EXPERIMENTS ON THE LOWER LETHAL SALINITY FOR NASSARIUS OKSOLETUS 

The lethal salinity for a species may be determined either experimentally in the 
laboratory or from observations in the field. In the laboratory, organisms may be 
subjected to different salinities and their behavioral or physiological responses 
measured (e.g., Blum, 1922), while in the field unusual natural conditons, such 
as sudden changes in runoff, may, by large mortalities, show when salinity limits 
tolerated by a particular species have been surpassed (e.g., Beaven, 1946). 

A criterion to be used in the laboratory by which the effect of salinity upon an 
organism may be quantitatively measured is difficult to find. The methods adopted 
here were unsophisticated, but the results were reasonably reproducible. 

Salinity lethal to adult snails of N. obsoletus was determined by placing either 
15 or 20 organisms from a collection made on the intertidal flats in one of a series 
of four-liter tanks. Salinities in the series systematically descended in value from 
full-strength sea water to about 5% . Reduced salinities were obtained by diluting 
sea water with tap water. The resulting salinity in each tank \vas checked w r ith 
a hydrometer. The interval between tanks in the initial experiments was 5%o. In 
subsequent experiments this was reduced to 2%c as values approached the lethal 
limit of the organisms. All the experiments were performed at room temperature 
(ca. 20 C.). The animals were collected from a brackish- water estuary near the 
laboratory and held overnight at a salinity of lS%c before use. At the beginning of 
the experiment the snails were directly transferred to the salinity being tested. 
The effect of salinity was appraised by watching the behavior of the snails. Snails 
completely withdrawn into the shell were considered "inactive," whereas those not 
completely withdrawn were regarded as "active." At each observation the per- 
centage of active and inactive snails was recorded. 

The results of these simple experiments are shown in Figure 1. The graph 
illustrates that the region of stress lay between \2.S%o and 13.5% salinity. The 
results are after four hours, but when the experiments were extended over a period 
of three days the values do not differ significantly. A slight increase in the per- 
centage activity was evident, but the lower lethal limit was not markedly shifted, 
nor was the value at which stress was first observed altered. 



342 



RUDOLF S. SCHELTEMA 



The lower lethal limit of salinity among veliger larvae of N. obsoletus was 
determined by methods similar to those used for the adult snails. The criterion 
used to determine the effect of salinity upon the larvae was their swimming activity. 
Fifty larvae shortly after their emergence from the egg capsule were placed in 
400 ml. of sea water, the salinity of which was adjusted by the addition of tap water. 
The salinities tested ranged between 8.5% and 33.0% and were spaced at intervals 
of roughly 6%o in the earlier and 3%o in the later experiments. The temperature 
throughout was between 25 and 26 C., which is near the optimum for growth. 



100 r 



80 



jg 

<0 

^ 



60 



^40 



20 



10 20 

SALINITY % 



30 



FIGURE 1. Inhibition of activity in adult Nassariits obsoletus resulting from reduced 
salinity at about 20 C. The graph illustrates the percentage snails active following a four-hour 
exposure period. 



RELATIONS! I IF' OF SALINITY TO LARVAL SURVIVAL 



343 



100r 



80 



60 



<0 
^ 

kj 



40 



20 




10 20 

SALINITY % 



30 



FIGURE 2. Inhibition of swimming in larvae of Nassarius obsolctus resulting from reduced 
salinity at 25-26 C. The larvae are those taken shortly after their emergence from the egg 
capsule. The results are following a 10-hour exposure period. 

Larvae for all the experiments originated from water of 32% c salinity. At the 
end of 10 hours the dish containing the larvae was placed under a bright light and 
those swimming were counted. The results of these experiments are shown in 
Figure 2. The greatest decrease in the percentage of swimming larvae falls be- 
tween I4%o and 15.S%c salintity. Below 10%c no larvae were ever seen swimming 
after the 10 hours of exposure. 

The response of the late stage creeping-swimming larva of N. obsoletus was com- 
pared with that of the early veliger just after its emergence from the egg capsule. 



344 



RUDOLF S. SCHELTEMA 



These further experinirnts differed only in detail from those already described. 
Five-centimeter petri dishes we- re tilled with dilutions of sea water ranging from 
6.6%o to 33.0%o and in each, 10 veligers were placed. Into one sequence of 
dilutions were pipetted the early larvae; into the other, veligers which had com- 
pleted their development to the creeping-swimming stage. The latter were reared 
in the laboratory for 19 days at 24 C. according to the method already described 
(Scheltema, 1962a). 

TABLE I 

Activity indexes* of Xassarius obsolctus veliger larvae <;.s n function of salinity 



Early pelagic larvae 
(1-3 days after emergence from egg capsule; 



Salinity 


1 min. 


20 min. 


1 hour 


3 hours 


Mean for 3 hours 


6.6 





1 








0.3 


9.0 








1 


1 


0.5 


11.0 





1.5 


1 


2 


1.1 


13.2 





2 


2 


3 


1.8 


16.5 


1 


3 


3 


3 


2.5 


19.8 


3 


4 


4 


4 


3.8 


26.4 


4 


4 


4 


4 


4.0 


33.0 


4 


4 


4 


4 


4.0 



Creeping-swimming stage**' 



6.6 


0.0 




0.0 


0.0 


0.0 


0.0 


9.0 


0.4 




0.0 


0.0 


0.0 


0.1 


11.0 


0.4 




0.0 


0.0 


1.0 


0.4 


13.2 


0.0 




0.4 


0.2 


2.6 


0.8 


16.5 


3.0 




2.8 


3.4 


3.9 


3.3 


19.8 


3.0 




2.0 


3.8 


4.0 


3.2 


26.4 


4.0 




4.0 


3.9 


3.5 


3.9 


32.0 


4.0 


4.0 


3.9 


3.5 


3.9 



* The definition of this term is given in the text. 

** The values in these experiments were based on evaluation of each individual larva's per- 
formance, while those of the r.u-lv larvae were simultaneously estimated by assigning a value to 
all the larvae in the dish. 



The results of these experiments were expressed in terms of "activity indexes" 
which were recorded at the beginning of the experiment and after 20 minutes, one 
hour and three hours. The following numerical values were used to describe the 
responses of the larvae and to compute the "activity indexes": 0, no movement of 
velar cilia; 1, cilia of velum moving but not vigorously enough to allow the larva 
to swim; 2, larva moving sluggishly along the bottom and sides of dish or, if 
creeping-swimming stage, ilicn responding immediately to the touch of a pin; 3, 
actively swimming or creeping; and 4, very actively swimming or creeping. All 
examinations were made under bright light. 

Results of one such experiment are summarized in Table I. A mean activity 
index of between 2.5 and 4.0 indicates the "normal" range of behavior. The 



RELATIONSHIP OF SALINITY TO LARVAL SURVIVAL 



345 



activity indexes of the ne\v and old larvae as a function of salinity have been 
plotted together in Figure 3. This graph shows that there is no significant 
difference in the activity of the two ages of larvae relative to the salinity. A sharp 
decrease in activity occurred hetween 13.5',, and 16. S/^ salinity. Similar experi- 
ments of longer duration (38 hours) fully confirmed the results shown here. 



I 

X 

K 



fc: 
<o 
^ 







/ 



/ 



/ 



8 EARLY LARVAE 

'CREEPING SWIMMING 
LARVAE 



' 



10 20 

SALINITY % 



30 



Fiut'KK 3. Relationship of the "activity index" of early and creeping-swimming larvae of 
Nassarius nbsolctus to salinity. The graph illustrates the average "activity index" resulting from 
a series of observations over a period of three hours. 

EXPERIMENTS ON THE EFFECT OF SALINITY n-ox LARVAL GROWTH OF 

NASSARIUS OBSOLETUS 

The larval life of Nassarius obsoletus can be divided into two periods. The 
first of these is a phase of rapid growth and external development leading to the 
creeping-swimming or veliconcha stage (Scheltema, 1962a). During this period 
the growth rate is essentially constant (Fig. 4). This is followed by a second 
period of very slow growth and no apparent further external morphological change. 
The beginning of the second period is evident from the completion of the develop- 



346 



RUDOLF S. SCHELTEMA 



ment of the foot and from the behavior of the larvae, namely, frequent creeping on, 
and inspection of, the bottom . 

The length of the first period is determined by those conditions which control 
larval growth. The length of the second period varies greatly, at least two-fold in 
N. obsolctus, and depends upon the encounter by the larva of a sediment suitable 
for post-larval life (Scheltema, 1961 ). Under favorable conditions metamorphosis 
may occur very near the beginning of the second period. The total length of 



700 r 




>100% MORTALITY 



O LARVAE GROWN AT 32.9 % 

A LARVAE GROWN AT 24.2% 

LARVAE GROWN AT 20.9% 

LARVAE GROWN AT 17.7% 



6 9 12 

TIME IN DAYS 



15 



18 



FIGURE 4. Increase in length of Nassarius obsolctus veliger larvae from the time of 
emergence from egg capsules to the completion of their development to the creeping-swimming 
stage. This is the first pelagic phase, during which growth occurs at a rapid and nearly 
constant rate. Each curve shows the result at a different salinity as indicated by the 
conventions. 



RELATIONSHIP OF SALINITY TO LARVAL STRYIVAL 



347 



larval life consequently is not determined solely by the growth rate, but also by the 
opportunity for metamorphosis upon a favorable substratum. In investigating the 
effect of salinity on the larvae, I have confined my attention only to the period of 
rapid and constant growth rate. 

Laboratory experiments on the growth rate of veliger larvae are possible only 
after techniques for their mass culture are worked out (Scheltema, 1962a). The 
veliger larvae of N. obsolctus were grown in a series of 10-liter vessels with 
salinities ranging from that minimal for survival to that of full-strength sea water. 
It was soon found that the larvae held at a salinity of less than \6%o did not 
survive more than a few days. Consequently the minimum average salinity in the 
experiment reported here was \7.7% and the remainder of the series had salinities 
of 20.9%c, 24.2% , 26.2% and 32.8% c . All larval cultures were fed the euryhaline 
diatom, Phaeodactylum tricornutum, from the same algal culture. 

TABLE II 

Length in microns of Nassarius obsoletus throughout larval development 
as a function of the salinity 



Mean 
salinity 
%o 


Age in days 





3 


6 


9 


12 


15 


18 


17.7 


278 4* 


291 4 


363 8 


429 7 











20.9 


278 4 


312 6 


401 6 


433 7 


468 6 


501 6 


552 8 


24.2 


278 4 





458 7 


492 8 


556 9 


613 10 


665 10 


26.2 


278 4 





447 6 


487 9 


543 8 


586 6 





32.9 


278 4 


343 4 


446 5 


502 8 


615 8 


643 8 


683 9 



* One standard error is indicated on all values of this table. 

The mean temperature of the veliger cultures during this experiment, which 
extended over a period of 18 days, was 23.1 C. The maximum difference in 
water temperature measured during the course of the experiment was 1.1 C. 
However, since all cultures were kept together in the same temperature bath, the 
same fluctuations were experiencd by all. Maximum temperature differences 
between cultures were 0.5 C, but the mean difference was only 0.2 C. 

At the lower values, the salinity in the cultures never varied more than 0.5%c 
from the mean ; in cultures at salinities higher than 24%o, the maximum deviation 
from the mean value never exceeded 0.7%c. Growth of the larvae was deter- 
mined by measuring the maximum shell dimensions at three-day intervals with 
an ocular micrometer at a magnification of 100 X. This measurement, hereafter 
termed length, was made on an aliquot of 30 larvae from each culture. Previous 
measurements have shown this to be an adequate sample size. 

The results of the experiment (Table II) show that among salinities above 
24% c there is no statistically significant difference in length. As the salinity 
reached 2l.Q% c , the difference becomes significant. Below 21.0% C , the data indicate 
substantial decrease in shell growth rate. Under the conditions of the experiment, 
completion of larval development to metamorphosis did not occur at a salinity of 
177V 



.us 



RUDOLF S. SCHELTEMA 



The pertinent data are summarized by means of growth curves in the graph 
of Figure 4. Here the vertical lines indicate two standard errors. The difference 
between the two curves above 2\%c with those of 2\% c and less is quite evident, and 
the statistical significance is conspicuous. 

The results of two additional experiments, similar to the one just described 
above, together with the data from the first experiment, are summarized in Table 
III. In each of the additional experiments, two cultures of veligers were started 
simultaneously with larvae obtained randomly from the same collection of egg 
capsules. The initial size of the larvae in the two cultures of an experiment were 
consequently the same. Development previous to emergence of larvae from the 

TABLE 1 1 1 

Percentage inhibition of growth in Nassarius obsoletus larvae resulting from the lowest salinity 
at which development is completed to metamorphosis 



Expt. 
no. 


Temp, 
range 
C. 


Age of 
larvae 
at end 
of expt. 


Length 
n at 
begin 
expt. 


A 


B 


Percent- 
age inhi- 
bition 
(I) 


Mean 
salinity 


Length 
n at end 
expt. (A) 


Total 
growth* 

(A A) 


Mean 
salinity 
%o 


Length 
/i at end 
expt. (B) 


Total* 
growth 
(AB) 


1 
II 
III 


23.0-24.0 
19.5-21.3 
26.1-28.0 


18 
19 
12 


278 

270** 
249 


32.9 
33.1 
33.2 


6839*** 
7014 

5455 


405 
431 
296 


20.9 
21.5 
21.3 


5528 

634 6 
504 8 


274 
364 
255 


32.1 
15.5 

13.8 



Sum 
Mean 



61.4 
20.5 



* This denotes the difference between the initial length at the time of emergence from the 
egg capsule and the length at the end of the experiment. 

** In this experiment only the length of the larvae at the termination of the experiment is 
known; the assumed length of 270 /* is the usual length of larvae at the time of emergence from 
the egg capsule. However, even if extreme values are assumed, the percentage difference in the 
final column is altered by no more than 1%. 

*** One standard error is indicated. 

egg capsules was at room temperature. The unusually small initial size of the 
larvae in experiment III is a peculiarity of that particular collection of egg capsules 
and is probably related to the time at which they were deposited within the breeding 
cycle of the female snails. 

In one of the two cultures in each experiment, larvae were grown at the 
salinity of normal sea water. This culture is designated as "A" in each of the 
experiments of Table III. In the other culture, designated "B" in Table III, the 
larvae were grown at a reduced salinity near 2\%o. Both cultures in each experi- 
ment were terminated at the same time. This was done near the end of the period 
of constant growth rate in the high salinity culture "A" of each experiment. 

