(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "The Biological bulletin"

THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 



E. G. CONKLIN, Princeton University 

E. N. HARVEY, Princeton University 

SELIG HECHT, Columbia University 

LEIGH HOADLEY, Harvard University 

L. IRVING, Swarthmore College 

M. H. JACOBS, University of Pennsylvania 

H. S. JENNINGS, Johns Hopkins University 



FRANK R. LILLIE, University of Chicago 
CARL R. MOORE, University of Chicago 
GEORGE T. MOORE, Missouri Botanical Garden 
G. H. PARKER, Harvard University 
A. C. REDFIELD, Harvard University 
F. SCHRADER, Columbia University 
DOUGLAS WHITAKER, Stanford University 



H. B. STEINBACH, Washington University 
Managing Editor 



VOLUME 91 

AUGUST TO DECEMBER, 1946 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE & LEMON STS. 

LANCASTER, PA. 



11 



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

Subscriptions and similar matter should be addressed to The 
Biological Bulletin, Marine Biological Laboratory, Woods Hole, 
Massachusetts. Agent for Great Britain: Wheldon and Wesley, 
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, 
W. C. 2. Single numbers, $1.75. Subscription per volume (three 
issues), $4.50. 

Communications relative to manuscripts should be sent to the 
Managing Editor, Marine Biological Laboratory, Woods Hole, 
Massachusetts, between July 1 and September 1, and to the De- 
partment of Zoology, Washington University, St. Louis, Missouri, 
during the remainder of the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, 
Pa., under the Act of August 24, 1912. 



1ANCASTFR PRESS. INC., LANCASTER, PA 



CONTENTS 



No. 1. AUGUST, 1946 

PAGE 

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY ... 1 

POMERAT, C. M., AND C. M. WEISS 

The influence of texture and composition of surface on the attachment 

of sedentary marine organisms 57 

SCOTT, SISTER FLORENCE MARIE 

The developmental history of Amaroecium constellatum. II. Organo- 

genesis of the larval action system 66 

GIESE, ARTHUR C. 

Comparative sensitivity of sperm and eggs to ultraviolet radiations . 81 
CARRIKER, MELBOURNE ROMAINE 

' Observations on the functioning of the alimentary system of the snail 

Lymnaea stagnalis appressa Say . . 88 

CHEN, TZE-TUAN 

Temporary pair formation in Paramecium bursaria . 112 

No. 2. OCTOBER, 1946 

WEISZ, PAUL B. 

The space-time pattern of segment formation in Artemia salina. . 119 

JAKUS, M. A., AND C. E. HALL 

Electron microscope observations of the trichocysts and cilia in Para- 
mecium 141 

PEASE, DANIEL C. 

Hydrostatic pressure effects upon the spindle figure and chromosome 
movement. II. Experiments on the meiotic divisions of Tradescantia 
pollen mother cells 145 

BROWN, FRANK A. JR., AND LORRAINE M. SAIGH 

The comparative distribution of two chromatophorotropic hormones 
(CDH and CBLH) in Crustacean nervous systems 170 

MORRISON, PETER R. 

Physiological observations on water loss and oxygen consumption in 
Peripatus 181 

KOZLOFF, EUGENE N. 

Studies on ciliates of the family Ancistrocomidae Chatton and Lowff 
(order Holotricha, suborder Thigmotricha). III. Ancistrocoma pelse- 
neeri Chatton and Lwoff, Ancistrocoma dissimilis sp. nov., and Hypo- 
comagalma pholadidis sp. nov .... 189 

KOZLOFF, EUGENE N. 

Studies on ciliates of the family Ancistrocomidae Chatton and Lwoff 
(order Holotricha, suborder Thigmotricha). IV. Heterocineta janickii 
Jarocki, Heterocineta goniobasidis sp. nov., Heterocineta fluminicolae 

sp. nov., and Enerthecoma properans Jarocki 200 

iii 



60544 



iv CONTENTS 

ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL 
LABORATORY, SUMMER OF 1946 210 

PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GENERAL PHYSI- 
OLOGISTS . . 236 

No. 3. DECEMBER, 1946 

WHITING, P. W. 

A strongly intersexual female in Habrobracon . . 243 

TOBIAS, J. M., AND J. J. KOLLROS 

Loci of action of DDT in the cockroach (Periplaneta americana) . 247 

BEERS, C. D. 

Tillina magna: Micronuclear number, encystment and vitality in diverse 
clones; capabilities of amicronucleate races. . . . 256 

SCOTT, ALLAN 

The effect of low temperature and of hypotonicity on the morphology 

of the cleavage furrow in Arbacia eggs. . . 272 

BODENSTEIN, DIETRICH 

Developmental relations between genital ducts and gonads in Droso- 
phila 288 

LEHMAN, H. E. 

A histological study of Syndisyrinx franciscanus, gen. et sp. nov., an 
endoparasitic rhabclocoel of the sea urchin, Strongylocentrotus francis- 
canus 295 

SPOOR, W. A. 

A quantitative study of the relationship between the activity and oxygen 
consumption of the goldfish, and its application to the measurement of 
respiratory metabolism in fishes . 



Vol. 91, No. 1 August, 1946 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



THE MARINE BIOLOGICAL LABORATORY 
FORTY-EIGHTH REPORT, FOR THE YEAR 1945 FIFTY-EIGHTH YEAR 

I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 14, 1945) .... .1 

STANDING COMMITTEES 
II. ACT OF INCORPORATION 

III. BY-LAWS OF THE CORPORATION 

IV. REPORT OF THE TREASURER 

V. REPORT OF THE LIBRARIAN 

VI. REPORT OF THE DIRECTOR 11 

Statement 11 

Addenda : 

1 . Publications from this Laboratory during the years 1941-1945. . 13 

2. The Staff 33 

3. Investigators and Students 35 

4. Tabular View of Attendance, 1941-1945 41 

5. Subscribing and Co-operating Institutions 42 

6. Evening Lectures 42 

7. Shorter Scientific Papers 43 

8. Members of the Corporation 44 



I. TRUSTEES 

EX OFFICIO 

FRANK R. LILLIE, President Emeritus of the Corporation, The University of Chicago 
LAWRASON RIGGS, President of the Corporation, 120 Broadway, New York City 
E. NEWTON HARVEY, Vice President of the Corporation, Princeton University 
CHARLES PACKARD, Director, Marine Biological Laboratory 
OTTO C. GLASER, Clerk of the Corporation, Amherst College 
DONALD M. BRODIE, Treasurer, 522 Fifth Avenue, New York City 



EMERITUS 



E. G. CONKLIN, Princeton University 
B. M. DUGGAR, University of Wisconsin 
W. E. GARREY, Vanderbilt University 
R. A. HARPER, Columbia University 
Ross G. HARRISON, Yale University 

H. S. JENNINGS, University of California 

F. P. KNOWLTON, Syracuse University 



2 MARINE BIOLOGICAL LABORATORY 

R. S. LILLIE, The University of Chicago 

*C. E. McCLUNG, University of Pennsylvania 

S. O. MAST, Johns Hopkins University 

A. P. MATHEWS, University of Cincinnati 

*T. H. MORGAN, California Institute of Technology 

W. J. V. OSTERHOUT, Rockefeller Institute 

G. H. PARKER, Harvard University 

W. B. SCOTT, Princeton University 

TO SERVE UNTIL 1949 

W. R. AMBERSON, University of Maryland School of Medicine 

P. B. ARMSTRONG, Syracuse University 

L. G. BARTH, Columbia University 

S. C. BROOKS, University of California 

W. C. CURTIS, University of Missouri 

H. B. GOODRICH, Wesleyan University 

A. C. REDFIELD, Harvard University 

C. C. SPEIDEL, University of Virginia 

TO SERVE UNTIL 1948 

ERIC G. BALL, Harvard University Medical School 

R. CHAMBERS, Washington Square College, New York University 

EUGENE F. DuBois, Cornell University Medical College 

COLUMBUS ISELIN, Woods Hole Oceanographic Institution 

C. W. METZ, University of Pennsylvania 

H. H. PLOUGH, Amherst College 

E. W. SINNOTT, Yale University 

W. R. TAYLOR, University of Michigan 

TO SERVE UNTIL 1947 

W. C. ALLEE, The University of Chicago 

G. H. A. CLOWES, Lilly Research Laboratory 

P. S. GALTSOFF, U. S. Fish and Wild Life Service 

L. V. HEILBRUNN, University of Pennsylvania 

LAURENCE IRVING, Swarthmore College 

J. H. NORTHROP, Rockefeller Institute 

A. H. STURTEVANT, California Institute of Technology 

LORANDE L. WOODRUFF, Yale University 

TO SERVE UNTIL 1946 

DUGALD E. S. BROWN, New York University 

E. R. CLARK, University of Pennsylvania 

OTTO C. GLASER, Amherst College 

E. N. HARVEY, Princeton University 

M. H. JACOBS, University of Pennsylvania 

A. K. PARPART, Princeton University 
FRANZ SCHRADER, Columbia University 

B. H. WILLIER, Johns Hopkins University 

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES 

LAWRASON RIGGS, Ex officio. Chairman 
E. N. HARVEY, Ex officio 

* Deceased. 



ACT OF INCORPORATION 

D. M. BRODIE, Ex officio 
CHARLES PACKARD, Ex officio 

P. B. ARMSTRONG, to serve until 1947 

L. G. EARTH, to serve until 1946 

P. S. GALTSOFF, to serve until 1947 

WM. RANDOLPH TAYLOR, to serve until 1946 

THE LIBRARY COMMITTEE 
A. C. REDFIELD, Chairman 

E. G. BALL 
S. C. BROOKS 
M. E. KRAHL 
J. W. MAYOR 

THE APPARATUS COMMITTEE 

E. P. LITTLE, Chairman 

C. L. CLAFF 
G. FAILLA 
S. E. HILL 

A. K. PARPART 

THE SUPPLY DEPARTMENT COMMITTEE 

D. A. MARSLAND, Chairman 
P. B. ARMSTRONG 

P. S. GALTSOFF 
R. T. KEMPTON 
CHARLES PACKARD 

THE EVENING LECTURE COMMITTEE 

F. M. LANDIS, Chairman 
CHARLES PACKARD 

THE INSTRUCTION COMMITTEE 

H. B. GOODRICH, Chairman 
W. C. ALLEE 
S. C. BROOKS 
VIKTOR HAMBURGER 
CHARLES PACKARD, Ex officio 

THE BUILDINGS AND GROUNDS COMMITTEE 

E. G. BALL, Chairman 
D. P. COSTELLO 
MRS. E. N. HARVEY 
ROBERTS RUGH 

MRS. C. C. SPEIDEL 



II. ACT OF INCORPORATION 
No. 3170 

COMMONWEALTH OF MASSACHUSETTS 

Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. 
Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, 
William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves 
with the intention of forming a Corporation under the name of the Marine Biological 
Laboratory, for the purpose of establishing and maintaining a laboratory or station for 



4 MARINE BIOLOGICAL LABORATORY 

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

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

HENRY B. PIERCE, 
Secretary of the Commonwealth. 



III. BY-LAWS OF THE CORPORATION OF THE MARINE 

BIOLOGICAL LABORATORY 

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

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

III. The Annual Meeting of the members shall be held on the second Tuesday in 
August in each year, at the Laboratory in Woods Hole, Massachusetts, at 11:30 A.M., 
and at such meeting the members shall choose by ballot a Treasurer arid 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. 

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

VI. Inasmuch as the time and place of the Annual Meeting of members are fixed by 
these By-laws, no notice of the Annual Meeting need be given. Notice of any special 
meeting of members, however, shall be given by the Clerk by mailing notice of the time 
and place and purpose of such meeting, at least fifteen (15) days before such meeting, 
to each member at his or her address as shown on the records of the Corporation. 

VII. The Annual Meeting of the Trustees shall be held on the second Tuesday in 
August in each year, at the Laboratory in Woods Hole, Mass., at 10 A.M. Special 
meetings of the Trustees shall be called by the President, or by any seven Trustees, to be 
held at such time and place as may be designated, and the Secretary shall give notice 
thereof by written or printed notice, mailed to each Trustee at his address as shown on 
the records of the Corporation, at least one (1) week before the meeting. At such 
special meeting only matters stated in the notice shall be considered. Seven Trustees of 
those eligible to vote shall constitute a quorum for the transaction of business at any 
meeting. 

VIII. There shall be three groups of Trustees : 

(A) Thirty-two Trustees chosen by the Corporation, divided into four classes, each 
to serve four years ; and in addition there shall be two groups of Trustees as follows : 



REPORT OF THE TREASURER 5 

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

(C) Trustees Emeritus, who shall be elected from the 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, whereupon 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 Emeritus shall have all the rights of the Trustees except that Trustees Emeritus shall 
not have the right to vote. 

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

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

X. Any person interested in the Laboratory may be elected by the Trustees to a group 
to be known as Associates of the Marine Biological Laboratory. 

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

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

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



IV. THE REPORT OF THE TREASURER 
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY: 

Gentlemen: 

Herewith is my report as Treasurer of the Marine Biological Laboratory for 
the year 1945. 

The accounts have been audited by Messrs. Seamans, Stetson, and Tuttle, certi- 
fied public accountants. A copy of their report is on file at the Laboratory and 
inspection of it by members of the Corporation will be welcomed. 

The principal summaries of their report The Balance Sheet, Statement of 
Income and Expense, and Current Surplus Account are appended hereto as 
Exhibits A, B, and C. 

The following are some general statements and observations based on the de- 
tailed reports : 



6 MARINE BIOLOGICAL LABORATORY 

I. Assets 

1. Endowment Assets 

As of December 31, 1945, the total book value of all the Endowment Assets, 
including the Scholarship Funds, was $966,772.16, a loss for the year of $17,128.41. 
The decline was due, as in the last two years, to losses on the mortgage participa- 
tions on New York City realty held in the Trust Funds. 

At the end of the year $831,993.01 was invested in marketable securities (bonds, 
preferred stocks and common stocks) with a market value of $910,162.31. $125,- 
753.85 was invested in mortgage participations on New York City real estate. 
$9,025.30 was in uninvested principal cash. 

The Treasurer's estimate of the actual value of the $125,753.85 in mortgage 
notes and participations held on December 31 is $85,750.00. With the market 
value of $910,162.31 on marketable securities and the $9,025.30 in cash this makes 
a total current valuation of $1,004,937.61 compared with total book value of 
$966,772.16. 

The increase for the year in market values, $75,454.79, is largely due to the 
rise in common stock prices. 

t 

2. Plant Assets 

There were no changes of any consequence in Plant Assets during the year. 
The Reserve Fund was increased nearly $10,000.00 to a total of $26,830.71 by the 
transfer of the Crane Co. dividends, part of the General Biological Supply House 
dividends and other items of current income. 

3. Current Assets 

The total of current assets increased $10,730.68 during 1945 to a total of 
$212,970.35. Current Liabilities at the end of the year were $2,754.70. Current 
Surplus increased $12,277.94 to a total of $196,337.90. 

II. Income and Expenditures 

The return to more normal operations for the Laboratory last summer resulted 
in larger totals for both income and expense items. Total income was $182,818.23, 
total expense including depreciation reserves of $26,968.12 was $173,044.95, giving 
a net surplus for the year of $9,773.28. 

EXHIBIT A 

MARINE BIOLOGICAL LABORATORY BALACE SHEET, DECEMBER 31, 1945 

Assets 
Endowment Assets and Equities : 

Securities and Cash in Hands of Central Hanover Bank and 

Trust Company, New York, Trustee $ 950,130.04 

Securities and Cash in Minor Funds 16,642.12 

$ 966,772.16 



REPORT OF THE TREASURER 

Plant Assets : 

Land $ 111,425.38 

Buildings 1,326,345.54 

Equipment 187,837.87 

Library 337,266.01 



Less Reserve for Depreciation 

Reserve Fund, Securities and Cash 
Book Fund, Securities and Cash . 



$1,962,874.80 
677,140.22 



Current Assets : 

Cash 

Accounts Receivable 

Inventories : 

Supply Department $ 44,441.66 

Biological Bulletin 20,1 17.40 



$1,285,734.58 

26,830.71 
18,282.46 



$ 30,467.02 
20,396.05 



64,559.06 



$1,330,847.75 






Investments : 

Devil's Lane Property $ 46,556.99 

Gansett Property 1,749.92 

Stock in General Biological Supply House, 

Inc 12,700.00 

Other Investment Stocks 20,095.00 

Retirement Fund 11,517.82 



Prepaid Insurance 
Items in Suspense 



92,619.73 

4,033.08 
895.41 




$ 212,970.35 

Total Assets $2,510,590.26 

Liabilities 

Endowment Funds : 

Endowment Funds $ 948.646.S2 

Reserve for Amortization of Bond Premiums.. 1,483.22 



Minor Funds 



Plant Funds : 

Mortgage Notes Payable 

Donations and Gifts $1,172,564.04 

Other Investments in Plant from Gifts and 

Current Funds 153,283.71 



Current Liabilities and Surplus : 

Accounts Payable 

Items in Suspense 

Reserve for Repairs and Replacements 
Current Surplus 



$ 950,130.04 
16,642.12 



$ 5,000.00 



$1,325,847.75 



$ 2,754.70 

1,799.63 

12,078.12 

196,337.90 



$ 966,772.16 



$1,330,847.75 



$ 212,970.35 
Total Liabilities $2,510,590.26 



MARINE BIOLOGICAL LABORATORY 
EXHIBIT B 

MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE, 
YEAR ENDED DECEMBER 31, 1945 

Total Net 

Expense Income Expense Income 

Income : 



General Endowment Fund 




$ 32,214.07 




$ 32,214.07 


Library Fund 




9,479.18 




9,479.18 


Donations 




755.00 




755.00 


Instruction -. 


$ 9,554.39 


7,220.00 


$ 2,334.39 




Research 


4,550.59 


17,434.24 




12,883.65 


Evening Lectures 


86.35 




86.35 




Biological Bulletin and Membership Dues. 


6,393.65 


8.775.63 




2,381.98 


Supply Department 


39,255.03 


47,812.56 




8,557.53 


Mess 


24,146.52 


20,750.36 


3,396.16 




Dormitories 


27,443.23 


14,547.91 


12,895.32 




(Interest and Depreciation charged to above 










3 Departments) 


(25,574.03) 






25,574.03 


Dividends, General Biological Supply House, 










Inc 




14.732.00 




14.732.0C 


Dividends, Other Investment Stocks 




725.00 




725.00 


Rents : 










Bar Neck Property 


767.65 


6,000.00 




5,232.35 


Janitor House 


30.89 


360.00 




329.11 


Danchakoff Cottages 


240.86 


275.00 




34.14 


Sale of Library Duplicates, Micro Film, etc. 




344.74 




344.74 


Microscope and Apparatus Rental 




1,372.54 




1,372.54 


Sundry Income 




20.00 




20.00 


Maintenance of Plant : 










Buildings and Grounds 


23,642.27 




23,642.27 




Apparatus Department 


4,911.52 




4,911.52 




Chemical Department 


2,265.30 


. 


2,265.30 




Library Expense 


6,487.95 


/ 


6,487.95 




Workmen's Compensation Insurance .... 


526.63 




526.63 




Truck Expense 


238.60 




238.60 




Bay Shore Property 


92.78 




92.78 




Great Cedar Swamp 


21.00 




21.00 





General Expenses : 

Administration Expense 15,168.99 15,168.99 

Endowment Fund Trustee and Safe-Keep- 
ing 1,028.45 1,028.45 

Bad Debts 375.97 375.97 

Special Repairs on account of 1944 Hurri- 
cane Damage 4,297.24 4,297.24 

Interest 125.00 125.00 

Reserve for Depreciation 26,968.12 26,968.12 



$173,044.95 $182,818.23 $104,862.04 $114,635.32 
Excess of Income over Expense carried to 

Current Surplus 9,773.28 9,773.28 



$182,818.23 $114,635.32 



REPORT OF THE LIBRARIAN 
EXHIBIT C 

MARINE BIOLOGICAL LABORATORY. CURRENT SURPLUS ACCOUNT, 
YEAR ENDED DECEMBER 31, 1945 

Balance January 1, 1945 $184,059.96 

Add: 

Excess of Income over Expense for Year as shown, in Exhibit B . . $ 9,773.28 

Gain on Gansett Lots Sold 464.18 

Bad Debts Recovered 82.23 

Mortgage Payable, Transferred to Plant Funds 5,000.00 

Reserve for Depreciation Charged to Plant Funds 26.968.J2 42,287.81 



$226,347.77 
Deduct : 

Payments from Current Funds during Year for Plant Assets as 
shown in Schedule IV: 

Buildings $ 7,402.65 

Equipment 4,462.50 

Library 7,500.43 



$19,365.58 
Less Received for Plant Assets Sold 5,600.00 



$13,765.58 

Pensions Paid $ 3,460.00 

Loss on Retirement Fund Securities 847.32 




$ 4,307.32 
Less Retirement Fund Income 311.79 



$ 3,995.53 
Transfers to Reserve Fund : 

Portion of Dividends from General Biological Sup- 
ply House, Inc $ 2,500.00 

Dividends from Crane Co 625.00 

Income from Operation and Sale of Property 445- 

51 W. 23rd and 450-2 W. 24th Sts., N. Y. C. 8,947.72 
Gansett Property Profits, 1944 176.04 12,248.76 30,009.87 



Balance, December 31, 1945 $196,337.90 

Respectfully submitted, 

DONALD M. BRODIE, 

Treasurer 



V. REPORT OF THE LIBRARIAN 

The sum $12,262.54 appropriated to the library in 1945 was expended as 
follows: books, $469.99; serials, $2,625.79; binding, $577.60; express, $43.22; 
supplies, $147.84; salaries, $7,262.54 ($1,150 of this sum was contributed by the 
Woods Hole Oceanographic Institution) ; back sets, $1,104.98; insurance, $45.00; 



10 MARINE BIOLOGICAL LABORATORY 

sundries, $5.00 ; total, $12,281 .96. The cash receipts of the library totalled $344.74 : 
for microfilms, $220.34 ($62.17 expenses paid by the library and accounted above 
under "supplies") ; sale of duplicates, $122.74; sale of the "Serial List," Biological 
Bulletin supplement number, $1.66. This sum, $344.74, reverts to the laboratory 
and does not include rent payments for library readers which are collected by the 
main office. There were 49 library readers accommodated in the library during 
the summer of 1945. 

Of the Carnegie of New York Fund, $126.35 was expended for the completion 
of one journal and the partial completion of another. 

The sum appropriated by the Woods Hole Oceanographic Institution in 1945 
for purchases was $800. A balance of $949.39 remaining from 1944 made an 
available total of $1,749.39. Of this sum $947.12 was expended, leaving a balance 
of $802.27 towards future purchases. In addition to the above, the Woods Hole 
Oceanographic Institution contributed $1,150 (see above under salaries). 

During 1945 the library received 902 current journals: 279 (8 new) by sub- 
scription to the Marine Biological Laboratory; 30 (7 new) to the Woods Hole 
Oceanographic Institution; exchanges 352 (6 new; 145 reinstated foreign) and 58 
(35 foreign reinstated) with the Woods Hole Oceanographic Institution publica- 
tions; 177 as gifts to the former and 6 to the latter. The library acquired 206 
books : 77 by purchase of the Marine Biological Laboratory ; 44 by purchase of 
the Woods Hole Oceanographic Institution; 10 gifts by the authors; 46 gifts by 
the publishers ; 20 by miscellaneous donors and 9 from Miss Jane Strong. There 
were 22 back sets of serial publications completed: 14 purchased by the Marine 
Biological Laboratory (one with the Carnegie Fund) ; 2 by the Woods Hole 
Oceanographic Institution ; 5 by exchange of the "Biological Bulletin" and one by 
exchange of duplicate material. Partially completed sets were 54: purchased by 
the Marine Biology Laboratory, 27 (1 by the Carnegie Fund) ; purchased by the 
Woods Hole Oceanographic Institution, 2 ; by exchange with the "Biological 
Bulletin," 2; by gift and exchange of duplicate material, 23. 

The reprint additions to the library were 4,620; current of 1944, 604; current 
of 1945, 64; and of previous dates, 3,952. A total of 6,390, 2,130 not duplicates 
of our holdings, were presented to the library ; 4,295 by Mrs. Meigs ; 67 by Dr. 
H. G. Cassidy; 627 by the University of Utah; 26 by Dr. B. M. Davis; and 1,375 
by Dr. L. C. Wyman. The large collection of Dr. Carrey's reprints presented last 
year have not as yet been counted nor started on the way toward cataloguing. 

At the end of the year 1945 the library contained 53,990 bound volumes and 
137,674 reprints. 

Readers of the library report will be glad to note the number of foreign ex- 
changes that have already been reinstated during 1945, both for the "Biological 
Bulletin" and for the Woods Hole Oceanographic Institution publications : 145 for 
the former and 35 for the latter. Next year's report will probably show the pre-war 
number reinstated save only for Germany and perhaps for Russia since we get very 
poor response from that country. Nor have we heard anything in regard to the 
German journals on order with Otto Harrassowitz which are apparently stalled 
if not destroyed in Leipzig. The library committee that is working with the State 
Department to get these released has nothing so far to report to this library. 



REPORT OF THE DIRECTOR 

VI. REPORT OF THE DIRECTOR 

To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY: 
Gentlemen: 

I herewith submit a report on the fifty-eighth session of the Marine Biological 
Laboratory. 

1. Attendance 

Since 1943 our total' attendance has increased from the low point reached that 
year. In 1944 it was 53 per cent of the pre-war average of 490; in 1945 it was 
63 per cent. This increase is found among the independent investigators and the 
students; the beginning investigators and research assistants, who, as I explained 
in the last report, belong in one group, are still sparsely represented. In 1945 
there were only 36, whereas the pre-war average was 130. The advance registra- 
tion for 1946 shows an encouraging increase in this group. Many of the appli- 
cants are veterans who are taking advantage of Government funds provided under 
the G. I. Bill of Rights. 

2. Building Repairs 

One of the inevitable effects of the war has been the deterioration of our build- 
ings. Lack of materials and labor has up until now prevented all but the most 
essential repairs from being made. Fortunately, we are now able to begin to put 
our house in order, in spite of the shortage of some critical materials. To deter- 
mine what work should be done, a Committee on Special Repairs, under the able 
leadership of Mr. C. L. Claff, conducted a thorough survey of all of the buildings 
and made detailed recommendations. The report, a model of completeness as 
drawn up by Mr. Claff, calls for the ultimate expenditure of approximately $145,000 
for present repairs and future desirable improvements not only in the buildings but 
also in equipment for the Apparatus Department and the Supply Department. 

The Executive Committee voted to expend the entire Reserve Fund, amounting 
to $25,000, and all but a minimum of the current cash on hand for making the most 
urgently needed repairs at once. It also laid plans for securing outside funds with 
which to complete the changes called for in the report, and to purchase apparatus. 
In addition, funds for a new building and for additional endowment are to be 
sought. 

Many of the essential repairs have already been made. The Mess kitchen, 
never properly restored after the Navy occupation, is now in good condition, and 
improvements in the dining room have been made. The Botany Building, unused 
for several seasons, has been put to rights with new plumbing, wall tables, shelves, 
and other fixtures. Replacements have been made in the Supply Department and 
Rockefeller Building; hot water systems are installed in the residences heretofore 
not so provided ; and much painting has been done. This work was accomplished 
in large measure by our permanent staff, under the direction of Mr. MacNaught. 
All the men worked faithfully and energetically, and have completed the assigned 
tasks in a most satisfactory manner. It is hoped that waterproofing of the Crane 
and Brick Buildings may be completed before the 1946 season begins. As soon as 
this has been satisfactorily finished, those Laboratory rooms which have been dam- 
aged by water can be made presentable. 



12 MARINE BIOLOGICAL LABORATORY 

3. The Housing Problem 

An unexpected outcome of war-time activities is the housing shortage. Before 
the war our residences and the houses in the village could accommodate 450 to 500 
persons during the course of a summer. But when the Oceanographic Institution 
emharked on extensive defense projects, the number of its workers increased from 
comparatively few to upwards of 250, most of whom are year round residents. 
They now occupy most of the available houses in the village ; some are forced to 
live as far away as North Falmouth and Hyannis. As a result of this crowding 
we shall be unable, in the summer of 1946, to take care of more than 375 investi- 
gators and students that is, about 100 less than our pre-war average. Indeed, 
we can accommodate this number only because the authorities of the U. S. Fish 
and Wild Life Commission have granted us the use of a part of the Fisheries 
residence. For their cooperation in this, and in many other ways, the Laboratory 
is grateful. 

When it is possible once more to build houses, some of this pressure for living 
space will be relieved. To encourage investigators to have homes here in Woods 
Hole, the Laboratory has opened up the Devil's Lane tract, situated a mile and 
more from the center of the village, between the State Road to Falmouth and the 
railroad. About 100 lots will presently be available. 

In the meantime, the number of applicants for research space will undoubtedly 
increase, and we shall be unable to find places for all cmalified investigators who 
wish to come. The Administration thus faces the unwelcome prospect of having 
to choose between applicants. The Executive Committee has ruled that investi- 
gators, instructors, and students should have preference over Library readers in 
the residences and at the Mess. But some further method of selection must be 
followed until the housing shortage is relieved. 

4. Financial Problems 

The report of the Treasurer shows that our financial condition is sound; that 
is, we are free from debt, and have about $57,000 in the Reserve and Current Cash 
accounts. But most of this has already been ear-marked to pay for the most 
necessary repairs, and for foreign journals not yet delivered. We shall still need 
a larger amount for other needed repairs and replacements. When these have 
been made we can say that our regular income from all sources is sufficient to 
maintain the Laboratory on its present basis. But in order to expand our research 
facilities we must have additional funds. It is estimated that $30,000 each year 
should be spent for this purpose. 

5. Gifts 

Mr. Allen R. Memhard has provided a fund of $1,000, the income of which 
may be awarded to a qualified student who has completed the Embryology course. 

Mrs. Adele K. Strieker has presented to the Laboratory the sum of $50 in 
memory of her son, Capt. George J. Strieker, who worked here during the sum- 
mers of 1933 and 1934. 

Dr. A. C. Redfield contributed $100 for a hedge and trees to be planted to the 
east of the Stone Building. 

Donations for current purposes received during the year were as follows : 
Mrs. E. B. Meigs, $25.00; Dr. William D. Curtis, $100.00; M. B. L. Associates, 
$630.00. 



REPORT OF THE DIRECTOR 13 

6. Deaths 

This year \ve have sustained irreparable losses by death ; Dr. T. H. Morgan, 
Trustee since 1897, whose scientific achievements and devotion to this Laboratory 
from its earliest days contributed greatly to its growth in usefulness and influence, 
and Dr. C. E. McClung, elected Trustee in 1913, active in all Laboratory affairs, 
especially in the building up of our great Library. 

7. Election 'of Trustees 

At the meeting of the Corporation, held August 14, 1945. the following were 
elected Trustees Emeriti: Dr. F. P. Knowlton, elected Trustee in 1922; Dr. R. S. 
Lillie, elected Trustee in 1921. 