The percentage inhibition, /, due to the reduction of salinity is shown for each 
experiment in the right-hand column of Table III and was computed by the 
relationship 



A A 



x 



RELATIONSHIP OK SALINITY TO LARVAL SURVIVAL 

where &A is the change in length of shell between the beginning and end of the 
experiment among larvae maintained at sea-water salinities, and A# is the change 
in shell length of larvae held at a minimum salinity required for completion of 
development. Table III shows that maximum inhibition was obtained at a tem- 
perature range of between 23 and 24 C, which is near the optimum for growth 
of the larvae of N. obsolctus (Scheltema, 1963). At both higher and lower tem- 
perature ranges there \vas substantially less inhibition in growth. However, owing 
to the large differences in larval growth rate frequently obtained between experi- 
ments, a direct effect or interaction of temperature on growth inhibition by reduced 
salinity cannot be assumed without further experiments. The results show that 
at the lowest salinity at which development to metamorphosis was completed, an 
average of 20.5% inhibition of growth occurred. 

DISCUSSION 
Salinity as a limiting factor to distribution 

The upstream distribution of most organisms that live within estuaries is seem- 
ingly related to salinity. However, mere correspondence between salinity values 
and the distribution of a particular species cannot in itself be taken as sufficient 
evidence that salinity is limiting. To show this, it is necessary to discover the 
extremes tolerated by an organism throughout its life history. 

Thorson (1946, p. 472) has suggested that the larval stage may limit the dis- 
tribution of bottom species, as this stage is "the weakest link of the chain." Experi- 
ments upon the salinity lethal to larval and adult N. obsoletus, however, showed 
no large difference between their lower tolerances. Such differences which did 
appear might be accounted for by the previous acclimation of the adult snails to a 
lower salinity or by the inadequacy of the techniques in making such small dis- 
tinctions. The lower lethal salinity did not change significantly as larval develop- 
ment progressed (Figs. 2 and 3). 

The known upstream distribution of N. obsoletus into estuaries in many in- 
stances seems to correlate well with the lower lethal salinity determined in the 
laboratory. Hence, Pfitzenmeyer (1961) found the species at locations in Chesa- 
peake Bay where the summer salinity was 14.6/ce. On the other hand, in certain 
estuaries on Cape Cod snails do not ascend farther up than a summer bottom 
salinity of \7%o, and in such instances salinity is probably not the factor limiting 
distribution. In order to make valid comparisons, laboratory results must be 
related to bottom salinities in areas where the seasonal extremes are known. 

Salinity and larval growth rate 

Growth of N. obsoletus is inhibited only as the lower limit of salinity tolerance 
is approached (Scheltema, 1962b). Thus, it was not until the salinity was near 
2Q% that any significant effect on growth rate was noticed. Although not directly 
investigated, the decreased rate of growth in N. obsolctus is not likely to be re- 
lated in any simple way to osmotic activity because marine mollusks, insofar as 
known, have no active osmotic control involving the expenditure of energy 
(Prosser et al., 1961 ). The mortality of larvae at salinities below 20%o was high. 



350 



RUDO1 }' S. SCHELTEMA 



No larvae survived beyond the intermediate stage of development in laboratory 
culture, although growth proceeded up until the ninth day at 17 ' ,7%o salinity. The 
inhibition of growth attributable to the reduction of salinity amounted on the 
average to about 20%. 



100 



80- 



60- 



40- 



Nassarius Obsoletus 




60- 



40- 



20- 



Venus Mercenaria 



, 



30 



! 

35 



SALINITY % 



FIGURE 5. Percentage growth obtained at various reduced salinities relative to the 
maximum growth obtained at optimal salinity conditions in the gastropod, Nassarius obsolctu? 
Say and pelecypod, Venus mcrccnaria L. Data for V '. mcrccnaria are from Davis (1958), p. 301, 
Fig. 1. Growth data for each species were used only if more than 5% of the larvae in the 
culture completed development to metamorphosis. The data refer to 15 days after the beginning 
(jf planktotrophic life in N. obsolctus (i.e., creeping-swimming stage) and 12 days after 
fertilization in V. mcrccnaria. The different conventions indicate values for individual series 
of experiments. The curves are only intended to he suggestive. 



RELATIONSHIP OF SALINITY TO LARVAL SURVIVAL 



351 



TABLE IV 

Maximum differences of larval growth rate attributable to various ecological factors 
(Average values of I from experimental data} 





N. 


obsoletus 


V. 


mercenaria 



Physical characteristics 



Temperature 

Range within which development is completed 
Average difference in growth rate between optimum and 
minimum required for complete development 

Salinity 

Range within which development is completed 

Average difference in growth rate between optimum and 

minimum required for complete development 
Extreme difference in growth rate observed between opti- 
mum and minimum required for complete development 



17.5 to 30 C.* 



50%* 



20% 
13 to 40% 



18 to 30 C.** 
60%** 



M5.0V** 

34%*** 
22 to 45%*** 



Biological characteristics 



ca. 50% 
(preliminary 
estimation) 



17.7%**** 
51.2%**** 



29.7%****f 



Up to 75%ft 



Concentration of algal food 

(Differences between optimum and minimal growth 
obtained at concentrations between 2.5 X 10~ 3 and 
40 X 10~ 3 mm. 3 packed cells per 3-1. culture) 

Isochrysis galbana 

Monochrysis lutheri 

Chlorella sp. (causes inhibition of growth and death of 
larvae at highest concentrations) 

Phaeodactylum tricornutum (data from relative concentra- 
tions only) 

Species of algal food 

(based on 10 species of or combination of species when equal 
packed cell volumes were used) 

* Scheltema (1963), p. 17, Fig. 2. 

** Loosanoff et al. (1951), p. 71, Table III; Loosanoff (1959), p. 315, Fig. o. 
*** Davis (1958), p. 301, Fig. 1. 
**** Davis and Guillard (1958), p. 302, Fig. 6. 

f This figure represents difference in growth at concentrations between 2.5 X 10~ 3 and 
20 X 10~ 3 mm. 3 per 3-liter culture. At higher concentrations larvae did not survive. 
ft Davis and Guillard (1958), p. 298, Fig. 3; p. 299, Fig. 4. 

Because differences in the nutritional value of the algal food cells used in 
growth experiments are not readily controlled (Walne, 1963), it is not possible 
to compare the results from one series of experiments directly with the next without 
elaborate experimental procedures. A direct comparison between most series of 
growth experiments usually shows large discrepancies. Only cultures of larvae 
simultaneously grown using the same source of algal food can be directly com- 
pared with one another. It is possible, however, to compare differences in com- 
puted growth rates. Likewise the per cent inhibition, I, computed from the equa- 
tion given above, can be directly compared if the differences in length are derived 



352 Kl'DOI.K S. SCHELTEMA 

from samples of similar size. Using this kind of information it is also possible to 
compare the relative importance of reduced salinity to growth between species of 
mollusks. In Figure 5 this has been done for two species which show similar 
distributions within estuaries along the Atlantic coast of the United States : the 
data of Davis (1958) on the growth of larvae of the pelecypod, Venus mercenaria, 
are compared with growth data from the larvae of N. obsoletus. The conclusion 
may be made that salinity little affects larval growth in either species until the 
lower limit of salinity tolerance is approached. Only the lower third of the 
salinity range has any marked inhibitory effect on growth and there is no simple 
linear relationship between growth and the salinity level. The similarity in 
response of the two species, V . mercenaria and N. obsoletus, is striking. 

The importance of salinity relative to other factors affecting larval growth 

An indication of the relative importance of salinity to larval growth can be had 
by comparing its maximum effect relative to that of some other factors known to 
control growth rate of N. obsoletus and V '. mercenaria. By tabulating the values for 
maximum percentage difference obtained from that of optimum growth, the im- 
portance of various ecological factors in limiting growth rate becomes apparent. 
This is shown in Table IV. Here the range within which completion of develop- 
ment occurs is given for physical characteristics of the environment, and the 
percentage values are average maximum differences in growth attributable to these 
physical factors. Differences in growth rate under different biological conditions 
for which data are available, viz. concentration and species of algal food, are given 
within the limits of concentrations indicated. The maximum inhibition of growth 
varies with the algal species. On the basis of the figures given, the concentration 
and the species of algal food usually affect growth rate of V . mercenaria much 
more than any of the physical factors of the environment. There is preliminary evi- 
dence that this is also true for N. obsoletus larvae. The food value of algal cells 
to mollusk larvae in relation to the conditions under which the algal cells were 
grown is not yet known (see Walne, 1963). The table shows that the importance 
of low salinity in inhibiting growth rate, within the limits in which development 
of the larvae is completed, is certainly minimal, less than any other factor in the 
environment known to retard growth of the larvae. 

This work is dedicated to the memory of G. Francis Beaven, who in his quiet 
way first interested me in the relationship between salinity and the distribution 
and survival of estuarine organisms. 

SUMMARY 

1. There is no large difference between the lower lethal salinity for the veliger 
larva and the adult of Nassarins obsoletus. The region of stress in snails is be- 
tween I2.5%o and 13.5'vr. ; that of the early larva is between 14.0/c and I5.5%o. 
Throughout larval development no change occurred in the value of the lower 
lethal salinity. 

2. Difference in growth rate of X. ohsolctus larvae observed at salinities above 
24 r / ( ( are usually slight. However, at 21 ( ,< ( and less there is a statistically sig- 



RELATIONSHIP OF SALINITY TO LARVAL SURVIVAL 

nificant drop in growth rate, while at a mean salinity of \7.7% , it was not pos- 
sible to rear the larvae to the completion of development and metamorphosis. The 
maximum inhibition of growth attributable to the affects of salinity, within the 
range at which development of the larvae is completed, is between approximately 
13% and 40% and averages about 20%. This is less than that of other ecological 
factors known to retard growth. 

3. The net result of reduced salinity, within the lower third of the range at 
which the larval development of N. obsoletus is completed, is an increase in the 
length of time to reach the creeping-swimming stage which precedes metamorphosis, 
and an increased mortality of larvae as the limit of salinity tolerance is reached. 

4. A comparison of data from N. obsoletus with that of another molluscan 
species, the pelecypod Venus mercenaries, which is found at approximately the same 
salinities within Atlantic coast estuaries along the United States, shows striking 
similarities. 

LITERATURE CITED 

AMEMIYA, I., 1926. Notes on experiments on the early developmental stages of the Portuguese, 

American and English native oysters, with special reference to the effect of varying 

salinity. /. Mar. Biol. Assoc., 14: 161-175. 
BEAYEN, G. F., 1946. Effect of Susquehanna River stream flow on Chesapeake Bay salinities and 

history of past oyster mortalities on upper bay bars. Proc. Nat. Shellfish. Assoc.. 

37:38-41. 
BLUM, H. F., 1922. On the effect of low salinity on Teredo navalis. Univ. Cat. Pub. Zool, 22 : 

349-368. 
CARRIKER, M. R., 1951. Ecological observations on the distribution of oyster larvae in New 

Jersey estuaries. Ecol. Monogr., 21 : 19-38. 
CLARK, A. E., 1935. Effects of temperature and salinity on early development of the oyster. 

Prog. Kept., Atl. Biol. St., St. Andrews, N. B., No. 16 : 10. 
DAVIS, H. C, 1958. Survival and growth of clam and oyster larvae at different salinities. 

Biol. Bull, 114:296-307. 
DAVIS, H. C., AND A. D. ANSELL, 1962. Survival and growth of larvae of the European oyster. 

O. ednlis, at lowered salinities. Biol. Bull, 122: 33-39. 
DAVIS, H. C., AND R. R. L. GUILLARD, 1958. Relative value of ten genera of microorganisms 

as food for oyster and clam larvae. Bull. Fish and Wildl. Ser., 58: 293-304. 
HASKIN, H. H., 1964. The distribution of oyster larvae. Symposium on Experimental Ecology, 

Narragansett Mar. Lab., Grad. School Oceanogr., Univ. of R. L, Occ. Pub. No. 2. 
KORRINGA, P., 1952. Recent advances in oyster biology. Quart. Rev. Biol., 27: 266-308. 
KUNKLE, D. E., 1957. Vertical distribution of oyster larvae in Delaware Bay (Summary). 

Proc. Nat. Shellfish Assoc., 48: 90-91. 
LOOSANOFF, V. L., 1959. The size and shape of metamorphosing larvae of Venus (Mercenaria) 

mcrcenaria grown at different temperatures. Biol. Bull., 117: 308-318. 
LOOSANOFF, V. L., W. S. MILLER AND P. B. SMITH, 1951. Growth and setting of larvae of 

Venus merccnaria in relation to temperature. /. Mar. Res., 10: 59-81. 
XELSON, T. C., AND E. B. PERKINS, 1930. The reaction of oyster larvae to currents and to 

salinity gradients. Anal. Rec., 47: 288. 
PFITZENMEYER, H. T., 1961. Benthic shoal water invertebrates from tidewater of Somerset 

County, Maryland. Chesapeake Sci., 2: 89-94. 
PROSSER, C. L., AND F. A. BROWN, JR., 1961. Comparative Animal Physiology. W. B. Saunders 

Co., Philadelphia, ix + 688 pp. 
RAO, K. V., 1951. Observations on the probable effect of salinity on the spawning development 

and setting of the Indian backwater oyster, Ostrea madrasensis Preston. Proc. Indian 

Acad. Sci., 33B : 231-256. 

SCHELTEMA, R. S., 1961. Metamorphosis of the veliger larvae of Nassariits t >bs!ctns (Gastro- 
poda) in response to bottom sediment. Biol. Bull., 120: 92-109. 



354 RUDOLF S. SCHELTEMA 

SCHELTEMA, R. S., 1962a. Pelagic larvae of New England interticlal gastropods. /. Nassaritts 

obsolctus Say and Nassarnis vibcx Say. Trans. Amcr. Microsc. Soc., 81: 1-11. 
SCHELTEMA, R. S., 1962b. Environmental factors affecting length of pelagic development in the 

gastropod, Nassarius obsolctus. Amcr. Zoo!., 2: 445. 
SCHELTEMA, R. S., 1963. Larval development. Summary of Investigations conducted in 1962, 

Woods Hole Oceanographic Institution Ref. 63-18,, pp. 15-19. (Unpublished manu- 
script.) 
SCHELTEMA, R. S., 1964. Feeding and growth in the mud-snail Nassaritis obsolctus. Chesapeake 

Sci.,5: 161-166. 
SEND, H., J. HORI AND D. KUSAKABE, 1926. Effects of temperature and salinity on the 

development of the eggs of the common Japanese oyster, Ostrea gigas Thunberg. 

/. Fish. Inst. Tokyo, 22: 41-47. 
STICKNEY, A. P., 1964. Salinity, temperature and food requirements of soft-shell clam larvae 

in laboratory culture. Ecology, 45: 283-291. 
THORSON, G., 1946. Reproduction and larval development of Danish marine bottom invertebrates 

with special reference to the planktonic larvae in the Sound (0resund). Medd. Komm. 