The following were elected Trustees: Dr. P. B. Armstrong, Professor of 
Anatomy, College of Medicine, Syracuse University ; Dr. A. K. Parpart, Associate 
Professor of Biology, Princeton University. 

8. Publications 

The Executive Committee voted to print in this Report a list of papers, based 
wholly or in part on work done at this Laboratory, and published during the years 
1941-1945. A similar list, which appeared in the Annual Report of 1908, covered 
the years from the beginning of the Laboratory in 1888 to 1907. It is hoped that 
eventually a complete compilation of titles to include the intervening years may 
be made. 

Appended as parts of this Report are : 

1. Publications from this Laboratory during the years 1941-1945. 

2. The Staff. 

3. Investigators and Students. 

4. Tabular View of Attendance, 1941-1945. 

5. Subscribing and Cooperating Institutions. 

6. Evening Lectures. 

7. Shorter Scientific Papers. 

8. Members of the Corporation. 

Respectfully submitted, 

CHARLES PACKARD, 

Director 

( 

1. PUBLICATIONS FROM THE MARINE BIOLOGICAL LABORATORY, 1941-1945 

Note: An asterisk before a title indicates that the work was done only in part at this Laboratory. 

ABELL, R. G. On the comparative permeability of blood capillary sprouts, newly formed capil- 
laries, and older capillaries. Anat. Rcc., 82: 1942. 

ABELL, R. G. See also Zweifach, Abell, Chambers, and Clowes, 1945. 

ABELL, R. G. AND I. H. PAGE. *Behavior of the arterioles in hypertensive rabbits and in normal 
rabbits following injections of angiotonin. Biol. Bull., 81: 1941 (abs.). 

ABELL, R. G. AND I. H. PAGE. *The reaction of peripheral blood vessels to angiotonin, renin, 
and other pressor agents. Jour. E.rp. Med., 75 : 1942. 

ADDISON, W. H. F. *The distribution of elastic tissue in the arterial pathway to the carotid 
bodies in the adult dog. Bio!. Bull., 81: 1941 (abs.). 

ADDISON, W. H. F. *Histologic methods adapted for rat tissues. A Chapter in "The Rat in 
Laboratory Investigation," Lippincott, 1942. 

ADDISON, W. H. F. The hypophysis of the goose-fish. Anat. Rcc.. 85: 1943 (abs.). 



14 MARINE BIOLOGICAL LABORATORY 

ADDISON, W. H F. *The extent of the carotid pressoreceptor area in the cat as indicated by 

its special elastic-tissue wall. Anat. Rec., 91 : 1945. 
ADDISON, W. H. F. *The arterial relations of the glomus caroticum in the rabbit. Anat. Rec., 

88: 1944 (abs.). 
ALBAUM, H. G. AND BARRY COMMONER. The relation between the four-carbon acids and the 

growth of bat seedlings. Blol. Bull, 80: 1941. 

ALLEE, W. C. *Integration of problems concerning protozoan populations with those of gen- 
eral biology. Amer. Nat., 75: 1941. 
ALLEE, W. C. AND RUTH M. MERWIN. The effect of carbon dioxide on the rate of cleavage 

in frog's eggs. Anat. Rec. Suppl, 81: 1941 (abs.). 
ALLEE, W. C. AND RUTH M. MERWIN. The effect of low concentration of carbon dioxide on 

the cleavage rate in frog's eggs. Ecology, 24: 1943. 
ALLEE, W. C, A. J. FINKEL AND H. R. GARNER. Factors affecting rate of cleavage in Arbacia; 

the accelerating action of copper. Anat. Rec. SuppL, 81: 1941 (abs.). 
ALLEE, W. C., A. J. FINKEL AND H. R. GARNER. Copper and the acceleration of cleavage. 

Jour. Cell. Comp. Physiol, 20: 1942. 
ALLEE, W. C., A. J. FINKEL, H. R. GARNER, R. M. MERWIN AND G. E. EVANS. Some effects 

of homotypic extracts on the rate of cleavage of Arbacia eggs. Biol. Bull., 83 : 1942. 
ALLEE, W. C. AND MARJORIE B. DOUGLIS. A dominance order in the hermit crab, Pagurus 

longicarpus Say. Ecology, 26 : 1945. 
ALSUP, F. W. Photodynamic action in the eggs of Nereis limbata. Jour. Cell. Comp. Physiol., 

17: 1941. 

ALSUP, F. W. Photodynamic studies on Arbacia eggs. Biol. Bull., 81 : 1941. 
ALSUP, F. W. The effects of light alone and photodynamic action on relative viscosity of 

Amoeba protoplasm. Physiol. Zool., 15: 1942. 
ANDERSON, T. F. *The study of colloids with the electron microscope. Advances in Colloid 

Science, 1 : 1942. 
ANDERSON, T. F. *The application of the electron microscope to biology. Collecting Net, 17 : 

1942. 
ANDERSON, T. F. See also Harvey, E. B. and Anderson, 1943; Luria, Delbruck and Anderson, 

1943 ; Richards, Steinbach and Anderson, 1943. 
ANDERSON, T. F. AND A. G. RICHARDS. Nature through the electron microscope. Scientific 

Month., 55 : 1942. 
ANDREW, WARREN. The reticular nature of glia fibers in the cerebrum of the frog and in the 

higher vertebrates. Jour. Comp. Neural., 79 : 1943. 
ANGERER, C. A. Sec also Hartman, Lewis, Brownell, Sheldon and Angerer. Physiol. Zool., 

17: 1944. 
ANGERER, C. A. AND H. ANGERER. *Weight variations of muscles of adrenalectomized frogs in 

normal and hypotonic Ringer solutions. Amer. Jour. Physiol., 133: 1941. 
ANGERER, C. A. AND K. M. WILBUR. *The action of various types of electric fields on the 

relative viscosity of plasmagel of Amoeba proteus. Physiol. Zool., 16 : 1943. 
BAILEY, BASIL, P. KORAN AND H. C. BRADLEY. The autolysis of muscle of highly active and 

less active fish. Biol. Bull, 83 : 1942. 

BAKER, GLADYS E. *Studies in the genus Physalacria. Bull. Torrey Bot. Club, 68 : 1941. 
BAKER, H. D. *Notes on Salasiella from Mexico. Nautilus, 54: 1941. 
BAKER, H. D. *Zonitid snails from Pacific Islands. Bull. Bishop Museum, Honolulu, 166: 

1941. 

BAKER, H. D. *Some Haplotrematidae. Nautilus, 54: 1941. 
BAKER, H. D. *Puerto Rican Oleacinidae. Nautilus, 55 : 1941. 
BAKER, H. D. ^Outline of American Oleacininae and new species from Mexico. Nautilus, 

55: 1941. 

BAKER, H. D. *A new genus of Mexican helicids. Nautilus, 56: 1942. 
BAKER, H. D. *A new genus of Chinese Microcystinae. Nautilus, 56 : 1942. 
BAKER, H. D. *Some Antillean helicids. Nautilus, 56: 1943. 

BAKER, H. D. *The mainland genera of American Oleacininae. Proc. Acad. Nat. Sci., Phila- 
delphia, 95 : 1943. 

BAKER, R. F. See Cole and Baker, 1941. 
BALL, E. G. A blue chromoprotein found in the eggs of the goose-barnacle. Jour. Biol. Chcm., 

152: 1944. 



REPORT OF THE DIRECTOR 15 

BALL, ERNEST. The effects of synthetic growth substances on the shoot apex of Tropaeolum 

majus. Amer. Jour. Bot., 31 : 1944. 
BALLARD, W. W. The mechanism for synchronous spawning in Hydractinia and Pennaria 

B iol Bull, 82 : 1942. 

BALLENTINE, ROBERT. See Parpart and Ballentine, 1943. 
BARNES, W. A. AND O. B. FURTH. Roentgen rays in single and parabiotic mice. Amer. Jour. 

Roentgenol. and Radium Therap., 69: 1943. 
BARRON, E. S. G. AND J. M. GOLDINGER. Pyruvate metabolism in fertilized and non-fertilized 

sea urchin eggs. Biol. Bull., 81 : 1941. 

EARTH, L. G. *Neural differentiation without organizer. Jour. Exp. Zool., 87: 1941. 
BARTH, L. G. *Oxygen consumption of the amphibian gastrula. Physiol. Zool., 15 : 1942. 
BARTH, L. G. *Colloid chemistry of development. A chapter in Alexander's Colloid Chem- 
istry. New York, 1944. 
BARTH, L. G. The determination of the regenerating hydranth in Tubularia. Physiol. Zool., 

17: 1944. 

BARTH, L. G. Sec also Goldin and Earth, 1941. 

BARTLETT, J. H. *Transient Anode Phenomena. Trans. Electrochem. Soc., 87 : 1945. 
BARTLETT, J. H. *Periodic phenomena at anodes. Phys. Rev., 67 : 1945. 
BEAMS, H. W. See Evans, Beams and Smith, 1941. 
BEERS, C. D. The role of bacteria in the excystment of the ciliate Didinium nasutum. Biol. 

Bull., 89: 1945 (abs.). 
BELDA, W. H. *Permeability to water in Pelomyxa carolinensis. 1. Changes in volume of 

P. carolinensis in solutions of different osmotic concentration. The Salesianum, 37: 1942. 
BELDA, W. H. 2. The contractile vacuoles of P. carolinensis. The Salesianum, 37 : 1942. 
BELDA, W. H. 3. The permeability constant for water in P. caroliensis. The Salesianum, 

38: 1943. 
BERGER, C. A. *Some criteria for judging the degree of polyploidy of cells in the resting stage. 

Amer. Nat., 75: 1941. 

BERGER, C. A. *Reinvestigation of polysomaty in Spinacia. Bot. Gaz., 102: 1941. 
BERGER, C. A. ^Multiple chromosome complexes in animals and polysomaty in plants. Cold 

Spring Harbor Symposia, 9: 1941. 

BERGER, C. A. *Experimental studies on the cytology of Allium. Torreya (abs.) 44 : 1944. 
BERGER, C. A. AND E. R. WITKUS. *A cytological study of c-mitosis in the polysomatic plant 

Spinacia oleracea, with comparative observations on Allium cepa. Bull. Torrey. Bot. Club, 

70f: 1943. 
BERGER, C. A. AND E. R. WITKUS. *Veratrine, a new polyploidy inducing agent. Jour. Hered., 

35: 1944. 
BERGER, C. A., E. R. WITKUS AND B. J. SULLIVAN. *The cytological effects of benzene vapor. 

Bull. Torrey Bot. Club, 71 : 1944. 
BERTHOLF, L. M. Accelerating metamorphosis in the tunicate, Styela partita. Biol. Bull., 89 : 

1945 (abs.). 
BERTHOLF, L. M. AND S. O. MAST. Metamorphosis in the larva of the tunicate, Styela partita. 

Biol. Bull., 87: 1944 (abs.). 
BEVELANDER, GERRIT. ^Radioactive phosphate absorption by dentin and enamel. Jour. Dent. 

Res., 24: 1945. 
BEVELANDER, GERRIT. *The histochemical localization of alkaline phosphatase in the developing 

tooth. Jour. Cell. Comp. Physiol., 26 : 1945. 
BEVELANDER, GERRIT. *The localization of phosphatase in the cyclic growth of the hair,. Anat. 

Rcc., 91 : 1945. 
BIRMINGHAM, LLOYD. Regeneration in the early zooid of Amaroucium constellatum. Biol. 

Bull, 81: 1941 (abs.). 
BLISS, A. F. Derived photosensitive pigments from invertebrate eyes. Jour. Gen. Physiol, 

26: 1943. 
BOCHE, R. D. AND J. B. BUCK. Studies on the hydrogen ion concentration of insect blood and 

their bearing on in vitro cytological technique. Physiol. Zool, 15 : 1942. 
BODIAN, DAVID. *Cytological aspects of synaptic function. Physiol. Rev., 22 : 1942. 
BODIAN, DAVID. *Poliomyelitic changes in tnultinucleated neurons, with special reference to 

the site of action of virus in the cell. Bull. Johns Hopkins Hosp., 77 : 1945. 



16 MARINE BIOLOGICAL LABORATORY 

BODIAN, DAVID AND R. C. MELLORS. *The regenerative cycle of motor neurons, with special 

reference to phosphatase activity. Jour. E.\-p. Mcd., 81 : 1945. 
BOELL, E. J. AND L. L. WOODRUFF. Respiratory metabolism and mating types in Paramoecium 

calkinsi. Jour. E.vpcr. Zoo!., 87: 1941. 
BOTSFORD, E. FRANCES. The effect of physostigmine on the responses of earthworm body wall 

preparations to successive stimuli. Biol. Bull., 80: 1941. 
BRADLEY, H. C. Sec Bailey, Koran and Bradley, 1942. 
BROOKS, M. M. *Infrared spectrophotometric studies on hemoglobin as affected by cyanide, 

methylene blue, and carbon monoxide. Amcr. Jour. PhysioL, 132: 1941. 
BROOKS, M. M. Effects of CO and methylene blue upon O~ consumption of shark blood. Proc. 

Soc. Exp. Biol. Mcd., 46: 1941. 

BROOKS, M. M. Interpretations of effects of CO and CN on oxidations in living cells. Col- 
lecting Net, 16:' 1941. 
BROOKS, M. M. Further interpretations of the effects of CO and CN on oxidations in living 

cells. Biol. Bull., 81: 1941. 

BROOKS, M. M. The effect of thiamine chloride on the oxygen consumption and the develop- 
ment of Arbacia punctulata at different stages. Biol. Bull., 83 : 1942. 
BROOKS, M. M. Mechanism of fertilization of eggs. Federation Proc., 2 : 1943. 
BROOKS, M. M. Methylene blue, potassium cyanide, and carbon monoxide as indicators for 
studying the oxidation-reduction potentials of developing marine eggs. Biol. Bull., 84 : 
1943. 
BROOKS, M. M. *E1 mecanismo de accion del azul de metileno en las celulas vivas. Adas 

Acad. Nacional de Ciensa c.ractas, fisicas y naliialcs de Lima, Peru, 7 : 1944. 
BROOKS, M. M. *The effect of methylene blue on performance efficiency at high altitudes. 

Jour. Aviation Mcd., 16: 1945. 
BROOKS, M. M. *Oxidation-reduction studies on Penicillium notatum and other organisms. 

Biol. Bull., 89 : 1945. 
BROOKS, M. M. Electrode potential measurements of Penicillium notatum. Federation Proc., 

4: 1945. 

BROOKS, M. M. Mechanism of fertilization of eggs. Federation Proc., 5: 1945. 
BROOKS, M. M. AND S. C. BROOKS. ^Permeability of Living Cells. Gebriider Borntraeger, 

Berlin. Preprinted by Edwards Bros., 1945. 
BROOKS, S. C. Intake and loss of phosphate ions by eggs and larvae of Arbacia and Asterias. 

Biol. Bull., 83 : 1942. 

BROOKS, S. C. Intake and loss of ions by living cells. 1. Eggs and larvae of Arbacia punctu- 
lata and Asterias forbesi exposed to phosphate and sodium ions. Biol. Bull., 84: 1943. 
BROOKS, S. C. 2. Early changes of phosphate content of Fundulus eggs. Biol. Bull., 84 : 1943. 
BROOKS, S. C. *The permeability of cells. Science, 100: 1944. 
BROOKS, S. C. Sec also Brooks and Brooks, 1945. 
BROWN, D. E. S. Sec Marsland and Brown, 1941, 1942; Marsland, Johnson and Brown, 1941, 

1942; Hiatt. Brown, Quinn and MacDuffie, 1945. 
BROWN, F. A. AND O. CUNNINGHAM. Upon the presence and distribution of a chromatophoro- 

tropic principle in the central nervous system of Limulus. Biol. Bull., 81 : 1941. 
BROWN, F. A. AND V. J. WULFF. Chromatophore types in Crago and their endocrine control. 

Jour. Cell. Comp. PhysioL, 18: 1941. 
BROWNELL, K. A. Sec Hartman, Lewis, Brownell, Shelden and Walther, 1941 ; Hartman, 

Lewis, Brownell, Angerer and Shelden, 1944. 

BUCHSBAUM, RALPH AND R. WILLIAMSON. The rate of elongation and constriction of dividing 
sea urchin eggs as a test of a mathematical theory of cell division. PhysioL ZooL, 16 : 1943. 
BUCK, J. B. *Micromanipulation of salivary gland chromosomes. Jour. Hered., 33 : 1942. 
BUCK, J. B. Sec also Boche and Buck, 1942. 
BUCK, J. B. AND A. M. MELLAND. ^Methods for isolating, collecting, and orienting salivary 

gland chromosomes for diffraction analysis. Jour. Hercd., 33: 1942. 

BUDINGTON, R. A. The ciliary transport-system of Asterias forbesii. Biol. Bull., 83 : 1942. 
BULLOCK, T. H. Neuromuscular facilitation in scyphomedusae. Jour. Cell. Comp. PhysioL, 

22: 1943. 

BULLOCK, T. H. The giant nerve fiber system in balanoglossids. Jour. Comp. Neural. , 80: 
1944. 



REPORT OF THE DIRECTOR 17 

BULLOCK, T. H. Problems in the comparative study of brain waves. Yale Jour. Biol. Mcd., 

17: 1945. 
BULLOCK, T. H. Organization of the giant nerve fiber system in Neanthes virens. Biol. Bull., 

89 : 1945. 
'BULLOCK, T. H. Anatomical organization of the nervous system of Enteropneusta. Quart. Jour. 

Micr. Set., 86 : 1945. 
BULLOCK, T. H. AND D. NACHMANSOHN. Choline esterase in primitive nervous systems. 

Jour. Cell. Comp. Physiol, 20: 1942. 
CABLE, R. M. Sec also Hunninen and Cable, 1941, 1943. 

CABLE, R. M. AND A. V. HUNNINEN. Studies on Deropristis inflata, its life history and affi- 
nities to trematodes of the family Acanthocolpidae. Biol. Bull.. 82 : 1942. 
CABLE, R. M. AND A. V. HUNNINEN. Studies on the life history of Siphodera vinaledwardsii. 

Jour. Parasitol., 28 : 1942. 
CAHEN, R. L. *The effects of morphine on the cortical activity of the rat. Yale Jour. Biol. 

Mcd., 16: 1944. 
CAHEN, R. L. *The influence of morphine on tissue permeability and the spreading effect of 

hyaluronidase. Yale Jour. Biol. Mcd., 16 : 1944. 
CAHEN, R. L. *Urinary 17 ketosteroids in metabolism. 1. Standardized chemical estimation. 

Jour. Biol. Chem., 152: 1944. 
CANNAN, R. K. *The hydrogen ion dissociation curve of beta-lactoglobulin. Jour. Biol. Chem., 

142: 1942. 
CANNAN, R. K. *The dicarboxylic amino acids in protein hydrolysates. Jour. Biol. Chem., 

152: 1944. 
CARSON, H. L. A comparative study of the apical cell of the insect testis. Jour. Morph., 77 : 

1945. 
CHAMBERS, ROBERT. The intrinsic expansibility of the fertilization membrane of echinoderm 

ova. Jour. Cell. Comp. Physiol., 19: 1942. 
CHAMBERS, ROBERT. Electrolytic solutions compatible with the maintenance of protoplasmic 

structures. Biol. Symposia, 10: 1943. 

CHAMBERS, ROBERT. Post war Biology rehabilitation. Science. 100: 1944. 
CHAMBERS, ROBERT. Some physical properties of protoplasm. Chapter in Alexander's Colloid 

Chemistry, 1944. 
CHAMBERS, ROBERT. ^Rehabilitation of the biological sciences in the post-war period. Am. 

Nat., 79: 1945. 
CHAMBERS, ROBERT, B. W. ZWEIFACH, B. E. LOWENSTEIN AND R. E. LEE. Vaso-excitor and 

vaso-depressor substances as "toxic" factors in experimentally induced shock. Proc. Soc. 

Exp. Biol. Med., 56: 1944. 
CHAMBERS, ROBERT, B. W. ZWEIFACH AND B. E. LOWENSTEIN. ^Circulatory reactions of rats, 

traumatized in the Noble-Collip drum. Am. J. Physiol., 139: 1943. 
CHAMBERS, ROBERT, B. W. ZWEIFACH AND B. E. LOWENSTEIN. *The peripheral circulation 

during the tourniquet syndrome in the rat. Ann. Surg., 120: 1944. 
CHASE, A. M. *Effect of azide on Cypridina luciferin. Collecting Net, 16 : 1941. 
CHASE, A. M. Observations on luminescence in Mnemiopsis. Biol. Bull. 81: 1941 (abs.). 
CHASE, A. M. *The reaction of Cypridina luciferin with azide. Jour. Cell Comp. Physiol., 19 : 

1942. 
CHASE, A. M. *The absorption spectrum of luciferin and oxidized luciferin. Jour. Biol. Chem., 

150: 1943. 

CHASE, A. M. *The visible absorption band of reduced luciferin. Jour. Biol. Chem., 159: 1945. 
CHENEY, R. H. Myofibrillar modifications induced by caffeine in cardiac muscle of the frog. 

Jour. Comp. Physiol., 18: 1941. 
CHENEY, R. H. Caffeine effect in fertilization and development of Arbacia eggs. Biol. Bull., 

83: 1942 (abs.). 

CHENEY, R. H. Oxygen consumption of caffeinized Arbacia eggs. Biol. Bull., 83: 1942. (abs.). 
CHENEY, R. H. Development of Arbacia eggs in caffeinized sea water. Anat. Rec., 84: 1942 

(abs.). 
CHENEY, R. H. Inhibitory effect of caffeine on oxygen consumption in Arbacia eggs. Anat. 

Rec., 84: 1942 (abs.). 
CHENEY, R. H. *Variation in reproductive phenomena by caffeine. Federation Proc., 3 : 1944 

(abs.). 



18 MARINE BIOLOGICAL LABORATORY 

CHENEY, R. H. The effects of caffeine on oxygen consumption and cell division in the fertilized 
egg of the sea urchin. Jour. Gen. Physiol., 29: 1945. 

CHURNEY, LEON. The osmotic properties of the nucleus. Biol. Bull., 82 : 1942. 

CLAFF, C. L. *Glass electrode for determination of hydrogen ion activity of small quantities 
of culture media. Science, 94: 1941. 

CLAFF, C. L., VIRGINA C. DEWEY AND G. W. KIDDER. *Feeding mechanisms in three species 
of Bresslaua. Biol. Bull., 81 : 1941. 

CLAFF, C. L. AND O. SWENSON. *Micro glass electrode technique for determination of hydrogen- 
ion activity of blood and other biological fluids. Jour. Biol. Chcin., 152: 1944. 

CLARK, E. R. AND ELEANOR L. CLARK. ^Microscopic observations on the formation of cartilage 
and bone in the living mammal. Amcr. Jour. Anat., 70: 1942. 

CLARK, E. R. AND ELEANOR L. CLARK. *Caliber changes in minute blood vessels observed in 
living mammal. Amer. Jour. Anat., 73: 1943. 

CLARK, E. R. AND ELEANOR L. CLARK *The formation of venae comites. Anat. Rec., 85: 1943. 

CLARK, E. R. AND ELEANOR L. CLARK. *Growth and behavior of epidermis as observed mi- 
croscopically in the living, in chambers introduced in the rabbit's ear. Anat. Rcc., 88 : 1944. 

CLOWES, G. H. A. *Interactions of biologically significant substances in surface films, with 
especial reference to two-dimensional solutions and association complexes formed by car- 
cinogenic hydrocarbons and sterols. Publication 21. Amer. Assoc. Advance. Sci., 1942. 

CLOWES, G. H. A. See also Krahl, Keltch, Neubeck and Clowes, 1941 ; Keltch, Baker, Krahl 
and Clowes, 1941; Davis, Baker and Clowes, 1941, 1942; Krahl, Jandorff and Clowes, 1942; 
Powell, Krahl and Clowes, 1942; Hutchens, Keltch, Krahl and Clowes, 1942; Zweifach, 
Abell, Chambers and Clowes, 1945. 

COLE, K. S. Rectification and inductance in the squid giant axon. Jour. Gen. Ph\siol., 25: 1941. 

COLE, K. S. See also Guttman and Cole, 1941 ; Curtis and Cole, 1942, 1944. 

COLE, K. S. AND R. F. BAKER. Transverse impedance of the squid giant axon during current 
flow. Jour. Gen. Physiol., 24: 1941. 

COLE, K. S. AND R. F. BAKER. Longitudinal impedance of the squid giant axon. Jour. Gen. 
Physiol., 24 : 1941. 

COLE, K. S. AND H. J. CURTIS. Membrane potential of the squid giant axon during current flow. 
Jour. Gen. Physiol., 24: 1941. 

COLE, K. S. AND H. J. CURTIS. *Electrical Physiology : Electrical resistance and impedance of 
cells and tissues. Chapter in Medical Physics. Chicago, 1944. 

COLE, K. S. AND R. H. COLE. *Dispersion and absorption in dielectrics. 1. Alternating current 
characteristics. Jour. Cheni. Phys.,2: 1941. 

COLE, K. S. AND R. H. COLE. *2. Direct current characteristics. Jour. Chcm. Phys. 10 : 1942. 

COLWIN, LAURA H. Binary fission and conjugation in Urceolaria synapta, with special refer- 
ence to the nuclear phenomena. Jour. Morph., 75: 1944. 

COMMONER, BARRY. Sec Albaum and Commoner, 1941. 

CORNMAN, IVOR. Characteristics of the acceleration of Arbacia cleavage in hypotonic sea- 
water. Biol. Bull. 81: 1941 (abs.). 

CORNMAN, IVOR. Sperm activation by Arbacia egg extractives, with special reference to 
echinoehrome. Biol. Bull. 80: 1941. 

CORNMAN, IVOR. Acceleration of cleavage of Arbacia eggs by hypotonic sea water. Biol. Bull., 
84: 1943. 

COSTELLO, D. P. ^Advances in Zoology during 1940. hid. Engineer. Chcm., 19: 1941. 

COSTELLO, D. P. Segregation of ooplasmic constituents. Jour. Elisha Mitchell Sci. Soc., 61 : 
1945. 

COSTELLO, D. P. Experimental studies in germinal localization in Nereis. 1. The development 
of isolated blastomeres. Jour. E.rp. Zool., 100: 1945. 

COSTELLO, D. P. WITH G. I. LAVIN. Ultra violet photomicroscopy of the Nereis and Asterias 
egg. Anat. Rcc. Suppl., 87: 1943. (abs.). 

CROUSE, HELEN V. *Translocations in Sciara ; their bearing on chromosome behavior and sex 
determination. Univ. Missouri Res. Bull., 379: 1943. 

CROUSE, HELEN V. See also Ris and Crouse, 1945. 

CROWELL, SEARS. A comparison of shells utilized by Hydractinia and Podocoryne. Ecology, 
26 : 1945. 

CUNNINGHAM, O. Sec Brown and Cunningham, 1941. 



REPORT OF THE DIRECTOR 19 

CURTIS, H. J. See also Cole and Curtis, 1941. 

CURTIS, H. J. AND K. S. COLE. Membrane resting and action potentials' from the squid giant 

axon. Jour. Cell. Comfy. Physiol., 19: 1942. 
DAVSON-, HUGH. See also Shapiro and Davson, 1941. 
DAVSON, HUGH AND J. M. REINER. Ionic permeability; an enzyme-like factor concerned in 

the migration of sodium through the cat erythrocyte membrane. Jour. Cell. Comp. Physiol 

20: 1942. 

DELBRUCK, M. See Luria, Delbruck and Anderson, 1943. 

DENT, J. N. *The embryonic development of Plethodon cinereus as correlated with the dif- 
ferentiation and functioning of the thyroid gland. Jour. Morph. 71 : 1942. 
DENT, J. N. See also Lynn and Dent, 1941. 
DEWEY, VIRGINIA C. See also Claff, Dewey and Kidder, 1941. 
DEWEY, VIRGINIA C. AND G. W. KIDDER. The possibility of thiamin synthesis by ciliates. Biol. 

Bull., 81: 1941. (abs.). 

DOUGLIS, MARJORIE B. See Alice and Douglis, 1945. 
DZIEMIAN, A. J. *The permeability and the lipid content of immature red cells. Jour. Cell. 

Comp. Physiol., 20 : 1942. 
EVANS, T. C., H. W. BEAMS, AND M. E. SMITH. Effects of roentgen radiation on the jelly of 

the Arbacia egg. Biol. Bull. 80: 1941. 
EVANS, T. C. AND J. C. SLAUGHTER. Effect of sea water on the radiosensitivity of Arbacia 

sperm. Biol. Bull., 81 : 1941 (abs.). 
EVANS, T. C. AND J. C. SLAUGHTER, E. P. LITTLE AND G. FAILLA. Influence of the medium on 

radiation injury of sperm. Radiology, 39: 1942. 
FAILLA, G. See Evans, Slaughter, Little and Failla, 1942. 

FAUST, E. C. *Clinical Parasitology. Co-author with C. F. Craig, Lea and Febiger, 1945. 
FINKEL, A. J. See Alice, Finkel and Garner, 1941, 1942; Alice, Finkel, Garner, Merwin and 

Evans, 1942 
FISHER, K. C. The fractionation of cellular respiration by the use of narcotics. Biol. Bull., 81 : 

1941 (abs.). 

FREEDMAN, W. B. AND R. WALKER. *Size, development, and innervation of labyrinth sensory 
areas in Squalus. Jour. Comp. Neur., 77 : 1942. 

FREIS, E. F. B. Some neurohumoral evidence for double innervation of xanthophores in kill- 
fish. Biol. Bull., 82 : 1942. 

FRISCH, J. A. *The rate of pulsation of the posterior contractile vacuole in Paramoecium 
woodruffi and P. calkinsi. Anat. Rec. 89: 1944 (abs.). 

FRISCH, J. A. *The rate of adaptation of P. caudatum to sea water. Anat. Rec. 89: 1944 (abs.). 

FROEHLICH, ALFRED. The influence of drugs on heat narcosis. Biol. Bull. 89: 1945 (abs.). 

FURTH, JACOB. *The teaching of experimental pathology. Arch. Path., 34 : 1942. 

FURTH, JACOB. Neoplastic growth. Ann. Rev. Physiol., 6: 1944. 

FURTH, JACOB, R. K. COLE AND M. C. BOON. *The effect of maternal influence upon spontane- 
ous leukemia of mice. Cancer Research, 2 : 1942. 

FURTH, JACOB AND M. C. BOON. ^Enhancement of leukemogenic action of methylcholanthrene 
by pre-irradiation with X-rays. Science, 98 : 1943. 

FURTH, JACOB, M. C. BOON AND N. KALISS. *On the genetic character of neoplastic cells as 
determined in transplantation experiments : with notes on the somatic mutation theory. 
Cancer Research, 4 : 1944. 

GABRIEL, M. L. The effect of temperature on vertebral variations is Fundulus. Biol. Bull., 83 : 

1942 (abs.). 

GABRIEL, M. L. Factors affecting the number and form of vertebrae in Fundulus. Jour. Exp. 

Zoo!., 95 : 1944. 
GALTSOFF, P. S. Storage and distribution of manganese in Ostrea virginica. Collecting Net, 16 : 

1941. 
GALTSOFF, P. S. Accumulation of manganese and the sexual cycle in Ostrea virginica. Physiol. 

Zool., 15 : 1942. 

GALTSOFF, P. S. Reaction of the oyster to free chlorine. Biol. Bull., 89: 1945 (abs.). 
GARNER, H. R. See Alice, Finkel and Garner, 1941, 1942. 
CARREY, W. E. Action of acetylcholine on the heart of Limulus. Amer. Jour. Physiol., 133 : 

1941. 



20 MARINE BIOLOGICAL LABORATORY 

CARREY, W. E. An analysis of the action of acetylcholine on the cardiac ganglion of Limulus. 

Amer. Jour. Physiol. 136: 1942. 

GATES, R. R. Tests of nuceoli and cytoplasmic granules in marine eggs. Biol. Bull., 81 : 1941. 
GATES, R. R. *Nucleoli and phylogeny. Collecting Net, 17 : 1942. 
GATES, R. R. *Chromosome numbers in mammals and man. Science, 96 : 1942. 
GATES, R. R. *Symbols for human genes. Science, 95 : 1942.t 
GATES, R. R. *Nucleoli and related nuclear structures. Bot. Rev., 8 : 1942. 
GATES, R. R. *Our ancestors, ourselves, our descendants. Medical Genetics and Eugenics, 2 : 

1943. 
GATES, R. R. (with G. N. PATHAK). *Variations in the offspring. of tetraploid Oenotheras. 

Amer. Naturalist, 78 : 1944. 
GIDGE, NATALIE M. -AND S. M. ROSE. The role of larval skin in promoting limb regeneration 

in adult Anura. Jour. Exp. Zool., 97 : 1944. 
GIESE, A. C. *Studies on nutrition of dim and bright variants of a species of luminous bacteria. 

Jour. Bact., 46: 1943. 
GIESE, A. C. *The action of azide on luminescence, respiration, and growth of the luminous 

bacteria. Jour. Cell. Comp. Physiol., 26 : 1945. 

GIESE, A. C. ""Ultraviolet radiations and life. Physiol. Zool., 18 : 1945. 
GIESE, A. C. *Effects of ultraviolet radiations on luminescence and respiration of Achromobac- 

ter fischeri. Jour. Cell. Comp. Physiol., 17: 1941. 
GIESE, A. C. AND E. L. TATUM. *The effect of some vitamins of the B-complex on respiration 

of mutants of Neurospora. Biol. Bull., 83: 1942 (abs.). 
GILBERT, P. W. *The urogenital system of the male frilled shark, Chlamydoselachus anguineus. 

Anat. Rec., 84: 1942 (abs.). 

GILBERT, P. W. *The morphology of the male urogenital system of the frilled shark Chlamy- 
doselachus anguineus. Jour. Morph., 3: 1943. 
GILMAN, L. C. *Mating types in diverse races of Paramoecium caudatum. Biol. Bull., 80: 

1941. 

GLASER, O. C. Protein metabolism and embryonic growth rate. Biol. Bull., 83: 1942 (abs.). 
GOLDIN, A. Factors influencing regeneration and polarity determination in Tubularia crocea. 

Biol. Bull, 82 : 1942. 

GOLDIN, A. A quantitative study of the interrelationships of oxygen and hydrogen ion concen- 
tration in influencing Tubularia regeneration. Biol. Bull., 82 : 1942. 
GOLDIN, A. See also Spiegelman and Goldin, 1944. 
GOLDIN, A. AND L. G. BARTH. Regeneration of coenosarc fragments removed from the stem 

of Tubularia crocea. Biol. Bull, 81 : 1941. 
GOLDINGER, J. M. Sec Barren and Goldinger, 1941. 
GOODCHILD, CHAUNCEY. *Additional observations on the life history of Gorgodera amplicava. 

Jour. Parasitol, 31 : 1945. 
GOODRICH, H. B., N. D. JOSEPHSON, J. P. TRINKHAUS AND JEANNE M. SLATE. *The cellular 

expression and genetics of two new genes in Lebistes reticulatus. Genetics, 29 : 1944. 
GRANICK, SAM. See Michaelis and Granick, 1945. 

GRAVE, B. H. The sexual cycle of the shipworm, Teredo navalis. Biol. Bull, 82: 1942 (abs.). 
GRAVE, CASWELL. Further studies of metamorphosis of ascidian larvae. Biol. Bull, 81 : 1941 

(abs:). 
GRAVE, CASWELL. The "eye spot" and light response of the larva of Cynthia partita. Biol Bull, 

81: 1941 (abs.). 

GRAVE, CASWELL, AND S. O. MAST. The larva of Styela (Cynthia) partita; structure, ac- 
tivities, and duration of life. Jour. Morph., 75 : 1944. 
GRAY, I. E. *Changes in weight and water content during the life cycle of the wood-eating 

beetle, Passalus cornutus. Biol Bull, 86: 1944. 
GROSCH, D. S. *The relation of cell size and organ size to mortality in Habrobracon. Growth, 

9: 1945. 
GUTTMAN, RITA. *Action of potassium and narcotics on rectification in nerve and muscle. 

Jour. Gen. Physiol, 28 : 1944. 
GUTTMAN, RITA AND K. S. COLE. The rectifying property of the giant axon of the squid. 

Collecting Net, 16: 1941. 
GUTTMAN, RITA AND K. S. COLE. Electrical rectification in single nerve fibers. Proc. Soc. 

Exp. Biol.Afed.,48: 1941. 



REPORT OF THE DIRECTOR 21 

HAGEK, R. P. *Sex linkage of stubby in Habrobracon. Biol. Bull., 81: 1941 (abs.). 
HAMILTON, H. L. *The influence of hormones on the differentiation of melanophores in birds. 

Biol. Bull, 81: 1941 (abs.). 
HAMILTON, H. L. AND B. H. WILLIER. ^Developmental Physiology. Ann. Rev. Physiol 4- 

1942. 
HARNLY, M. H. *Wing form and gene function in nine genotypes of Drosophila melanogaster. 

Biol. Bull., 82 : 1942. 
HARRIS, D. L. The osmotic properties of cytoplasmic granules of the sea urchin egg. Biol. 

Bull., 85 : 1943. 
HARTMAN, F. A., L. A. LEWIS, K. A. BROWNELL, F. F. SHELDEN AND R. F. WALTHER. Some 

blood constituents of the normal skate. Physiol. Zool. 14: 1941. 
HARTMAN, F. A., F. F. SHELDEN, AND E. L. GREEN. Weights of interrenal glands of elasmo- 

branchs. Anat. Rcc., 87 : 1943. 
HARTMAN, F. A., L. A. LEWIS, K. A. BROWNELL, C. A. ANGERER AND F. F. SHELDEN. Effect of 

interrenalectomy on some blood constituents in the skate. Physiol. Zool., 17 : 1944. 
HARVEY, ETHEL BROWNE. Relation of the size of "halves" of the Arbacia punctulata egg to 

centrifugal force. Biol. Bull., 80: 1941. 
HARVEY ETHEL BROWNE. The cytology of fertilization and cleavage of Arbacia punctulata. 

Turtox Neivs, 19: 1941. 

HARVEY ETHEL BROWNE. Cross fertilization of echinoderms. Science, 94 : 1941. 
HARVEY ETHEL BROWNE. Vital staining of the centrifuged Arbacia egg. Biol. Bull., 81 : 1941. 
HARVEY ETHEL BROWNE. Maternal inheritance in echinoderm hybrids. Biol. Bull., 81 : 1941. 

(abs.). 
HARVEY ETHEL BROWNE. Rate of breaking and size of the "halves" of the Arbacia egg when 

centrigued in hypo- and hypertonic sea water. Biol. Bull., 85 : 1943. 
HARVEY, ETHEL BROWNE. *Early biological photomicrographs. Jour. Biol. Photograph. Assoc., 

13: 1945. 
HARVEY ETHEL BROWNE. Stratification and breaking of the Arbacia egg when centrifuged in 

single salt solutions. Biol. Bull., 89 : 1945. 
HARVEY ETHEL BROWNE. Development of granule-free fractions of Arbacia eggs. Biol. Bull., 

89: 1945 (abs.). 
HARVEY ETHEL BROWNE AND T. F. ANDERSON. The spermatozoan and fertilization membrane 

of Arbacia, as shown by the electron microscope. Biol. Bull., 85: 1943. 
HARVEY ETHEL BROWNE AND G. I. LAVIN. The chromatin in the living Arbacia egg; and the 

cytoplasm of the centrifuged egg as photographed by ultraviolet light. Biol. Bull., 86 : 

1944. 
HARVEY, E. N. Stimulation of cells by intense flashes of ultraviolet light. Jour. Gen. Physiol., 

25 : 1942. 
HARVEY, E. N. Note on the red luminescence and the red pigment of the "railroad worm." 

Jour. Cell. Comp. Physiol., 25 : 1945. 

HARVEY, E. N. AND H. SHAPIRO. The recovery period (relaxation) of marine eggs after de- 
formation. Jour. Cell. Comp. Physiol., 17 : 1941. 
HARVEY, E. N. AND F. J. M. SICHEL. The response of single striated muscle fibers to intense 

flashes of ultraviolet light. Jour. Cell. Comp. Physiol., 19: 1942. 
HARVEY, E. N. AND F. J. M. SICHEL. A method of recording the dimensions of muscle fiber 

striations during contraction. Federation Proc., 1 : 1942. 
HARVEY, E. N. AND F. J. M. SICHEL. High speed linear photography. Jour. Cell. Comp. 

Physiol., 25 : 1945. 
HARVEY, E. N., D. K. BARNES, W. D. MCELROY, A. H. WHLTELY, D. C. PEASE AND K. W. 

COOPER. *Bubble formation in animals. I. Physical factors. Jour. Cell. Comp. Physiol., 

24: 1944. 
HASSETT, C. C. *Photodynamic action in the flagellate Peranema trichophorum with special 

reference to motor response to light. Physiol. Zool., 17 : 1944. 
HAYASHI, TERU. Dilution medium and survival of the spermatozoa of Arbacia punctulata. 

I. Effect of the medium on fertilizing power. Biol. Bull., 89: 1945. 
HAYWOOD, CHARLOTTE. The permeability of the toadfish liver to inulin, with and without 

choleretics. Federation Proc., 2: 1943 (abs.). 
HAYWOOD, CHARLOTTE, VIRGINIA C. DICKERSON, AND MARGARET C. COLLINS. *The secretion 

of dye by the fish liver. Jour. Cell. Comp. Physiol., 25 : 1945. 



MARINE BIOLOGICAL LABORATORY 

HEILBRUNN, L. V. *An Outline of General Physiology. 2nd Ed. Saunders. 1943. 

HEWATT, W. G. A method of narcotizing Holothurians. Science, 97 : 1943. 

HIATT, E. P. AND G. P. QUINN. *The distribution of quinine, quinidine, cinchonine, and 

cinchonidine in fluids and tissues of dogs. Jour. Pharmacol. Exp. Thcrap., 83 : 1945. 
HIATT, E. P., D. E. S. BROWN, G. P. QUINN AND K. MACDUFFIE. *The blocking action of 

the cinchona alkaloids and certain related compounds on the cardioinhibitory vagus endings 

of the dog. Jour. Pharmacol. Exp. Therap., 85 : 1945. 
HIBBARD, HOPE AND G. I. LAVIN. *A study of the Golgi apparatus in chicken gizzard epithelium 

by means of the quartz microscope. Biol. Bull., 89: 1945. 
HIESTAND, W. A. Oxygen consumption of the sea cucumber as a function of oxygen tension 

and hydrogen ion concentration of the surrounding medium. Trans. Wisconsin A cad. Sci- 
ences, Arts and Letters, 32 : 1941. 
HIESTAND, W. A. Action of certain drugs on the sea star, Asterias forbesii. Proc. Soc. Exp. 

Biol. Med., 52 : 1943. 
HILL, S. E. The relation between protoplasmic streaming and the action potential in Nitella 

and Chara. Biol. Bull., 81: 1941 (abs.). 
HOLLINGSWORTH, JOSEPHINE. Activation of Cumingia and Arbacia eggs by bivalent cations. 

Biol. Bull, 81: 1941. 

HOPKINS, D. L. See Mast and Hopkins, 1941. 
HORN, ANNABELLE. *Proof for multiple allelism of sex differentiating factors in Habrobracon. 

Amer. Nat., 77: 1943. 
HOUCK, C. R. The effects of bichloride of mercury upon the luminescence and respiration of 

the luminous bacterium, Achromobacter fischeri. Jour. Cell. Comp. Physiol., 20 : 1942. 
HUNNINEN, A. V. See also Cable and Hunninen, 1941, 1942. 
HUNNINEN, A. V. AND R. M. CABLE. Life history of Lecithaster confusus. Jour. ParasitoL, 

29: 1943. 
HUNNINEN,- A. V. AND R. M. CABLE. The life history of Podocotyle atomon. Trans. Amcr. 

Microscop. Soc., 62: 1943. 
HUTCHENS, J. O. The utilization of ammonia by Chilomonas paramoecium. Biol. Bull., 81 : 

1941 (abs.). 

HUTCHENS, J. O., A. K. KELTCH, M. E. KRAHL AND G. H. A. CLOWES. Studies on cell metab- 
olism and cell division. VI. Observations on the glycogen content, carbohydrate consump- 
tion, lactic acid production, and ammonia production of eggs of Arbacia punctulata. Jour. 

Gen. Physiol., 25 : 1942. 
HUTCHENS, J. O., M. J. KOPAC AND M. E. KRAHL. The cytochrome content of centrifugally 

separated fractions of unfertilized Arbacia eggs. Jour. Cell. Comp. Physiol., 20 : 1942. 
IRVING, LAURENCE. See Root and Irving, 1941. 

JACOBS, M. H. Sec also Netzky and Jacobs, 1941 ; Stewart and Jacobs, 1941. 
JACOBS, M. H. AND DOROTHY R. STEWART. Catalysis of ionic exchange by bicarbonates. Biol. 

Bull., 81 : 1941. 
JACOBS, M. H. AND DOROTHY R. STEWART. The role of carbonic anhydrase in certain exchange 

involving the erythrocyte. Jour. Gen. Physiol., 25 : 1942. 

JACOBS, M. H. AND DOROTHY R. STEWART. *A biological method for the quantitative estima- 
tion of certain organic bases. Amer. Jour. Med. Sci., 206 : 1943. 
JACOBS, M. H. AND DOROTHY R. STEWART. *Osmotic equilibria between the erythrocyte and 

a complex external solution. Amer. Jour. Med. Sci., 209: 1945. 
JACOBS, M. H. AND J. D. HELM. *Some apparent differences between the erythrocytes of white 

and negro subjects. Jour. Cell. Comp. Physiol., 22: 1943. 
JACOBS, M. H., DOROTHY R. STEWART AND MARY K. BUTLER. *Some effects of tannic acid on 

the cell surface. Amer. Jour. Med. Sci., 205 : 1943. 
JAEGER, LUCENA. Glycogen utilization by the amphibian gastrula in relation to invagination 

and induction. Jour. Cell. Comp. Physiol., 25 : 1945. 
JANDORF, B. J. AND M. E. KRAHL. Studies on cell metabolism and division. VIII. The diphos- 

phopyridine nucleotide content of eggs of Arbacia punctulata. Jour. Gen. Physiol., 25 : 

1942. 

JONES, E. R. *The morphology of Enterostomula gram. Jour. Morph., 68: 1941. 
JONES, E. R. AND W. J. HAYES, JR. *Microdalyellia gilesi, a new Turbellarian worm from 

Mountain Lake, Va. Amer. Midland Naturalist, 26: 1941. 



REPORT OF THE DIRECTOR 23 

KALISS, NATHAN. Sec Furth, Boon and Kaliss, 1944. 

KAWATA, N. See Steinbach, Spiegelman and Kawata, 1944. 

KAYLOR, C. T. *Studies on experimental haploidy in salamander larvae. Blol. Bull., 81 : 1941. 

KAYLOR, C. T. *Sex differentiation in two androgenetic salamander larvae. Anat Rec 87 

1943. 
KELTCH, ANNA K. See Krahl, Keltch, Neubeck and Clowes, 1941 ; Hutchens, Keltch, Krahl 

and Clowes, 1942. 
KIDDER, G. W. *Growth studies on ciliates. VII. Comparative growth characteristics of four 

species of sterile ciliates. Biol. Bull., 80 : 1941. 

KIDDER, G. W. See also Claff, Dewey and Kidder, 1941 ; Burt, Kidder and Gaff, 1941. 
KNOWLTON, F. P. Observations on the dual contraction of crustacean muscle. Biol. Bull., 82 : 

1942. 
KNOWLTON, F. P. An investigation of inhibition by direct stimulation of the turtle's heart. 

Amer. Jour. Physiol., 135: 1942. 
KNOWLTON, F. P. The action of certain drugs on crustacean muscle. Jour. Exp. Pharmacol. 

Exp. Therap., 75: 1942. 
KNOWLTON, F. P. """Inhibition of the turtle's atria by single induction shocks applied directly. 

Amer. Jour. Physiol., 140: 1943. 
KOPAC, M. J. Disintegration of the fertilization membrane of Arbacia by the action of an 

"enzyme." Jour. Cell. Comp. Physiol., 18: 1941. 
KOPAC, M. J. See also Hutchens, Kopac and Krahl, 1942. 
KRAHL, M. E. See also Hutchens, Keltch, Krahl and Clowes, 1942; Jandorf and Krahl, 1942; 

Hutchens, Kopac and Krahl, 1942. 

KRAHL, M. E., A. K. KELTCH, C. E. NEUBECK AND G. H. A. CLOWES. Studies on cell metab- 
olism and cell division. V. Cytochrome oxidase activity in eggs of Arbacia punctulata. 

Jour. Gen. Physiol., 24: 1941. 
KRAHL, M. E., B. J. JANDORF AND G. H. A. CLOWES. Studies on cell metabolism and cell 

division. VII. Observations on the amount and possible function of diphosphothiamine in 

eggs of Arbacia punctulata. Jour. Gen. Physiol., 25 : 1942. 
KRASNOW, FRANCES. *The physiological significance of phospholipid in human saliva. Jour. 

Dental Res., 24: 1945. 

LANCEFIELD, REBECCA C. *Studies on the antigenic composition of Group A hemolytic strepto- 
cocci. I. Effects of proteolytic enzymes on streptococcal cells. Jour. Exp. Med., 78 : 1943. 
LANCEFIELD, REBECCA C. AND W. A. STEWART. *II. The occurrence of strains in a given type 

containing M but no T antigen. Jour. Exp. Med., 79: 1944. 
LAVIN, G. J. Some observations with a simplified quartz microscope. Biol. Bull., 83: 1942 

(abs.). 

LAVIN, G. J. *Simplified ultraviolet microscopy. Rec. Scientific Instruments, 14: 1943. 
LAVIN, G. J. See also Costello and Lavin, 1943; Harvey and Lavin, 1944; Hibbard and Lavin, 

1945. 

LAZAROW, ARNOLD. *The chemical organisation of the cytoplasm. Biol. Bull., 87: 1944. 
LEE, R. E. The occurrence of female sword-fish in southern New England waters, with a 

description of their reproductive condition. Copeia, 1942. 
LEE, R. E. The hypophysis of the broad-billed sword-fish, Xiphias gladius. Biol. Bull 82 : 

1942. 
LEE, R. E. Notes on the color changes of the sea robin, with special reference to the erythro- 

phores. Jour. Exp. Zool, 91 : 1942. 
LEE, R. E. Pituitary function in the chromatic physiology of Opsanus tau. Biol. Bull 83 

1942 (abs.). 
LEE, R. E. *A quantitative survey of the invertebrate bottom fauna in Menemsha Bight. Biol 

Bull. 86: 1944. 
LEE, R. E. See also Chambers, Zweifach, Lowenstein and Lee, 1944; Zweifach, Lee, Hyman 

and Chambers, 1944. 
LEFEVRE, P. F. Certain chemical factors influencing artificial activation of Nereis eggs Biol 

Bull, 89 : 1945. 

LEVY, MILTON AND A. H. PALMER. *Amino peptidase. Jour. Biol. Chein., 150 : 1943. 
LEWIS, L. A. See Hartman, Lewis, Brownell, Shelden and Walther, 1941 ; Hartman, Lewis, 

Brownell, Angerer and Shelden, 1944. 



24 MARINE BIOLOGICAL LABORATORY 

LEWIS, MARGARET R. *The failure of purified penicillin to retard the growth of sarcoma in 

mice. Science, 100: 1944. 
LEWIS, MARGARET R. *A study of inducement and transplantability of sarcoma in rats. 

Growth, 9 : 1945. 

LEWIS, W. H. The superficial gel layer and its role in development. Blol. Bull., 87 : 1944. 
LIEBMAN, EMIL. *The coelomocytes of Lumbricidae. Jour. Morph., 71 : 1942. 
LILLIE, F. R. Further experiments on artificial parthenogenesis in starfish eggs, with a review. 

Physiol. Zool, 14: 1941. 

LILLIE, F. R. The Woods Hole Marine Biological Laboratory. Univ. Chicago Press, 1944. 
LILLIE, R. S. *The problem of synthesis in Biology. Philosoph. Science, 9: 1942. 
LILLIE, R. S. *Living systems and non-living systems. Philosoph. Science, 9 : 1942. 
LILLIE, R. S. *The. psychic factor in living organisms. Philosoph. Science, 10: 1943. 
LILLIE, R. S. *Vital organization and the psychic factor. Philosoph. Science, 11 : 1944. 
LILLIE, R. S. *General Biology and Philosophy of the Organism. Univ. Chicago Press, 1945. 
LITTLE, E. P. See Evans, Slaughter, Little and Failla, 1942. 
LLOYD, D. P. C. *Activity in neurons of the bulbospinal correlation system. Jour. Neuro- 

physiol., 4: 1941. 

LOEWI, OTTO. *Chemical transmission of nerve impulses. Science Progress, 4 : 1945. 
LOEWI, OTTO. ^Aspects of the transmission of the nervous impulse. Jour. Mt. Sinai Hasp., 

12: 1945. 
LUCAS, A. M. AND J. SNEDECOR. Coordination of ciliary movement in the Modiolus gill. Biol. 

Bull., 81: 1941 (abs.). 
LUCRE, BALDUIN, A. K. PARPART AND R. A. RICCA. Failure of choleic acids in carcinogenic 

hydrocarbons to alter permeability of marine eggs and of mammalian erythrocytes. Cancer 

Research, 1: 1941. 
LURIA, S. E., M. DELBRUCK AND T. F. ANDERSON. Electron microscope studies of bacterial 

viruses. Jour. Bad., 46 : 1943. 
LYNN, W. G. *The embryology of Eleutherodactylus nubicola, an anuran which has no tadpole 

stage. Carnegie Contrib. to Embryol., 30: 1942. 
LYNN, W. G. AND J. M. DENT. Notes on Plethodon cinereus and Hemidactylium scutatum on 

Cape Cod. Copeia, 1941. 
MACLEAN, BERNICE. Sec Shapiro, 1945. 

MARSLAND, D. A. *Protoplasmic streaming. Chronica Botan., 6: 1941. 
MARSLAND, D. A. ^Protoplasmic streaming. Collecting Net, 16: 1941. 
MARSLAND, D. A. *Protoplasmic streaming in relation to gel structure in the cytoplasm. 

Chapter in The Structure of Protoplasm, Iowa State College Press, 1942. 
MARSLAND, D. A. The contractile mechanism in unicellular chromatophores. Biol. Bull., 83 : 

1942 (abs.). 

MARSLAND, D. A. ^Quieting Paramoecium for the elementary student. Science, 98 : 1943. 
MARSLAND, D. A. Mechanism of pigment displacement in unicellular chromatophores. Biol. 

Bull., 87 : 1944. 

MARSLAND, D. A. *Principles of Modern Biology. Holt and Co., New York, 1945. 
MARSLAND, D. A. AND D. E. S. BROWN. The action of pressure on sol-gel equilibria. Anat. 

Rec., 81: Suppl., 1941. 
MARSLAND, D. A. AND D. E. S. BROWN. The effects of pressure on sol-gel equilibria, with 

special reference to myosin and other protoplasmic gels. Jour. Cell. Comp. Physiol., 20 : 

1942. 

MARSLAND, D. A. AND R. RUGH. *Effects of pressure on maturation, cleavage, and early de- 
velopment of the frog's egg. Anat. Rec., 82 : 1942. 

MARSLAND, D. A. AND R. RUGH. *The effect of hydrostatic pressure upon the early develop- 
ment of the frog's egg. Proc. Amer. Philosoph. Soc., 86: 1943. 
MARTIN, W. E. Cerama solemyae, probably a blood fluke from the marine pelecypod, Solemya 

velum. Jour. Parasitol, 30 : 1944. 
MARTIN, W. E. Studies on trematodes of Woods Hole. IV. Additional observations upon 

Cercaria loossi developing in an Annelid. Trans. Amcr. Micro. Soc., 63: 1944. 
MARTIN, W. E. Two new species of marine cercariae. Trans. Amcr. Micro. Soc., 64: 1945. 
MAST, S. O. *The hydrogen ion concentration of the content of the food vacuoles and the 

cytoplasm in Amoeba and other phenomena concerning the food vacuoles. Biol. Bull.. 83 : 

1942. 



REPORT OF THE DIRECTOR 25 

MAST, S. O. A new peritrich belonging to the genus Ophridium. Trans. Amer. Mic. Soc., 63 : 

1944. 

MAST, S. O. Sec also Bertholf and Mast, 1944; Grave and Mast, 1944. 
MAST, S. O. AND D. L. HOPKINS. *Regulation of the water content of Amoeba mira and 

adaptation to changes in the osmotic concentration of the surrounding medium. Jour. Cell. 

Comp. Physiol, 17: 1941. 
MAST, S. O. AND D. M. PACE. *The effect of phosphorus on metabolism in Chilomonas para- 

moecium. Jour. Cell. Comp. Physiol., 20 : 1942. 
MAST, S. O. AND W. G. BOWEN. *The food vacuole in the Peritricha, with special reference 

to the hydrogen ion concentration of its content and of the cytoplasm. Biol. Bull., 87 : 1944. 
MCELROY, W. D. See Harvey, Barnes, McElroy, Whitely, Pease and Cooper, 1944. 
MELLAND, A. M. See Buck and Melland, 1942. 

MENKIN, VALY. *Studies on the chemical basis of fever. Biol. Bull.. 87 : 1944. 
MERWIN, RUTH M. See Alice and Merwin, 1941 ; Alice, Finkel, Garner, Merwin and Evans, 

1942; Alice and Merwin, 1943. 
METZ, C. B. *The inactivation of fertilizin and its conversion to the "univalent" form by 

X-rays and ultraviolet light. Biol. Bull., 82 : 1942. 

METZ, C. B. *The agglutination of starfish sperm by fertilizin. Biol. Bull., 89 : 1945. 
MEYERHOF, BETTINA. See Nachmansohn and Meyerhof, 1941. 

MEYERHOF, OTTO. *Nature, function, and distribution of the phosphagens in the animal king- 
dom. Collecting Net, 16: 1941. 
MICHAELIS, L. AND SAM GRANiCK. *Metachromasy of basic dye stuffs. Jour. Amer. Chem. 

Soc., 67 : 1945. 
MILLER, J. A. Some effects of covering the perisarc upon tubularian regeneration. Biol. Bull., 

83: 1942. 

MIRSKY, A. E. See Pollister and Mirsky, 1942, 1943. 
MOOG, FLORENCE. The influence of temperature on reconstitution in Tubularia. Biol. Bull., 

81: 1941 (abs.). 
MOOG, FLORENCE. Some effects of temperature in the regeneration of Tubularia. Biol. Bull., 

83: 1942 (abs.). 

MOOG, FLORENCE. See also Spiegelman and Moog, 1944. 
MOOG, FLORENCE AND S. SPIEGELMAN. Effects of some respiratory inhibitors on respiration 

and reconstitution in Tubularia. Proc. Soc. Exp. Biol. Med., 49: 1942. 
MORGAN, T. H. *Further experiments in cross- and self-fertilization of Ciona at Woods Hole 

and Corona del Mar. Biol. Bull., 80: 1941. 

MORGAN, T. H. Cross- and self-fertilization in the Ascidian, Styela. Biol. Bull., 82 : 1942. 
MORGAN, T. H. Cross- and self-fertilization in the Ascidian, Molgula manhattensis. Biol. 

Bull., 82 : 1942. 
MUIR, R. M. Effect of radiation from radioactive isotopes on the protoplasm of Spirogyra. 

Jour. Cell. Comp. Physiol., 19: 1942. 
NACHMANSOHN, DAVID. On the mechanism of transmission of nerve impulses. Collecting 

Net, 17 : 1942. 
NACHMANSOHN, DAVID. On the energy source of the nerve action potential. Biol. Bull., 87: 

1944. 
NACHMANSOHN, DAVID AND B. MEYERHOF. Relation between electrical changes during nerve 

activity and concentration of choline esterase. Jour. Neurophysiol., 4: 1941. 
NACHMANSOHN, DAVID AND H. B. STEINBACH. On the localization of enzymes in nerve fibers. 

Science, 95: 1942. 
NACHMANSOHN, DAVID AND H. B. STEINBACH. Localization of enzymes in nerves. 1. Suc- 

cinic dehydrogenase and vitamin B!. Jour. Neurophysiol., 5 : 1942. 
NAVEZ, A. E., J. D. CRAWFORD, D. BENEDICT AND A. B. DuBois. On the metabolism of the 

heart of Venus mercenaria. Biol. Bull., 81 : 1941. 

NETSKY, M. G. AND M. H. JACOBS. Some effects of desoxycorticosterone and related com- 
pounds on the mammalian red cell. Biol. Bull., 81 : 1941. 
NEUBECK, C. E. See Krahl, Keltch, Neubeck and Clowes, 1941. 
O'BRIEN, J. P. ^Studies on the effects of X-rays on regeneration in the fragmenting oligo- 

chaete, Nais paraguayensis. Growth, 6 : 1942. 
OLSON, MAGNUS. Histology of the radula protractor muscles of Busycon canaliculatum. Biol. 

Bull, 82 : 1942. 



26 MARINE BIOLOGICAL LABORATORY 

OPPENHEIMER, JANE M. The anatomical relationships of abnormally located Mauther's cells 

in Fundulus embryos, four. Com p. Neural., 74: 1941. 

ORMSBEE, R. A. *The normal growth of Tetrahymena geleii. Biol. Bull., 82 : 1942. 
OSBORN, C. M. Studies on the growth of integumentary pigment in the lower vertebrates. 

*I. The origin of artificially developed melanophores on the normally unpigmented ventral 

surface of the summer flounder. Biol. Bull., 81: 1941. 
OSBORN, C. M. II. The role of the hypophysis in melanogenesis in the common catfish. Biol. 

Bull., 81: 1941. 
OSTERHOUT, W. J. V. Some properties of protoplasmic gels. I. Tension in the chloroplast of 

Spirogyra. Jour. Gen. Physiol., 29 : 1945. 
PACE, D. M. See Mast and Pace, 1942. 