Damn. Fiskcriog Ilavundcrs., Ser. Plankton, 4: 1-523. 
TURNER, H. J., AND C. J. GEORGE, 1955. Some aspects of the behavior of the quahaug, Venus 

incrccnaria, during the early stages. Eighth Rept. Inv. Shell Fish. Massachusetts, 

pp. 5-14. 
WALNE, P. R., 1963. Observations on the food value of seven species of algae to the larvae of 

Ostrea cdulis. J. Mar. Biol. Assoc., 43: 767-784. 
WELLS, HARRY W., 1961. The fauna of oyster beds, with special reference to the salinity 

factor. Ecol. Monogr.. 31 : 239-266. 



PHYSIOLOGICAL SALT SOLUTION FOR THE LAND CRAB, 

GECARCINUS LATERALIS 

DOROTHY M. SKINNER, DONALD J. MARSH AND JOHN S. COOK 

Department of Physiology and Biophysics, Nezv York University Medical Center, New York, 
Neiv York 10016, and The Lerncr Marine Laboratory, Bimini, Bahamas 

The land crab, Gecarcinus later alls, is an active responsive animal until it 
approaches ecdysis ; in the few weeks before and after ecdysis, however, the animal 
becomes lethargic (Bliss, 1962). These variations in activity may well be related 
to the marked changes in the metabolism of somatic muscle during molting (Skinner, 
1962; 1963a, 1963b). In order to investigate the physiology of this muscle it has 
first been necessary to develop a Ringer's solution appropriate to the animal. This 
paper describes analyses performed on Gecarcinus serum. Observations on osmo- 
regulation are included. From these data a Ringer's solution has been devised 
and tested. 

MATERIALS AND METHODS 

1. Animals 

Some specimens of Gecarcinus lateralis were collected in the field at Bimini 
and were used immediately at the Lerner Marine Laboratory. Other specimens 
were shipped from the Bermuda Biological Station to New York and were housed 
in covered aquaria containing sand moistened with tap water. A fingerbowl of sea 
water was available in each tank ; where indicated, tap water was substituted for 
sea water. 

2. Preparation of blood serum 

Blood was collected from the cut appendage of an animal which had been 
acutely chilled to prevent autotomy of the cut limb. The clot was mechanically 
disrupted and sedimented by centrifugation. 

3. Osmolality, sodium, potassium and chloride 

Immediately after preparation, the osmolality of the serum was measured in a 
Fiske osmometer. Concentrations of sodium and potassium in the serum were de- 
termined by standard flame-photometric methods, using LiCl as an internal 
standard. 

Chloride was determined by the Cotlove titrimetric method (Cotlove et al.. 
1958). Initial attempts to determine chloride concentration of untreated blood 
serum led to results varying by as much as 15% on five replicate samples. Since the 
protein concentration of Gecarcinus serum is high and variable (2% to 10%, 
unpublished data), we thought that protein might be interfering with the analyses. 

355 



SKINNER, MARSH AND COOK 

Consequently, the protein precipitated by the nitric-acetic acid reagent used in the 
analysis was homogenized to free any trapped chloride and removed by centrifuga- 
tion. Ali(|uots of the supernatant were used for the titration. This procedure 
reduced the variability between replicate samples to less than 2%. 

4. Calcium and magnesium 

(a) Preparation and characterization of an ultrafiltrate. Blood was ultrafiltered 
to obtain a value for free calcium and magnesium without including divalent ions 
associated with proteins. Three-inch dialyzer tubing (average pore diameter 48 A) 
was cut along its edge, giving a piece 6 inches wide. This was shaped into a sack 
and inserted into the top of a 12-ml. conical centrifuge tube. One to 2 ml. of blood 
were introduced into the sack which was then tightly stoppered and centrifuged. 
The first fluid collected after bringing the centrifuge to speed was set aside as 
possible condensate from the tubing. TCA was added to each of these initial 
collections. In the rare event that any precipitate formed (indicating the presence 
of protein and hence a leak in the system), the sample was transferred to another 
dialysis sack. The tubes were then spun at 3000 rpm in a model CM International 
centrifuge for two hours. Heating was prevented by packing the drive shaft of 
the centrifuge in dry ice during the centrifugation. The rate of ultrafiltration was 
about 0.025 nil. hr.' 1 ml. serum' 1 . The ultrafiltrate obtained was colorless and 
contained no more than .06% protein (as determined by the method of Lowry et al, 
1951), representing about \% of the protein initially present in the serum. Within 
the limits of experimental error, the alkali metal concentrations in the ultrafiltrate 
were the same as those in whole serum. Since the small correction for serum water 
would be opposite to that applied for the Donnan equilibrium, the similarity was an 
expected result, and indicated that there was no significant evaporation of the 
ultrafiltrate during preparation. 

(b) Assay method. The dye Eriochrome Black T is pink when chelated to 
divalent cations and blue when free in solution after the cations have been removed 
by a stronger chelating agent. With this dye as an indicator, the sum of calcium 
and magnesium was titrated with EDTA at a basic pH in the presence of cyanide 
( Ames and Nesbett, 1958). Calcium alone was determined titrimetrically on sepa- 
rate aliquots of each sample, using 2-hydroxy-l-(2-hydroxy-4-sulfo-l-naphthyl-azo)- 
3-naphthoric acid (HHSNN, Fisher Scientific Company) as indicator and EGTA 
ictliylene glycol bis ( /^-aminoethyl ether )-N, N-tetraacetic acid) as the titrant 
i : Weber and Her/., 1963). Magnesium was obtained by subtracting the calcium 
value from the total. Standard curves were run with each set of experimental 
samples. 

5. Sul fate 

Proteins were precipitated from serum with perchloric acid (0.7 M final concen- 
tration). The supernatant was neutralized with KOH and the concentration of 
inorganic sulfate measured according to the method of Jones and Letham (1956V 
In four experiments, where- known amounts of sulfate were added to crab serum. 
101.5% of the added sulfate was recovered. 



LAND CRAB SERUM ELECTROLYTES 



357 



6. pH, pCOt, pO*, and bicarbonate 

Blood was collected by immersing a cut appendage below the surface of paraffin 
oil saturated with water. The clot was mechanically disrupted and the serum 
transferred anaerobically to a cuvette housing a Clark polarographic O 2 electrode, 
a Severinghaus CCX electrode, and a glass pH electrode, all of which were read out 
by means of a Beckman model 160 gas analyzer. The pH of any sample which 
differed significantly from the others was measured independently with a Radio- 



300 



450 



U) 



400 





o 



330 



I 



300 



No*-. 




700 800 900 

MILLIOSMOLES/KO. HO 



1000 



FIGURE 1. Sodium and chloride as functions of osmolality in the serum of Gecarcinus 
latcralis. Sodium data: closed circles; chloride data: open circles. Regression lines were 
fitted to the data by the method of least squares. 

meter pH meter ; in such cases the two readings always checked within 0.02 pH unit. 
In addition pH measurements were made after equilibration of samples with varying 
concentrations of CO 2 in air, in order to obtain the apparent pK. With this pK and 
the measured pH and pCO 2 , the bicarbonate concentrations were calculated. 

7 '. Inorganic phosphate 

Protein was precipitated from serum with TCA (trichloroacetic acid) at a 
final concentration of 5%. Inorganic phosphate was determined by the method of 
Fiskeand SubbaRow (1925). 



358 



SKINNER, MARSH AND COOK 



RESULTS AND DISCUSSION 
1. Osmolality, sodium, potassium and chloride 

The osmolality of Gecarcinus serum varied from 610 to 1060 mosm/kg. H 2 O, 
depending on environmental conditions. Sodium varied from 310 to 480 meq/L. 
and, in any given animal, accounted for approximately one-half the total osmolality 
(Fig. 1). Chloride, the principal serum anion, was also linearly related to the 
osmolality hut was present at concentrations about 35 meq/L. less than the sodium 
in the 12 sera analyzed for both ions. 

Serum potassium varied from 7 to 15 meq/L. Figure 2 shows that the potas- 
sium concentration also tended to vary with osmolality, but in this case the data were 
proportionately more scattered and the interdependence was not as evident until 
we obtained the data on osmoregulation described below. 



13 



ul 



13 

'in 
co 



o 

CL 



10 



1000 



600 700 800 900 

MILLIOSMOLES/KG. HgO 
FIGURE 2. Potassium as a function of osmolality in the serum of Gecarcinus lateralis. 



During the initial phases of this work at Bimini, 21 animals sampled immediately 
after they were caught in the field had an average serum sodium of 369 28 ( S.D.) 
meq/L., while 8 animals kept on moistened sand (but with no other source of water) 
for 48 hours before sampling had an average serum sodium of 456 26.3 (S.D.) 
meq/L. This difference was highly significant and prompted further investigation 
of the effects of environmental conditions. 

In Bimini, Gecarcinus burrows in the sand some distance from the sea in an 
area where the ground water is salty. Animals shipped to New York have, 
shortly after arrival, a serum osmolality of approximately 830 mosm/kg. H..O. 
A group of animals was kept from arrival in an aquarium with sea water x avail- 
able in the water bowls ; after several weeks a blood sample was taken for osmolality, 
sodium, potassium and chloride determinations. The animals were replaced in tanks 

1 The sea water used was obtained from the New York Aquarium (Coney Island) and had 
the following measured composition (in meq/L.) : Sodium, 375; potassium, 8.1; chloride, 460; 
magnesium, 79.0 ; calcium, 16.4 ; osmolality = 860 mosm/kg. H 2 O ; salinity 27.\%c. 



LAND CRAB SERUM ELECTROLYTES 



359 



with tap water in the bowls, and after 8 days (three animals) or 27 days (two 
animals), the hlood collections and determinations were repeated. The results 
(Table I) show that all four parameters decreased when only fresh water was 
available, and that the decrease was greater after the longer exposure. These ex- 
periments show that potassium, as well as sodium and chloride, does vary in the 
same sense as the osmolality, a relationship not readily seen from the data in 
Figure 2. 

2. Calcium and magnesium 

The mean value for free calcium in serum water of intermolt animals was 
17. 2 2.4 (S.D.) meq/L. ; the mean value for free magnesium was 13.8 2.2 
(S.D.) meq/L. (Table II). 



TABLE I 

Osmolality, sodium, potassium and chloride of Gecarcinus serum in animals given access 
to sea water only for several weeks (I) and thereafter given access to tap water only (II) 



Animal 


Days of access 
to tap water 


Osmolality 
(mosm/kg. HzO) 


Sodium 
(meq./L.) 


Chloride 
(meq./L.) 


Potassium 
(meq./L.) 


1 


8 


I 1000 


467 


435 


13.0 






II 890 


423 


385 


10.8 


2 


8 


I 965 


456 


414 


11.7 






II 890 


438 


410 


10.5 


3 


8 


I 990 


456 


422 


11.1 






II 960 


452 


415 


11.3 


4 


27 


I 948 


430 


396 


11.2 






II 825 


388 


362 


8.0 


5 


27 


I 1058 


457 


418 


11.5 






II 895 


430 


385 


10.0 



These values are distinctly lower than those found by Gross (1963) for 
Gecarcinus. The difference probably reflects the fact that Gross dialyzed the serum 
against distilled water (for his method, see Gross, 1959), a procedure which 
would be expected to release cations normally bound to protein. 

We found that two premolt animals had calcium concentrations higher than the 
average intermolt level, whereas the magnesium levels were the same at both stages 
(Table II). After ecdysis, the calcium level fell while the magnesium level rose 
more than 40%. Travis (1955) has described a similar pattern for calcium in the 
pre- and postmolt periods for another crustacean, the spiny lobster. Although these 
changes are of interest in the overall electrolyte metabolism of molting, we con- 
sidered them too small to influence significantly the physiological effectiveness of a 
Ringer's solution ; therefore we did not sample a larger series of pre- and postmolt 
animals. 



Son 



SKINNER. MARSH AND COOK 



I \BIJ- 11 
i'J i ale in in and magnesium in Gecarcinus serum ultrafiltratc 



Stage 


Animal 


Calcium 

(meq./L. ) 

i 


Magnesium 
(meq./L.) 


Intermolt 


1 


13.2 


15.2 




2 


18.5 


11.5 




3 


17.4 


15.7 




4 


24.8 


1(1. (i 




5 


1S.4 


17.6 




6 


17.1 


15.3 




7 


19.2 


13.2 




8 


17.6 


12.S 




9 


16.8 


12.4 




10 
11 
12 


16.8 
14.3 
13.5 


14.4 


Avg: 13.8 db 2.2 (S.D. 


Avg: 17.2 2.4 (S.D.i 


Premolt 


1 


22.d 


14.11 




2 


24. S 


13.6 


Avg: 23.4 


Avg: 13.8 


Post molt 


1 


18.4 


17.6 




2 


20.11 


18.0 




3 


18.8 

Avg: I'M 


23.2 


Avg: 19.6 



3. Inorganic snlfate 

The results of 17 analyses are listed in Table III. Sera from 13 intermolt 

j 

animals had an inorganic snlfate concentration of 11. 18 0.66 (S.D.) meq/L. 
Two premolt and two postmolt animals had similar values, indicating no variation 
during the molt cycle. 



4. pH, pCO z , pOn and bicarbonate 

Gecarcinus blood serum has a relatively constant pH of 7.2 and a pCO 2 of 14 
mm. Hg (Table IV). The wide fluctuations observed in the oxygen tension are 
unexplained. They are probably not due to the mixing of "arterial" with "venous" 

TABLE 1 1 1 
Inorganic snlfate in Gecarcinus serum 



Stage 


Number of animals 


Inorganic sulfate 


Range 

(meq./L.) 


Average 
(meq./L.) 


Intermolt 
Premolt 
Postmoll 


13 

2 
2 


10.20-11.94 
10.86-11.06 
10.92-11.68 


11.18 0.66 (S.n.) 
10.96 

11.30 



[.AND CRAP, SERUM ELECTROLYTES 



361 



pH, pCO-2, 



TABLE IV 

bicarbonate in Gecarcinns serum 



Animal 


I>H 


pCOz 
(mm. Hg) 


pO 2 

(mm. Hg) 


Bicarbonate 
(meq./L.) 


1 


7.20 


14 


24 


7.40 


2 


7.22 


13 


29 


5.14 


3 


7.43 


12 


28 


7.70 


4 


7.14 


12 


72 


3.95 


5 


7.05 


16 


56 


4.15 


6 


7.08 


15 


36 


4.99 


7 


7.26 


16 


30 


6.94 


8 


6.95 


14 


46 


2.97 


Averages 


7.17 


14 


40 


5.40 



blood (if such terms can be used to describe the hemolymph of an arthropod), 
since if mixing were the cause of the variability, we would expect low pO 2 values to 
lie correlated with high pCO 2 values. 

The pCO, is considerably higher than that of the sera of many other inverte- 
brates (Spector, 1956; p. 270). The low pCO 2 of insects is probably due to the 
direct oxygenation of every cell by tracheole penetration, while the low pCO 2 of 
various marine Crustacea is probably due to the solubility of CO 2 in the sea water 
bathing the gills. 

To determine the site of the diffusion barrier for CO 2 in Gccarcinus, the 
branchial chamber of an animal was flushed with 100% O 2 for 10 minutes before and 
throughout the collection of the blood sample. The pO 2 of that serum was only 52 
mm. Hg, while the pCO 2 was 13.5 mm. Hg. The maintenance of this high pCCX 
in the blood despite the fact that the gill chamber was flushed free of CO 2 indicates 
that the barrier lies between the gill chamber and the branchial chamber. Further 
experiments will be performed to test this possibility. 

5. Inorganic phosphate 

The inorganic phosphate content of 31 serum samples collected from animals 
in the field averaged 0.76 but ranged from 0.21 to as high as 2.08 mmols/liter 
(Table V). 

TABLE V 

Inorganic phosphate in Gecarcinus serum 



Stage 


Conditions 


Number of 
animals 


Inorganic phosphate 
range 
(mmoles/L.) 


Average 


Intermolt 


Collected in field 


31 


0.21-2.08 


0.76 


Intermolt 


Starved >3 dav-; 


7 


0.29-0.53 


0.42 


Premolt (D - D 4 ) 1 
Day of ecdysis 
Postmolt (A - Ci) J 


Did not eat 
after onset 
of Do 


8 
1 
12 


0.33-0.78 
0.42 
0.34-0.66 


0.50 
0.42 
0.51 



362 SK1NXKK. MARSH AND COOK 

Travis (1955) found that under controlled feeding conditions the level of in- 
organic phosphate in the serum of the spiny lobster remained relatively constant 
throughout the molt cycle. A decrease of 25% in the postmolt period was the 
greatest fluctuation she observed. According to Travis, diet was the principal 
factor which determined serum phosphate levels. 

\Ye took our blood samples in the field within a few hours after the animals 
had been collected ; therefore, the time and content of each specimen's most recent 
meal probably accounted for the 10-fold variation in inorganic phosphate level. 
More recently we have analyzed blood from a group of animals maintained in the 
laboratory, where feeding conditions could be controlled. Sera from these animals 
starved for three or more days showed much less variation in the inorganic phos- 
phate concentration and were in the lower range of those collected in the field 

(Table V). 

There was no correlation of inorganic phosphate concentration with the molting 
cycle. These data appear to be similar to those obtained by Travis for the spiny 
lobster. 

TABLE VI 

Composition of Ringer's solution for Gecarcinus 



Compound 


mmols./L. 


NaCl 


430 


K 2 SO 4 


5 


MgCl 2 


7 


CaCl 2 


9 


"Tris" buffer 


10 



The final pH is adjusted to 7.2 with 0.2 N maleic acid. 

6. Preparation and physiological efficacy of the Ringer's solution 

Based on the measurements above a Ringer's solution has been devised 
(Table VI). 2 Regardless of the serum osmolalities (and corresponding ion con- 
centrations) within the range of 610 to 1060 mosm/kg. H 2 O, there were no gross 
behavioral differences in the specimens of Gecarcinus. Hence it appeared that a 
Ringer's solution within this range should support normal neuromuscular activities. 
We have selected a sodium concentration (430 meq/L.) and osmolality close to the 
values observed in animals shortly after their arrival in the laboratory. The use of 
chloride salts raised the concentration of chloride somewhat higher than any ob- 
served in the animals. Since the final Ringer supported prolonged neuromuscular 
activity, the high chloride does not appear to exert any deleterious effect. 

2 Previous data published mi the ionic composition of Gecarcinus latcralis serum by Prosser 
and Brown (1962; p. 60) were preliminary data obtained by J. W. Green in collaboration with 
one of us (DAIS). Since for technical reasons we were not confident of the validity of some 
of the numbers obtained at the time, we did not publish the data. However, we did make them 
available to a few colleagues, one of whom submitted them for publication to Dr. Prosser. 

Dr. Prosser published them in good faith without knowledge of their source. Before under- 
taking the present work, we tested a Ringer's solution prepared from the values obtained 
earlier and found that it did not support nerve or muscle function. 



LAND CRAB SERUM ELECTROLYTES 363 

The solution is brought to pH 7.2 with Tris(hydroxymethyl) aminomethane/ 
maleic acid, a buffer commonly used in crustacean Ringer's solution (Elliott an.l 
Florey, 1956). The concentration of inorganic phosphate in Gecarcinus plasma 
was too low to use it as an effective buffer. Indeed, the small concentration 
prompted us to omit inorganic phosphate from the solution entirely. We have also 
omitted bicarbonate from the Ringer since its buffering capacity at 5 X 10~ 3 M 
would be small and would require in any case the maintenance of a constant pCCX. 

The measured osmolality of the final solution was 850 mosm/kg. HoO. 

A chela of an intermolt animal was removed and the nerve trunk in the merus 
was freed of all surrounding tissue. Forty ml. of the Ringer were perfused through 
the cut end of the propus to wash out blood. The nerve trunk was stimulated and 
the contraction of the adductor muscles in the propus was observed intermittently 
over a four-hour period. During the same period of time, sensory stimulation (i.e., 
light taps) in the region of the mechanoreceptors in the leg joints elicited action 
potentials which could be recorded approximately 4 cm. down the sensory nerve. 
Thus, axonal conduction, neuromuscular transmission, and muscular contraction 
appear normal for up to four hours, at which time the experiments were terminated. 

CONCLUSIONS 

Summing the inorganic ions for an animal with an osmolality in the median 
range, e.g., 850 mosm/kg. H 2 O, we find that the inorganic cations total about 450 
meq/L. and the inorganic anions about 385 meq/L., leaving 65 meq/L. anionic 
charge unaccounted for. Acidic amino acids contribute little to this charge since 
they are present in low concentrations (ca. 0.05-0.10 mmol/liter total) and are 
more than balanced by basic amino acids (ca. 0.13-0.74 mmol/liter, unpublished 
data). The protein concentration in Gecarcimis serum is high and the isoelectric 
points of all the proteins are not kno\vn. Most should be negatively charged at the 
pH of the animal's plasma unless the isoelectric points are unusually basic. There- 
fore negative charges on protein probably account for many of the undetermined 
anions. 

In a serum of osmolality 850 mosm/kg. H 2 O the total of all inorganic ions 
is about 815 mmol/L. If we assume a rational osmotic coefficient of about 0.9 
for these electrolytes, only 13% of the total osmotic pressure is unaccounted for. 
Much of this will be made up by proteins, amino acids, glucose, and other commonly 
occurring organic solutes. Therefore it is unlikely that any single organic compound 
makes up an important fraction of the total osmotic pressure, as does urea in 
elasmobranchs (Prosser and Brown, 1962; p. 142) and glycerol in some insects 
(Wyatt and Meyer, 1959). 

The clear dependence of sodium, chloride and potassium concentrations and of 
osmolality on the nature of the available water supply indicates that Gecarcinus does 
not regulate these concentrations about a critical set point. A corollary of this con- 
clusion is the observation that all animals showed similar motor behavior regardless 
of their plasma ion concentrations. It is of interest that the serum osmolality is al- 
ways greater than that of the available water supply. Gecarcinus is a land animal 
and evaporation at its gills undoubtedly leads to the observed hemoconcentration. 
Gross' data (1963) show that concentrations of alkali metal cations are essentially 
the same in urine as in blood over a wide range of blood concentrations. This 



364 SKINNHIv. MARSH AND COOK 

observation precludes the possibility that renal mechanisms compensate blood 
changes. 

Gross measured sodium and potassium in groups of Gccarcinns exposed to a 
variety of environments; he concluded, as have we, that the concentrations of these 
two ions are not closely regulated. Flemister (1958) immersed the animals in 
aqueous solutions of various chloride concentrations and found that even after 
several days the blood chloride did not equal environmental chloride. Under these 
conditions Flemister noted that animals immersed in hypotonic sea water had 
blood chloride concentrations greater than that of the environment, whereas animals 
immersed in hypertonic sea water had blood chloride less than that of the environ- 
ment. In his experimental situation the normal evaporative processes are pre- 
vented. However, his data indicate, as do ours, that the plasma chloride concen- 
tration decreases in a hypotonic medium and increases in a hypertonic environment. 

Whether one concludes that Gecarcinus is capable of osmoregulation depends 
to some extent on one's definition of the term. Serum ion and osmolality levels 
are not maintained constant independent of the environment ; but not even in the 
case of total immersion do they equilibrate with the environment. In their normal 
terrestrial habitat evaporative losses can and do occur, and the animals appear to 
compensate for these in the laboratory by spending some time in the available water 
supply (personal observations; see also Gross, 1963). Thus we may conclude 
that the animals are capable of osmoregulation only to a limited extent (in part 
by behavioral mechanisms), and that the resulting fluctuations in serum concentra- 
tions are readily tolerated. 

We wish to express our appreciation to the staff of the Lerner Marine Labora- 
tory, Bimini, where this work was initiated ; to Arnold Davidson for excellent 
technical assistance ; to Dr. E. Bergofsky for performing some of the analyses and 
Dr. M. Mendelson for performing some of the tests on the efficacy of the Ringer. 
This work was supported by USPHS grant #AM 06268 to one of us (l)MS) 
and by ONR assistance which made the preliminary work in the field possible. 

SUMMARY 

1. From determinations of the principal electrolytes and respiratory gases in 
the serum of the land crab, Gecarcinus lateralis, a Ringer's solution has been 
devised and found to be effective in supporting neuromuscular activity for at least 
four hours in isolated preparations. 

2. The animal is capable of a limited osmoregulation. 

LITERATURE CITED 

AMES, A., AND F. B. NESBETT, 1958. A method for multiple electrolyte analyses on small 

samples of nervous tissues. /. Neurochem., 3: 116-126. 
BLISS, D. E., 1962. Neuroenclocrine control of locomotor activity in the land crab Gecarcinus 

Ititcmlis. Mem. Soc. Endocrinol., no. 12, Neurosecretion : 391-410. 
COTI.OVE, E., H. V. TRANBRAM AND R. L. BOWMAN, 1958. An instrument and method for 

automatic, rapid, accurate, and sensitive titration of chloride in biologic samples. 

J. Lab. Clin. Mcd.. 51: 461-468. 
KI.I.IOTT, K. A. C, AND E. FLOREY, 1956. Factor I inhibitory factor from brain. Assay. 

Conditions in brain. Simulatint!, and antagonizing substances. J. Neurochem., 1: 

181-192. 



LAND CRA1J SERUM ELECTROLYTES 

FISKE, C. H., AND V. SruK.\Ro\v, 1925. The colorimetric determination of phorphorus. ./. 

Biol. Chem., 66: 375-400. 
FLEMISTER, L. J., 1958. Salt and water anatomy, constancy and regulation in related crabs from 

marine and terrestrial habitats. Biol. Bull., 115: 180-200. 
GROSS, W. J., 1959. The effect of osmotic stress on the ionic exchange of a shore crab. Biol. 

Bull, 116: 248-257. 
GROSS, W. J.. 1963. Cation and water balance in crabs showing the terrestrial habitat. 

Physiol ZouL, 36: 312-320. 
JONES, A. S., AND D. S. LETHAM, 1956. A spectrophotometric method for the determination 

of sub-micro quantities of sulphur with 4-amino-4'-chlorodiphenyl. Analyst, 81: 15-18. 
LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, 1951. Protein measure- 
ment with folin phenol reagent. /. Biol. Chem., 193: 265-275. 
PROSSER, C. L., AND F. A. BROWN, JR., 1962. Comparative Animal Physiology. W. B. Saunders 

Co., 2nd Ed., Philadelphia. 
SKINNER, D. M., 1962. The structure and metabolism of a crustacean integumentary tissue 

during a molt cycle. Biol. Bull., 123: 635-647. 

SKINNER, D. M., 1963a. Growth in a crustacean. Amer. Zool., 3: 71. 
SKINNER, D. M., 1963b. In I'iro synthesis of protein in fully formed and developing muscle 

during the molting cycle in the land crab Gecarcimis. J. Cell Biol., 19: 66A. 
SPECTOR, W. S., 1956. Handbook of Biological Data. W. B. Saunders Co., Philadelphia. 
TRAVIS, D. F., 1955. The molting cycle of the spiny lobster, PanuUrus argus Latreille. III. 

Physiological changes which occur in the blood and urine during the normal molting 

cycle. Biol. Bull., 109: 484-503. 
WEBER, A. M., AND R. HERZ, 1963. The binding of calcium to actomyosin systems in relation 

to their biological activity. /. Biol. Chem,, 238: 599-605. 
WYATT, G. R., AND W. L. MEYER, 1959. The chemistry of insect hemolymph. III. Glycerol. 

/. Gen. Phvsiol.. 42: 1005-1011. 



CHLOROPLAST PIGMENTS AND THE CLASSIFICATION OF 
SOME SIPHONALEAN GREEN ALGAE OF AUSTRALIA 1 

HAROLD H. STRAIN 
Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60440 

With respect to their chloroplast pigments, the siphonaceous green algae of the 
order Siphonales (class, Chlorophyceae) differ slightly from the common uni- 
cellular and multicellular green algae (Strain, 1958, pp. 37, 162, 167). From this 
standpoint of pigment composition, the Siphonales are not so closely related to the 
other green algae as all the other uninucleate and multinucleate Chlorophyceae are 
related to one another (Fritsch, 1935, pp. 60, 368; Smith, 1955, p. 101). 

From the nature and the proportions of their chloroplast pigments, the 
Siphonales are remotely related to the other siphoneous or coenocytic algae such 
as the Vaucheriaceae (Strain, 1958, pp. 51, 169), which are alternatively classified 
as Siphonales (Fritsch, 1935, p. 368) or as Heterosiphonales (class, Xanthophyceae 
or Heterokontae) (Smith, 1955, p. 177; Taylor, 1960, p. 190). With respect to 
their pigments, the Heterosiphonales are similar to a group of multicellular 
filamentous algae known as the Heterotrichales (Tribonemataceae), also of the 
class Heterokontae (Smith, 1955, p. 174; Strain, 1951, p. 254, 1958, p. 169). 
This relationship, based on pigments, supports the contention that the Hetero- 
siphonales (Vaucheriaceae) and the Heterotrichales (Monociliaceae and Tri- 
bonemataceae) belong to the same class (Heterokontae) (Smith, 1955, p. 166). 
With respect to pigments, Dichotomosiplwn tuberosus (A.Br.) Ernst, a fresh-water, 
siphonaceous species, is related to the Siphonales (Strain, 1958, pp. 37, 167) (pre- 
dominantly marine species), not to the siphonaceous Heterosiphonales (pre- 
dominantly fresh-water species) (Fritsch, 1935, p. 369; Smith, 1955, pp. 101, 115; 
Taylor, 1960, p. 190). 