PACKARD, CHARLES. *Roentgen radiation in biological research. Radiology, 45 : 1945. 
PACKARD, CHARLES AND F. M. EXNER. *Comparison of physical and biological methods of 

depth dose measurement. Radiology, 44 : 1945. 
PARK, THOMAS. The laboratory population as a test of a comprehensive ecological system. 

Quart. Rev. Biol., 16: 1941. 
PARK, THOMAS, ELLA V. GREGG AND CATHERINE Z. LUTHERMAN. *Studies in population 

physiology. X. Interspecific competition in populations of granary beetles. Physiol. Zool., 

14: 1941. 
PARKER, G. H. Melanophore bands and areas due to nerve cutting, in relation to the protracted 

activity of nerves. Jour. Gen. Physiol., 24: 1941. 
PARKER, G. H. The methods of excitation of melanophores in the skin of the catfish Ameiurus. 

Science, 93: 1941. 

PARKER, G. H. Limited responses of melanophores as determined by activating agents. Sci- 
ence, 93: 1941. 

PARKER, G. H. The responses of catfish melanophores to ergotamine. Biol. Bull., 81 : 1941. 
PARKER, G. H. The organization of the melanophore system in bony fishes. Biol. Bull., 81 : 

1941. 
PARKER, G. H. Hypersensitization of catfish melanophores to adrenalin by denervation. Biol. 

Bull., 81: 1941. 
PARKER, G. H. The method of activation of melanophores and the limitations of melanophore 

responses in the catfish Ameiurus. Proc. Amcr. Philosoph. Soc., 85: 1941. 
PARKER, G. H. The activity of peripherally stored neurohumors in catfishes. Jour. Gen. 

Physiol., 25: 1941. 
PARKER, G. H. *Color changes in Mustelus and other elasmobranch fishes. Jour. Exp. Zool., 

89: 1942. 
PARKER, G. H. *Sensitization of melanophores by nerve cutting. Proc. Nat. Acad. Sci., 28: 

1942. 
PARKER, G. H. Methods of estimating the effects of melanophore changes in animal coloration. 

Biol. Bull., 84: 1943. 

PARKER, G. H. Color changes in the American eel, Anguilla rostrata. Anat. Rcc., 87: 1943. 
PARKER, G. H. *The time factor in chromatophore responses. Proc. Amer. Philosoph. Soc., 

87 : 1944. 
PARKER, G. H. Melanophore activators in the common American eel Anguilla rostrata. Jour. 

Exp. Zool., 98: 1945. 

PARPART, A. K. Lipoprotein complexes in the egg of Arbacia. Biol. Bull., 81 : 1941. 
PARPART, A. K. The preparation of red cell membranes. Jour. Cell. Comp. Physiol., 19: 1942. 
PARPART, A. K. See also Lucke, Parpart and Ricca, 1941 ; Chase, Lorenz, Parpart and Gregg, 

1944. 
PARPART, A. K. AND R. BALLENTINE. *Hematocrit determination of red cell volume. Science, 

98: 1943. 

PEASE, D. C. Sec Harvey, Barnes, McElroy, Whitely, Pease and Cooper, 1944. 
PIERCE, MADELENE E. Response of melanophores of the skin to injections of adrenalin, with 

special reference to body weight of the animal. Jour. Exp. Zool., 86: 1941. 
PLOUGH, H. H. *Spontaneous mutability in Drosophila. Cold Spring Harbor Svmposia, 9 : 

1941. 

PLOUGH, H. H. ^Temperature and evolution. Biol. Symposia, 6 : 1942. 
POLLISTER, A. W. *Mitochondrial orientations and molecular patterns. Physiol. Zool., 14 : 

1941. 



REPORT OF THE DIRECTOR 27 

POLLISTER, A. W. AND A. E. MiRSKY. *Nucleoproteins of cell nuclei. Proc. Nat. Acad. Sci., 

28: 1942. 
POLLISTER, A. W. AND A. E. MIRSKY. *Studies on the chemistry of chromatin. Trans. N. Y. 

Acad. Sci., 5 : 1943. 
POLLISTER, A. W. AND A. E. MIRSKY. *Fibrous nucleoproteins of chromatin. Biol. Symposia, 

10: 1943. 
POLLISTER, A. W. AND P. F. POLLISTER. *Relation between centriole and centromere in a 

typical spermatogenesis of viviparid snails. Ann. N. Y. Acad. Sci., 45 : 1943. 
PRICE, DOROTHY. *A comparison of the reactions of male and female rat prostate transplants. 

Anat. Rec., 82 : 1942. 
PROSSER, C. L. An analysis of the action of acetylcholine on hearts, particularly in Arthopods. 

Biol. Bull., 83 : 1942. 
PROSSER, C. L. Single unit analysis of the heart ganglion discharge in Limulus polyphemus. 

Jour. Cell. Comp. Physiol., 21 : 1943. 
PROSSER, C. L. AND G. L. ZIMMERMAN. Comparative pharmacology of myogenic and neuro- 

genic hearts. Biol. Bull., 81: 1941 (abs.). 
QUINN, GERTRUDE P. Sec Hiatt and Quinn, 1945. 
RECKNAGEL, RICHARD. See Wilbur and Recknagel, 1943. 
REID, W. M. *The relationship between glycogen depletion in the nematode Ascaridia galli 

and the elimination of the parasite by the host. Amcr. Jour. Hyg., 41 : 1945. 
REID, W. M. *In vivo and in vitro glycogen utilization in the fowl nematode Ascaridia galli. 

Biol. Bull., 89: 1945 (abs.). 
REINHARD, E. G. A hermit crab as intermediate host of Polymorphus. Jour. Parasitol., 30 : 

1944. 
REINHARD, E. G. Paguritherium alatum, an entoniscian parasite of Pagurus longicarpus. Jour. 

Parasitol., 31 : 1945. 

RICCA, R. A. See Lucke, Parpart and Ricca, 1941. 
RICHARDS, A. G. The interfibrillar material in the central nervous system of mosquito larvae. 

Biol. Bull,, 83: 1942 (abs.). 
RICHARDS, A. G. *Lipid nerve sheaths in insects and their probable relation to insecticide 

action. Jour. N. Y. Entomol. Soc., 51 : 1943. 
RICHARDS, A. G. *The structure of living insect nerves and nerve sheaths as deduced from the 

optical properties. Jour. N. Y. Entomol. Soc., 52 : 1944. 
RICHARDS, A. G. AND JANE L. WEYGANT. *The selective penetration of fat solvents into the 

nervous system of mosquito larvae. Jour. N. Y. Entomol. Soc., 53 : 1945. 
RICHARDS, A. G., H. B. STEINBACH AND T. F. ANDERSON. Electron microscope studies of 

squid giant nerve axoplasm. Jour. Cell. Comp. Physiol., 21 : 1943. 
Ris, HANS. *A cytological and experimental analysis of the meiotic behavior of the univalent 

X-chromosome in the bearberry aphid Tamalia. Jour. Exp. Zool., 90 : 1942. 
Ris, HANS. *A quantitative study of anaphase movement in the aphid Tamalia. Biol, Bull., 

85: 1943. 
Ris, HANS AND HELEN GROUSE. *Structure of the salivary gland chromosomes of Diptera. 

Proc. Nat. Acad. Sci,, 31 : 1945. 
ROBBIE, W. A. Balanced centerwell solutions for manometric experimentation. Biol. Bull., 89 : 

1945 (abs.). 

ROGICK, MARY D. Resistance of fresh water Bryozoa to dessication. Biodynamica, 3: 1941. 
ROGICK, MARY D. Supplementary note on the effect of the 1938 hurricane. Ohio J. Sci,, 41 : 

1941. 
ROGICK, MARY D. ^Studies on fresh-water Bryozoa XV. Hyalinella punctata growth data. 

Ohio Jour. Sci., 45 : 1945. 

ROGICK, MARY D. Field trips with a long-range purpose. Amcr. Biol. Teacher, 7 : 1945. 
ROGICK, MARY D. Studies on marine Bryozoa. I. Aeverrillia setigera. Biol. Bull., 89 : 1945. 
ROGICK, MARY D. *Studies on fresh-water Bryozoa. XVI. Fredericella australiensis. Biol. 

Bull., 89 : 1945. 

ROGICK, MARY D. "Calcining" specimens. Amcr. Biol. Teacher, 8 : 1945. 
ROOT, R. W. AND LAURENCE IRVING. The equilibrium between hemoglobin and oxygen in 

whole and hemolyzed blood of the tautog, with a theory of the Haldane effect. Biol. Bull., 

81 : 1941. 



MARINE BIOLOGICAL LABORATORY 

ROSE, S. M. *A method for inducing limb regeneration in adult Anura. Proc. Soc. Exp. Biol. 

Med., 49 : 1942. 

ROSE, S. M. *Causes for loss of regenerative power in adult Anura. Anat. Rec., 89: 1944. 
ROSE, S. M. *Methods for initiating limb regeneration in adult Anura. Jour. Exp. Zool., 95 : 

1944. 
ROSE, S. M. *The effect of NaCl in simulating regeneration of limbs of frogs. Jour. Morph., 

77: 1945. 
ROSE, S. M. AND FLORENCE C. ROSE. The role of a cut surface in Tubularia regeneration. 

Physiol. Zool., 14: 1941. 
ROSE, S. M. See also Gidge and Rose, 1944. 
RUGH, ROBERT. See Marsland and Rugh, 1942. 
RUNYON, E. H. Aggregation of separate cells of Dictyostelium to form a multicellular body. 

Biol. Bull, 83: 1943 (abs.). 
SANDOW, ALEXANDER. Studies of the muscle twitch recorded by electronic methods. Biol. 

Bull, 89: 1945 (abs.). 
SAYLES, L. P. Regeneration in the polychaete, Clymenella torquata. Biol Rev. of the City 

College, N. Y ., 3 : 1941. 
SAYLES, L. P. Buds induced in Clymenella torquata by implants of nerve cord and neighboring 

tissues derived from the mid-body region of worms of the same species. Biol. Bull, 82 : 

1942. 

SAYLES, L. P. Implants consisting of young buds, formed in anterior regeneration in Cly- 
menella, plus the nerve cord of the adjacent old part. Jour. Exp. Zool, 94: 1943. 
SCHAEFFER, MORRIS. *Preparation of influenza virus. Proc. Soc. Exp. Biol Med., 51 : 1942. 
SCHALLEK, WILLIAM. The reaction of certain Crustacea to direct and to diffuse light. Biol 

Bull, 84: 1943. 
SCHALLEK, WILLIAM. Action of potassium on bound acetylcholine in lobster nerve cord. 

Jour. Cell Comp. Physiol, 26 : 1945. 
SCHARRER, BERTA. *Neurosecretion. II. Neurosecretory cells in the central nervous system of 

cockroaches. Jour. Comp. Neural, 74: 1941. 

SCHARRER, BERTA. III. The cerebral organ of the nemerteans. Jour. Comp. Neurol, 74: 1941. 
SCHARRER, BERTA. IV. Localization of neurosecretory cells in the central nervous system of 

Limulus. Biol Bull, 81: 1941. 

SCHARRER, BERTA. *Endocrines in Invertebrates. Physiol. Re^'., 21 : 1941. 
SCHARRER, BERTA. *Experimental tumors after nerve section in an insect. Proc. Soc. Biol. 

Med., 60: 1945. 

SCHARRER, BERTA. See also Scharrer and Scharrer, 1945. 
SCHARRER, BERTA AND E. SCHARRER. Neurosecretion VI. A comparison between the inter- 

cerebralis-cardiacumallatum system of the insects and the hypothalamo-hypophyseal system 

of the vertebrates. Biol Bull, 87: 1944. 
SCHARRER, ERNST. Neurosecretion I. The nucleus preopticus of Fundulus. Jour. Comp. 

Neurol, 74: 1941. 
SCHARRER, ERNST. The capillary bed of the central nervous system of certain invertebrates. 

Biol Bull, 87 : 1944. 

SCHARRER, ERNST. *The blood vessels of the nervous tissue. Quart. Rev. Biol, 19 : 1944. 
SCHARRER, ERNST. Capillaries and mitochrondria in neurophil. Jour. Comp. Neurol, 1945. 
SCHARRER, ERNST. See also Scharrer and Scharrer, 1944. 

SCHARRER, ERNST, S. L. PALAY AND R. G. NILGES. Neurosecretion VIII. The Nissl sub- 
stance in secreting nerve cells. Anat. Rec., 92 : 1945. 

SCHARRER, ERNST AND BERTA SCHARRER. Neurosecretion. Physiol Rev., 25: 1945. 
SCHECHTER, VICTOR. Experimental studies upon the egg cells of the clam, Mactra solidissima, 

with special reference to longevity. Jour. Exp. Zool, 86: 1941. 
SCHECHTER, VICTOR. *Oxygen as a factor in the polarity of Corymorpha palma. Physiol. 

Zool, 14: 1941. 
SCHECHTER, VICTOR. *Tolerance of the snail, Thais floridana to waters of low salinity and 

the effect of size. Ecology, 24 : 1943. 
SCHECHTER, VICTOR. *Two flatworms from the oyster-drilling snail, Thais floridana. Jour. 

Parasitol, 29 : 1943. 
SCHMITT, F. O. *Structural proteins of cells and tissues. In Advances in Protein Chemistry, 

1: 1945. 



REPORT OF THE DIRECTOR 29 

SCOTT, ALLAN. Reversal of sex production in Micromalthus. Biol. Bull., 83: 1942 (abs.). 
SCOTT, SISTER FLORENCE MARIE. The early embryonic development of Amaroecium constel- 

latum. Biol. Bull, 83: 1942 (abs.). 
SCOTT, SISTER FLORENCE MARIE. The developmental history of Amaroecium constellatum. 

1. Early embryonic development. Biol. Bull., 88: 1945. 
SEVAG, M. G. *Immuno-catalysis. C. C. Thomas, Springfield, 111., 1945. 
SHAEFFER, A. A. A fourteen day rhythm in the left-right spiralling ratio of Flabellula citata. 

Biol. Bull. ,83: 1942 (abs.). 
SHANES, A. M. Current, voltage, and resistance characteristics of injured nerves. Biol. Bull 

87: 1944 (abs.). 
SHANES, A. M. Frog nerve as a generator of current and voltage. Jour. Cell. Comp. Ph\sioL, 

24: 1944. 
SHANES, A. M. Evidence of a metabolic effect by potassium in lowering the injury potential 

of invertebrate nerve. Biol. Bull, 89: 1945 (abs.). 
SHAPIRO, H. H. AND BERNICE L. MACLEAN. *Transplantation of developing tooth germs in 

the mandible of the cat. Jour. Dental Res., 24: 1945. 

SHAPIRO, HERBERT. Oxygen utilization by starfish eggs. Amcr. Jour. Physiol, 133: 1941. 
SHAPIRO, HERBERT. Metabolism and fertilization in the starfish egg. Collecting Net, 16: 1941. 
SHAPIRO, HERBERT. Centrifugal elongation of cells, and some conditions governing the return 

to sphericity, and cleavage time. Jour. Cell Comp. Physiol, 18 : 1941 
SHAPIRO, HER^ERT. Water permeability of the Chaetopterus egg before and after fertilization. 

Jour. Cell Comp. Physiol, 18 : 1941. 
SHAPIRO, HERBERT AND HUGH DAVSON. Permeability of the Arbacia egg to potassium. Biol 

Bull, 81: 1941. 

SHAPIRO, HERBERT. Metabolism and fertilization in the starfish egg. Biol Bull, 81 : 1941. 
SHAPIRO, HERBERT. The speed of membrane formation. Anat. Rcc., 81 : 1941. 
SHAPIRO, HERBERT. See also Harvey and Shapiro, 1941. 
SHAW, MYRTLE. Sec Sickles and Shaw, 1941. 
SHELDEN, F. F. See Hartman, Lewis, Brownell and Shelden, 1941 ; Hartman, Shelden and 

Green, 1943; Hartman, Lewis, Brownell, Angerer, and Shelden, 1944. 
SICHEL, F. J. M. *The relative elasticity of the sarcolemma and of the entire skeletal muscle 

fiber. Amer. Jour. Physiol, 133 : 1941. 
SICHEL, F. J. M. Sec also Harvey and Sichel, 1942, 1945. 
SICKLES, GRACE M. AND MYRTLE SHAW. The production of specific antisera for enzymes that 

decompose pneumococcus carbohydrates types 3 and 8. Jour. Bacterial, 42: 1941. 
SLAUGHTER, J. C. See Evans, Slaughter, Little, and Failla, 1942. 
SLIFER, ELEANOR H. *A mutant stock of Drosophila with extra sex-combs. Jour. Exp. Zool 

90: 1942. 
SLIFER, ELEANOR H. *The internal genitalia of some previously unstudied species of female 

Acrididae. Jour. Morph. 72: 1943. 
SLIFER, ELEANOR H. *The internal genitalia of female Tetrigidae, Eumastacidae, and Proscopi- 

idae. Jour. Morph., 73 : 1943. 

SMELSER, G. K. The oxygen consumption of eye muscles of thyroid-ectomized and thyroxin in- 
jected guinea pigs. Amer. Jour. Physiol, 142: 1944. 
SMITH, M. E. See Evans, Beams, and Smith, 1941. 
SOSA, J. M. *Woods Hole, Acropolis de los biologos. El. Dia., 1943. 
SOSA, J. M. *Quince meses en los Estados Unidos de Norte America. Anales Facultad de 

Med. Montevideo, 30: 1945. 
SPIEGELMAN, S. Mass and time relationships in the regeneration of Tubularia. Biol Bull, 83 : 

1942 (abs.). 

SPIEGELMAN, S. See also Moog and Spiegelman, 1942 ; Steinbach and Spiegelman, 1943, 1944. 
SPIEGELMAN, S. AND A. GOLDIN. A comparison of regeneration and respiration rates of 

Tubularia. Proc. Soc. Exp. Biol Med. 55 : 1944. 
STEINBACH, H. B. Chloride in the giant axons of the squid. Jour. Cell Comp. Physiol, 17 : 

1941. 
STEINBACH, H. B. See also Nachmansohn and Steinbach, 1942 ; Richards, Steinbach and 

Anderson, 1943. 



30 MARINE BIOLOGICAL LABORATORY 

STEIXBACH, H. B. AND S. SPIEGELMAN. The sodium and potassium balance in squid nerve axo- 

plasm. Jour. Cell. Comp. Physiol, 22 : 1943. 
STEINBACH, H. B. AND N. KAWATA. The recovery of the cut surface of the scallop muscle. 

Fed. Proc., 3 : 1944. 
STEINBACH, H. B., S. SPIEGELMAN AND N. KAWATA. Rectification and injury potential in 

squid axons. Fed. Proc., 3 : 1944. 
STEINBACH, H. B., S. SPIEGELMAN AND N. KAWATA. The effects of potassium and calcium on 

the electrical properties of squid axons. Jour. Cell. Comp. Physiol. 24: 1944. 
STERN, K. G. *Oxidases, Peroxidases, and Catalase. Symposium on respiratory enzymes. 

Madison, Wis., 1942. 
STERN, K. G. Physical-chemical studies on chromosomal nuceloproteins. Biol. Bull., 89 : 1945 

(abs.). 
STERN, K. G. AND S. F. VELICK. The effect of centrifugation upon the oxygen consumption of 

Arbacia eggs. Biol. Bull, 81 : 1941. 
STEWART, DOROTHY R. See Jacobs and Stewart, 1941, 1942, 1945 ; Jacobs, Stewart and Butler, 

1943. 
STEWART, DOROTHY R. AND M. H. JACOBS. The role of carbonic anhydrase in the catalysis of 

ionic exchanges by bicarbonates. Biol. Bull., 81 : 1941 (abs.). 
STEWART, W. A. See Lancefield and Stewart, 1944. 
STILES, K. A. *Handbook of microscopic characteristics of tissues and organs. Blakiston, 

1943. 

STILES, K. A. ^Laboratory explorations in general zoology. Macmillan, 1943. 
STOREY, ALMA G. *Gametophytes of Marattia sambucina and Macroglossum Smithii. Bot. 

Gas., 103 : 1942. 

STOKEY, ALMA G. *The gametophyte of Dipteria conjugata. Bot. Gas., 106: 1945. 
STUNKARD, H. W. Specificity and host-relations in the trematode genus Zoogonus. Biol. Bull., 

81 : 1941. 
STUNKARD, H. W. Pathology and immunity to infection with heterophyid trematodes. Biol. 

Bull., 81 : 1941. 
STUNKARD, H. W. Studies on pathology and resistance in terms and dogs infected with the 

heterophyid trematode, Cryptocotyle lingua. Trans. Amer. Microscop. Soc., 61 : 1942. 
STUNKARD, H. W. The morphology and life history of the digenetic trematode, Zoogonoides 

laevis. Biol. Bull., 85 : 1943. 
TAFT, C. H. The effects of a mixture of high molecular alkyl-dimethyl-benzyl-ammonium 

chlorides on the isolated heart of Limulus polyphemus. Proc. and Trans. Texas A cad. 

Set., 28 : 1945. 

TAFT, C. H. The action of amino acids on color changes in Fundulus. Science, 101 : 1945. 
TAFT, C. H. *Action of quitenine on the living Tautog and Toadfish. Biol. Bull., 89: 1945 

(abs.). 
TAFT, C. H. AND J. A. PLACE. *The comparative effects of the subcutaneous injection of 

quitenine on the kidneys of glomerular and aglomerular fish. Texas Rep. on Biol. Mcd. 2 : 

1944. 

TAYLOR, W. R. Reappearance of rare New England marine algae. Rhodora, 43: 1941. 
TAYLOR, W. R. *Notes on the marine algae of Texas. Mich. Acad. Sci. Artes. and Letter, 26 : 

1941. 
TAYLOR, W. R. Tropical marine algae of the Arthur Schott Herbarium. Field Mus. Nat. 

Hist. Bot., 230 : 1942. 
TAYLOR, W. R. *Marine algae of the Allan Hancock Expedition to the Caribbean, 1937. Allan 

Hancock Atlantic Exped. 2: 1942. 

TAYLOR, W. R. *Marine algae from Haiti. Mich. Acad., 28 : 1943. 
TAYLOR, W. R. *The collecting of seaweeds and fresh water algae. Instructions to naturalists 

in the armed forces for botanical work. 1944 2nd ed. 1945. 
TAYLOR, W. R. *William Gilson Farlow, promotor of phycological research in America. Far- 

lowia, 2 : 1945. 
TAYLOR, W. R. *Pacific marine algae of the Allan Hancock Expeditions to the Galapagos 

Islands. Allan Hancock Pacific Exp. 12 : 1945. 
TEWINKEL, Lois E. Structures concerned with yolk absorption in Squalus acanthias. Biol. 

Bull, 81: 1941 (abs.). 



REPORT OF THE DIRECTOR 31 

TE\VINKEL, Lois E. Observations on later phases of embryonic nutrition in Squalus acanthias. 

Jour. Morph., 73: 1943. 
TE\VINKEL, Lois E. Embryonic nourishment in the spiny dogfish. Wards Nat. Hist. Bull., 

19: 1945. 

THIVY, FRANCESA. A new species of Ectochaete from Woods Hole. Biol. Bull., 83 : 1942. 
THIVY, FRANCESA. New records of some marine Chaetophoraceae and Chaetosphaeridiaceae 

for North America. Biol. Bull., 85 : 1943. 

TRACER, WILLIAM. *The nutrition of invertebrates. Physiol. Rev., 21 : 1941. 
TRACER, WILLIAM. *Studies on conditions affecting the survival in vitro of a malarial para- 
site. Biol. Bull., 81: 1941 (abs.). 

TRINKHAUS, J. P. See Goodrich, Josephson, Trinkhaus and Slate, 1944. 
TROMBETTA, VIVIAN (Mrs. Roland Walker). The cytonuclear ratio. Bot. Rev., 8: 1942. 
VON DACH, HERMAN. Respiration of a colorless flagellate, Astasia klebsii. Biol. Bull., 82 : 

1942. 
VON SALLMAN, L. J. K. *Hydrogen ion concentration of the vitreous in the living eye. Arch. 

Ophthalmol., 33 : 1945. 
WALKER, ROLAND AND G. C. BENNET. *Size relations in the optic system of telescope-eyed 

goldfish. Trans. Connecticut Acad. Art and Sci., 36: 1945. 
WALKER, ROLAND. See also Freedman and Walker, 1942. 

WARREN, C. O. *The role of bicarbonate in the action of serum in supporting tissue respira- 
tion. Jour. Biol. Chem., 156 : 1944. 
WARREN, C. O. *The effect of thiouracil on the respiration of bone marrow and leucocytes 

in vitro. Amcr. Jour. Physiol., 71 : 1944. 

WATERMAN, A. J. The action of drugs on the compound ascidian, Perophora viridis, as in- 
dicated by the activity of the intact heart. Physiol. Zool., 15: 1942. 
WATERMAN, A. J. Further study of the action of drugs on the heart of the compound ascidian. 

Physiol. Zool., 16 : 1943. 
WATTERSON, R. L. *Some aspects of pigment deposition in feather germs of chick embryos. 

Biol. Bull., 81: 1941 (abs.). 
WATTERSON, R. L. Asexual reproduction in the colonial tunicate Botryllus schlosseri, with 

special reference to the developmental history of intersiphonal band of pigment cells. Biol. 

Bull. 88 : 1945. 
WEIDENREICH, FRANZ. *The brachycephalization of recent mankind. Southwestern. Jour. 

Anthropol, 1 : 1945. 
WENRICH, D. H. *The morphology of some protozoan parasites in relation to microtechnique. 

Jour. Parasitol., 27: 1941. 
WENRICH, D. H. *Observations on the food habits of Entamoeba muris and Entamoeba ra- 

narum. Biol. Bull., 81: 1941. 
WENRICH, D. H. Morphology of the intestinal trichomonad flagellates in man and of similar 

forms in monkeys, cats, dogs, and rats. Jour. Morph., 74 : 1944. 
WENRICH, D. H. Comparative morphology of the trichomonad flagellates of man. Amcr. Jour. 

Trap. Med., 24 : 1944. 
WENRICH, D. H. Nuclear structure and nuclear division in Dientamoeba fragilis. Jour. 

Morph., 74: 1944. 
WENRICH, D. H. Studies on Dientamoeba fragilis. IV. Further observations with an outline 

of present day knowledge of this species. Jour. Parasitol., 30 : 1944. 
WHALEY, W. G. AND C. Y. WHALEY. A developmental analysis of inherited leaf patterns in 

Tropaeolum. Amcr. Jour. Botany, 29 : 1942. 
WHITING, ANNA R. *X-ray sensitivity of first meiotic prophase and metaphase in Habrobracon 

eggs. Genetics, 27 : 1942. 
WHITING, ANNA R. *Effects of X-rays on hatchability and on chromosomes of Habrobracon 

eggs treated in first meiotic prophase and metaphase. Amcr. Nat., 79 : 1945. 
WHITING, P. W. *The cytogenetics of sex determination. Proc. 7th Internat. Genctical Cong., 

1941. 
WHITING, P. W. *Sex determination in Habrobracon. Proc. 7th Internat. Genctical Cong., 

1941. 
WHITING, P. W. *Multiple alleles in complementary sex determination of Habrobracon. 

Genetics, 28: 1943. 



32 MARINE BIOLOGICAL LABORATORY 

WHITING, P. W. *Intersexual females and intersexuality in Habrobracon. Biol. Bull., 85 : 

1943. 

WHITING, P. W. *Androgenesis in the parasitic wasp Habrobracon. Jour. Hered., 34: 1944. 
WHITING, P. W. *The problem f reversal of male haploidy by selection. Biol. Bull., 89: 

1945 (abs.). 

WHITING, P. W. *The evolution of male haploidy. Quart. Rev. Biol., 20 : 1945. 
WICHTERMAN, RALPH. *Pure line mass cultures for demonstrating the mating reaction and 

conjugation in Paramoecium. Turtox News, 22 : 1944. 
WICHTERMAN, RALPH. *Recent discoveries of nuclear processes and sexual phenomena in 

Paramoecium. Turtox News, 22 : 1944. 
WICHTERMAN, RALPH. A modified petri dish for continuous temperature observation. Science, 

101: 1945. 
W T IERCINSKI, F. J. An experimental study of intracellular pH in the Arbacia egg. Biol. Bull., 

81: 1941. 
WIERCINSKI, F. J. An experimental study of protoplasmic pH determination. I. Amoebae and 

Arbacia punctulata. Biol. Bull., 86 : 1944. 
WILBUR, K. M. The stimulating action of citrates and oxalates on the Nereis egg. Physiol. 

Zoo/., 14: 1941. 

WILBUR, K. M. See also Angerer and Wilbur, 1943. 
WILBUR, K. M. AND R. O. RECKNAGEL. The radiosensitivity of eggs of Arbacia punctulata in 

various salt solutions. Biol. Bull., 85 : 1943. 
WILHELMI, R. W. *The application of the precipitin technique to theories concerning the origin 

of vertebrates. Biol. Bull., 82 : 1942. 

WILLIAMSON, R. R. See Buchsbaum and Williamson, 1943. 
WILLIER, B. H. *Melanophore control of the sexual dimorphism of feather pigmentation in 

the Barred Plymouth Rock. Biol. Bull., 87 : 1944. 

WILLIER, B. H. AND MARY E. RAWLES. *Genotypic control of feather color pattern as demon- 
strated by the effects of a sex-linked gene upon the melanophores. Genetics, 29 : 1944. 
WILLIER, B. H. AND MARY E. RAWLES. Melanophore control of the sexual dimorphism of 

feather pigmentation pattern in the Barred Plymouth Rock fowl. Yale Jour. Biol. Med., 

17: 1944. 

WITKUS, ELEANOR R. *Some hints on smear technique. Turtox News, 23 : 1945. 
WITKUS, ELEANOR R. *Endomitotic tapetal cell divisions in Spinacia. Amer. Jour. Botany, 

32: 1945. 

WITKUS, ELEANOR R. *Endomitosis in plants. Biol. Bull., 89: 1945 (abs.). 
WITKUS, ELEANOR R. Sec also Berger and Witkus, 1943 ; Berger, Sullivan and Witkus, 1944. 
WOODRUFF, L. L. Sec Boell and Woodruff, 1941. 
WOODWARD, ALVALYN E. AND J. M. CONDRIN. *Physiological studies on hibernation in the 

chipmunk. Physiol. Zool, 18: 1945. 
WRINCH, DOROTHY. *The native protein theory of the structure of protoplasm. Cold Spring 

Harbor Symposia, 9: 1941. 

WRINCH, DOROTHY. Proteins in action. Collecting Net, 16: 1941. 
WRINCH, DOROTHY. Further implication of flexible protein frameworks. Collecting Net, 16 : 

1941. 
WRINCH, DOROTHY. The structure of biologically active membranes. Biol. Bull., 83: 1942 

(abs.). 
WRINCH, DOROTHY. *Native proteins, flexible frameworks and cytoplasmic organization. 

Nature, 150: 1942. 