Like all the common green algae, and the higher plants as well, the species of 
the Siphonales examined thus far contained chlorophyll a plus chlorophyll b. The 
heterosiphonalean and heterotrichalean species contained chlorophyll a unaccom- 
panied by other chlorophylls (Strain, 1958, pp. 37, 51, 167, 169). Like the com- 
mon green algae, the coenocytic species of the order Siphonales contained the 
principal xanthophylls neoxanthin, violaxanthin and lutein plus traces of zeaxanthin. 
They also contained an additional xanthophyll, called siphonaxanthin, and an ester 
of this pigment called siphonein. The heterosiphonalean and the heterotrichalean 
species contained a group of unnamed xanthophylls not observed elsewhere (Allen 
et al., 1964; Strain, 1958, p. 51). The green algae of the order Siphonales con- 
tained much more a-carotene than ^-carotene. The algae of the heterokontaen 
groups contained principally /^-carotene. Dichotomosiphon contained the same 
chlorophylls and xanthophylls found in the Siphonales, but the principal carotene 
was the /3-isomer (Strain, 1958, p. 167). 

1 Based on work performed under the auspices of the U. S. Atomic KniT^y (.' 
and the Australian Academy of Science. 

366 



PIGMENTS OF SOME SIPHONALEAN ALGAE 367 

The siphonalean green algae available for the earlier studies (Strain, 1958, p. 
167) included about 24 varieties and species. They represented 9 genera and 5 
families. Except for Dichotomosiphon, obtained from Lake Michigan, these species 
were indigenous to the coastal waters of California, Hawaii and Puerto Rico. 

MATERIALS AND OBSERVATIONS 

As an extension of the earlier studies, the pigments of several Australian 
species of siphonaceous, green algae have now been isolated and compared with 
the pigments described before (Strain, 1958). The organisms were collected at 
various remote locations in the Southern Hemisphere (Womersley, 1959) at low 
tide during the spring months, August through November, 1963. They were 
examined soon after collection. The pigments were separated and compared by 
chromatography in columns of powdered sugar with petroleum ether plus 0.5% 
w-propanol as the wash liquid (Strain, 1958, p. 27). In the United States the 
powdered sugar is sold as Confectioners Powdered Sugar. In Australia it is 
available as Soft Icing Mixture and as Icing Sugar. The carotenes were separated 
in columns of activated magnesia [Micron Brand magnesium oxide No. 2641 
(Strain, 1958, p. 30) also sold as Sea Sorb 43 by Fisher Scientific Company]. 
Before the columns were packed, the magnesia was mixed with twice its weight 
of heat-treated siliceous earth (Celite 545). 

The individual pigments with their sequence (top to bottom) and color in 
the columns of powdered sugar were : 

Siphonaxanthin, red-orange 
Neoxanthin, light yellow 
Violaxanthin, light yellow 
Siphonein, red-orange 
Chlorophyll b, yellow-green 
Lutein zeaxanthin, yellow 
Chlorophyll a, green 
a-Carotene + /2-carotene, yellow 

The location of siphonein was previously reported below the chlorophyll b due 
to mislabelling of the chlorophyll b and siphonein zones (Strain, 1958, p. 38). 
Separate chromatographic experiments also revealed that siphonein and fucoxanthin, 
the principal xanthophyll of diatoms and brown algae, are adsorbed together in 
the sugar columns. Both pigments form zones of nearly the same color above the 
chlorophyll b zone. When adsorbed together in sugar columns, these two xantho- 
phylls are incompletely separated, even after prolonged washing with the petroleum 
ether-propanol solvent. Reaction of the fucoxanthin, in ether solution, with con- 
centrated hydrochloric acid, to yield a blue color in the acid layer, serves to dis- 
tinguish the fucoxanthin from the siphonein, which does not yield a blue color. 

The siphonalean algae that were examined are listed in Table I along with 
the regions where they were collected. 

All the species included in Table I yielded much more a-carotene than ^-carotene. 
All these species except Caulerpa filifonnis contained siphonaxanthin and siphonein 
in addition to the other chloroplast pigments found in green algae and higher 



368 



HAROLD H. STRAIN 



plants (Strain, 1958, pp. 130, 157, 162). They yielded these pigments in the 
chromatographic sequence indicated above. Chlorodesmis comosa yielded very 
little of the siphonaxanthin and siphom'in. 

Because of the absence of siphonaxanthin and siphonein in Caulerpa filijormis, 
analyses were repeated several times. The organism was also collected from two 
different stands on neighboring rocks, but the siphonaxanthin and siphonein, 
found in other species of Caulerpa as well as in all the other species of the Siphonales, 
could not be detected. 

TABLE I 

Algae of the order Siphonales, whose chloroplast pigments were determined. 
and the locations where they were collected in Australia 



Bryopsidaceae 

Bryopsis sp. 

Caulerpaceae 

Caulerpa cupressoides (West) C.Ag. 

Caulerpa distichophylla Sender 

Caulerpa filiform is (Harv.) C.Ag. 

Caulerpa lentillij'era ]. Ag. 

Caulerpa racetnosa (Forssk.) J.Ag. 

Caulerpa racemos/t var. laetcvircns (Mont.) Weber-van 

H/ilinieda discniden I Vvai^ne 

Codiaceae 

Chlorodesmis onin^a Harv. & Bail. 

Codiuni ditthieae Silva 

Codimn fragile (Suring) Hariot 

Codiiini lurasii St.-tch. 

Codimn liicus/i Setch. 

Codium spongiosum Harv. 



Cottesloe Beach ( Perth i 



Heron Id. (Gt. Barrier Reef) 
Cottesloe Beach (Perth) 
Cronulla Beach (Sydney) 
Redcliff (Brisbane) 
Heron Id. (Gt. Barrier Rt.vi "> 
Pt. Peron (Freemantle) 
Heron Id. (Gt. Barrier Reef) 



Heron Id. (Gt. Barrier Reef) 

Pt. Peron (Freemantle) 

Hungry Pt. Cronulla (Sydney) 

Cronulla (Sydney) 

Redcliff (Brisbane) 

Heron Id. (Gt. Barrier Reef) 



Cladoplwropsis herpestica (Mont.) Howe (Valoniaceae), collected at Redcliff 
(Brisbane), yielded the pigments characteristic of the uninucleate green algae. 
Siphonaxanthin and siphonein were absent, and /3-carotene was the principal 
carotene isomer. These results conform to those obtained with other species of 
the Valoniaceae, Boodleaceae and Anadyomenaceae (order, Siphonocladales) 
(Strain, 1958, p. 164). 

CONCLUSIONS 

On the basis ot the species available thus far, siphonaxanthin and siphonein 
are constituents of siphonalean green algae indigenous to remote regions both of 
the Northern and Southern Hemispheres. The absence of these two pigments in 
Caulerpa filijormis indicates that siphonaxanthin and siphonein are not definitive 
characters for the classification of all the Siphonales. The preponderance of 
a-carotene relative to /^-carotene is without exception in the marine Siphonales. 
The fre.sh- \\ater Dichotomosiphon is an exception (Strain, 1958, p. 157). 

The wide distribution of siphonaxanthin and siphonein in the species examined 
(Table I) indicates that the Siphonales comprise a distinct group. This conclusion 



PKiMKXTS OF SO.MK SIPHONALKAX 

is supported by the preponderance of a-carotene over /3-carotenc in all the marine, 
species. 

The absence of siphonaxanthin and siphonein and the preponderance of /2-caro 
tene in Cladophoropsis hcrpcstica agree with earlier analyses of other species 
the Siphonocladales (Strain, 1958, p. 164). This result indicates that the 
Siphonales stand apart from the Siphonocladales. 

The universal occurrence of chlorophyll b along with chlorophyll a supports the 
view that the Siphonales and the Siphonocladales are major divisions of the 
Chlorophyceae. This relationship is supported by the occurrence of the same 
xanthophylls, namely, lutein, violaxanthin and neoxanthin, in the Siphonales, in the 
Siphonocladales and in all the other species of the Chlorophyceae. 

There appears to be no relationship among the occurrence of particular 
chloroplast pigments, the calcification of the organisms and the composition of 
the structural polysaccharide in the marine Siphonales (Feldmann, 1946; Fritsch, 
1935, p. 368; Miwa et al, 1960; Smith, 1955, p. 12). 

The occurrence of chlorophyll a and /2-carotene in the Siphonales and in the 
Hetero siphonales indicates a basic relationship between these two groups. This 
relationship is akin to that existing among all autotrophic organisms in which 
oxygen production is always associated with the presence of chlorophyll a and - or 
^-carotene (Strain, 1951, 1958, p. 83, 1964). 

In conjunction with the earlier pigment studies (Strain, 1958), the current 
results support the classification of the Heterosiphonales with the Heterotrichales 
in a class apart from the Chlorophyceae. 

This work was made possible by a Senior Fellowship of the Australian Academy 
of Science and by assistance from many Australian scientists. Dr. A. B. Cribb 
identified the algae collected near Brisbane and Cronulla. Miss A. M. Baird, Dr. 
Mary A. Pocock, Dr. G. G. Smith and Dr. H. B. S. Womersley identified species 
collected near Perth and Freemantle. Prof. D. A. Herbert permitted use of the 
laboratory facilities of the Department of Botany at the University of Queensland 
at Brisbane. Prof. B. J. Grieve provided a laboratory in the Department of Botany 
at the University of Western Australia at Nedlands (Perth). Dr. G. F. Humphrey 
arranged for the use of a laboratory at the Fisheries Station of the Commonwealth 
Scientific Industrial and Research Organization at Cronulla (Sydney). Mr. J. 
Madgwick assembled equipment at Cronulla. Mrs. Harold H. Strain contributed 
indispensable laboratory assistance. 

SUMMARY 

1 . The chloroplast pigments of twelve species of siphonalean green algae native 
to the marine waters of Australia were isolated by chromatography. 

2. All these siphonalean species but one contained the same pigments found 
in the siphonalean Chlorophyceae of the Northern Hemisphere. 

3. Caulcrpa filijonnis lacked the siphonaxanthin and siphonein associated with 
the chlorophylls and carotenoids in the other Siphonales, but it contained a pre- 
ponderance of a-carotene relative to /3-carotene as did all the other Siphonales. 



370 MAN'' >LD JI. STRAIN 

4. The.M' oliM-natioiis indicate ;liat the Siphonales arc significantly different 
from the other screen al-ac, hut they are more closely related to the C'hlorophyceac 
than to any other al^al ^roiip. 

5. The pigment distrihution supports the classification of the Siphonales with 
the Chlorophyceae. the Meterosiphonales (' Yaucheriaeeae) and the Heterotrichales 
(Tribonematareae i with the Xanthophyceae or Heterokontae. 

LITERATURE CITED 

ALLEN, M. 1!., I.. Fun-;.-, T. \V. (iooinvix AND D. M. THOMAS, 1964. The carotenoids of alga-: 

pigments from >ome cryptomonads, a heterokont and some Rhodophyseae. /. Gen. 

Microbiol, 34: J5" 267. ' 
FKUIMANN, ].. l"4o. Sur I'heU'niplaMe de certaines Siphonales et leur classification. (". l\. 

Acad. Set., 222: 752-753. 
FRITSCH, F. !"., 1 ( '35 ( Reprinted 1%1). The Structure and Reproduction of the Algae. Vol. I, 

pp. xvii i 7 () 1. _45 tigs.. University Press, Cambridge. 
MIXVA. 'I'.. Y. IKIKI AND T. SIV.UKI, 1960 (Pub. 1961). Mannan and xylan as essential cell 

\vall coiistitncnls of some siphonous green algae. Colloq. Intern. Centre Nat'l. Rcch. 

Set.. ( Paris) No. 103: 135-144 (English). 
SMITH, G. M.. 1955. Cryptogamic Botany. Vol. I, pp. vii + 546, 311 figs., McGraw-Hill Book 

Co., Xeu York. 
STRAIN, H. H., 1951. Manual of Phycology, pp. 243-262. Smith, G. M., ed., Chronica Botanica 

Co., pp. xi + 375, 48 figs., Waltham, Mass. 
STRAIN, II. H., 1958. Chloroplast Pigments and Chromatographic Analysis. 32nd Annual 

Priestley Lectures, pp. 180, 41 figs., Pennsylvania State University, University Park. 
STRAIN, H. H., 1964. Chloroplast pigments of various plants. Argonne National Laboratory 

Reviews, Vol. 1, No. 3, 6-7. 
TAYLOR, W. R., 1960. Marine Algae of the Eastern Tropical and Subtropical Coasts of the 

Americas. Pp. xi + 870, 14 photos, 80 pi., University of Michigan Press, Ann Arbor. 
WOMK.RSI.KV. H. R. S., 1959. The marine algae of Australia. Baton. Rcr., 25: 545-614. 



A MICROSPORIDIAN INFECTION OF THE DIGESTIVE TRACT 
THE WINTER FLOUNDER, PSEUDOPLEURONECTES 

AMERICANUS x 

HORACE W. STUNKARD AND FRED E. LUX 

The American Museum of Natural History, Nezv York, N. Y ., and The U. S. Deft, of the Interior, 
Bureau of Commercial Fisheries, Biological Laboratory. U'oods Hole, Massachusetts 

Linton (1901) reported sporozoan infections in two small winter flounders, 
Pseudopleuronectes auiericanns. taken from Katama Bay, Martha's Vineyard, and 
examined at Woods Hole on August 28, 1900. His account, although brief, is 
adequate for recognition of the parasite and reads (p. 487), "The walls of tin- 
intestine of one throughout almost the entire length and of the other for a short 
distance were completely covered with sporocysts. The cysts were irregular where 
crowded together ; where not crowded together, which was in but few places, they 
were elliptical or spherical, of various sizes, but comparatively few reaching 1 mm. 
in diameter and none much exceeding that. Spores oblong-ovate about 0.003 mm. 
in length and 0.0015 mm. in diameter. Intestine where affected was chalky-white 
in color." The accompanying figure shows a "Piece of intestine of Pseudopleuro- 
nectes aniericanits, serous coat covered with cysts due to sporosperms (sic} ." There 
was no attempt at identification of the parasite, but Linton recognized that it was 
distinct from another, reported in the same publication (pp. 438 and 439), found 
in the muscles of the back and sides of the herring, Cliipea harentjus, and the alewife, 
Pomolobus pseudoharengus, 

The latter species was identified as a myxosporidian and almost one-half of the 
young fishes were infected. Tyzzer (1900) reported the discovery and prevalence 
of this infection in young P. pseudoharengus; Auerbach (1910) assigned the species 
to the genus Chloroiny.rwn Mingazzini, 1890; and Halm (1917) proposed the 
specific name, Chloromyxum clupeidae. The allocation to Chloromyxum was based 
on the spore, which has a quadrilateral apical end and bears four polar capsules. 
Kudo (1920, p. 94) examined the slides prepared by Tyzzer, and others made from 
'various species of fishes, and reported the infection in Clupea harengus, Pomolobus 
pseudoharengus, P. acstivalis, P. mediocris, Brevoortia tyrannus, Stenotomus 
chrysops, and Tautogolabrus adspcrsus, taken at Woods Hole. The parasites from 
the muscles of these fishes were regarded as specifically identical and referred to 
Chloromyxum, clupeidae Hahn, 1917. 

A third sporozoan was reported by Linton (1901; p. 455) from the liver 
nf the butterfish, Poronotus triacanthus (syn. Rhombus triacanthus}. The cyst was 
white and globular, about 1.5 mm. in diameter; when compressed it liberated im- 
mense numbers of spores, often aggregated in globular or oblong clusters, as large 
as 0.02 mm. in diameter. The spores were short and thick, with bluntly rounded 
ends, about 0.0025 mm. in length and a little less than that in breadth and thickness. 

1 This investigation was supported in part by NSF-GB-3606, Continuation of G23561. 

371 



372 HORACE \\ . STUNKARD AND FRED E. LUX 

The parasite is obviously a microsporidian and Woodcock (1904; p. 54) regarded it 
as a species of Pleistophora. 

A fourth sporozoan was uhservt'd by Linton (1901 ; p. 433) : an enormous 
number of small elliptical bodies. 14 by 6 microns, were found in the intestinal 
contents of a sting-ray, Dasvafis coitronra. Since the spores were in the lumen 
of the gut, it is apparent that they were ingested in food and were parasitic in some 
animal other than the ray. 

The parasites reported by Linton from the wall of the digestive tract of P. 
aincricanus are similar to and possibly identical with others reported about the 
>ame time from flat-fishes of Europe. Hagenmiiller (1899) observed the infection 
in at least one-half ("IS fois sur 30") of the small fishes, Fle.vns passer Moreau 
(== Plcnroncctt's passer] from littoral pools in the area of Endoume, Bouches-du- 
Rhone, France. The parasite was named Nosenm stephani in honor of M. Pierre 
Stephan. who first found the cysts and called them to the attention of the author. 
He wrote (p. S37, "Cette Myxosporidie appartient au genre Glitgca Thelohan. 
aujourd'hui !\'<>scniu ; elle infeste, sous forme d'infiltration diffuse ou de kystes, les 
parois du tube digestif . . . L'infiltration diffuse represente plus particulierement 
un mode de pullulation endogene, tandis que les kystes assurent la dissemination du 
parasite a 1'exterieur. Kystes et amas d'infiltration s'observent depuis la partie 
superieure de 1'oesophage jusqu' a 1'extremete du rectum, loges dans les tissus ou 
.simpk>ment reconverts par le peritoine. II n'existe ni amas ni kystes dans le 
parenchyme d'aucun organe, rein, rate, foie, coeur, etc. Cependant, sous le peritoine 
a la surface du foie et dans les replis peritoneaux ou cheminent des vaisseaux, les 
kystes sont assez nombreux; j'en ai trouve jusque sur le conduit choledoque pres 
de son abouchement avec 1'intestin. Dans la paroi intestinale, les kystes siegent 
dans les couches musculaires et surtout dans la couche conjonctive. J'en ai vu 
jusque dans la charpente conjonctive des replis de la muqueuse et des villosites. 
mais jamais, non plus que d'infiltration diffuse, dans la couche epitheliale de 
1'intestin. 

"Ce.s k \\stes apparaissent a 1'oeil nu comme de petits grains d'un blanc de lait. 
ovoides ou plus rarement spheriques, ne depassant guere 1 mm. en diametre. 
n'atteignant meme pour la plupart que quelques dixiemes de millimetre, ou moins 
encore." I fagenmuller discussed the formation of the cyst and concluded that the 
membranous wall is produced by the host as a reaction to invasion by the parasite. 

A similar and presumably identical species was reported by Johnstone (1901 i 
from the plaice, 1'lcitronectes platessa, taken in the Irish Sea along the coast of 
Lancashire, fhe author recognized the parasite as a protozoan, probably a sporo- 
zoan, but further identification was not attempted. The infection was limited to 
the digestive tract and the intestine, from the pylorus to the anus, was thickened and 
superficially looked like a ripe ovary. The external surface was studded with small. 
round, white, opaque bodies; the internal surface was disposed in irregular, longi- 
tudinal folds, covered with projecting, round white bodies; the lumen was reduced 
and the inucosa often lost ; and the wall measured 3 to 4 mm. in thickness. The 
cysts were about 0.60 mm. in diameter, with a capsule composed of an outer cuticular 
and an inner fibrous layer. The spores were oval with a maximum length of 0.005 
mm. Figures portrayed ti i<>pearance of the intestine, the structure of the 

wall, and the form of the spores. 



MICROSPORIDIOSIS IN WINTER FLOUNDER 

Woodcock (1904) described a second infection in the digestive tract of the 
plaice, P. platessa, taken near Plymouth, England, and discussed the Myxosr 
in flatfishes. For him, the Myxosporidia Biitschli, 1881 were "characterized 
by the fact that reproduction by spores goes on throughout the growing or 'trophic' 
period, and (b) by the complicated process of spore-formation and the natur. 
the spores." The group was, thus, the equivalent of the Neosporidia Schaudinn, 
1900 and the Cnidosporidia Doflein, 1901, and included the Microsporidia Balbiana, 
1882. Woodcock reviewed the papers by Hagenmuller, Linton, and Johnstone; 
he noted that the infection reported by Johnstone was a "ripe, well-matured one," 
extensively distributed, whereas the infection he studied was only a slight and 
limited one, from a fish that superficially was quite healthy in appearance. In this 
specimen, the gut showed little oval patches, 1.00 mm. in diameter, usually 
projecting slightly on the outer, coelomic side together with other small pyriform 
appendages, 1.50 to 2.00 mm. in length, attached to the gut by the narrow end. 
These enlargements were all on the side of the gut to which the mesentery was 
attached, and in which the blood vessels ran. The functional activity of the 
intestine was not impaired ; the mucosa was intact and normal in appearance. 
Woodcock compared sections made from the lightly infected intestine with others 
made from material sent by Dr. Johnstone. He discussed endogenous multi- 
plication ("multiplicative reproduction" of Doflein) in young forms, the spread 
of the infection into neighboring tissue by diffuse infiltration and the formation 
of cysts and pseudocysts. He stated (p. 57), "Quite probably 'multiplicative 
reproduction' is, here, simply a separation of the pansporoblast rudiments, as 
daughter individuals. Indeed the whole nature of diffuse infiltration in Glugea 
seems to me to support this idea. There is no question of the individual parasites 
attaining size, still less of any continuity of a protoplasmic mass ramifying in and 
between host's tissue-cells. It is far rather a cell-infection, visible, when ripe, as 
separate clumps of spores, each formed from, and representing, one pansporoblast, 
and either still surrounded by a hypertrophied host-cell, or else free, but only 
owing to the latter's breakdown." The infections reported by Hagenmuller, 
Linton and Johnstone were referred to the same species, here designated as 
Glugea stephani (Hagenmuller, 1899) Woodcock, 1904. 

Stempell (1904) studied the development of Nosctna auoinala Moniez. 1887, a 
species from the connective tissue in subcutaneous loci and in the gut-wall, liver, 
and gonads of the fresh-water stickleback, Gastcrostcus aculeatus. This species 
had been transferred by Gurley (1893) to the genus, Glugea Thelohan, 1891. 
Stempell noted that recent investigations had disclosed a series of protozoan species 
in which the life-cycles consisted of a limited period of vegetative, asexual 
multiplication, after which different, "ycartete" forms appear, whose further 
multiplication is conditional on the conjugation or copulation of two individuals. 
After citing essentials of these investigations he stated (p. 31), "Bedenkt mann 
dagegen, dass die allgemeinen Grundziige der Entwicklung, soweit sie sich feststellen 
liessen, in alien Fallen dieselben sind, so darf man wohl mit Recht schliessen, dass 
samtliche beschriebenen Parasitenformen der Spezies Nosema anomalum Monz. 
angehoren. In der Tat, ein treffender Name fiir eine so variable Spezies !" Ac- 
cordingly, he returned the species, anoniala, to the genus Nosema. In this species 
he reported growth of the protoplasmic masses with rapid, asexual multiplication of 



374 HORACE \\ STUNKARD AND FRED E. LUX 

nuclei, followed l.\ tin- differentiation of .-poronts, the admitted progenitors of the 
>e\ual generation. His accounl re; ds (p. 33), "Wir sehen, wie in der enzystierten 
Parasitenma.vse /unachst ein \Vachsunn des Protoplasmas und eine starke Vermeh- 
rung der Kerne ;inf rein ungeschlechtlichem Wege erfolgt, wie sich aber schon 
sehr bald ans dieser vegetal Parasitenmasse die als Vorfahren der Geschlechts- 
generation auf/ufassendcn Sporonten differenzieren. Nur dadurch unterscheiden 
sich die vorliegenden Mic oridien und so viele phanozyste Myxosporidien von 
der Mehrzahl des anderen Sporozoen, dass diese Geschlechtgeneration durch 
endogene Knospung im Korper der vegetativen Individuen entsteht." 

Weissenberg (1911) reported that about 2% of the smelt. Osnicrus cpcrlaints. 
taken from sources near Berlin and from inlets of the Baltic Sea, were infected 
with a microsporidian parasite, similar to but distinct from Gluyca anomala, which 
he described as a new species, Glugca hcrtwici'i. He observed no difference in the 
infections of fishes from fresh and salt water. In a second paper, Weissenberg 
(1913) reviewed the work of Stempell (1904) and other authors on microsporidian 
species and reported on the life-cycles of G. anotnala and G. heiiu'igi. Since the 
time of Pasteur it has been known that certain microsporidians invade the ovary 
and penetrate the ova, with hereditary transmission of infection. Stempell de- 
scribed Mich infected ova, but Weissenberg (1913) declared the evidence was not 
convincing. To test the matter, he raised sticklebacks, Gastcrosteits aculeatus, 
from eggs. When the yolk-sacs were resorbed he fed small copepoda and daphnids 
but the fishes did not grow 7 . Fine emulsions of spores were added to the water, 
but no infection resulted. With other fishes raised in aquaria but fed plankton 
with an abundance of plant and animal food, growth was good and two young 
-licklebacks daily were fed plankton mixed with an emulsion of spores. Three 
weeks after the beginning of the experiment, one of the fishes had a G I it yea-cyst, 
300 microns in diameter, on the wall of the throat. This result demonstrates that 
a fish raised in the laboratory became infected and provides information on the rate 
of cyst formation. Weissenberg concluded (p. 157),"Wenngleich die oben dargeleg- 
ten I'.efunde beziiglich der Entwicklungsprozesse von GliKjca anoinala in zahl- 
reichen I'nnkten von den Ergebnissen der Voruntersucher abweichen, so gelange 
id i doch y.n der gleichen Gesamtauffassung wie die alteren Autoren, insbesonder 
Stempell Audi nach meinen Befunden koinmt Ghujca ein grosser eigener Plas- 
makorper mil xablreichen vegetativen Kernen zu. Die ganze Cyste gehort zum 
Protozoon. Wirtszellen oder hypertrophische Wirtskerne sind am Cystenaufbau 
nicht beteiligt." In a subsequent paper, Weissenberg (1921) discussed the prob- 
l ( 'm whether 01 nol the large Plasmakorper, with its large vesicular nuclei, is de- 
rived from host tissue or is of protozoan origin. After presenting new evidence 
he concluded (p. 420) 'An der Wirtsgewebsableitung des Plasmakorpers und 
der blaschenformigei i rne der G'litf/ca-Cysten kann nun nicht mehr gezweifelt 
werden. Aufgabe kunfii ''orschung wird es sein, die Art der phagocytenartigen 
verstreut im Bind ret< nden I'ischzellen, die somit den Alutterboden fiir 

die Glitfii'it Cysten al i genauer zu eruieren." 

Meanu-liile, Mavor i L915 . reported that about 50% of tin- I'sciuluplcnn>ncflcs 
americanus examined In the er and autumn of 1910 at Woods Hole, Mass., 

were infected with (,'/n ,///. Me also found Osincnis inonia.v at Woods 

Hole frequently infected with a ui!cro>])oridian, apparently G. stcplidiii. These find- 



MICROSPORIDIOSIS IN WINTER FLOUNDi 

ings are in marked contrast to others made in the summer of 1912, \\hen no infec 
tions were found on examination of 82 P. americanus and 22 0. mordax ta' 
the region of St. Andrews, New Brunswick. Kudo (1924) suspected thai: the 
parasite of 0. mordax was G luge a hertwigi. 

Schrader (1921) found 28% to 53% of the smelts, 0. mordax from lakes in New 
Hampshire, and 1.5% to 16% of those from the coast of Maine were infected with a 
species which he identified as 0. hertwigi Weissenberg, 1911. The intestine was 
the primary seat of infection although cysts were present in the liver and gonads. 
The cysts ranged in size from microscopic to 3 mm. in diameter, but were similar 
in size in each fish. The highest incidence of infection was in immature fishes, 
about 10 cm. long; adult fishes were rarely parasitized and Schrader predicated that 
the majority of infected fish die while immature. Unlike G. ano/mila, G. hertwigi 
was regarded as specific for smelts since other fishes in the same area were not in- 
fected. Furthermore, connective tissue and muscles were not infected, which ap- 
parently served to distinguish G. hertwigi from G. anomala. 

Reichenow (1929) described Glugca stephani from infections of Pleuronectes 
liuianda at Helgoland. He found white cysts, 0.5 mm. in diameter, in the sub- 
mucosa of the intestine and reported (p. 1099), "Die Parasiten bilden zuerst In- 
fektionsherde in der Darmwand, die von Hagenmuller und Woodcock als Zustand 
diffuser Infiltration (vgl. S. 1046) bezeichnet warden. Die Parasiten haben 
jedoch keinen interzellularen Sitz, vielmehr befallen sie im Laufe ihrer Vermehrung 
zahllose benachbarte Zellen (entweder Bindegewebszellen oder vielleicht Leuko- 
cyten, die sich an der Infektionsstelle ansammeln). Um den ganzen Herd herum 
bildet sich eine dicke Bindegewebskapsel, und so entstehen die Cysten, deren Inhalt 
also in diesem Falle nicht durch eine einzige Riesenzelle, sondern durch viele infi- 
zierte Zellen dargestellt wird. Die fertig ausgebildeten Cysten findet man haupt- 
sachlich von ungeheuren Sporenmassen erfiillt, zwischen denen verstreut Zell- 
und Kernreste vorkommen. Eine paarige Anlage der Sporen, welche die Stellung 
dieser Art zu der Gattung Glugca begriinden wiirde, ist von keinem der Untersucher 
beschrieben worden. Ich babe in dem von mir beobachteten Falle eher den Ein- 
druck gewonnen, dass die Sporen einzeln entstehen, so dass die Art also zu Nosema 
zu rechnen w r are. Doch wird sich dies erst bei Beobachtung friiherer Infektions- 
stadien, die iibersichtlichere Bilder geben, entscheiden lassen. In meinem Falle, 
in dem die Cysten dicht gedrankt in der Darmwand sassen, war die Schleimhaut auf 
weite Strecken vollig abgestossen; es ist daher zu vermuten, dass die Fische an 
starken Infektionen zugrunde gehen." 