WRINCH, DOROTHY. *Growth and form. Isis, 34: 1943. 
WRINCH, DOROTHY. Native protein crystallography and diffraction patterns. Biol. Bull., 87 : 

1944. 

WRINCH, DOROTHY. Fourier transforms and structure factors. Phys. Rev., 67 : 1945. 
WRINCH, DOROTHY. A tetrahedral framework for native proteins? Biol. Bull., 89: 1945. 
WULFF, V. J. Sec Brown and Wulff, 1941. 
YNTEMA, C. L. *An experimental study on the origin of the sensory neurones and sheath cells 

of the IXth and Xth cranial nerves in Amblystoma punctatum. Jour. Exp. Zool., 92: 1943. 
YNTEMA, C. L. ^Experiments on the origin of the sensory ganglia of the facial nerve in the 

chick. Jour. Comp. Ncur., 81 : 1944. 



REPORT OF THE DIRECTOR 33 

YNTEMA, C. L. AND W. S. HAMMOND. ""Depletions and abnormalities in the cervical sympa- 
thetic system of the chick following extirpation of neural crest. Jour. E.r/>. Zoo/., 100 : 

1945. 
ZINN, D. J. AND R. W. PERNAK. Mystacocarida, a new order of Crustacea from intcrtidal 

beaches in Massachusetts and Connecticut. Smithsonian Miscellaneous Collections, 103: 

1943. 
ZWEIFACH, B. J., R. E. LEE, C. HYMAN AND R. CHAMBERS. *Omental circulation in morph- 

inized dogs subjected to graded hemorrhage. Annals Surg., 120: 1944. 
ZWEIFACH, B. J. See also Chambers and Zweifach, 1944. 
ZWEIFACH, B. J., R. G. ABELL, R. CHAMBERS AND G. H. A. CLOWES. Role of the decompensa- 

tory reactions of peripheral blood vessels in tourniquet shock. Surg. Gyn. Obstct., 80 : 1945. 

2. THE STAFF, 1945 

CHARLES PACKARD, Director, Marine Biological Laboratory, Woods Hole, Massachusetts. 

SENIOR STAFF OF INVESTIGATION 

E. G. CONKLIN, Professor of Zoology, Emeritus, Princeton University. 

FRANK R. LILLIE, Professor of Embryology, Emeritus, The University of Chicago. 

RALPH S. LILLIE, Professor of General Physiology. Emeritus. The University of Chicago. 

C. E. McCLUNG, Professor of Zoology, Emeritus, University of Pennsylvania. 

S. O. MAST, Professor of Zoology, Emeritus, Johns Hopkins University. 

A. P. MATHEWS, Professor of Biochemistry, Emeritus. University of Cincinnati. 

T. H. MORGAN, Director of the Biological Laboratory, California Institute of Technology. 

G. H. PARKER, Professor of Zoology, Emeritus, Harvard University. 

ZOOLOGY 

I. CONSULTANTS 

T. H. BISSONNETTE, Professor of Biology, Trinity College. 
L. L. WOODRUFF, Professor of Protozoology, Yale University. 

II. INSTRUCTORS 

F. A. BROWN, Associate Professor of Zoology, Northwestern University, in charge of 

course. 

T. H. BULLOCK, Assistant Professor of Neurology University of Missouri Medical School. 
W. D. BURBANCK, Associate Professor of Biology, Drury College. 
C. G. GOODCHILD, Associate Professor of Biology, Southwest Missouri State Teachers 

College. 

JOHN H. LOCH HE AD, Instructor in Zoology, University of Vermont. 
MADELENE E. PIERCE, Assistant Professor of Zoology, Vassar College. 
W. M. REID, Assistant Professor of Biology, Monmouth College. 
MARY D. ROGICK, Professor of Biology, College of New Rochelle. 

III. LABORATORY ASSISTANT 
ANTOIN BACA, Duke University Medical School. 

EMBRYOLOGY 

I. CONSULTANTS 
H. B. GOODRICH, Professor of Biology, Wesleyan University. 



34 MARINE BIOLOGICAL LABORATORY 

II. INSTRUCTORS 

W. W. BALLARD, Professor of Zoology, Dartmouth College. 

DONALD P. COSTELLO, Professor of Zoology, University of North Carolina. 

VIKTOR HAMBURGER, Professor of Zoology, Washington University, in charge of course. 

JANE M. OPPENHEIMER, Assistant Professor in Biology, Bryn Mawr College. 

III. RESEARCH ASSISTANT 
MARJORIE HOPKINS, University of California. 

IV. LABORATORY ASSISTANTS 

CATHERINE HENLEY, The Johns Hopkins University. 
ELEANOR LERNER, Washington University. 

PHYSIOLOGY 

I. CONSULTANTS 

WILLIAM R. AMBERSON, Professor of Physiology, University of Maryland, School of 

Medicine. 

HAROLD C. BRADLEY, Professor of Physiological Chemistry, University of Wisconsin. 
WALTER E. CARREY, Professor of Physiology, Vanderbilt University Medical School. 
MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania. 

II. INSTRUCTORS 

ROBERT BALLENTINE, Lecturer in Zoology, Columbia University (absent in 1945). 

AURIN CHASE, Assistant Professor of Biology, Princeton University. 

ARTHUR C. GIESE, Associate Professor of Biology, Stanford University (absent in 1945 I. 

E. S. GUZMAN BARROX, Associate Professor of Biochemistry, The University of Chicago. 

RUDOLF T. KEMPTOX, Professor of Zoology, Vassar College (absent in 1945). 

ARTHUR K. PARPART, Associate Professor of Biology, Princeton University, in charge 

of course. 
ROBERT RAMSEY, Associate Professor of Physiology, Medical College of Virginia. 

BOTANY 

I. CONSULTANTS 

S. C. BROOKS, Professor of Zoology, University of California. 

B. M. DUGGAR, Professor of Plant Physiology. University of Wisconsin. 

II. INSTRUCTORS 

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

course. 
HANNAH CROASDALE, Technical Assistant, Dartmouth College. 

EXPERIMENTAL RADIOLOGY 

G. FAILLA, Memorial Hospital, New York City. 

L. ROBINSON HYDE, Phillips Exeter Academy, Exeter, N. H. 

LIBRARY 

PRISCILLA B. MONTGOMERY (MRS. THOMAS H. MONTGOMERY, JR.), Librarian 
DEBORAH LAWRENCE MRS. ELON H. JESSUP MARY A. ROHAN 



REPORT OF THE DIRECTOR 

APPARATUS DEPARTMENT 

E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager 
J. D. GRAHAM DOROTHY LEFEVRE 

CHEMICAL DEPARTMENT 
E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager 

SUPPLY DEPARTMENT 

JAMES Mclxxis, Manager 

D. J. Zixx. Naturalist 

RUTH CROWELL GRACE M. KENNERSON 

M. B. GRAY W. E. KAHLER F. N. WHITMAN 

A. M. HILTON G. LEHY 

GENERAL OFFICE 

F. M. MACNAUGHT, Business Manager 
POLLY L. CROWELL MRS. LILA S. MYERS 

* 

GENERAL MAINTENANCE 

T. E. LARKIN, Superintendent 

W. C. HEMENWAY G. T. NICKELSON, JR. 

R. W. KAHLER T. E. TAWELL 

A. J. PIERCE 

THE GEORGE M. GRAY MUSEUM 
GEORGE M. GRAY, Curator Emeritus 



3. INVESTIGATORS AND STUDENTS 
Independent Investigators, 1945 

ABELL, RICHARD G., Assistant Professor of Anatomy, University of Pennsylvania. 

ADDISON, WILLIAM H. F., Professor of Normal Histology and Embryology, University of 

Pennsylvania. 

ANDERSON, RUBERT S., Assistant Professor of Physiology, University of Maryland. 
ANFINSON, CHRISTIAN B., JR., Instructor in Biological Chemistry, Harvard Medical School. 
ARMSTRONG, PHILIP B., Professor of Anatomy, Syracuse University. 
ARONOFF, SAMUEL, Instructor, University of Chicago. 
AXELRAD, ARTHUR A., Investigator, McGill University. 

BALL, ERIC G., Associate Professor of Biological Chemistry, Harvard Medical School. 
BALLARD, W. W., Professor of Zoology, Dartmouth College. 
BALLENTINE, ROBERT, Instructor, Columbia University. 

BARRON, E. S. GUZMAN, Associate Professor of Biochemistry, The University of Chicago. 
EARTH, L. G., Associate Professor of Zoology, Columbia University. 
BEERS, CHARLES DALE, Professor of Zoology, University of North Carolina. 
BERGER, CHARLES A., Professor of Cytology, Fordham University. 
BERTHOLF, LLOYD M., Professor of Biology, Western Maryland College. 
BEVELANDER, GERRIT, Associate Professor of Anatomy, New York University. 
BLISS, ALFRED F., Instructor in Physiology and Pharmacology, Union University. 
BODIAN, DAVID, Associate in Epidemiology, Johns Hopkins University. 



36 MARINE BIOLOGICAL LABORATORY 

BRONK, DETLEV W., Professor of Biophysics, Johnson Foundation. 

BROOKS, MATILDA M., Research Associate in Biology, University of California. 

BROOKS, SUMNER C., Professor of Zoology, University of California. 

BROWN, DUGALD E. S., Professor of Physiology, New York University. 

BROWN, FRANK A., JR., Associate Professor of Zoology, Northwestern University. 

BROWNELL, KATHARINE A., Research Associate, Ohio State University. 

BUDINGTON, ROBERT A., Professor of Zoology, Emeritus, Oberlin College. 

BULLOCK, THEODORE H., Assistant Professor of Anatomy, University of Missouri. 

BURBANCK, WILLIAM D., Associate Professor of Biology, Drury College. 

BURKHOLDER, PAUL R., Professor of Botany. Yale University. 

CHAMBERS, ROBERT, Research Professor of Biology, New York University. 

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

CHENEY, RALPH H., Chairman Biology Department, Long Island University. 

CHIDESTER, F. E., Research Worker, Lee Foundation. 

CLAFF, C. LLOYD, Research Fellow in Surgery, Harvard Medical School. 

CLARK, ELEANOR L., Voluntary Research Worker, University of Pennsylvania. 

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

CLAUDE, ALBERT, The Rockefeller Institute for Medical Research. 

CLEMENT, A. C., Associate Professor in Biology. College of Charleston. 

CLOWES, G. H. A., Director of Research, Lilly Research Laboratories. 

CONKLIN, EDWIN G., Professor of Zoology, Emeritus, Princeton University. 

COPELAND, MANTON, Professor of Biology, Bowdoin College. 

COSTELLO, DONALD P., Professor of Zoology, University of North Carolina. 

CRAMPTOX, HENRY E., Professor Emeritus. Columbia L'niversity. 

CROASDALE, HANNAH T., Technical Assistant, Dartmouth College. 

CROUSE, HELEN V., Research Associate, University of Pennsylvania. 

CROWELL, SEARS, Assistant Professor of Zoology, Miami University. 

FROEHLICH, ALFRED, Associate, May Institute for Medical Research. 

FURCHGOTT, ROBERT F., Research Associate, Cornell L^niversity. 

FURTH, JACOB, Professor of Pathology, Cornell University. 

GAFFRON, HANS, Assistant Professor of Biochemistry, Research Associate, University of Chicago. 

GALTSOFF, PAUL S., Senior Biologist, U. S. Fish and Wildlife Service. 

CARREY, W. E., Professor of Physiology, Emeritus, Vanderbilt University, School of Medicine. 

GLASER, OTTO C., Professor of Biology, Amherst College. 

GOODCHILD, DR. C. G., Associate Professor of Biology, State Teachers College. 

GORBMAN, AUBREY, Instructor in Biology, Wayne University. 

GOULD, HARLEY N., Professor of Biology, H. Sophie Newcomb College. 

GRAND, C. G., Research Associate, New York University. 

HAMBURGER, VIKTOR, Professor of Zoology, Washington University. 

HARTMAN, FRANK A., Professor and Chairman of Department of Physiology, Ohio State 

University. 

HARVEY, ETHEL BROWNE, Independent Investigator Biology Department, Princeton University. 
HARVEY, E. NEWTON, Professor of Physiology, Princeton University. 
HAYASHI, TERU, Instructor in Zoology, University of Missouri. 
HAYWOOD, CHARLOTTE, Professor of Physiology, Mount Holyoke College. 
HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania. 
HIBBARD, HOPE, Professor of Zoology, Oberlin College. 
HICKSON, ANNA KELTCH, Research Chemist, Eli Lilly & Company. 
HOPKINS, HOYT S., Associate Professor of Physiology, New York University, College of 

Dentistry. 

HUBER, WOLFGANG, Senior Research Chemist, Winthrop Chemical Company. 
JACOBS, M. H., Professor of General Physiology, University of Pennsylvania. 
JAEGER, LUCENA, Research Associate, Columbia University. 

JENKINS, GEORGE B., Professor of Anatomy, Emeritus, George Washington University. 
JOHLIN, J. M., Associate Professor of Biochemistry, Vanderbilt University. 
JOHNSON, FRANK H., Assistant Professor of Biology, Princeton University. 
KRAHL, M. E., Instructor in Pharmacology, Columbia University. 
LANDIS, EUGENE M., Professor of Physiology and Head of Department, Harvard Medical 

School. 



REPORT OF THE DIRECTOR 37 

LAVIN, GEORGE I., In charge of Spectroscopic Laboratory, Rockefeller Institute for Medical 

Research. 

LEE, RICHARD E., Student, Columbia University. 
LIEBEN, FRITZ, Research Fellow, Johns Hopkins University. 
LILLIE, RALPH S., Professor of Physiology, Emeritus, University of Chicago. 
LOCH HEAD, JOHN H., Assistant Professor of Zoology, University of Vermont. 
McCLUNG, C. E., Professor of Zoology, Emeritus, University of Pennsylvania. 
MACLEAN, BERNICE L., Assistant Professor, Department Biological Sciences, Hunter College. 
MAC.ALHAES, HULDA, Instructor in Zoology, Duke University. 
MARKS, MILDRED H., Student, Massachusetts Institute of Technology. 
MARSLAND, DOUGLAS A., Associate Professor of Biology, New York University. 
MAST, S. O., Professor of Zoology, Emeritus, Johns Hopkins University. 
MATHEWS, ALBERT P., Professor of Biochemistry, Emeritus, University of Cincinnati. 
MATTHEWS, SAMUEL A., Professor of Biology, Williams College. 
MEMHARD, ALLEN R., Crescent Road, Riverside, Connecticut. 
MENKIN, VALY, Assistant Professor of Pathology, Duke University. 
METZ, CHARLES W., Director Zoological Laboratory, University of Pennsylvania. 
MICHAELIS, LEONOR, Member Emeritus, Rockefeller Institute for Medical Research. 
NACHMANSOHN, DAVID, Research Associate in Neurology, Columbia University. 
NORTHROP, JOHN H., Member of the Institute, Rockefeller Institute for Medical Research. 
OPPENHEIMER, JANE M., Assistant Professor of Biology, Bryn Mawr College. 
OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research. 
PARPART, ARTHUR K., Associate Professor of Biology, Princeton University. 
PIERCE, MADELEXE E., Associate Professor of Zoology, Vassar College. 
RAMSEY, ROBERT W., Associate Professor of Physiology, Medical College of Virginia. 
RANKIN, JOHN S., JR., Assistant Professor of Zoology, University of Connecticut. 
REID, W. MALCOLM, Assistant Professor of Biology, Monmouth College. 
RIKER, WALTER F., JR., Instructor in Medicine and Pharmacology, Cornell University Medical 

School. 

Ris, HANS, Assistant in Physiology, Rockefeller Institute for Medical Research. 
ROBBIE, WILBUR A., Research Associate, State University of Iowa. 
ROGICK, MARY DORA, Professor of Biology, College of New Rochelle. 
SAMPSON, MYRA M., Professor of Zoology, Smith College. 
SANDOW, ALEXANDER, Assistant Professor of Biology, New York University. 

SCHAEFFER, A. A., Professor of Biology, Temple University. 

SCHARRER, ERNST A., Assistant Professor of Anatomy, Western Reserve University School of 
Medicine. 

SCOTT, SISTER FLORENCE MARIE, Professor of Zoology, Seton Hill College. 

SCOTT, GEORGE T., Instructor, Oberlin College. 

SHANES, ABRAHAM M., Assistant Professor of Physiology. New York University College of 
Dentistry. 

SHAPIRO, HARRY H., Assistant Professor of Anatomy, Columbia University. 

SLIFER, ELEANOR H., Assistant Professor of Zoology, State University of Iowa. 

SMITH, DIETRICH CONRAD, Associate Professor of Physiology, University of Maryland, School 
of Medicine. 

STERN, KURT G., Lecturer in Department of Chemistry, Polytechnic Institute of Brooklyn. 

STEWART, DOROTHY R., Fellow in Physiology, University of Pennsylvania. 

STOKEY, ALMA G., Professor of Plant Science, Emeritus, Mount Holyoke College. 

STUNKARD, H. W., Professor of Biology, New York University. 

TAFT, CHARLES H., Associate Professor of Pharmacology, Medical Branch, University of Texas. 

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

TEWINKEL, Lois E., Associate Professor of Zoology, Smith College. 

THIVY, FRANCESCA, Professor of Biology, Women's Christian College. 

VILLEE, CLAUDE A., Assistant Professor of Zoology, University of North Carolina. 

WAINIO, WALTER W., Assistant Professor of Physiology, New York University, College of 
Dentistry. 

WARREN, CHARLES O., Assistant Professor of Physiology, Cornell University Medical College. 

WENRICH, D. H., Professor of Zoology, University of Pennsylvania. 

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



MARINE BIOLOGICAL LABORATORY 

WHITING, P. W., Associate Professor of Zoology. University of Pennsylvania. 

WICHTERMAN, RALPH, Assistant Professor of Biology, Temple University. 

WILLIER, B. H., Professor of Zoology and Director of the Biological Laboratories, Johns 

Hopkins University. 

WINSOR, CHARLES P., Research Associate, Princeton University. 
WITKUS, ELEANOR R., Instructor in Botany and Bacteriology, Fordham University. 
WOODWARD, ALVALYN E., Assistant Professor, University of Michigan. 
WOODWARD, ARTHUR A., JR., Research Assistant, University of Pennsylvania. 
WOOLEY, D. W., Associate. Rockefeller Institute for Medical Research. 
WRINCH, DOROTHY, Lecturer in Physics, Smith College. 

YNTEMA, CHESTER L., Assistant Professor of Anatomy, Cornell University Medical College. 
ZWEIFACH, BENJAMIN W., Research Associate in Biology, New York University. 

Beginning Investigators, 1945 

BROWN, ELLEN, Commonwealth Fellow, University of California Medical School. 

BROWN, VIRGINIA H., Graduate Student, Ohio State University. 

COYNE, CHRISTOPHER J., Student, University of Pennsylvania. 

DAVIDSON, MARGARET E., Demonstrator and Assistant to Dr. Berrill, McGill University. 

KRUGELIS, EDITH J., Graduate Student, Columbia University. 

LERNER, ELEANOR, Fellow in Zoology, Washington University. 

LOVELACE, ROBERTA, Teaching Fellow, University of North Carolina. 

MILLER, HELMA C., Assistant, Johns Hopkins University. 

SCHNEYER, LEON H., Instructor, New York University, College of Dentistry. 

WILSON, WALTER, Graduate Student, University of Pennsylvania. 

Research Assistants, 1945 

ABRAMSKY, TESS, Research Assistant, Rockefeller Institute for Medical Research. 

BRUNELLI, ELEANOR L., Research Assistant, New York University, College of Dentistry. 

DEFALCO, ROSE H., Research Assistant, University of Pennsylvania. 

FISCHL, MATHILDA, Research Assistant in Medicine, Cornell University. 

FRANZ, RUTH ESTELLE, Research Assistant, Yale University. 

GARZOLI, RAY F., Graduate Student, University of California. 

GOULD, DAVID, Research Technician, New York University. 

HARLOW, JANET, Technician, Syracuse University. 

HELFMAN, MYRNA, Technician, New York University. 

HENLEY, CATHERINE, Graduate Teaching Assistant, Johns Hopkins University. 

HONEGGER, CAROL, Student, Temple University. 

LAWLER, H. CLAIRE, Research Assistant, New York University. 

LEVIN, ISAAC, Research Assistant, Princeton University. 

LEVY, BETTY, Research Assistant, Rockefeller Institute for Medical Research. 

LOOFBOURROW, G. N., Instructor, Rhode Island State College. 

MCVEIGH, IDA, Research Assistant in Botany, Yale University. 

METZ, DELILAH B., Research Assistant, Eli Lilly & Co. 

MINER, KARYL, Research Assistant, New York University. 

MITCHELL, CONSTANCE, Research Assistant, University of Pennsylvania. 

PETTENGILL, OLIVE S., Student, Temple University. 

QUINN, GERTRUDE P., Research Assistant, New York University. 

ROTHENBERG, M. A., Research Assistant in Biochemistry, College of Physicians and Surgeons. 

UHLMAN, GLORIA E., Research Assistant, Yale University. 

WALTERS, C. PATRICIA, Research Assistant, Eli Lilly & Co. 

WARNER, CHARLOTTE, Medical Student, University of Pennsylvania. 

ZACKS, SUMNER L, Student, Brookline High School. 

Library Readers, 1945 

AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland. 
BECK, LYLE V., Associate Professor of Physiology, Hahnemann Medical College. 



REPORT OF THE DIRECTOR 39 

BENDICH, AARON, Member War Research Division, Neurological Institute, New York. 

BLOCK, RICHARD J., Associate, New York Medical College. 

BREHME, KATHERINE S., Lecturer, Cornell University Medical College. 

CAHEN, RAYMOND L., Research Assistant, Yale University, Medical School. 

CARSON, HAMPTON L., Instructor in Zoology, Washington University. 

CASSIDY, HAROLD G., Assistant Professor of Chemistry, Yale University. 

COLWIN, LAURA HUNTER, Instructor, Pennsylvania College for Women. 

FRIEDEMANN, ULRICH H., Head of Department of Bacteriology, Brooklyn Jewish Hospital. 

FRISCH, JOHN A., Professor of Biology, Canisius College. 

GATES, R. RUGGLES, Professor Emeritus, University of London. 

GUREWICH, VLADIMIR, Assistant Visiting Physician, Bellevue Hospital. 

KABAT, ELVIN A., Research Associate in Biochemistry, College of Physicians and Surgeons. 

KAYLOR, CORNELIUS T., Assistant Professor of Anatomy, Syracuse University. 

KELLER, RUDOLPH, Researcher, Robinson Foundation, New York. 

KRASNOW, FRANCES, Head of Department of Research, Guggenheim Dental Foundation. 

LANGE, MATHILDE M., Professor of Zoology, Head of Department of Biology, W'heaton College. 

LOEWI, OTTO, Research Professor of Pharmacology, New York University, College of Medicine. 

MARINELLI, LEONIDAS, Physicist, Memorial Hospital. 

MAVOR, JAMES W., Professor of Biology, Union College. 

MAYER, MANFRED M., Scientific Staff, War Research Division, Columbia University. 

METZ, CHARLES B., Instructor in Biology, Wesleyan University. 

MEYERHOF, DR. OTTO, Research Professor of Biochemistry, University of Pennsylvania. 

MOLDAVER, JOSEPH. Research Associate in Neurology, Columbia University. 

MOORE, JOHN A., Assistant Professor of Zoology, Barnard College. 

MOSCHCOWITZ, ELI, Assistant Professor of Chemical Medicine, Columbia University. 

OSEASOHN, ROBERT O., Long Island College of Medicine. 

PERRY, BARBARA H., Graduate Student and Teaching Fellow in Zoology, Smith College. 

PONDER, ERIC, Research, Nassau Hospital. 

RAMSDEN, ETHEL J., Instructor in Biology, Montclair Teachers Colle;-ir. 

ROBINSON, MILES H., Instructor in Pharmacology, University of Pennsylvania. 

RYAN, FRANCIS J., Assistant Professor, Columbia University. 

SCOTT, ALLAN, Assistant Professor of Biology, Union College. 

STRAUSS, WILLIAM L., JR., Associate Professor of Anatomy, Johns Hopkins University. 

VoxDACH, HERMAN, Assistant Professor of Physiology, Georgetown Medical School. 

WALLACH, JACQUES B., Long Island College of Medicine. 

ZORZOLI, ANITA, Assistant Instructor, New York University. 

Students, 1945 
BOTANY 

BARRACLOUGH, MARY EDITH, Student, Smith College. 

DIETZ, ALMA, Assistant in Biology, American International College. 

GARDNER, ELIZABETH B., Radcliffe College. 

MOUL, EDWIN THEODORE, Botany Assistant, University of Pennsylvania. 

SMITH, MATTIE Lot', Student, Radcliffe College. 

EMBRYOLOGY 

BEACH, JANET, Student, University of Connecticut. 

BERNIER, GERMAINE, University of Montreal, Quebec, Canada. 

BERRY, BETH SINCLAIR, Student, Rockford College, Illinois. 

CARTER, MARJORIE ESTELLE, Teacher, Georgia State Women's College. 

CHIRICO, ANNA MARIE, Student. Seton Hill College. 

CLARK, CARL CYRUS, Student, Amherst College. 

COPINGER, ANNE STEVENS, Goucher College. 

EHRLICH, MIRIAM, Knox College. 

Izzo, MARY JANE, University of Rochester. 

KELL, AMY, University of Illinois. 

LEVIN, ILANE B., Goucher College. 



40 MARINE BIOLOGICAL LABORATORY 

LODICO, DOROTHY GERALDINE, University of Rochester. 

LOVELACE, LOLLIE ROBERTA, Teaching Fellow, University of North Carolina. 

MARKER, MURIEL JOSEPHINE, Student, Colby College. 

MEZGER, LISELOTTE, Student, Bryn Mawr College. 

MILLER, HELMA C., Graduate Assistant, Johns Hopkins University. 

PERKINS, BARBARA BURNHAM, University of Connecticut. 

RAYMOND, BARBARA, Student, Swarthmore College. 

RICE, MARY ESTHER, Assistant in Biology Laboratory, Drew University. 

ROBERTS, ELIZABETH S., Assistant in Biology, Wilson College. 

RUDERMAN, CLAIRE, Teaching Assistant, University of Rochester. 

THORBY, JEAN ADELAIDE, Student, Rockford College. 

UPHOFF, DELTA E v University of Rochester. 

PHYSIOLOGY 

BRUST, MANFRED, Student, New York University. 

COOK, JOHN ALFRED, George Washington University. 

FERGUSON, ALICE HOWARD, Graduate Assistant, Louisiana State University. 

FLINKER, MARIE-LOUISE M., Assistant in Physiology, Vassar College. 

FOGERSON, VIRGINIA LEE, Student, Drury College. 

FOSTER, ELIZABETH JANE, Student, University of Illinois. 

GOLDSMITH, YVETTE, Perth Amboy, New Jersey. 

HAJEK, NORMA MARY, Cornell University. 

HECHT, LISELOTTE ISABELLA, Student, University of Michigan. 

RESNICK, OSCAR, Resident Scholar, Harvard University. 

WEISS, MICHAEL S., Student, Washington Square College. 

WOLFF, MARY LYDA, Instructor, Cedar Crest College. 

WORKEN, BARNEY, 3400 Wayne Avenue, New York City. 

ZOOLOGY 

ARONOWITZ, OLGA, New York University. 

BATES, MARY FLORENCE, Student, Vassar College. 

BAYORS, WINIFRED M., Student Seton Hill College. 

BEAL, JUDITH D., Vassar College. 

BENJAMIN, MRS. REZSIN C., Undergraduate Student, University of Rochester. 

BERNARD, SISTER MARIE, Fordham University. 

BERNIER, GERMAINE, Instructor, University of Montreal. 

BEZILLA, HELEN, Student, Seton Hill College. 

BRADIN, JOHN L., Northwestern University. 

CALVERT, JULIE NEIL, Student, Wilson College. 

CARLSON, ALICE MARIE, Laboratory Assistant, University of Minnesota. 

CHAFFIN, EVELYN L., Student. Drury College. 

CLARK, CARL CYRUS, Amherst College. 

CUMMINGS, REV. GEORGE W., Graduate Student, Catholic University. 

DAILEY, DOROTHY HELEN, Depamv University. 

DAWSON, MARY JEAN, Student, Mt. Holyoke College. 

DEMPSEY, ELLEN, Oberlin College. 

DICKASON, MARY ELIZABETH, Student, Smith College. 

FARNHAM, CAROL JEAN, Student, Drury College. 

FREITAG, JANET FAITH, Student, University of Connecticut. 

GOLDIS, BERNICE RUTH, Graduate Student, University of Pennsylvania. 

HANLON, REV. JAMES J., Graduate Student, Fordham University. 

HILL, SHIRLEY B., Student, Vassar College. 

HINES, EILEEN BARBARA, State University of Iowa. 

JONES, DOROTHY B., Student, University of Connecticut. 

JOSITA, SISTER M., Student, Fordham University. 

JULIER, EDITH VAILLANT, Student, Vassar College. 



REPORT OF THE DIRECTOR 



41 



KREKELER, CARL H., Student, Washington University. 

KUHN, ALICE ROBERTS, Western-Maryland College. 

LOWENS, MARY DOROTHY, Student, Swarthmore College. 

McCLAiN, MARYLOW, Student, Swarthmore College. 

MCGREGOR, ELIZABETH, Instructor, Mount Holyoke College. 

McVicKER, SISTER MAUREEN, Teacher of Biology, St. Joseph's College for Women 

MALLOCH, JEAN, Vassar College. 

MEIHACK, HELEN LLOYD, Student, Oberlin College. 

MINA, FRANK A., Laboratory Instructor, Fordham University. 

OSBORN, JOAN A., Student, Barnard College. 

PETERS, REV. JOSEPH J., Graduate Student, Fordham University. 

RAYMOND, BARBARA, Student, Swarthmore College. 

RIGGS, AUSTIN F., Student, Harvard University. 

ROGERS, HENRY CRAMPTON, Deerfield Academy. 

SCHAEFER, GERTRUDE, Undergraduate, Temple University. 

SEAMAN, ARLENE, Zoology Assistant, Cornell University. 

SNIPES, ANNE, Wheaton College. 

STEES, NANCY, Teacher, West Chester State Teachers College. 

SURRARRER, THOMAS C, Professor of Biology, Baldwin-Wallace College. 

THORNTON, DOROTHY GOLDEN, Assistant in Zoology Dept., Wellesley College. 

TUPPER, LYLA, Graduate Student, Northwestern University. 

UBER, VIRGINIA M., Student, Pennsylvania College for Women. 

WAX, FLORENCE SIMA, Student, Oberlin College. 

WHYTE, MARJORIE ANN, Assistant, Cornell University. 

WILCOX, BARBARA L., Student, Radcliffe College. 

WILLIAMS, OLWEN, Teacher of Biology and Chemistry, The Putney School. 

WILSON, FAITH EVELYN, Johns Hopkins University. 

WILSON, MARIE ELLEN, Student, Western Maryland College. 