Recent accounts have added little information on microsporidian infections of 
fishes. Bond (1938) identified cysts found in sections of the stomach of Fundulus 
het.eroditus taken in Chesapeake Bay as G. hertwigi, but the determination may 
not be correct. Fantham et al. (1941) described an infection in the hindgut of a 
specimen of O. mordax taken from Lake Edward, Quebec, and listed the parasite as 
G. hertwigi var. canadensis. Also, they reported G. stephani in the submucosa 
of the intestine of P. americanus and Limanda jerruginea, taken near Halifax, Nova 
Scotia; L. jerruginea was recognized as a new host of the parasite. Haley (1952) 
described a severe epidemic of microsporidiosis in 0. mordax in Loon Pond, Gil- 
manton, New Hampshire, and 16 of 20 O. mordax from the Oyster River taken at 



376 HORACE W. STVXK.WD AND FRED E. LUX 

Durham, N. II.. were infected i<\ m -pecies, which he identified as G. 

hertwigi. 

The Micnporidia arc chiell\ sites of invertebrates, especially crustaceans 

and insect-. The classilicatii Microsporidia or Microspirida is based pri- 

marily upon the form and strucum of the spores and to a lesser degree upon differ- 
ences in the details of sport/genesis. The parasites of P. americanus belong to the 
family Nosematidae, characterized by small, oval or ovate spores, each with one 
IK ilar filament. The genera are distinguished by the number of spores that are 
produced by each sporont. According to Poisson (1953), in Nosema each sporont 
develops into a sporoblast and produces a single spore; in other genera the numbers 
of spores produced are: Glugea Thelohan, 1891 and Perczia Leger et Duboseq. 
1909, two .-pores; in Gitrleya Doflein, 1898 and Pyrotheca Hesse, 1935, four 
sporoblasts and four spores; but in Stempellia Leger et Hesse, 1910, the numbers 
of spores produced are: Glugea Thelohan, 1891 and Pcrczia Leger et Duboscq, 
number varies from 8 to 32; and in Plistophora Gurley, 1893, each sporont (pan- 
.-poroblast) produces more than 16 spores. It is generally believed that the micro- 
.-poridia are narrowly host-specific. According to Poisson (1953) some 40 spe- 
cies in the genera Plistophora, Glugea, and Noscina occur in fishes and one species, 
Glugea danilewsky, occurs in the muscles and connective tissue of Rana jnsca, 
liiuys orbicularis, Natrix natri.v and other hosts. If this determination is correct, 
the distribution of G. danilewsky belies the opinion that species of Glugea are host- 
specific. 

The life-history of the Microsporidia, as conceived by Debaisieux (1928), com- 
prises two distinct phases : a multiplicative stage, schizogony, beginning with the 
liberation of the uninucleate or binucleate sporoplasm or planont from the spore 
and its entry into a host-cell, and sporogony, a spore-forming stage, in which spo- 
ronts produce sporoblasts that give rise to resistant spores, the infective agents that 
serve for dispersal of the parasite and the infection of new hosts. According to 
Kudo (1924, p. 34), "No intermediate host animals have up to date been found 
for Microsporidia. The infection of a new host animal takes place when the latter 
ingests spores of a specific microsporidian capable of germinating in its gut." A 
similar statement was made by Dogiel, Petrushevski and Polyanski (1961) but no 
reference to experimental evidence was cited. 

It is generally agreed that the life-cycle of the microsporidian involves sexual 
phenomena but there is wide disagreement concerning the location in the cycle where 
meio-is and > jamy occur. Meiotic phenomena have never been observed in the 
Microsporidia and j amy has been reported by autogamy of nuclei in the sporo- 
plasm before or after n from the spore, and also by nuclear fusion preceding 
sporont formation. Writing on sexual phenomena in Protozoa, Hall (1953, p. 80) 
stated, "A reduction of the chromosomes to the haploid number may occur in ga- 
metogenesis (yin dosis), in an early division of the zygote (sygotic inciosis), 
or in one of the pregami ;ons in conjugation (conjugated uieiosis). The type 
of meiosis varies in different Protozoa. Available data indicate that the Heli- 
ozoida, Foraminifera, Cnido>poridia, and Ciliophora are diploid throughout most 
of the life-cycle.' An opposite opinion was stated by Cheissin and Poljansky 
(1963, p. 343), "In the life-cycles of the Sporozoa the alternation of sexual process 
and sporogony or that of sexual process, sporogony and repeated asexual multipli- 



MICROSPORIDIOSIS IN WINTER FLOUNDER 

cation by means of schizogony occurs. All the developmental stage 
haploid ones because the meiosis usually appears during the process of sp; 
followed by formation of sporozoites." The statement by the Russian an: 
parently is based on the situation in the malarial parasites, but the Micro^ 
are distinct from the Haemosporidia and the life-cycles may be quite different. 
deed. Kudo (1944, p. 50) reporting on the life-cycle of Noscina votabilis Kudo. 
1939, stated, "Schizogony is by binary fission. No sexual process has been observed 
in the development of Nosema notabilis." 

The small size of the amoeboid stages and of the spores, usually less than 4 
microns in length, together with the inability to obtain early stages by controlled 
experimental infections of fishes, has made it impossible to describe the develop- 
mental cycle of these microsporidian species with assurance. The time and place of 
chromosome-reduction in meiosis and of syngamy are controversial. Cells with 
two nuclei may represent a stage before fusion of gametes or the first division of a 
zygote. Specific distinctions are often precarious and even generic diagnoses are 
unsatisfactory. In his monographic treatise, Poisson (1953) stated (p. 1043), 
"Mais bien des especes de Microsporidies sont msuffisamment etudiees ; trop d'es- 
peces ont ete decrites comme nouvelles parce qu'elles etaient trouvees dans des 
hotes nouveaux. D'apres Steinhaus et Hughes par example, Noscina destructor 
S. et H, a ete observee chez au moins 10 especes dTnsectes apparentant a trois 
groupes differents : la chenille de Gnorimo schema opcrculclla (Zeller) (Lepi- 
doptere), des Hymenopteres, des Nevropteroides. II est done des Microsporidies 
qui ne manifestent qu'une specificite toute relative. D'autre part, les caracteres 
distinctifs utilises pour separer les especes, et meme les genres, n'offrent peut-etre 
toujours la precision desirable et certains genres, tels les genres Nosema, Plis- 
tophora, Glugca, Perezia, devront etre revises." 

Microsporidian infections of Psendoplcuronectes americanus have long been 
known by members of the staffs of the New York Aquarium and the New York 
State Conservation Department, but precise and detailed records of incidence and 
intensity are not available. Dr. Ross F. Nigrelli, at the Aquarium of the New York 
Zoological Society, has observed the frequent occurrence of the parasite in fishes of 
the New York area and Mr. John C. Poole of the Conservation Department reports 
that the infection has a "spotty" distribution, e.g., in one year over 25% of the 
young of that year taken in Shinnecock Bay were infected and no infection was 
found in the same location the following year. 

MATERIALS AND METHODS 

The present investigation was begun in the summer of 1961 and has been con- 
ducted more or less continuously since that time. Over 1000 fishes. P. americanus, 
taken from different locations in New England, have been inspected for micro- 
sporidian infection. Data have been compiled (Tables I-VI) on the number of 
fishes examined, the time of year and area where they were caught, their size, sex, 
and the incidence and intensity of infection. Fishes taken on Georges Bank, off 
Yarmouth, Nova Scotia, were caught on the August, 1963, cruise and those from 
Nantucket shoals on the April, 1964, cruise of the Albatross IV. Records denote 
the organs involved and the extent of infection. Winter flounder are present from 
April to November in Woods Hole harbor. The stomachs and intestines of 751 



378 



HORACE W. STUNKARD AND FRED E. LUX 



i i I 
Incidence of inicrosf)oridia>i < hi icintt r flounder from Woods Hole Harbor in 1962 



Month 




Number 
infected 


Per cent 
infection 


April 




3 


3.4 


May 


67 


2 


2.3 


|une 


86 


1 


1.2 


July 


102 


7 


6.9 


August 


156 


6 


3.8 


September 


86 





().() 


October 


125 


3 


2.4 


\i i\ I'mluT 


49 


4 


S.2 


Total 


751 


26 


3.5 



fishes were removed and preserved for food analysis, and patent sporozoan infec- 
tions were noted. Very light infections may have been missed, so the recorded in- 
tensity is minimal. Analysis of the stomach-contents was made to determine the 
kinds and amounts of food ingested. In November, 1964, about 300 young fishes 
that measured from 40 to 110 mm. in length were taken in Lake Tashmoo, Martha's 
Vineyard, where the infection-rate was known to be high. Eighty-five of these 
fishes, which died at the time of collecting or a few hours later, were examined and 
the results are given in Table VI. Heavy infections included those where the infil- 



TABLI-; II 

Length-distribution of microsporidian-infected winter flounder com pared icitli Hint 
of nil winter flounder examined, in Woods Hole Harbor during 1962 



Length 
m.) 


Number of fish 


Length 
(cm.) 


Number of fish 


Total 


IllIiM Iril 


Total 


Infected 


12 


1 




28 


64 


6 


13 


1 




29 


70 




11 


1 




30 


66 


2 


15 


(t 




31 


70 


1 


L6 


3 




32 


66 


1 


17 


4 




33 


48 


2 


18 


i 


1 


34 


45 


1 


1<) 


11 


1 


35 


33 


2 


20 


11 




36 


20 




21 


7 




37 


16 




22 


15 




38 


(, 


1 


23 


1') 


2 


39 


6 




24 






10 


1 




25 


33 


2 


41 


2 




26 


is 




1.' 


2 




, 


51 










751 


26 



MICROSPORIDIOSIS IN WINTER FLOUN ' 



379 



TABLE III 

Mean weights of infected and non-infected winter flounder (row Woods Hole 1;. 





Infected 


Non-infected 


Mean length 






cm. 












Number 


Mean weight 


Number 


Mean weight 


July-August 


males 










23 


2 


193 


5 


211 


30 


2 


366 


9 


351 


33 


1 


443 


7 


439 


34 


1 


402 


4 


513 


females 










18 


1 


76 








23 


1 


150 


3 


165 


27 


1 


232 


6 


272 


28 


4 


278 


10 


293 



October-November 



males 










19 


1 


70 


1 


64 


26 


2 


194 


6 


223 


females 










28 


1 


280 


9 


270 


31 


1 


404 


9 


368 


35 


1 


541 


8 


551 


38 


1 


652 


3 


774 



tration was massive and the gut was partly or largely destroyed; light infections 
included those with from one to 20 cysts in the wall of the intestine. By the time 
that cysts are formed the infection is already well established. 

In Table VI, the winter flounders less than 100 mm. in total length were of 
the 1964 year class, i.e., less than a year old. Those of 100 mm. or more in length 
probably were of the 1963 year class, but final age-determination was not made. 

Since the microsporidiosis is located primarily in the intestine, and the infective 
agent was presumably taken in with food, the stomach-contents of 751 fishes from 

TABLE IV 

Amount of food in stomachs of infected and non-infected icinter jiminder 
from Woods Hole Harbor in July-August, 1962 



Month 


Infected 


Non-infected 


Number 


Mean length 
cm. 


Food 
grams 


Number 


Mean length 
cm. 


Food 
grams 


July 
August 


7 
6 


28 
26 


1.25 
1.02 


102 
156 


30 

27 


2.19 

0.82 



I!()R.\( I \\ -|i ' > i k! l> I- I I \ 



i V 
Incidence of microsporidian inffcti<>n i>; ' d> i 



different Acw England fishing gr 



Location 


( it-ill -41-- Hank 


Ofl luth, 
Nova Scotia 


X.mtiiekrt Shoals 


Ol'f Plymouth, 
Mass. 


Length 

nil. 


r.it.,i 

number 


Nui 

inte. 


Total 
number 


Number 
infected 


Total 
number 


Number 
intccted 


Total 
number 


Number 
infected 


11-15 


1 























16-20 


1 





1 


1 














21 J5 














16 


1 


1 





26- SO 


1 











25 


1 








31-35 


6 





1 





53 


11 


12 


1 


36 to 


6 





3 





23 


7 


5 


2 


41-45 


16 





2 





8 


1 


1 





46-50 


2 











1 











51-55 


3 




















56 60 


1 























(>1 65 
Total 


1 








8 




1 












3 


38 


126 


21 


1') 


1 'IT renl 










infected 





1.2 


16.7 


15.8 



Woods Hole harbor have been examined in an attempt to discern the source or 
sources of the infective agent or agents. Also, since Microsporidia are presumed to 
be one-host parasites, microsporidian cysts from winter flounder gut-wall embedded 

TABU; VI 

Incidence <>j microsporidian injection in >/;/</// winter flounder 
from Lake Tushinoo, Martha's Vineyard 






Degree of infection 



(mm.) 


Heavy 


Light 


None 


11 -15 


2 








lo 50 


1 





1 


51 55 


6 


1 


1 


50 Mi 


6 


3 


3 


61 65 


2 


3 


4 


66 70 





2 


7 


71 75 


1 


6 


8 


76 Ml 


1 


2 


4 


xi 85 





2 


4 


86 'MI 


1 


4 


1 


91- 'J5 


o 





2 


'JO 100 











101 105 








2 


100 110 








2 


1 lll.ll 


23 


23 


39 



I Vr (in iiilVi-trd, 51.1. 



MICROSPORIDIOSIS IN WINTER FLOUNDER 381 

in pieces of clam, Mercenaria mercenaries, have been fed to other winter Bounder 
kept in aquaria in attempts to induce experimental infections, 
two fish in the summer of 1962. They were examined six weeks later and tli< 
was no evidence of infection. Five fish were fed cysts in November, 1964. Three 
were examined in April, 1965, and the other two were autopsied in June, 1965. 
No infection resulted from these experiments. It appears that direct infection does 
not occur, that either the sporoplasms do not emerge from the spores or they fail 
to invade the intestinal epithelium. 

Tissues from natural infections were fixed in different fluids, cut in serial sec- 
tions at 5 and 10 microns in thickness, and stained for particular effects. Haema- 
toxylin and erythrosin were used for general purposes and routine pathological 
staining. Heidenhain's iron technique was employed on thin sections for cyto- 
logical details and azan trichrome for special histology. 