4. TABULAR VIEW OF ATTENDANCE 



1942 1943 1944 1945 



INVESTIGATORS Total 337 

Independent 197 

Under instruction / 59 

Library readers 31 

Research assistants 50 

STUDENTS Total 131 

Zoology 55 

Embryology 37 

Physiology 24 

Botany 15 

TOTAL ATTENDANCE 468 

Less persons registered as both students and investigators 7 



461 

INSTITUTIONS REPRESENTED Total 144 

By investigators 102 

By students 72 

SCHOOLS AND ACADEMIES REPRESENTED 

By investigators 5 

By students 2 

FOREIGN INSTITUTIONS REPRESENTED 

By investigators 3 

By students 1 



201 

132 

16 

28 

25 

74 

36 

24 

6 

8 

275 
2 

273 

126 

83 

43 



160 
89 
19 
35 
17 
68 
47 
13 
8 

228 
6 

222 

116 

70 

41 

2 
1 



2 



193 

112 

11 

50 
20 

75 
37 
23 
10 

5 
276 

1 

275 

106 

74 

41 

1 
2 

2 
3 



212 

138 

10 

38 

26 

96 

55 

23 

13 

5 

308 



124 

100 

49 

2 
2 



42 



MARINE BIOLOGICAL LABORATORY 



5. SUBSCRIBING AND COOPERATING INSTITUTIONS 



1945 



Albany Medical College 

Amherst College 

Biological Institute, Philadelphia, Pennsylvania 

Bowdoin College 

Bryn Mawr College 

Cathedral College 

The Catholic University of America 

Columbia University 

Cornell University 

Cornell University Medical College 

Duke University 

Fish and Wild Life Service, U. S. Dept. of 

the Interior 
Fordham University 
Goucher College 
Harvard University 
Harvard University Medical School 
Industrial and Engineering Chemistry, of the 

American Chemical Society 
Johns Hopkins University 
Johns Hopkins Medical School 
Lee Foundation 
Eli Lilly & Company 
Long Island University 
Macy Foundation 

Massachusetts Institute of Technology 
McGill University 
Miami University 
Mount Holyoke College 
New York University 

New York University College of Medicine 
New York University School of Dentistry 
New York University Washington Square 

College 



Oberlin College 

Ohio State University 

Pennsylvania College for Women 

Princeton University 

Radcliffe College 

Rockefeller Institute for Medical Research 

St. Joseph College for Women 

Smith College 

State University of Iowa 

Syracuse University 

Syracuse University Medical School 

Temple University 

University of Chicago 

University of Connecticut 

University of Illinois 

University of Maryland Medical School 

University of Michigan 

University of Missouri 

University of Pennsylvania 

L'niversity of Pennsylvania School of Medicine 

University of Rochester 

Vanderbilt University Medical School 

Vassar College 

Washington University 

Wayne University 

Wellesley College 

Wesleyan University 

Western Maryland College 

Western Reserve University 

Wheaton College 

Williams College 

Wilson College 

Woods Hole Oceanographic Institution 

Yale University 



6. EVENING LECTURES, 1945 

Friday, June 29 

PROF. P. W. WHITING "The Development of Hymenopteran Ge- 
netics." 
Friday, July 6 

DR. R. R. GATES "Human Heredity in Relation to Animal 

Genetics." 
Friday, July 13 

DR. I. FANKUCHEN "X-Ray Diffraction and Protein Structure." 

Friday, July 20 

PROF. S. C. BROOKS "Our Interrelationships with South Ameri- 
can Universities, together with Illustrated 
Travel Notes." 
Friday, July 27 

PROF. E. G. BUTLER "Problems of Differentiation and Dediffer- 

entiation in Amputated Urodele Limbs." 



REPORT OF THE DIRECTOR 43 

Friday, August 3 

DR. BOSTWICK H. KETCHUM "The Prevention of Ship Bottom Fouling." 

Friday, August 10 

DR. DANIEL MERRIMAN "A Study in Pure and Applied Marine Bi- 
ology. The Life History and Economic 
Importance of the Ocean Pout." 
Friday, August 17 

DR. DETLEV W. BRONK "Biological Research During the War and 

Postwar Periods." 
Friday, August 24 

DR. F. L. HISAVV "Endocrines and the Evolution of Vivi- 

parity among the Vertebrates." 
Monday, August 27 

GEORGE G. LOWER "Local Invertebrates." 


Wednesday, August 29 

DR. PAUL S. GALTSOFF "Impressions of a Biologist at the San 

Francisco Conference." 
Thursday, August 30 

MAJOR A. H. NEUFELD "Medical Research Organization in the Ca- 
nadian Army." 
Thursday, August 30 

CAPT. W. R. DURYEE "Medical Military Training." 

7. SHORTER SCIENTIFIC PAPERS, 1945 

Tuesday, July 24 

DR. M. M. BROOKS "The Redox Potential of Penicillium rota- 
turn Medium under Some Different Con- 
ditions of Growth." 

DR. WILBUR ROBBIE "The Use of Cyanide in Manometric Ex- 
perimentation." 

DR. SEARS CROWELL "The Displacement of Terns by Gulls at 

Weepecket Island." 
Tuesday, July 31 

DR. P. W. WHITING "The Problem of Reversal of Male Hap- 

loidy by Selection." 

DR. BERTA SCHARRER "Experimental Tumors after Nerve Section 

in an Insect." 

DR. P. S. GALTSOFF "Reactions of Oysters to Free Chlorine." 

Tuesday, August 7 

DR. T. H. BULLOCK "Organization of Giant Nerve Fibers in cer- 
tain Polychaetes." 

DR. ERNST SCHARRER "The Origin of Neurosecretory Granules 

from Basophil Substances in the Nerve 
Cells of Fishes." 

DR. C. H. TAFT "The Action of Quitenine on the Livers of 

Tautog and Toadfish." 

DR. A. M. SHANES "Evidence of a Metabolic Effect by Potas- 
sium in Lowering the Injury Potential of 
Nerve." 



44 MARINE BIOLOGICAL LABORATORY 

Tuesday, August 14 

DR. R. CHAMBERS "Interrelations between Sperm-Nucleus, 

Egg-Nucleus and Cytoplasm in Asterias 
Egg." 

DR. KURT G. STERN "Physical-chemical Studies on Chromosomal 

Nucleoproteins." 
Tuesday, August 21 

DR. DOROTHY WRINCH "Hemoglobin and other Native Proteins." 

DR. E. R. WIT.KUS "Endomitosis in Plants." 

DR. C. A. BERGER "Recent Cytological Studies in Culex." 

Thursday, August 23 

DR. ETHEL B. HARVEY "Development of Granule-free Fractions of 

Arbacia eggs." 

DR. ALEXANDER SANDOW "Studies of the Muscle Twitch by Methods 

of Electronic Recording." 

DR. C. D. BEERS "The Role of Bacteria in the Excystment of 

the Ciliate Didinium." 
Monday, August 27 

DR. ANNA R. WHITING "Differences in Sensitivity, Hatchability 

Curves and Cytological Effects between 
Eggs X-rayed in First Meiotic Prophase 
and Metaphase." 

DR. W. W. WAINIO "Aerobic Oxidation of Simple sugars by 

Mammalian Liver." 

DR. DUGALD E. S. BROWN "The Role of Myosin and Myosin Triphos- 

phatase ;';; Vitro and in Muscle." 
Tuesday, August 28 

DR. LLOYD M. BERTHOLF "Accelerating Metamorphosis in the Tuni- 
cate Styela." 

DR. ALFRED FROELICH "The Influence of Drugs on Heat-narcosis." 

DR. W. MALCOLM REID "In Vivo and in Vitro Glycogen Utiliza- 
tion in the Avial Nematode Ascardia 
Galli." 

8. MEMBERS OF THE CORPORATION, 1945 

1. LIFE MEMBERS 

ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France. 

BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York. 

BILLINGS, MR. R. C., 66 Franklin Street, Boston, Massachusetts. 

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

COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin. 

CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New Jersey. 

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

JACKSON, MR. CHAS. C., 24 Congress Street, Boston, Massachusetts. 

JACKSON, Miss M. C., 88 Marlboro Street, Boston, Massachusetts. 

KING, MR. CHAS. A. 



REPORT OF THE DIRECTOR 45 

KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York. 

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

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

MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Missouri. 

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

MORGAN, MRS. T. H., Pasadena, California. 

MORGAN, PROF. T. H., Director of Biological Laboratory, California Institute of 

Technology, Pasadena, California. 
NOYES, Miss EVA J. 

PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania. 
SCOTT, DR. ERNEST L., Columbia University, New York City, New York. 
SEARS, DR. HENRY F., 86 Beacon Street, Boston, Massachusetts. 
SHEDD, MR. E. A. 

STRONG, DR. O. S., Columbia University, New York City, New York. 
WAITE, PROF. F. C., 144 Locust Street, Dover, New Hampshire. 
WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, California. 

2. REGULAR MEMBERS 

ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts. 

ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia, 
Pennsylvania. 

ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New 
York. 

ALBAUM, DR. HARRY G., Biology Dept., Brooklyn College, Brooklyn, N. Y. 

ALBERT, DR. ALEXANDER, 383 Harvard Street, Cambridge, Mass. 

ALLEE, DR. W. C., The University of Chicago, Chicago, Illinois. 

AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland, 
School of Medicine, Lombard and Greene Streets, Baltimore, Maryland. 

ANDERSON, DR. RUBERT S., University of Maryland School of Medicine, Depart- 
ment of Physiology, Baltimore, Maryland. 

ANDERSON, DR. T. F., University of Pennsylvania, Philadelphia, Pennsylvania. 

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

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

BAITSELL, DR. GEORGE A., Yale University, New Haven, Connecticut. 

BAKER, DR. H. B., Zoological Laboratory, University of Pennsylvania, Philadelphia, 
Pennsylvania. 

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

BALLENTINE, DR. ROBERT, Columbia University, Department of Zoology, New York 
City, New York. 

BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medi- 
cal School, Boston, Massachusetts. 

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

BARRON, DR. E. S. GUZMAN, Department of Medicine, The University of Chicago, 
Chicago, Illinois. 

BARTH, DR. L. G., Department of Zoology, Columbia University, New York City, 
New York. 







46 MARINE BIOLOGICAL LABORATORY 

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

BEADLE, DR. G. W., School of Biological Sciences, Stanford University, California. 

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

BECK, DR. L. V., Hahnemann Medical College, Philadelphia, Pennsylvania. 

BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana. 

BERTHOLF, DR. LLOYD M., Western Maryland College, Westminster, Maryland. 

BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge, Massachusetts. 

BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cambridge, Massa- 
chusetts. 

BINFORD, PROF. RAYMOND, Guilford College, North Carolina. 

BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut. 

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

BODINE, DR. J. H., Department of Zoology, State University of Iowa, Iowa City, 
Iowa. 

BORING, DR. ALICE M., Dickinson House, South Haclley, Massachusetts. 

BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wisconsin. 

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

BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, Washington Uni- 
versity Medical School, St. Louis, Missouri. 

BROOKS, DR. MATILDA M., University of California, Department of Zoology, Berke- 
ley, California. 

BROOKS, DR. S. C., University of California, Berkeley, California. 

BROWN, DR. DUGALD E. S., New York University, College of Dentistry, 209 East 
23d Street, New York City, New York. 

BROWN, DR. FRANK A., JR., Department of Zoology, Northwestern University, 
Evanston, Illinois. 

BUCK, DR. JOHN B., Industrial Hygiene Research Lab., National Institute of 
Health, Bethesda, Maryland. 

BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts. 

BUDINGTON, PROF. R. A., Winter Park, Florida. 

BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia. 

BULLOCK, DR. T. L., University of Missouri, Columbia, Missouri. 

BURBANCK, DR. WILLIAM D., Department of Biology, Drury College, Springfield, 
Missouri. 

BURKENROAD, DR. M. D., Yale University, New Haven, Connecticut. 

BUTLER, DR. E. G., Princeton University, Princeton, N. J. 

BYRNES, DR. ESTHER F., 1803 North Camac Street, Philadelphia, Pennsylvania. 

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

CANNAN, PROF. R. K., New York University College of Medicine, 477 First Ave- 
nue, New York City, New York. 

CARLSON, PROF. A. J., Department of Physiology, The University of Chicago, Chi- 
cago, Illinois. 

CAROTHERS, DR. E. ELEANOR, 134 Avenue C. East, Kingman, Kansas. 

CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massachusetts. 

CARROLL, PROF. MITCHELL, Franklin and Marshall College, Lancaster, Pennsyl- 
vania. 



REPORT OF THE DIRECTOR 47 

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

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

CATTELL, MR. WARE, 1621 Connecticut Are., Washington, D. C. 

CHAMBERS, DR. ROBERT, Washington Square College, New York University, Wash- 
ington Square, New York City, New York. 

CHASE, DR. AURIN M., Princeton University, Princeton, New Jersey. 

CHENEY, DR. RALPH H., Biology Department, Long Island University, Brooklyn, 
New York. 

CHIDESTER, PROF. F. E., Auburndale, Massachusetts. 

CHILD, PROF. C. M., Jordan Hall, Stanford University, California. 

CHURNEY, DR. LEON, 155 Powell Lane, Upper Darby, Pennsylvania. 

CLAFF, MR. C. LLOYD, Research Fellow in Surgery, Harvard Medical School, 
Boston, Mass. 

CLARK, PROF. E. R., University of Pennsylvania Medical School, Philadelphia, 
Pennsylvania. 

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

CLARKE, DR. G. L., Harvard University Biol. Lab., 16 Divinity Ave., Cambridge 
38, Mass. 

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

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

COE, PROF. W. R., Yale University, New Haven, Connecticut. 

COHN, DR. EDWIN J., 183 Brattle Street, Cambridge, Massachusetts. 

COLE, DR. ELBERT C., Department of Biology, Williams College, Williamstown, 
Massachusetts. 

COLE, DR. KENNETH S., University of Chicago, Chicago, Illinois. 

COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio. 

COLTON, PROF. H. S., Box 601, Flagstaff, Arizona. 

COOPER, DR. KENNETH W., Department of Biology, Princeton University, Prince- 
ton, New Jersey. 

COPELAND, PROF. MANTON, Bowdoin College, Brunswick, Maine. 

COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, 
Chapel Hill, North Carolina. 

COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro- 
lina, Chapel Hill, North Carolina. 

CRAMPTON, PROF. H. E., American Museum of Natural History, New York City, 
New York. 

CRANE, JOHN O., Woods Hole, Massachusetts. 

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

CROASDALE, HANNAH T., Dartmouth College, Hanover, New Hampshire. 

CROUSE, DR. HELEN V., University of Pennsylvania, Philadelphia, Pennsylvania. 

CROWELL, DR. P. S., JR., Department of Zoology, Miami University, Oxford, Ohio. 

CURTIS, DR. MAYNIE R., 377 Dexter Trail, Mason, Michigan. 

CURTIS, PROF. W. C., University of Missouri, Columbia, Missouri. 

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

DAVIS, DR. DONALD W., College of William and Mary, Williamsburg, Virginia. 

DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts. 



48 MARINE BIOLOGICAL LABORATORY 

DAWSON, DR. J. A., The College of the City of New York, New York City, New 
York. 

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

DEMEREC, DR. M., Carnegie Institution of Washington, Cold Spring Harbor, Long 
Island, New York. 

DILLER, DR. WILLIAM F., 1016 South 45th Street, Philadelphia, Pennsylvania. 

DODDS, PROF. G. S., Medical School, University of West Virginia, Morgantown 
West Virginia. 

DOLLEY, PROF. WILLIAM L., University of Buffalo, Buffalo, New York. 

DONALDSON, DR. JOHN C, University of Pittsburgh, School of Medicine, Pitts- 
burgh, Pennsylvania. 

DuBois, DR. EUGENE F., Cornell University Medical College, 1300 York Avenue, 
New York City, New York. 

DUGGAR, DR. BENJAMIN M., c/o Lederle Laboratories Inc., Pearl River, New 
York. 

DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota. 

DURYEE, DR. WILLIAM R., Surgeon General's Office, Washington, D. C. 

EDWARDS, DR. D. J., Cornell University Medical College, 1300 York Avenue, New 
York City, New York. 

ELLIS, DR. F. W., 1175 Centre Street, Newton, Massachusetts. 

EVANS, DR. TITUS C., College of Physicians and Surgeons, 630 West 168th Street, 
New York City, New York. 

FAILLA, DR. G., College of Physicians and Surgeons, 630 West 168th Street, New 
York City, New York. 

FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France. 

FAUST, DR. ERNEST C., Tulane University of Louisiana, New Orleans, Louisiana. 

FERGUSON, DR. JAMES K. W., Department of Pharmacology, University of Toronto, 
Ontario, Canada. 

FIGGE, DR. F. H. J., 4636 Schenley Road, Baltimore, Maryland. 

FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Rich- 
mond, Virginia. 

FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, To- 
ronto, Canada. 

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

FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Massachu- 
setts. 

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

FURTH, DR. JACOB, Cornell University Medical College, 1300 York Avenue, New 
York City, New York. 

GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy Chase, Mary- 
land. 

GARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee. 

GATES, DR. REGINALD R., Woods Hole, Massachusetts. 

GEISER, DR. S. W., Southern Methodist University, Dallas, Texas. 

GERARD, PROF. R. W., The University of Chicago, Chicago, Illinois. 

GLASER, PROF. O. C., Amherst College, Amherst, Massachusetts. 



REPORT OF THE DIRECTOR 49 

GOLDFORB, PROF. A. J., College of the City of New York, Convent Avenue and 139th 
Street, New York City, New York. 

GOODCHILD, DR. CHAUNCEY G., State Teachers College, Springfield, Missouri. 

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

GOTTSCHALL, DR. GERTRUDE Y., 919 20th Street, Washington, D. C. 

GRAHAM, DR. J. Y., Roberts, Wisconsin. 

GRAND, CONSTANTINE G., Biology Department, Washington Square College, New 
York University, Washington Square, New York City, New York. 

GRAVE, PROF. B. H., DePauw University, Greencastle, Indiana. 

GRAY, PROF. IRVING E., Duke University, Durham, North Carolina. 

GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City, 
New York. 

GUDERNATSCH, DR. J. FREDERICK, New York University, 100 Washington Square, 
New York City, New York. 

GUTHRIE, DR. MARY J., University of Missouri, Columbia, Missouri. 

GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin. 

HAGUE, DR. FLORENCE, Sweet Briar College, Sweet Briar, Virginia. 

HALL, PROF. FRANK G., Duke University, Durham, North Carolina. 

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

HANCE, DR. ROBERT T., The Cincinnati Milling Machine Co., Cincinnati 9, Ohio. 

HARGITT, PROF. GEORGE T., Department of Zoology, Duke University, Durham, 
North Carolina. 

HARMAN, DR. MARY T., Kansas State Agricultural College, Manhattan, Kansas. 

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

HARPER, PROF. R. A., R. No. 5, Bedford, Virginia. 

HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut. 

HARTLINE, DR. H. KEFFER, University of Pennsylvania, Philadelphia, Pennsylvania. 

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

HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New Jer- 
sey. 

HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey. 

HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachusetts. 

HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie University, Halifax, 
Nova Scotia. 

HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts. 

HECHT, DR. SELIG, Columbia University, New York City, New York. 

HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, Phila- 
delphia, Pennsylvania. 

HENDEE, DR. ESTHER CRISSEY, Russell Sage College, Troy, New York. 

HENSHAW, DR. PAUL S., National Cancer Institute, Bethesda, Maryland. 

HESS, PROF. WALTER N., Hamilton College, Clinton, New York. 

HIATT, DR. E. P., Duke University, Durham, North Carolina. 

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

HILL, DR. SAMUEL E., 18 Collins Avenue, Troy, New York. 

HINRICHS, DR. MARIE, Department of Physiology and Health Education, Southern 
Illinois Normal University, Carbondale, Illinois. 



50 MARINE BIOLOGICAL LABORATORY 

HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts. 

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

HOBER, DR. RUDOLF, University of Pennsylvania, Philadelphia, Pennsylvania. 

HODGE, DR. CHARLES, IV, Temple University, Department of Zoology, Philadelphia, 
Pennsylvania. 

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

HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Laboratory of In- 
dustrial Hygiene, Bethesda, Maryland. 

HOPKINS, DR. D.WIGHT L., Mundelein College, 6363 Sheridan Road, Chicago. Illi- 
nois. 

HOPKINS, DR. HOYT S., New York University, College of Dentistry, New York 
City, New York. 

HOWLAND, DR. RUTH B., Washington Square College, New York University, 
Washington Square East, New York City, New York. 

HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Virginia. 

HYMAN, DR. LIBBIE H., American Museum of Natural History, New York City, 
New York. 

IRVING, LT. COL. LAURENCE, Wright Field, Dayton, Ohio. 

ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts. 

JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsylvania, Phila- 
delphia, Pennsylvania. 

JENKINS, DR. GEORGE B., 1336 Parkwood Place, N.W., Washington, D. C. 

JENNINGS, PROF. H. S., Department of Zoology, University of California, Los An- 
geles, California. 

JOHLIN, DR. J. M., Vanderbilt University Medical School, Nashville, Tennessee. 

JONES, DR. E. RUFFIN, JR., College of William and Mary, Williamsburg, Virginia. 

KAUFMANN, PROF. B. P., Carnegie Institution, Cold Spring Harbor, Long Island, 
New York. 

KEMPTON, PROF. RUDOLF T., Vassar College, Poughkeepsie, New York. 

KIDDER, DR. GEORGE W., Brown University, Providence, Rhode Island. 

KIDDER, JEROME F., Woods Hole, Massachusetts. 

KILLE, DR. FRANK R., Carleton College, Northfield, Minnesota. 

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

KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, 36th Street and 
Woodland Avenue, Philadelphia, Pennsylvania. 

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

KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York. 

KOPAC, DR. M. J., Washington Square College, New York University, New York 
City, New York. 

KRAHL, DR. M. E., College of Physicians and Surgeons, 630 West 168th Street, 
New York 32, New York. 

KRIEG, DR. WENDELL J. S., 303 East Chicago Ave., Chicago, Illinois. 

LANCEFIELD, DR. D. E., Queens College, Flushing, New York. 

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

LANDIS, DR. E. M., Harvard Medical School, Boston, Massachusetts. 

LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts. 



REPORT OF THE DIRECTOR 51 

LAVIN, DR. GEORGE I., Rockefeller Institute, 66th Street and York Avenue, New 
York City, New York. 

LEWIS, PROF. I. F., University of Virginia, Charlottesville, Virginia. 

LILLIE, PROF. FRANK R., The University of Chicago, Chicago, Illinois. 

LILLIE, PROF. RALPH S., The University of Chicago, Chicago, Illinois. 

LITTLE, DR. E. P., Phillips Exeter Academy, Exeter, New Hampshire. 

LOCHHEAD, DR. JOHN H., Department of Zoology, University of Vermont, Bur- 
lington, Vermont. 

LOEB, PROF. LEO, 40 Crestwood Drive, St. Louis, Missouri. 

LOEB, DR. R. F., 180 Ft. Washington Avenue, New York City, New York. 

LOEWI, PROF. OTTO, 155 East 93d Street, New York City, New York. 

LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York 
City, New York. 

LUCAS, DR. ALFRED M., Regional Poultry Research Laboratory, East Lansing, 
Michigan. 

LUCRE, PROF. BALDUIN, University of Pennsylvania, Philadelphia, Pennsylvania. 

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

LYNCH, DR. RUTH STOCKING, Dept. of Zoology, University of California, Los 
Angeles 24, California. 

LYNN, DR. WILLIAM G., Department of Biology, The Catholic University of Amer- 
ica, Washington, D. C. 

MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia. 

MACNAUGHT, MR. FRANK M., Marine Biological Laboratory, Woods Hole, Massa- 
chusetts. 

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

MCGREGOR, DR. J. H., Columbia University, New York City, New York. 

MACKLIN, DR. CHARLES C., School of Medicine, University of Western Ontario, 
London, Canada. 

MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, Bos- 
ton, Massachusetts. 

MALONE, PROF. E. F.. 153 Cortland Avenue, Winter Park, Florida. 

MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York. 

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

MARTIN, PROF. E. A., Department of Biology, Brooklyn College, Bedford Avenue 
and Avenue H, Brooklyn, New York. 

MAST, PROF. S. O., Johns Hopkins University, Baltimore, Maryland. 

MATHEWS, PROF. A. P., Woods Hole, Massachusetts. 

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

MAYOR, PROF. JAMES W., Union College, Schenectady, New York. 

MAZIA, DR. DANIEL, Department of Zoology, Gowen Field, Boise, Idaho. 

MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania. 

MEIGS, MRS. E. B., 1736 M Street, N.W., Washington, D. C. 

MEM HARD, MR. A. R., Riverside, Connecticut. 



52 MARINE BIOLOGICAL LABORATORY 

MENKIN, DR. VALY, Duke University, School of Medicine, Durham, North Caro- 
lina. 

METZ, PROF. CHARLES W., University of Pennsylvania, Philadelphia, Pennsylvania. 

MICHAELIS, DR. LEONOR, Rockefeller Institute, 66th Street and York Avenue, New 
York City, New York. 

MILLER, DR. J. A., Division of Anatomy, College of Medicine, University of Ten- 
nessee, Memphis, Tennessee. 

MINNICH, PROF. D. E., Department of Zoology, University of Minnesota, Minne- 
apolis, Minnesota. 

MITCHELL, DR. PHILIP H., Brown University, Providence, Rhode Island. 

MOORE, DR. CARL R., The University of Chicago, Chicago, Illinois. 

MOORE, DR. J. A., Barnard College, New York City, New York. 

MORGAN, DR. ISABEL M., Poliomyelitis Research Center, 1901 E. Madison Street, 
Baltimore 5, Maryland. 

MORRILL, PROF. C. V., Cornell University Medical College, 1300 York Avenue, 
New York City, New York. 

MULLER, PROF. H. J., Department of Zoology, Indiana University, Bloomington, 
Indiana. 

NACHMANSOHN, DR. D., College of Physicians and Surgeons, 630 W. 168th Street, 
New York City, New York. 

NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Milton, Massa- 
chusetts. 

NEWMAN, PROF. H. H., 173 Devon Drive, Clearwater, Florida. 

NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania. 

NONIDEZ, DR. JOSE F., Cornell University Medical College, 1300 York Avenue, 
New York City, New York. 

NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New Jersey. 

OCHOA, DR. SEVERQ, New York University, College of Medicine, 477 First Avenue, 
New York 16, New York. 

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

OSBURN, PROF. R. C., Ohio State University, Columbus, Ohio. 

OSTERHOUT, PROF. W. J. V., Rockefeller Institute, 66th Street and York Avenue, 
New York City, New York. 

OSTERHOUT, MRS. MARIAN IRWIN, Rockefeller Institute, 66th Street and York 
Avenue, New York City, New York. 

PACKARD, DR. CHARLES, Marine Biological Laboratory, Woods Hole, Massachu- 
setts. 

PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio. 

PAPPENHEIMER, DR. A. M., 5 Acacia Street, Cambridge, Massachusetts. 

PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts. 

PARMENTER, DR. C. L., Department of Zoology, University of Pennsylvania, Phila- 
delphia, Pennsylvania. 

PARPART, DR. ARTHUR K., Princeton University, Princeton, New Jersey. 

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

PAYNE, PROF. F., University of Indiana, Bloomington, Indiana. 

PEEBLES, PROF. FLORENCE, Lewis and Clark College, Portland, Oregon. 



REPORT OF THE DIRECTOR 53 

PIERCE, DR. MADELENE E., Vassaf College, Poughkeepsie, New York. 

PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wisconsin. 

PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts. 

POLLISTER, DR. A. W., Columbia University, New York City, New York. 

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

PRATT, DR. FREDERICK H., Wellesley Hills 82, Massachusetts. 

PROSSER, DR. C. LADD, University of Chicago, Chicago, Illinois. 

RAND, DR. HERBERT W., Harvard University, Cambridge, Massachusetts. 

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

REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massachusetts. 

REID, DR. W. M., Monmouth College, Monmouth, Illinois. 

RENN, DR. CHARLES E., Harvard University, Cambridge, Massachusetts. 

RENSHAW, DR. BIRDSEY, Rockefeller Institute for Medical Research, 66th Street 
and York Avenue, New York City, New York. 

DERENYI, DR. GEORGE S., Department of Anatomy, University of Pennsylvania, 
Philadelphia, Pennsylvania. 

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

RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio. 

RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma. 

RICHARDS, DR. A. GLENN, Entomology Department. University Farm, Univ. of 
Minnesota, St. Paul 8, Minnesota. 

RICHARDS. DR. O. W.. Research Dept. American Optical Co., 19 Doat Street, 
Buffalo, New York. 

RIGGS, LAWRASON, JR., 120 Broadway, New York City, New York. 

ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio. 

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

ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts. 

ROOT, DR. R. W., Department of Biology, College of the City of New York, Con- 
vent Avenue and 139th Street, New York City, New York. 

ROOT, DR. W. S., College of Physicians and Surgeons, Department of Physiology, 
630 West 168th Street, New York City, New York. 

RUEBUSH, DR. T. K., Dayton, Virginia. 

RUGH, DR. ROBERTS, Department of Biology, Washington Square College, New 
York University, New York City, New York. 

SAMPSON, DR. MYRA M., Smith College, Northampton, Massachusetts. 

SASLOW, DR. GEORGE, Washington University Medical School, St. Louis, Missouri. 

SAUNDERS, LAWRENCE, W. B. Saunders Publishing Company, Philadelphia, Penn- 
sylvania. 

SAYLES, DR. LEONARD P., Department of Biology, College of the City of New York, 
139th Street and Convent Avenue, New York City, New York. 

SCHAEFFER, DR. ASA A., Biology Department, Temple University, Philadelphia, 
Pennsylvania. 

SCHARRER, DR. ERNST A., Western Reserve University, School of Medicine, 2109 
Adelbert Road, Cleveland 6, Ohio. 

SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street and Con- 
vent Avenue, New York City, New York. 



54 MARINE BIOLOGICAL LABORATORY 

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

SCHMITT, PROF. F. O., Department of Biology, Massachusetts Institute of Tech- 
nology, Cambridge, Massachusetts. 

SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massa- 
chusetts. 

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

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

SCHRAMM, PROF. J. R., University of Pennsylvania, Philadelphia, Pennsylvania. 

SCOTT, DR. ALLAN C., Union College, Schenectady, New York. 

SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, New Jersey. 

SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College, Greens- 
burg, Pennsylvania. 

SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New York. 

SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and 
Surgeons, 630 West 168th Street, New York City, New York. 

SHANES, DR. ABRAHAM M., New York University, College of Dentistry, New 
York. 

SHAPIRO, DR. HERBERT, Radiation Laboratory, Massachusetts Institute of Technol- 
ogy, Cambridge, Massachusetts. 

SHELFORD, PROF. V. E., Vivarium, Wright and Healey Streets, Champaign, Illinois. 

SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Michigan. 

SHUMWAY, DR. WALDO, University of Illinois, Urbana, Illinois. 

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

SICHEL, MRS. F. J. M., 35 Henderson Terrace, Burlington, Vermont. 

SINNOTT, DR. E. W., Osborn Botanical Laboratory, Yale University, New Haven, 
Connecticut. 

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

SMITH, DR. DIETRICH CONRAD, Department of Physiology, University of Mary- 
land School of Medicine, Lombard and Greene Streets, Baltimore, Maryland. 

SNYDER, PROF. L. H., Ohio State University, Department of Zoology, Columbus, 
Ohio. 

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

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

STARK, DR. MARY B., 1 East 105th Street, New York City, New York. 

STEINBACH, DR. H. BURR, Department of Zoology, Washington University, St. 
Louis, Missouri. 

STERN, DR. CURT, Department of Zoology, University of Rochester, Rochester, 
New York. 

STERN, DR. KURT G., Polytechnic Institute, Department of Chemistry, 85 Living- 
ston Street, Brooklyn, New York. 

STEWART, DR. DOROTHY R., University of Pennsylvania Medical School, Depart- 
ment of Physiology, Philadelphia 4, Pennsylvania. 

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



REPORT OF THE DIRECTOR 55 

STUNKARD, DR. HORACE W., New York University, University Heights, New 

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

California. 
SUMMERS, DR. FRANCIS MARION, Univ. of California, College of Agriculture, 

Davis, California. 

TAFT, DR. CHARLES H., JR., University of Texas Medical School, Galveston, Texas. 
TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati, Ohio. 
TAYLOR, DR. C. V., Leland Stanford University, Leland Stanford, California. 
TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michigan. 
TE\VINKEL, DR. L. E., Department of Zoology, Smith College, Northampton, 

Massachusetts. 

TURNER, DR. ABBY H., Mt. Holyoke College, South Hadley, Massachusetts. 
TURNER, PROF. C. L., Northwestern University, Evanston, Illinois. 
TYLER, DR. ALBERT, California Institute of Technology, Pasadena, California. 
UHLENHUTH, DR. EDUARD, University of Maryland, School of Medicine, Balti- 
more, Maryland. 

VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland, Ohio. 
WAINIO, DR. W. W., New York University, College of Dentistry, New York City. 
WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge, 

Massachusetts. 

WARBASSE, DR. JAMES P., Woods Hole, Massachusetts. 
WARD, PROF. HENRY B., 1201 W. Nevada, Urbana, Illinois. 

WARREN, DR. HERBERT S., 1405 Greywall Lane, Overbrook Hills, Pennsylvania. 
WATERMAN, DR. ALLYN J., Department of Biology, Williams College, Williams- 
town, Massachusetts. 
WEISS, DR. PAUL A., Department of Zoology, The University of Chicago, Chicago, 

Illinois. 

WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pennsylvania. 
WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, North Dakota. 
WHITAKER, DR. DOUGLAS M., P. O. Box 2514, Stanford University, California. 
WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania. 
WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania, 

Philadelphia, Pennsylvania. 

WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Nebraska. 
WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, 

Pennsylvania. 

WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, Ohio. 
WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore, 

Maryland. 

WILSON, DR. J. W., Brown University, Providence, Rhode Island. 
WITSCHI, PROF. EMIL, Department of Zoology, State University of Iowa, Iowa 

City, Iowa. 
WOLF, DR. ERNST, Biological Laboratories, Harvard University, Cambridge, 

Massachusetts. 

WOODRUFF, PROF. L. L., Yale University, New Haven, Connecticut. 
WOODWARD, DR. ALVALYN E., Zoology Department, University of Michigan, Ann 

Arbor, Michigan. 



56 



MARINE BIOLOGICAL LABORATORY 



WRINCH, DR. DOROTHY, Smith College, Northampton, Massachusetts. 

YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medical College, 

1300 York Avenue, New York City, New York. 
YOUNG, DR. B. P., Cornell University, Ithaca, New York. 
YOU-NG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland. 

9. ASSOCIATES OF THE MARINE BIOLOGICAL LABORATORY 



BARTOVV. MRS. FRANCIS D. 

BEHNKE. JOHN. 

BROWN, MR. AND MRS. THEODORE. 

CALKINS, MRS. GARY N. 

COOPER, CHARLES P. 

CROSSLEY, MR. AND MRS. ARCHIBALD. 

CROWELL, PRINCE. 

CURTIS, DR. WILLIAM D. 

FAY, MRS. H. H. 

FOSTER, RICHARD W. 

GARFIELD, IRVIN McD. 

GREEN, GEORGE S. 

GREEN, Miss GLADYS. 

HARRISON, R. G., JR. 

HUNT, MRS. REID. 

JANNEY, MRS. WALTER. 



KNOWER, MRS. H. McE. 
LILLIE, MRS. F. R. 

McMlTCHELL, MRS. J. McC. 

MURPHY, DR. W. J. 
NEWTON, Miss HELEN. 
NIMS, MRS. E. D. 
NORMAN, EDWARD A. 
RIGGS, MRS. LAWRASON. 
RUDD, MRS. H. W. D. 
SAUNDERS, MRS. LAWRENCE. 
STOCKARD, MRS. C. R. 
SWOPE, MR. AND MRS. GERARD. 
TEBBETS, WALTER. 
WEBSTER, MR. AND MRS. E. S. 
WICK, MRS. MYRON T. 
WILSON, MRS. E. B. 



^ v 



C 



<*D 



LIBRA*\ 



J*4S* 

o * 

* 



THE INFLUENCE OF TEXTURE AND COMPOSITION OF 

SURFACE ON THE ATTACHMENT OF SEDENTARY 

MARINE ORGANISMS* 

C. M. POMEKAT AND C. M. WEISS 
Medical Branch, University of Tc.vas and the IToods Hole Oceanographic Institution * 

Marine installations of various kinds necessitate exposure of construction mate- 
rials under sea water. Data dealing with the amount of fouling accumulated by 
such materials are not abundant. Information which might be of aid to the scien- 
tist seeking the most favorable material upon which to collect sedentary organisms 
for study is also scanty. The present study was undertaken to determine the effect 

TABLE I 

Effect of surface texture of glass on attachment of sedentary organisms. (Numbers of individuals 

on each surface of 80 square inches of plate) 



Surface number 


Plain 



Sand- 
blasted 
1 


Factrolite 
2 


Prestlitt- 
.? 


Ribbed 
4 


Pentecor 

5 


Series No. 1, Tahiti Beach 1 














39 days (8/22/42-9/30/42) 














Hydroides sp. 


143 


265 


152 


506 


349 


197 


Spirorbis sp. 


85 1 88 


122 


90 


163 


110 


Barnacles 


1,948 1.072 1,162 


975 


1,674 


2,140 


Total 


2,176 1,525 


1 ,436 


1,571 


2,186 


2,447 


Average pop. 


725.3 508.3 


478.7 


523.7 ' 728.7 


815.7 


Average/square inch 


9.1 


6.4 


6.0 


6.5 


9.1 


10.2 


Series No. 2, Miami Beach- 














17 days (8/22/42-9/8/42) 














Wet weight (grams) 


5 1 .0 


45.5 


50.0 


41.0 


50.0 


4 1 .0 


Drv weight (grams) 


8.5 


6.4 


7.9 


5.5 


7.5 


8.8 


Barnacles 


308 


227 


268 


213 


263 


331 


Series No. J, Miami Bcach- 














30 days (9/15/42-10/15/42) 














Wet weigh 1 (grams) 


164.5 


174.0 


149.0 


1 50.0 


126.0 


155.5 


1 )ry weight (grams) 


51.5 


50.0 


30.0 


24.0 


24.0 


33.0 


Barnacles 


642 


515 


554 


778 


798 


977 



1 Subtropical testing service. 



2 Beach boat slips. 



* The observations described here were made while the authors were engaged by the Woods 
Hole Oceanographic Institution in an investigation of fouling, under contract with the Bureau 
of Ships, Navy Department, which has given permission for their publication. The opinions 
presented here are those of the authors and do not necessarily reflect the official opinion of the 
Navy Department or the naval service at large. Contribution No. 349 from the Woods Hole 
Oceanographic Institution. 

57 



58 



C. M. POMERAT AND C. M. WEISS 



of surface irregularities and of substrate composition on the establishment of sessile 
populations. The experiments were conducted in Biscayne Bay at Miami, Florida, 
where subtropical conditions favor the attachment of fouling organisms throughout 
the year. 

Grateful acknowledgment is made to Dr. A. C. Redfield and Dr. F. G. Walton 
Smith for many helpful suggestions. 












FIGURE 1. Glass surfaces used in testing the relation of surface irregularities to fouling. 0. Plain. 
1. Sandblasted. 2. Factrolite. 3. Prestlite. 4. Ribbed. 5. Pentecor. 

EFFECT OF SURFACE IRREGULARITY 

Commercial glasses, manufactured by the Pittsburgh Glass Company, with 
various surface irregularities were used for this study. Six 8 X 10 inch glass plates 
were assembled, irregular surface down, in a rack suitable for floating on the surface 
of the water. The floats were constructed in such a way that sea water could move 
freely on both sides of the exposed surface. The backs of the panels which were 
all relatively smooth were placed upward. The fouling on the back surfaces was 



ATTACHMENT OF MARINE ORGANISMS 59 

not recorded. The surface irregularities of the panels are shown in Figure 1 and 
may be described as follows : 

Surface Number: 

0. Plain Smooth glass, polished. 

1 . Sandblasted Glass sandblasted on lower side. 

2. Factrolite Surface consisted of pyramidal depressions of which there were 

about 144 per square centimeter. 

3. Prestlitc Approximately nine pyramidal depressions per square centi- 

meter. 

4. Ribbed Surface of V-shaped grooves, nine grooves per centimeter of 

width. 

5. Pcntecor Approximately three V-shaped grooves per centimeter of 

width. 

Results obtained from three series of experiments in which the glass surfaces 
were exposed are shown in Table 1. 

The Sessile populations which grew on the glass plates were composed pri- 
marily of barnacles and tubeworms, with irregular, perhaps seasonal, appearances 
of tunicates and Anoinia sp. Barnacles (B. iinprorisns and B. amphitritc niveus in 
order of relative abundance) were numerous in both locations, but those at Tahiti 
Beach were always very small compared to those at Miami Beach. Many more 
barnacles attached to the lower (shaded) surfaces of the panels than to the upper 
surfaces where light was more abundant. This is in agreement with the experience 
of Pomerat and Reiner ( 1 ( M2). who report that larger numbers of barnacles accu- 
mulate on dark surfaces than on light surfaces. The shaded undersides of the glass 
panels, being darker than the upper sides, appear to attract more cyprids and hence 
show a greater barnacle accumulation. 

The various surface textures of glass had little influence on the number of at- 
tached organisms. In these experiments barnacles were consistently slightly more 
numerous on Pentecor than on smooth glass. This behavior was confirmed in the 
experiment reported in the following section although conditions of exposure were 
not exactly parallel. In the first experiment the glass panels were floated at the 
surface in a shaded location, while in the second they hung vertically below low tide 

TABLE II 

Influence of substrate on fouling, sixty days' exposure at the beach boat slips, September 25, 1942- 

November 25, 1942 

Weight of fouling on panel area of 264 sq. in., that of wood 

panels employed 
Substrate Wet weight grams Dry wt. grams 

1. Dade County pine 675.1 346.5 

2. Gum 1127.6 531.4 

3. Magnolia 1165.4 446.4 

4. White pine 968.7 446.8 

5. Cypress 954.8 392.0 

6. Tile 980.1* 487.3 

7. Cement 1033.0* 534.1 

8. Glass 386.1 167.3 

* Corrected to an area, 264 sq. in., equal to that of the wood panels. 



60 



C. M. POMERAT AND C. M. WElSS 



under sun exposure. Counts of tubeworms were made on only one set of expo- 
sures. H \droidcs sp. was most abundant on Prestlite and Sf>irorbis sp. was most 
abundant on sandblasted gla.ss. 

COMPOSITION OF THE SURFACE 

Unpainted panels of wood of five species, clay roofing tiles, cement roofing plates, 
and a glass panel were exposed for 60 days at the Reach Roat Slips in Miami Reach. 

TABLE 1 1 1 

Effect of substrate on fouling, exposures of three months at South Dock, Belle Isle, Miami Beach, 
Florida, January 9, 1943-April 9, 1943, all materials applied to, or mounted on, wood unless 

otherwise noted 



Composition of surfaces 


\Yet 
weight* 
(grams) 


Dry 

weight* 
(grams) 


Number* 
of bar- 
nacles 


Notes 


Plastics 










1. Celluloid 


3.8, 


2.2 


11 


Thin coat of algae. 


2. Plasticel 


24.3 


12.2 


124 


Barnacles' bases easily removed. 


3. Lucite 


5.6 


1.7 


41 




4. Formica 


6.9 


3.2 


11 




5. Isobutyl 


15.4 


7.2 


70 


Film applied to glass panel. 


Methacrylate 








Plastic peels intact with barnacles. 


Glass 










6. Prestlite 


57.0 


25.2 


176 


Some barnacles 12 mm. across. 


7. Pentecor 


46.0 


25.0 


148 


Some barnacles 12 mm. across. 


8. Sandblasted 


23.6 


7.0 


46 


6 calcareous tubeworms; tunica tes. 


9. Smooth 


4.5 


1.7 


16 


Green slime may have caused fish to 










remove young barnacles. 


Paints and ingredients** 




- 






Coatings applied to steel 










panels 










10. Ester gum vehicle 


36.3 


8.1 


58 


Tunica tes and bryozoa. 


1 1 . Rosin vehicle 


2.7 


0.4 





Fish spawn both sides. 


12. Anticorrosive paint 42-A 


2.7 


0.5 


9 


Baracles very small. 


13. Vehicle of 15RC 


6.1 


3.3 


43 




14. Antifouling paint 7C 


0.0 


0.0 





Some slime film. 


15. Antifouling paint 8C 


0.6 


0.3 


14 


Small barnacles close to edge. 


Coatings Applied to Wood 










16. Ceraloid 


57.6 


38.5 


183 




17. Paraffin 


11.3 


6.1 


59 


Lomnoria active in breaking paraffin. 


18. Asphalt u m 


121.4 


34.3 


768 


Barnacles onlv. 


19. Asphaltum varnish 


67.8 


13.8 


256 


Some bryozoa. 


20. Spar varnish 


45.1 


7.0 


304 




2 1 . Navy grev 


41.6 


5.6 


150 


Algae. 


22. Anti-corrosive 42-A 


48.2 


10.7 


156 





* Corrected to an area of 144 square inches. 

* Anticorrosive 42A is a standard Navy formula. Vehicle of 15RC is the non-pigmented 
portion of a standard Navy antifouling paint. Antifouling paints 7C and 8C are experimental 
modifications of a standard Navy antifouling paint of the cold plastic type in which the toxic 
pigment is reduced to 50 and 60 percent of the normal value. 



ATTACHMENT OF MARINE ORGANISMS 



61 



TABI.K I II Continued 





Wet 


Dry 


Number* 




Composition of surfaces 


weight* 


weight* 


of bar- 


Notes 




(grams) 


(grams) 


nacles 




Woods 










23. Dade County pine 


395.2 


120.7 


748 


Bryozoa. 


(soaked 60 days) 










24. Gum (soaked 60 days) 


452.1 


133.4 


686 




25. Hade County pine 


144.3 


27.3 


125 


Hydrozoa, bryozoa. 


(unsoaked) 










26. Gum (unsoaked) 


249.8 


43.5 


222 




27. Soft pine 


57.6 


11.5 


184 




28. Teak 


143.8 


88.7 


306 


Large barnacles. 


29. Maderia 


173.7 


84.2 


358 


Manv lish eggs. 


30. Greenheart 


77.0 


40.8 


342 




31. Balsa 


2.9 


1.6 


5 


Wood verv soft. 


Metals 










32. Steel 


224.4 


42.8 


88 




33. Galvanized iron 


2.6 


0.7 


6 


Barnacles easily removed. 


34. Zinc 


1.0 


0.2 





Active corrosion. 


35. Lead 


30.6 


50.9 


396 


Large barnacles. 


36. Monel 


1.6 


0.5 


6 


Manv fish eggs. 


37. Nickel 


43.2 


10.7 


126 




38. Galvanized iron pipe 


4.7 


3.0 


27 


Barnacle on rusted threads and dam- 










aged edges. 


Miscellaneous 










39. Linoleum 


79.7 


23.0 


193 




40. Deck canvas no. 10 


5.1 


2.3 


7 


Sagging-algae eaten by fish. 


41 . Sole leather 


32.4 


12.4 


66 




42. Masonite, heat tempered 


138.6 


31.8 


594 


Brown tunicates. 


43. Asbestos 


284.2 


65.9 


980 


Bryozoa, Anoniia, hydrozoa, calcar- 










eous tubeworm. 



All panels were suspended in a vertical position approximately two feet beneath the 
mean low water mark. The site was well shaded by a protecting roof. The results 
obtained are presented in Table II. 

The weights of the populations (barnacles, tubeworms, tunicates, bryozoa, and 
algae) which accumulated on the woods, tile, and cement were of the same order of 
magnitude, though variations as great as 59 per cent were observed. The weight 
of organisms accumulated on glass was approximately 30 per cent of that collected 
from other substrate materials. 

A much larger number of materials were tested in a second experiment, the re- 
sults of which are given in Table III. Exposure was made for three months at 
South Dock, Belle Isle, in Miami Beach, where conditions of bright sunlight, active 
current movement, and moderate fouling incidence were found. Growth on the 
panels consisted primarily of barnacles (B. hnprovisus and B. amphitrite niveus) 
with occasional tufts of hydrozoa and patches of colonial tunicates. A blanket of 
algae having very short filaments grew on panels of light color or shaded back- 
ground. Large sets of fish eggs were found on the rosin vehicle and Monel. Bor- 
ings of Liuuwria sp. were everywhere evident in unprotected wood. 



62 



C. M. POMERAT AND C. M. WEISS 



The substrates accumulating heaviest populations were asbestos, asphaltum, 
Dade County pine (pre-soaked 60 days), gum wood (pre-soaked 60 days) and 
Masonite. Asbestos shingle, commonly used as clapboarding, yielded the richest 
harvest as measured by the number of barnacles. A comparison of asbestos and 
Masonite, two of the best collectors, is shown in Figure 2. The asphaltum used 
was of the type employed as aquarium cement. It accumulated barnacles only. 

Panels of gum and Dade County pine, which had been exposed for 60 days in 
the earlier test reported in Table II, were included for comparison with unsoaked 
specimens of these woods. The unsoaked woods developed much less fouling. 



' 



' 

'- 






, Jf B 




j 

W&* I 

< , ;, 
i } i ' ;' -V i 

. * " > i / 

". - 1 ' ' /'- : J^ 

" 

^ ' <'' J; .^" 



' '.'.. ."..<'.: " v' 

' * 1^9" & 3 ~ft- ^B m > ' T . < 

.V. : '\ , ' - 

' . ' I : " ' - 

:*...,, *| -,"(- ',* 

'' : - ' '' ' "** 



': . "!* 



2. Accumulation oi fouling organisms on masonite and asbestos after 90 days' exposure 

at Belle Isle, Miami Beach, Florida. 



Intact galvanizing on iron was very resistant to marine life. No barnacles were 
obtained on zinc, on the experimental antifouling paint 7C or on rosin vehicle, a 
common paint component. 

Materials with hard non-porous and non-fibrous surfaces were in general rather 
poor collectors of fouling. The best accumulation of sedentary populations was 
found on surfaces which were porous and/or fibrous. Surface of paints, paint in- 
gredients and linoleum are in general non-porous and non-fibrous. Compared to 
the size and strength of the barnacle cyprid they are also smooth and hard. The 
histogram (Fig. 3) summarizes the collective efficiency of the substrates. 

Some results were undoubtedly spurious, and these should be noted. Fouling 
on the antifouling paint SC which occurred along the edges of the panel was probably 



ATTACHMENT OF MARINE ORGANISMS 



63 



Grams Wet Weight of Foulini 



lOO 



200 



400 



300 



PLASTICS 
GJ.ASSZS 



PA/HTS AND PAINT 



BALSA 



MSTALS (except Steel) - STEEL 

CAHVAS-LEATHIK.-LIMOLEUM - MASOMIT - ASBESTOS 



WOODS 



FICUKE 3. Relative amounts of foulins; on various classes ot materials used as test panels. 




FIGURE 4. Bases of barnacles grown on various substrates. A. Navy grey. B. Antifouling 
paint 15RC. C. Ester gum. D. Anticorrosive paint 42A. 



64 



C. M. POMERAT AND C. M. WEISS 



due to imperfections of the paint surface. In contrast, 7C which contained less 
copper was not fouled. Deck canvas and smooth glass both supported a culture of 
green algae which evidently served as food for fish. Active feeding on these panels 
unquestionably disturbed other fouling organisms. Balsa wood was apparently 
sloughing its surface and thus loosening attached forms. In spite of these minor 
qualifications, the results involve a range of population numbers sufficiently wide 
to indicate the relative merits of the substrates used. 

One of the most interesting of the results was observed when barnacles were 
removed from the various substrates. Some of the substrates bore barnacles with 
deeply scalloped margins (Fig. 4) instead of the typical smooth edges. These 
margins suggested that localized irregular marginal growth interruptions had taken 
place. Such barnacles were collected from : 

Spar varnish 

Linoleum 

Navy grey topside paint (P 50) 

Antifouling paint vehicle 15RC 

Ester gum paint vehicle 

Anticorrosive paint 42-. \ 



"' 



ft*, c? 




FIGURE 5. Bases of barnacles grown on various substrates. A. Isobutyl methacrylate. 
B. Plasticel. C. Soft paraffin. D. Ceraloid. 



ATTACHMENT OF MARINE ORGANISMS 65 

Barnacles growing on soft paraffin had distinctly concave bases. Mosaics of 
bases witb angular margins were typical of barnacles attached to lead but were also 
found on other overcrowded substrates. It was possible to remove barnacles with 
intact bases very easily from several materials, including plasticel, ceraloid, and iso- 
butyl methacrylate (Fig. 5). This finding might prove useful in designing experi- 
ments in which the minute anatomy of basal structures was to be studied. 

SUMMARY 

1. Submerged samples of 40 different construction materials were used as sub- 
strates for the collection of sedentary populations. The barnacle counts in the popu- 
lations ranged from 980 on asbestos shingles to zero on zinc and on two paint coat- 
ings, after three months' immersion in Biscayne Bay at Miami Beach, Florida. 

2. Various surface textures of glass plates were found to exert no significant 
influence on the accumulation and growth of sedentary marine organisms, although 
smooth clear glass accumulated smaller populations in the comparatively short expo- 
sure periods, 13 months . 

3. The results suggest that efficiency of a substrate as a fouling collector is in 
general correlated with porosity of surface or with fibrous nature of surface. 
Smooth, non-porous, non-fibrous surfaces, especially if also hard, seem to be poor 
accumulators of sedentary organisms. 

4. Further testing of substrates is greatly to be desired in this connection. 

REFERENCES 

POMERAT, C. M., AND E. R. REINER, 1942. The influence of surface angle and of light on the 
attachment of barnacles and other sedentary organisms. Biol Bull., 82 (1) : 14. 



THE DEVELOPMENTAL HISTORY OF AMAROECIUM CONSTEL- 
LATUM. II. ORGANOGENESIS OF THE LARVAL 

ACTION SYSTEM 

SISTER FLORENCE MARIE SCOTT 

The Marine Biological Laboratory, Woods Hole, Mass.. and the Biology Department, Scion Hill 

College. Greensburg, Pennsylvania 

INTRODUCTION 

The early development of the embryo of A-maroecium constellation has been pre- 
sented in a previous paper (Scott, 1945). The accumulation of yolk modifies the 
pattern of mosaic development characteristic of Tunicates to the extent that gastru- 
lation is accomplished in an atypical manner. Convergence of the cells of the lateral 
margins of the posterior blastoporal lip is accomplished to the left of the mid-line. 
The neural plate elongates posteriorly at the place where the lateral blastoporal lips 
meet and close. The chordal cells are inflected at the anterior lip and lie in the 
median axis. The potential muscle cells of the morphological right side lie dorsal 
to the notochord as a result of their growth across the mid-dorsal plane, the muscle 
cells of the morphological left side lie below the level of the notochord on the curved 
left side of the embryo. The two groups of muscle cells are separated by the poste- 
rior extension of the neural plate. 

MATERIALS AND METHODS 

Amaroecium constellation is abundant along the eastern coast of the United 
States. The breeding season lasts throughout the summer months. Material for 
this study was collected at Woods Hole, Massachusetts. The embryos, squeezed 
from adult colonies, were selected and arranged into a progressive series of stages 
for study. They were fixed in Bouin's fluid. Some were stained by Conklin's 
modification of Delafield's haematoxylin, others with borax-carmine, then cleared 
according to the benzyl-benzoate method described in Romeis' "Taschenbuch der 
Mikroscopischen Technik." Corresponding stages were sectioned serially, stained 
in Mayer's or Gallagher's or iron haematoxylin, and counterstained with eosin or 
triosin. All drawings were made with the aid of a camera lucida. The photo- 
micrographs were made with a Leitz "Macca" camera using Zeiss apochromat, 20 X , 
and fluorite oil immersion, 100 X, objectives with a Zeiss microscope. 

Later embryonic development 

It seems advisable to present a descriptive series of developmental stages that 
may be used as points of reference for structures differentiating during the organ- 
forming period. For convenience the developmental period following gastrulation 
is divided into four stages; 1) the tail bud stage, 2) early tadpole stage, 3) pre- 
hatching stage, and 4) the free-swimming tadpole stage. The free-swimming larva 

66 



AMAROECIUM CONSTELLATUM. II 



67 



or tadpole has been described thoroughly by Grave (1921) and shall be presented 
here in brief summary since reference to it is necessary. A short description of the 
external appearance of these stages will be given first and referred to in subsequent 
treatment of organogenesis as Stages I, II, III, and IV. The terms, larval action 
system and adult action system, used by Grave (1935, 1944) will be adopted for 
the structures functioning during larval life and those functioning during adult life 
respectively. 

The tail bud stage 

By the end of gastrulation the embryo is approximately spherical except for a 
shallow postero-ventral invagination of the ectoderm constricting tail from trunk 
region. The furrow appearing first on the right side is deeper there, and less deep 
as it extends to the left side. The tail bud is short and rounded, curving immedi- 
ately toward the ventral side of the trunk. Through the thin epidermis quadruple 
rows of large muscle cells can be seen lying dorsal and ventral to the notochord. 
The neural plate is elevated at the periphery to form the neural groove, enclosing; 
anteriorly a wide depression, the presumptive brain region, posteriorly a narrow, 
trough-like depression lying to the left of the notochord, the presumptive neural 
tube (Fig. I A). 



h.v 




FIGURE 1. A. Stage I, embryo before neural folds close. 160 X. B. Stage II, early tad- 
pole; beginning of differentiation of digestive and nervous systems. 160 X. b. v., brain vesicle; 
d. ph., dorsal diverticulum of pharynx; m. bd., muscle band; n. t., neural tube; ph., pharynx; 
y. m., yolk mass. 

A transverse section through the tail bud stage discloses that the embryo is solid. 
A single layer of definitive endoderm lies under the concave neural plate (Fig. 5). 
This layer of cells develops from the cells that form the superficial "pseudo- 
invagination" cavity of gastrulation. The depression closes by a reversal in change 
of shape of the cells involved rather than by approximation of the lips of the blasto- 
pore thus producing a solid archenteron (Scott, 1945). The endodermal cells 
spread under and anterior to the neural plate. Ventral to them is located the mass 
of heavily yolk-laden cells derived from the macromeres. Wedged between the 



68 FLORENCE MARIE SCOTT 

thin ectoderm and the solid endoderm on either side is a mass of mesenchyme, small, 
polygonal cells with prominent nuclei (Fig. 5). 

Posteriorly the definitive endoderm lies adjacent to the chordal cells which are 
beginning to interdigitate in the base of the tail bud. The mesenchyme terminates 
abruptly in this region against the muscle cells of the tail. 

Early tadpole stage 

The embryo increases in size and acquires the shape that justifies its being called 
"tadpole." The trunk region elongates slightly in the antero-posterior axis remain- 
ing curved at the anterior end. The tail encircles the body meridionally as it grows 
in length. The embryo is still opaque. 

The neural folds are closed, the position of the sensory vesicle being marked by 
aggregations of black pigment which show through the surface of the body. The 
neural tube is faintly visible along the side of the tail. More conspicuous are the 
large muscle cells dorsal and ventral to the prominent notochord which forms the 
axis of the tail throughout its length. Dorsally, on either side of the sensory vesicle 
there is a slight ectodermal invagination, rudiments of the atrial chambers. The 
embryo is confined within a test the cells of which are arranged in a compact layer 
(Fig. IB). 

Pre-hatching stage 

Changes in the external appearance of the later embryo depend on the develop- 
ment of siphons and adhesive papillae and the secretion of a tunic. As body growth 
continues and organs of the larval action system differentiate, the body becomes 
transparent except where the mass of yolk is lodged in the pharynx. 

The trunk region continues to elongate antero-posteriorly becoming elliptical in 
shape. Posteriorly the body narrows to the base of the tail ; anteriorly it flares in 
the dorso-ventral axis in relation to the vertical position of the adhesive papillae. 
Laterally the body is compressed. A thickening layer of tunic invests the entire 
trunk. It is indented at the junction of trunk and tail and continues over the sur- 
face of the tail. The tail encircles the body meridionally being pressed into a groove 
in the tunic. The tunic of the tail projects laterally into fins. 

The sensory vesicle occupies a dorsal position at the posterior end of the trunk. 
Two masses of pigment project into its cavity. Immediately in front of it lies the 
elevation of the oral siphon ; behind it and on the posterior curve of the body lies the 
atrial siphon. Much of the internal structure is visible through the tunic and 
mantle. The incipient adhesive papillae appear as three disc-like projections in 
verticle series at the rounded anterior end (Fig. 2). 

The free-swimming tadpole stage 

The trunk of the tadpole of Amaroecium at its release measures about 600 micra 
in length; it measures about 270 micra in depth. The tubular atria with their triple 
rows of gill slits are pressed into the dorsal pharynx through half of its length 
posteriorly. An obvious structure in the pharynx is the dorsal, heavily ridged endo- 
style which seems to rest on the lateral masses of yolk that form the wall of the 
pharynx. The transparent pericardium occupies a large space below the yolk ante- 



AMAROECIUM CONSTELLATUM. II 



69 



riorly in front of the loop of alimentary tract. On the right side of the body the 
stomach extends along the posterior and ventral curvature of the yolk. On the left 
the narrow intestine curves along the side of the stomach up to the left atrium where 
it terminates. The root of the tail lies in the posterior third of the length of the 
body. 

Anteriorly, the adhesive papillae project into the tunic in a vertical row slightly 
to the right of the median plane. The test vesicles lie loosely within the tunic or 
many of them, even at the time of hatching, retain a slender connection with the 
cone or ridge from which they originate. Where the tail is continuous with the 
trunk the tunic dips clown into an abrupt pocket. The epidermis secretes a thin 
sheath of tunic about the tail. Laterally it expands into wide sail-like fins (Fig. 3). 