RESULTS 

The incidence of infection in 751 fishes taken in the Woods Hole harbor and 
examined each month, April through November, 1962, and data on the size and sex 
of the fishes are presented in Tables I-III. There was no apparent effect of sea- 
sonal or sexual differences. Table IV records the amount of food in the stomachs 
of infected and non-infected fishes of comparable sizes taken from Woods Hole 
harbor in July and August, 1962. There was no obvious relationship between in- 
fection and amount of food in the stomach. Stomach-contents of 386 fishes col- 
lected in weekly samples in September, October, and November, 1961, consisted 
by weight of algae, 42% ; mollusks, 25% ; polychaetes, 24% ; crustaceans, 5% ; and 
other (mostly unidentified), 4%. The results of analyses made in 1962 are similar, 
with less algae eaten in the spring and summer. No fish were found in any of the 
flounders examined, thus confirming the statements by Bean (1903), Breder (1929) 
and Bigelow and Schroeder (1953) that young flounders feed exclusively on algae 
and invertebrates, chiefly crustaceans and polychaete annelids. The account of 
Bigelow and Schroeder is very complete and includes the findings of Breder and 
Linton as well as their own observations. It is generally agreed that the small 
mouths of these flounders preclude the ingestion of fishes as food, and it appears 
certain that fish are of no significance in the diet of P. americanus in the Woods 
Hole area. 

The incidence of infection in winter flounder of different sizes taken from dif- 
ferent coastal areas and from Georges Bank, which is offshore, is presented in Table 
V. Although the number of fishes from Georges Bank is small, the absence of 
infection there may be significant. There is evidence that Georges Bank winter 
flounder are geographically isolated from those on inshore grounds and that they 
have no contact with the shore at any time during their lives. Results from the 
release on inshore grounds of over 10,000 tagged winter flounder indicate that 
only one was re-caught on Georges Bank (Perlmutter, 1947; Bigelow and Schroe- 
der, 1953). Perlmutter also reported that winter flounder from Georges Bank have 
more fin rays than those from inshore grounds north and south of Cape Cod. Win- 
ter flounder from inshore subpopulations, on the other hand, are closely associated 
with the shore, spending their first year in estuaries and bays where much of the 
spawning occurs, and where infection may take place. 



382 Ilok.U'K \V. ST1 ^ ECARD AND FRED E. LUX 

The data from tlu- small fishes taken in November. 1 ( '(>4, at Martha's Vineyard 
< Table VI i are particularly interesting !n addition to the information presented 
in the table. 4 ( > individuals in the same si/e-range died in the period November 20 
to December 7, 1 ( >M. Nineteen of these fishes, mostly 65 to 85 mm. in length, were 
infected, with an incidence o! Seven of the infections were heavy; 12 of 

them were light. Fifteen of the remaining fishes were killed March 12, 1965, and 
8 of them, i.e., 53. 3 %, were infected. Three of these infections were heavy; five 
\\ere light. Inspection of the data from the 149 fishes examined shows that infec- 
tion was greatest in small fishes Almost all of the heavily infected ones were less 
than 80 mm. in length and some of them were less than 50 mm. in length. Since 
development of such massive infections must take some time, it is apparent that 
infection occurred very early in life, when the food consisted of small invertebrates. 
Comparison of the findings recorded in Tables II and VI, indicates strongly that 
fishes heavily infected during the first year of life do not survive into their second 
year. 

The site of infection is primarily the wall of the intestine and pyloric ceca, but 
in moderate and heavy infections, other structures adjacent to or in contact with the 
gut may be involved. These include the bile-duct, liver, mesenterial lymph-nodes 
and the ovary. The infections observed were already well advanced and were 
manifest by cysts (Figs. 4, 5) embedded in the connective tissue of the organs af- 
fected. In larger fishes, most of the infections were light and apparently did not 
seriously affect the hosts. In light infections, the cysts were usually on the external 
wall of the intestine, but in heavy ones, the gut wall was largely supplanted by 
layers of cysts. In such instances the intestine had a chalk-white, pebbled appear- 
ance and the wall was rigid, thickened and hard. Photographs of intact normal 
and parasitized digestive tracts and of sections of the intestine and cecum of infected 
fishes portray the effects of massive infection. Figure 1 is of a normal digestive 
tract. In the specimen shown in Figure 2, the anterior end of the digestive tract 
is the principal site of infection, whereas in Figure 3, it is the rectal end of the 
specimen that is most heavily parasitized. Figure 4 is a photograph of a cross- 
section ot the intestine shown in Figure 2. and Figure 5 is a photograph of a cross- 
section of one of the pyloric ceca taken from the specimen shown in Figure 2. The 
epithelium of the intestine in Figure 4 is denuded and the lumen of the cecum (Fig. 
5) is almost occluded. 

The cysts are spherical to oval unless deformed by pressure. They measure 
0.6 to 1.0 mm. in diameter and the wall (Figs. 6, 7) is composed of laminated layers 
that have the structural appearance and staining reactions of the connective tissue 
of the host. In addition to those in the cysts, there are masses or strands of spores 
scattered about in the tissue of the gut wall, often associated with or paralleling 
blood vessels. The material at present available for study consists of relatively 
mature infections multiplicative phases have largely been completed. 

Rarely, near the wall oi rst or in the intercystal areas there is a cell, which may 
be a pansporoblast, which contains a large number of bodies that color deeply with 
nuclear stains. Whether or not th< se structures are the nuclei of sporoblasts could 
not be determined. I'.elow the connective capsule of the cyst there is often a nar- 
row layer of stainable material, termed endoplasm by Woodcock, which contains 
large oval, apparently pycnotic, nuclei with fragmented chromatin and distinct nu- 



MICROSPORIDIOSIS IN WINTER FLOUNDER 



383 



cleoli. Their presence suggests that the cyst is formed around a number of host- 
cells, whose cytoplasm has been consumed and whose nuclei persist below the wall 
of the cyst. The spores are oval to ovate, and when fixed and stained measure 
about 4 by 2.5 microns. Precise and accurate measurements of such minute and 
refractive bodies are difficult. The basal, wider end of the spore contains a vesicle 




PLATE I 

FIGURE 1. Digestive tract of P. americanus, normal condition, in a Petri-dish, 9.5 cm. 
outside diameter. 

FIGURE 2. Pyloric ceca and intestine of infected fish, same magnification as Figure 1. 
FIGURE 3. Digestive tract of infected fish, same magnification as Figure 1. 



384 



HORACE W. STUNKARD VXD FRED E. LUX 



- 




i \TE II 



MICROSPORIDIOSIS IN WINTER FLOUNDER 

that may occupy almost one-half the length of the spore. The apical end alsf 
tains a smaller vesicle, while the central portion contains a band of chromatic n ate- 
rial, often in the form of strings of particles or granules, and a single strand 
tends to the apical end of the spore. 

DISCUSSION 

The most comprehensive account of the Microsporidia is the monograph by 
Kudo (1924). He gave a review of morphology and life-cycles, with a description 
and taxonomic survey of all previously described species. In a later study, Kudo 
(1944) stated (p. 38), "The early phases of the development of Microsporidia have 
not been seen in many species. In a few instances of experimental infection, cer- 
tain portions of the development have been seen, but in no case has observation in 
life been carried through." It is generally agreed, however, that the life-cycle of 
a microsporidian species consists of two distinct phases or stages : a multiplicative 
stage, schizogony, and a spore-forming stage, sporogony. In the multiplicative 
phase, cell division is rapid and according to certain investigators it may result 
from binary fission following nuclear division or multiple fission if nuclear division 
is rapid and cytoplasmic division is delayed. It has been suggested (Kudo, 1924) 
that in certain species the schizonts (meronts of Stempell, 1902) are not motile 
and as a consequence that the progeny of a sporoplasm remains in the host-cell and 
that all the spores formed in that cell are derived from the initial parasite. But 
usually the infection is invasive, with diffuse infiltration of tissue, and such a con- 
dition could result from either the penetration and dispersal of enormous numbers 
of planonts or by the liberation of schizonts and their ingestion by leukocytes or 
macrophages which accumulate at sites of inflammation and which could transport 
the schizonts to other areas and extend the infection. At the end of the schizogonic 
phase, sporonts are formed but the factors involved and details of the phenomena 
which result in the formation of sporonts are equivocal. Supporting the observa- 
tions of Weissenberg (1914), Debaisieux (1920) and Guyenot and Naville (1922), 
Kudo (1946, p. 162) stated, "In the Microsporidia, autogamy appears to initiate 
the spore-formation at the end of schizogonic activity." 

In the present study, the inability to obtain experimental infection of fishes has 
precluded observations on the multiplicative phases of the life-cycle. But this in- 
ability has raised important and perplexing problems. Since fishes become in- 
fected when only 50 mm. in length and when the food consists of small invertebrates, 
it seems probable that a second or intermediate host may be required in the life- 
cycle of the parasite. Such an invertebrate may be merely a paratenic or transport 
host, which ingests spores from a dead fish and is then eaten by a small flounder, 
or it may be essential in the completion of the life-cycle of the parasite. Since 

FIGURE 4. Photomicrograph of cross-section of the intestine shown in Figure 2. Note lack 
of digestive epithelium and disintegration of the gut wall. 

FIGURE 5. Photomicrograph of cross-section of one of the pyloric ceca shown in Figure 2. 
The infection is more intense in this area than in the intestine. 

FIGURE 6. Photomicrograph of section of pyloric cecuin, greater magnification, to show 
connective tissue capsular wall of the cyst and number of spores. 

FIGURE 7. Photomicrograph of section of pyloric cecum, showing walls of cysts and 
adjacent nuclei and cells. 



386 HORACE W. STUNKARD AND FRED E. LUX 

small crustaceans are carnivorous and constitute a considerable part of the food of 
small fishes, they become suspect. According to the account of Frederick E. Smith, 
(The Benthos of Block Island Sound : Ph.D. thesis, Yale University, 1950, 213 pp. 
and appendices), 75% of the food of the winter flounder consisted of amphipods 
and 437o of the amphipods were Lcptoclicints pinguis. 

Other questions also arise : why are certain infections mild while others become 
massive? Do older fishes develop resistance to infection and restrict the invasive 
activity of the parasite: \Yhy do the cysts manifest such uniformity in size? 
Finally, in view of the statements of Reichenow (1929) and Poisson (1953) that 
generic concepts are tenuous, what is the status of Gluc/ca and does the species. 
stcphani, belong in that genus? The answers to these and other questions await 
further investigation on the life-cycle of the species. 

SUMMARY 

A inicnporidian infection of the blackback or winter flounder. Pseudopleuro- 
ncctcs aincrianius. has been investigated. It was first noted at Woods Hole. 
Massachusetts by I.inton (1901) and may be identical with similar infections of 
Furopean flounders reported by Hagenmuller (1899), who described the parasite 
as Xoscnia stcphani. Woodcock (1904) transferred the species to Glmjca. a genus 
erected by Thelohan (1891) to contain a parasite of the striated muscle in Coitus 
scorpio and Cullion\iiuis lynt, which he described as a new species, Glugca inicro- 
spora. Gurley (1893) predicated that G. niicrospora is identical with Noscnnt 
(inoiinihi ( Moniez, 1877), although he recognized Gluc/ca as a valid genus, distinct 
from Nosema. In New England the infection is common in P. aiiicricanus. The 
incidence and intensity of infection in fishes of different sizes and from different 
geographical regions are reported, together with an account of the resultant 
pathology. Attempts to obtain experimental infection of fishes have not been 
-uccessful and the life-cycle of the parasite remains unknown. 

LITERATURE CITED 

\ ii KiiAen, M., 110. Die- Cnidosporidien (Myxosporidien, Actinomyxidien, Microsporidien). 

Eine Monographische Studie. 261 pp., Leipzig. 
BEAN, T. II.. l'H)3. Catalonia- of the Eishes of New York. Bull. Xo. 60, Zool. ( ). Xe\v York 

State MiiM'iim, 7X4 pp. 
BIGELOW, 11. 1'... AMI \\'. C. >< MKoKi.'KK, 1 ( )53. Fishes of the Gulf of Maine. U. S. Gov't. 

Fishery Mull., \ T o. 74, 577pp. 
BOND, E. l ; ., l''3X. Cnidosporidia from Fitndiilns Jictcniclihis Liu. Trans. Aincr. Micros. Sue.. 

57: in/ !-'_'. 
BREDER, C. M.. JR., 1"-"'. Held l><>k of Marine Fishes of the Atlantic Coast. G. P. Putnam'* 

Sons Xeu York. 
CHEISSIX, K. M., AMI G. 1. POI.JAXSKV. 1%3. On the taxonomic system of Proto/oa. Ada 

Proto >o t, 1 (31) : 327-352. 

I )i.i',Ais]Krx. P.. P'20. fctudes >ur les microsporidies. IV. Cilin/cd anmihihi Monx. I .a Ccllnl-.-, 

30: 215-245. 
I )i-.H.M.siKrx. I'., l''J. v fUiui' i \ lolo.nifnu-s sur <|iu-l(|ues microsporidies. In ('cllnlr. 38: 

387-451 
hooiEL, V. A., G. K. Pi '.IM SHEVSK] AND Y. 1. Poi.YA x SK i, 1961. Parasitology of Fislu-s. 

English edit., 3X4 pp. ' Iliver X- P-oyd, Edinburgh & London. 

FANTHAM, H. 1'., A.NNIE PORTED MI L. R. RICHAKDSON, 1941. Some Microsporidia found in 
tain fishes and insects in eastern Canada. I \n-asitol., 33: 186-208. 



MICROSPORID1OSIS IN WINTER FLOUNDER 

GURLEY, R. R., 1893. On the classification of the Myxosporidia, a group of pro oan par;. 

infesting fishes. Bull. U. S. Fish Comm. (for 1891), 11: 407-420. 
GUYENOT, E., AND A. NAVILLE, 1922. Recherches sur le parasitisme et revolution d'une micro 

sporidie, Ghtqca danilewski L. Pfr. (?) parasite de la couleuvre. AVr. Sirissc '/ . 

30: 1-61. 
HAGENMULLER, M., 1899. Sur une nouvelle myxosporidie, Noscma stephani, ])arasite du Plexus 

passer Moreau. C. R. Acad. Set., 129:' 836-839. 
HAHN", C. W., 1917. On the sporozoan parasites of fishes of Woods Hole and vicinity. III. 

On the Chloromy.rinn clupcidae of Chipea harengus (Young), Pomolobns pseiul>- 

harcngus (Young) and P. aestivalis (Young). /. Parasit., 4: 13-20. 
HALEY, A. J., 1952. Preliminary observations on a severe epidemic of microsporidiosis in the 

smelt, Osmcrus mordax (Mitchell). /. Parasit., 38: 183. 
HALL, R. P., 1953. Protozoology. Prentice-Hall, New York. 
JOHNSTONE, J., 1901. Note on a sporozoan parasite of the plaice (Pleuronectes platessa). 

Proc. Trans. Liverpool. Biol. Soc., 15: 184-187. 

KUDO, R., 1920. Studies on Myxosporidia. Illinois Biol. Monogr., 5 (3 and 4) : 1-265. 
KUDO, R., 1924. A biologic and taxonomic study of the Microsporidia. Illinois Biol. Monogr., 

9 (2 and 3) : 1-268. 
KUDO, R., 1944. Morphology a