,5.V. 




ad. 



p s ~ 



FIGURE 2. Stage III, lateral view of tadpole with incipient adhesive papillae. About 120 X. 
ad. p., adhesive papillae; end., endostyle ; ep., epidermis; /. a., left atrium; >i. t., neural tube; oes., 
oesophagus; p. c., pericardia! cavity; ph., pharynx; st., stomach-intestine rudiment; s. v., sensory 
vesicle ; tu., tunic ; y. i., yolk mass. 

ORGANOGENESIS OF THE LARVAL ACTION SYSTEM 
Digestive system 

The pharyngeal cavity develops in Stage II by delamination between the layer 
of definitive endoderm and the mass of yolk-laden cells, appearing first below the 
brain and spreading from that point (Fig. IB, 5F, 6E}. It extends back to the 
base of the notochord as an upwardly directed diverticulum. Ventral to the base 
of this projection a second invagination appears, the rudiment of the stomach and 
intestine located a little to the right of the median plane on the inner side of the 
visceral ganglion (Fig. 2, 6F). 

The pharynx deepens in Stage III encroaching upon the mass of yolk cells. 
Gradually thin septa of epithelium divide the yolk mass into four compact longi- 
tudinal columns, the two on each side being continuous at the bottom. The central 




70 



FLORENCE MARIE SCOTT 



two are lower than the outer two, thus providing greater depth for the limited 
pharyngeal cavity (Fig. 6A, F). This supply of nutritive material in the pharynx 
remains to be digested during the active life of the larva and throughout the critical 
period of metamorphosis. All other tissues lose their meager supply of yolk almost 
entirely, leaving their cytoplasm clear. 

Along the roof of the pharynx, anterior to the place of origin of the oral siphon, 
the epithelium rises up into a double fold enclosing the endostyle, restricted to the 
dorsal side above and between the lateral masses of yolk and passing to the anterior 
end of the yolk mass (Fig. 2, 6A}. Before the tadpole is released from its test, the 
cells in the floor of the groove develop long cilia. The pharynx grows out above 
and below the atrial sacs, bringing the mesial atrial and lateral pharyngeal walls into 
intimate contact (Fig. 65). 

Due to the combined activity of atrial and pharyngeal epithelia, three horizontal 
rows of gill slits are formed, each consisting of seven or eight perforations. The 



oral Siphon 




FIGURE 3. Stage IV, tadpole at hatching. About 120 X. end., endostyle; cp., epidermis: 
int., intestine; p. c., pericardia! cavity; st., stomach; s. v., sensory vesicle; te. v., test vesicles: 
tit., tunic ; y. m., yolk mass. 

bordering cells of each gill develop a heavy brush of cilia, precocious equipment from 
the functional point of view. Even though the mouth breaks through to the bran- 
chial chamber, the tunic fills up the oral and atrial siphons until metamorphosis is 
completed. 

The rudiment of the oesophagus grows forward along the curvature of the yolk 
and dilates to form the stomach. The diverticulum extends to the midventral region 
of yolk where it turns sharply upon itself and continues backward as the slender 
intestine. With a gradual slope upward the intestine retraces the course of the 
stomach on its left side terminating ventral to the posterior end of the left atrium 
(Fig. 3, 6B). Later the anus opens into the atrium here. 

There are no cilia evident in the intestine or stomach during this period of de- 
velopment. The wall of the stomach is thicker than the wall of the remaining parts 
of the digestive tract although the alimentary epithelium, throughout its length, con- 
sists of a single layer of cells. 



AMAROECIUM CONSTELLATUM. II 71 

With rapid general growth of the body, the loop of intestine and stomach in- 
creases in length anteriorly, extending through the posterior half of the body cavity 
below and behind the yolk-laden pharynx (Fig. 3). The pericardium lies directly 
in front of it. Between the arms of the loop posteriorly are lodged the bases of 
the axial organs of the tail. 

Atrium During Stage II the atrium or peribranchial sac appears as a 
pair of ectodermal invaginations, one on either side of the sensory vesicle (Fig. 
6). At the place of its origin the neck of each depression constricts and separates 
from the surface. 

In the transition from Stage II to Stage III, the atria, in contact with the lateral 
endodermal wall of the pharynx, grow in an anterior direction only, with the result 
that the atrial chambers are horizontal capsular cavities located dorsally, one on 
either side of the pharynx (Fig. 6B). They extend through the posterior two- 
thirds of the trunk, curving gently upward posteriorly where they grow towards 
each other and unite behind the pharynx (Fig. 3). The atrial siphon opens 
through the dorsal wall of this connecting canal between the two cavities. 

The atrial walls are characteristically thin and the cells lose their intercell mem- 
branes. Occasional yolk granules are scattered through the cytoplasm. During 
Stage III the gill slits perforate the walls in three horizontal rows on the inner side 
in direct contact with the wall of the pharynx. The lowermost row develops first, 
the atrial and pharyngeal fusing first in these regions. The slits number between 
seven and nine in each row. Later in the free-swimming period the cells bordering 
the gill aperture produce long cilia. The endoderm has no part in atrial formation 
except insofar as the gill slits are the product of joint activity of atrial and pharyn- 
geal walls (Caullery, 1895). 

Oral and Atrial Siphons Late in Stage III the dorsal ectoderm in front 
of the sensory vesicle thickens and invaginates, pushing the endoderm of the pharynx 
before it. The circle of epidermis around the invaginated area becomes elevated, 
giving the oral siphon a crater-like appearance (Fig. 68} . The floor of the invagi- 
nation thins out in a flat layer against the pharyngeal roof with which it is in con- 
tact. The lower part of the cavity projects outward from the center and produces 
a ring-shaped extension on the mouth opening. The oral cavity assumes the shape 
of a flask with a long neck and a flattened base (Fig. 3). Into this ectodermal 
cavity, or stomodaeum, the hypophysial duct opens, just before hatching of the tad- 
pole. Although the oral plate breaks through late in the tadpole's development, 
the tunic fills up the stomodaeal portion and prevents the passage of both food and 
water during larval life. 

The atrial siphon, like the oral, is formed by ectodermal invagination. The 
thickened mantle is elevated, raising the siphon above the level of the rest of the 
mantle in knob-like fashion (Fig. 6C). The floor of the invagination fuses with 
the dorsal wall of the connecting arm of the atrium. The atrial siphon is situated 
on the downward curve of the dorsal surface just posterior to the sensory vesicle 
and anterior to the insertion of the tail (Fig. 3, 6G, H). The epithelial lining of 
the oral and atrial siphons projects into each opening at several points forming 
small tentacles. The mesenchymatous muscles in the mantle in this region provide 
the contractile elements that control the apertures when the siphons begin to function. 



FLORENCE MARIE SCOTT 

Heart and pericardium 

Towards the end of Stage III, the endodermal cells extend completely around 
the yolk mass as a definite epithelium. Mid-ventrally it evaginates into the body 
space and constricts off from the yolk epithelium. The bladder-like vesicle is the 
pericardium which invaginates mid-dorsally into an inner enclosed vesicle, the heart. 
The cells lose their inter-cell membranes and the nuclei bulge irregularly in both 
cardial and pericardial walls (Fig. 6 A). The heart does not develop beyond this 
point at present, the circulatory system not functioning during larval life. 

The nervous system 

The neural folds of Stage I close in the early phase of Stage II thus forming the 
hollow nervous system typical of chordates except in one point, the curving of the 
neural tube through 90 to the left of the brain region. The anterior portion of the 
nervous system produces the sensory vesicle with its sensory organs, the hypophysis, 
definitive ganglion, and the so-called subneural gland. The intermediate part in- 
cluding the origin of curvature and a small contribution from the brain region gives 
rise to the visceral ganglion and the spinal enlargement, the posterior part becomes 
the neural tube. 

The cavity of the brain region is slightly dilated and its wall uniformly thick. 
The neural tube consists, in section, of four cuboidal cells surrounding a small lumen 
(Fig. 4C). Cell membranes in both regions are distinct at this stage, the nuclei 
are large and contain heavily staining nucleoli. The cytoplasm is reticular in ap- 
pearance and has occasional yolk granules. 

During Stage III the brain vesicle differentiates into two structures, the sensory 
vesicle in the entire right side and the rudiment of the hypophysis on the left poste- 
rior side (Fig. 4A, 5 A}. The vesicle expands, its walls becoming thin; the rudi- 
ment of the hypophysis remains small with thick walls. This secondary cavity is 
separated completely from the sensory vesicle at the region of evagination but their 
walls remain attached throughout subsequent development (Fig. 5B, C). 

The sensory vesicle Two sensory structures develop in the sensory vesi- 
cle, the statolith and the eye. The left posterior wall of the vesicle thickens, the 
right wall expands dorsally and laterally ; all the cells lose their inter-cell mem- 
branes. The left wall of the cavity remains thick and constitutes the sensory 
ganglion of the brain. One cell on the ventro-anterior wall projects into the cavity 
and large pigment granules are deposited in its cytoplasm ; these coalesce to form 
the statolith (Fig. 4B, SB). In Stage IV the statolith is a spherical mass of pig- 
ment confined within the cell membrane and attached to the ganglionic wall by a 
stout stalk, the remaining part of the cell (Fig. 4D, SC, D}. 

A group of cells situated dorso-laterally at the left posterior limit of the vesicle 
initiates the development of the eye by the deposition of pigment granules of much 
smaller size than those that form the statolith. Absence of cell membranes makes 
it difficult to ascertain the number of cells that participate in this activity. The pig- 
ment is deposited in the shape of a cup, its concavity facing dorso-laterally and to 
the right within the vesicle. Three ganglionic cells which retain their membranes 
fill up the concavity in series. They secrete globules of liquid which increase in size 
both by the gradual addition of the secretion and by the fusion of globules. The 
globules of liquid form the so-called lens cell (Fig. 4A, B, D, 5D). The nuclei 



AMAROECIUM CONSTELLATUM. II 



73 



..ret 




FIGURE 4. .4. Transverse section through brain after the neural folds close, Stage II. 750 X. 
B. Longitudinal section through brain of same stage. 750 X. C. Cross section through tail of 
tadpole, Stage III. 300 X. D. Section through brain of tadpole just before hatching, oblique to 
include both sensory organs. 750 X. E. Longitudinal section through tail of tadpole in Stage 
III. 300 X. F. Section through epidermis and test of Stage III. 750 X. G. Reconstruction 
of brain and related structures of Stage III, viewed from left side. 300 X. b. v., brain vesicle; 
con. fib., contractile fibrils; dcf. g., definitive ganglion; cp., epidermis; hyp., hypophysis; /. c., 
lens cell; m. bd., muscle band; n. c., neural canal; nch., notochord ; n. t., neural tube; ret., retinal 
cells of eye; s. gn., sensory ganglion; sn. gl., subneural gland; stat., statolith ; .?. c., sensory cell; 
s. e., spinal enlargement ; .y. in., smooth muscle cells of mantle ; j. p., sensory pigment ; t. c., test 
cells ; tu., tunic ; v. g., visceral ganglion ; y. g., yolk granules. 



74 FLORENCE MARIE SCOTT 

which at first occupy a central position in the cells are pushed to the periphery as 
the lenses, increasing in size, come eventually to monopolize the entire cell. 

The pigment granules of the eye always remain discrete, not coalescing as do 
those of the otolith. Extending through the concentrated pigment are small rods 
of clear cytoplasm. They run from the hase of the cup back towards the ganglion. 
Seven or eight of them may be seen in embryos of Stage IV that are mounted in a 
mixture of benzyl-benzoate and oil of wintergreen. 

The hypophysis The rudiment of the hypophysis early in Stage III ap- 
pears as an extension or small evagination of the brain cavity (Fig. 4A, 5 A). The 
cells retain their membranes, their nuclei are smaller than those of the adjoining 
part of the brain. Histologically they present the appearance of epithelial tissue. 
Upon its separation from the primary cavity during Stage III it elongates antero- 
posteriorly along the left side of the sensory ganglion (Fig. 4G). In Stage IV it 
ends blindly at the posterior wall of the oral siphon. Later these walls fuse and the 
hypophysis communicates with the posterior region of the stomodaeum, extending 
along the side of the ganglion with a gentle slope upward as far as the atrial siphon 
where it terminates blindly. The floor of the duct, corresponding in position to 
the region of the eye, deepens abruptly (Fig. 4D, G). The ventral wall of the 
pocket becomes slightly thicker, the indentation with its thickened floor constituting 
the subneural gland. Hjort (1896) reviews the opposing views concerning this 
structure in the early works on Tunicates. 

The definitive ganglion By a proliferation of cells in the mid-region of 
its roof in Stage II the hypophysial duct produces an oval mass containing small 
nuclei similar to those in the hypophysial duct itself. The cell membranes dis- 
appear and the nuclei wander out toward the periphery where they collect in several 
rows with the granular cytoplasm concentrated in the center (Fig. 4D, 4G, SC). 
This part of the nervous system, the definitive ganglion, persists through meta- 
morphosis and together with the hypophysis gives rise to the permanent nervous 
system of the adult. 

Visceral ganglion The visceral ganglion originates in that part of the 
neural plate that curves toward the left in Stage I. The lumen is obliterated, the 
large nuclei migrate to the periphery leaving the medulla mass of interlacing fibrils 
and granules (Fig. 4D). The visceral ganglion lies posterior to and ventral to the 
sensory vesicle. Dorsally where it merges with the sensory ganglion, it exceeds the 
sensory vesicle in diameter but it gradually diminishes in diameter towards the base 
of the tail where it is continuous with the neural tube. At the junction there is a 
slight enlargement called the spinal enlargement (Fig. 4G). The neural tube re- 
tains its lumen. It runs through the length of the tail to the left of the notochord. 
In Stage IV a single nerve emerges from the visceral ganglion on its right side just 
below the hypophysis. It runs anteriorly and sends out branches to the smooth 
musculature of the mantle. 

THE NOTOCHORD 

At the end of the gastrulation period the chordal cells lie under the posterior part 
of the neural plate. Anteriorly adjacent to them are endodermal cells; dorsally, the 
potential muscle cells of the right lateral margin of the blastopore ; ventro-laterally, 
the potential muscle cells of the left lateral margin of the blastopore. Posteriorly 
the chordal cells extend into the rudiment of the tail. 



AMAROECIUM CONSTELLATUM. II 



75 



. Vi 

., "I 



* 




FIGURE 5. A. Transverse section corresponding to Figure 4 A. 225 X. B. Transverse sec- 
tion through brain of Stage III, hypophysis separated from brain vesicle. 650 X. C, D. Sec- 
lions through sensory vesicle and definitive ganglion of Stage IV; oblique, thus including both 
sensory organs. 650 X. E. Transverse section through Stage I; anterior region. 150 X. F. 
Transverse section through early Stage II; neural folds closed. 150 X. G. Longitudinal sec- 
tion through adhesive papilla of Stage III. 650 X. d. c., definitive endoderm ; dcf. <;., definitive 
ganglion; <//. c., gland cells; //y/ 1 ., hypophysis; inch., mesenchyme cells; 11. p., neural plate; stal., 
statolith. 



Some of the endodermal cells of the yolk mass lie along the right side of the 
chordal cells and when the tail is constricted from the trunk region these cells form 
the loose column of caudal endoderm. In Stage IV little of it remains (Fig. 4C, 
65). 

In Stage I the notochordal cells begin to shift in position. They interdigitate 
into a row of disc-shaped cells occupying the central axis of the short tail. The cells 



76 FLORENCE MARIE SCOTT 

resemble the endodermal cells of the yolk mass in possessing delicate membranes, 
nuclei smaller than those of adjoining muscle cells, and yolk granules. 

During Stages II and III the notochord elongates as the tail lengthens. The 
chordal cells lengthen ; the inter-cell membranes separate from each other converting 
them into hour-glass shaped cells with the nucleus resting in the constricted neck 
between the peripheral masses of protoplasm (Fig. 4E). 

In Stage IV the cell halves separate completely giving the chord the appearance 
of a tube with a scalloped lining. The proximal end retains its relationship with 
the hinder end of the pear-shaped mass of yolk between the atrial cavities and the 
arms of the digestive tract (Fig. 6C, H). Distally it corresponds in length to the 
neural tube and tail muscles. 

Muscle cells of the tail 

Mesoderm differentiates into three structures of the larva, one of which is re- 
stricted to the larval action system, two of which function in both the larval and adult 
action systems. The former includes the muscles of the tail, the latter the muscles 
of the mantle and mesenchymatous connective tissue in the body cavity. The asym- 
metry of the posterior lip of the blastopore at the end of gastrulation (Stage I) 
places the muscle cells of the right side dorsal to the chordal cells and to the right 
of the neural plate at its posterior end, the muscle cells of the left side to the left of 
the posterior neural plate but ventral to the notochord (Fig. 4C). Each band is 
made up of four cells in fairly regular rows. 

In Stage II the myoblasts are the most prominent cells in the body because of 
their size and heavy membranes. Each cell contains a large faintly reticular nucleus 
with a conspicuous nucleolus. The deeper cytoplasm is grossly reticular and retains 
an occasional yolk granule (Fig. 6D). 

In Stage III the peripheral cytoplasm elaborates in its cortex, in a slightly spiral 
direction, along the longitudinal axis rows of contractile fibrillae composed of minute 
granules so distributed that they resemble the individual myofibrillae of striated 
muscle of the higher chordates (Fig. 4E, 6D). The myofibrillae are continuous 
from one cell to another throughout the length of the muscle bands. Grave (1921) 
has described this in the free-swimming tadpole of Amaroecium. The bases of the 
muscle bands, like that of the notochord in Stage IV, are located well within the 
posterior part of the trunk just behind the mass of yolk (Fig. 6H). 

Muscles of the mantle 

In the late embryonic period (Stage III) many of the mesenchyme cells located 
directly under the ectoderm unite end to end to form the smooth fibres of the mantle 
(Fig. 4F). One set of such muscle fibres radiates from each of the siphons. The 
other set encircles the trunk obliquely from the dorsal to the ventral side. 

Mcscnclivinc of the body cavity 

In Stage I two compact lateral masses of mesenchyme cells lie pressed tightly 
between the nutritive endoderm and shallow' ectodermal cells. The one on the right 
side is disposed more dorsally than the one on the left side (Fig. 5E). They ex- 
tend from the posterior muscle cells towards the anterior end of the body. 



AMAROECIUM CONSTELLATUM. II 



77 



w 




/^ 

*} 




.---or.S 







& 



Ph *VA KF- ^V 

'it 7Kf5 f ^4 i 

. -)' fed 

, 

PC V-^ / /* 

v.,.. ' ... ^gjf 




"1 C 




' r 

kj - 



H 



FIGURE 6. A, B, C. Transverse sections through tadpoles of Stage IV; A, anterior. B. In 
region of oral siphon. C. In region of atrial siphon. About 200 X. D. Section through part 
of muscle hand ; middle cell through center of muscle cell, lateral cell through peripheral cyto- 
plasm where myonbrillae are formed. About 850 X. E. Transverse section through Stage II to 
show atrial invaginations. 300 X. / ; . Longitudinal section through Stage III. About 150 X. 
G. Tadpole just before hatching, chorion not ruptured. About 250 X. H. Tadpole at hatching. 
Note insertion of notochonl at posterior end of trunk. About 250 X. ad. p., adhesive papilla; 
at., atrium; at. in., atrial invagination ; at. .?., atrial siphon; end., endostyle ; int., intestine; i)i. c., 
muscle cell; ncli.. notochonl; or. .v.. oral siphon; ph.. pharynx; p. c., pericardial cavity; st.. 
stomach ; ^. v., sensor}- vesicle ; v, y., visceral ganglion. 



78 FLORENCE MARIE SCOTT 

In Stage II both masses of cells multiply and spread out under the ectoderm in 
all directions except posteriorly. A small amount of mesenchyme is found in the 
tail, probably derived from the cells in the mid-region of the posterior lip of the 
blastopore. Being crowded together the cells appear angular in section. The nu- 
clei are relatively large (Fig. 4F, 5(7). 

During the transition from Stage II to Stage III growth of the body and absorp- 
tion of the yolk effect a separation between the epithelial cells of the epidermis and 
the endodermal cells (Fig. SF, 6F). As the body cavity enlarges the cells round 
up, separate from each other, and wander freely about, dividing frequently and even- 
tually filling up all available space except in the area around the base of the tail ( Fig. 
6A,B,Q. 

Other mesenchyme cells assume stellate shape and send out long slender strain IN 
of protoplasm by means of which they form a reticulum of mesenchymatous tissue. 
This is the nearest approach to a coelomic epithelium that is found in Tunicates with 
the possible exception of the perivisceral cavity of Ciona. 

The mantle and tunic 

The epidermis in Stage I is a layer of thin cells small in surface view dorsally 
where they adjoin the neural plate, larger towards the ventral body region. In 
Stage II the cells are of uniformly small size and cuboidal in section except where 
they invaginate to form the atrium and are columnar in shape. In surface view all 
present the characteristic polygonal arrangement of epithelial tissue. 

During Stage III the protoplasm becomes vacuolated medially, the nuclei being 
pushed to the periphery where the cytoplasm is more granular ( Fig. 5B, 6E). The 
epidermal cells grow thinner as development progresses and the inter-cell mem- 
branes disappear. \Yhen the epidermis has assumed the characteristic appearance 
of the Tunicate mantle in Stage III it secretes a thick layer of structureless tunic. 
Occasional cells of the test of the ovum are trapped in the clear tunicin, the greater 
number, however, being pushed with the test ahead of the tunic (Fig. 6C). The 
tunic is grooved where it is secreted about the tail and when the tail is released, with 
the disappearance of the test, the groove remains in evidence marking the embryonic 
position of the tail. 

Dcrh'ati'i'cs <>j tJie epidermis 

Adhesive papillae A conspicuous feature of the Amaroecium larva is a 
vertical row of three adhesive papillae at the anterior end (Fig. 3, 6/ ; , 6", H). Each 
papilla first appears early in Stage III as a local thickening of ectoderm forming a 
pad of columnar cells. The cells at the periphery of the thickened pad form a stem 
which increases in length as the tunic thickens, the whole organ becoming goblet- 
shaped. It retains its connection with the body cavity through its slender hollow 
stem (Fig. 6G, H). The papillae extend through the thickness of the tunic and 
are exposed at its surface. Cells that constitute the functional portion become vacu- 
olated and reticular proximally and toward the center of the cup, where the long 
cells converge, they produce secretion granules which lodge in the concavity of the 
papilla (Fig. 56"). The bordering epidermis surrounds the disc forming a thin 
layer over the cup-shaped depression. During the free-swimming life of the larva 



AMAROECIUM CONSTELLATUM. II 79 

the secretion granules are converted into a viscid substance by means of which the 
tadpole becomes attached. The entire glandular structure is of ectodermal origin. 
Grave (1921) from his study of the fully formed tadpole supposed that mesenchyme 
cells gave rise to the glandular portion of the papilla. Mesenchyme cells wander 
from the body cavity into the hollow stalk but they are not incorporated into its 
structure. The tail encircling the body crowds the papillae a little to the right of 
the sagittal plane thus adding to the asymmetry of the larva. The three papillae 
cannot be homologized with the tactile papillae of Botryllus, which are integral parts 
of the peripheral nervous system. Here they serve only as gross organs of 
attachment. 

Test vesicles During Stage III, when the adhesive papillae are differ- 
entiating, the test vesicles originate as numerous small ectodermal evaginations in 
four distinct regions at the anterior end of the trunk. Two groups, separated from 
each other by the median papilla, are directed forward. The dorsal group is de- 
rived from a short ridge extending in the direction of the oral siphon. The ventral 
group, below the ventral papilla, is derived from a long ridge extending posteriorly 
through about a third of the length of the trunk (Fig. 5, 6G, H). The vesicles 
themselves originate as independent hollow slender projections of the ectoderm. 
The attached end of each evagination becomes narrow, finally constricting off and 
severing its connection at the base. Frequently this separation is not effected by the 
time of hatching of the vesicle still being attached to the epidermis by their stalk-like 
bases. When detached the slightly pear-shaped vesicle rounds up and becomes a 
sphere consisting of a single layer of cells which lose their definition on the proximal 
side where they are extremely thin. 

The use of the word "test" in connection with these vesicles is unfortunate. The 
chorion of the egg of Tunicates is called the test and the cells that either lie freely 
in the enclosed liquid or are resolved into pavement epithelium are called the test 
cells. The tunic of the tadpole is a purely ectodermal derivative. The tunic of the 
adult colonies being the product of secretory activity of these vesicles, the vesicles 
should, with greater accuracy, be called the "tunic vesicles." 

SUMMARY 

1. The digestive system of Amaroecium lacks an open archenteron at the end of 
gastrulation. The pharynx appears as a narrow incision with a thin roof and heavy 
floor. An oesophageal evagination differentiates into stomach and intestine. 

2. Heart and pericardium originate from the floor of the pharynx. 

3. Atrium and siphons are ectodermal structures that become associated with 
the digestive system. 

4. The nervous system consists of a sensory vesicle enclosing two sensory masses 
of pigment, a hypophysis lying beside two sensory ganglion, a visceral ganglion de- 
scending laterally to the neural tube which lies to the left of the notochord through- 
out the length of the tail. 

5. The notocord is derived from chordal cells invaginated at gastrulation. Its 
cells become vacuolated. The notochord is confined to the tail and posteriormost 
region of the trunk. 

6. Muscle cells differentiate from mesodermal cells of the blastoporal margins. 
Asymmetry of the blastopore places the cells of its right margin dorsal to the noto- 



80 FLORENCE MARIE SCOTT 

chord, the cells of the left margin ventral to the notochord. Each band of muscle 
cells consists of four longitudinal rows. Cells separate from the two lateral masses 
of mesenchyme and move into the body space of the developing tadpole. They give 
rise to muscles of the mantle. 

LITERATURE CITED 

CAULLERY, M., 1895. Contribution 1'fitude des Ascidies Composees. Bull, dc la France ct de la 

Belglquc, 27: 1-158. 
CONKLIN, E. G., 1905. Organization and cell lineage of the Ascidian egg. Jour. Acad. Nat. 

Sci. Phild., 13: 1-119. 
GRAVE, C., 1920. The origin, function and fate of the test vesicles of Amaroucium constellatum. 

Anat. Rcc., 17 : 350. 
GRAVE, C., 1921. Amaroucium constellatum The structure and organization of the tadpole 

larva. Jour. Morfh., 36: 71-101. 
GRAVE, C., 1935. Metamorphosis of Ascidian larvae. Papers from the Tortugas Laboratory, 

29 : 209-292. 
GRAVE, C., 1944. The larva of Styela (Cynthia) partita: Structure, activities and duration of 

life. Jour. Morph., 75 : 173-190. 

HJORT, J., 1896. Germ layer studies based upon the development of Ascidians. Cliristiania. 
MAURICE, C. ET M. SCHULGIN, 1884. Embryogenie de 1'Amaroecium proliferum (Ascidie com- 

posee). Ann. Sci. Nat., (6) 17: I^t6. 
SCOTT, SISTER FLORENCE M., 1945. The developmental history of Amaroecium constellatum. I. 

Early embryonic development. Biol. Bull., 88: 126-138. 




COMPARATIVE SENSITIVITY OF SPERM AND EGGS TO 
ULTRAVIOLET RADIATIONS 

ARTHUR C. GIESE * 

Marine Biological Laboratory, Woods Hole, Mass, and Hopkins Marine Station, 
Pacific Grove, Calif, and Stanford University, California 

The sperm of the sea urchin are more sensitive to ultraviolet radiations than 
the eggs when the effectiveness of the rays is compared by the retardation of cleav- 
age of unexposed eggs fertilized with irradiated sperm on the one hand and of ir- 
radiated eggs fertilized with unexposed sperm on the other (Giese, 1939c). It 
would be interesting to know whether sperm are generally more susceptible to these 
radiations than eggs ; therefore, the experiments were repeated on a number of 
marine forms. It is also desirable to find an explanation for this differential sus- 
ceptibility in those cases where it occurs. Insight might be gained by determining 
action spectra for the sperm and egg, therefore the relative efficiency of action of 
different wave-lengths of ultraviolet light in retarding cleavage of irradiated eggs 
and of eggs fertilized with irradiated sperm was determined as described below. 

MATERIALS AND METHODS 

Arbacia punctulata, Nereis limbata, Chaetoptcrus pcrgamcntaccus, and Mactra 
sp. were studied at Woods Hole, Mass. Strongylocentrotus franciscaims and S. 
purpuratus, collected at Moss Beach, and Urcchis caiipo collected at Bolinas Bay, 
California, were used at Stanford University, and Dendrastcr e.vccntricns and Pa- 
tcria miniata were studied at the Hopkins Marine Station, each type of egg being 
used during the active breeding season. 

The methods for studying the eggs were similar to those previously described 
(Giese, 1938). Except for the work on the action spectrum, the mercury argon 
discharge tube which emits about 85 per cent of its light at A 2537 A was used and 
the light intensity was measured with a Hanovia Ultraviolet Meter (No. 949). The 
dishes were kept in running sea water to attain a lower temperature than that of the 
room. The work on the action spectrum was done with a mercury arc and a natural 
quartz monochromator and the intensity of the light was measured with a thermopile 
as in previous studies (Giese, 1938). The eggs were kept in dishes in moist cham- 
bers and in a constant temperature room at 15 C. 

Sperm were used in dilutions of between 1 : 200 and 1 : 1 .000 of the spawn. Such 
dilution is necessary because in denser suspensions ultraviolet light is completely 
removed by the sperm first reached. Irradiated sperm lose their fertilizing power 
rapidly, therefore they must be used soon after exposure (see Hinrichs, 1927, for 
studies on inactivation). 

1 This work was supported in part by grants from the Rockefeller Foundation. The writer 
is indebted to Dr. C. Packard, Director of the Marine Biological Laboratory, and to Dr. L. R. 
Blinks, Director of the Hopkins Marine Station, for making available the facilities and for the 
many kindnesses extended to the author during the course of this work. 

81 



82 



ARTHUR C. GIESE 



EXPERIMENTAL 



Comparison of various eggs 



A summary of the general results obtained with all the eggs studied will be found 
in Table I. Not all the eggs respond to ultraviolet radiations in the same way. 
Thus cleavage of eggs of Arbacia and Strongylocentrotus is merely slowed up but 
remains normal after small and medium dosages so that comparisons of the effects 
of various dosages and wave-lengths is relatively easy. Abnormalities only appear 
after larger dosages. In Urechis, Nereis, and some of the other eggs the threshold 
for abnormal development is relatively low. While per cent of abnormal develop- 
ment could be used for analysis of effects of radiations, it would be much more 
difficult. 

It is readily apparent that with regard to ultraviolet susceptibility, there are two 
types of sperm : in the Echinoderms, especially Arbacia and Strongylocentrotus, the 

TABLE I 

Comparative action of ultraviolet radiation 2 on eggs and sperm of various marine animals 



Species 



Effects on eggs 



Effects on sperm 



Strongylocentrotus 
purpuratus 



Arbacia punctulata 



Dendr aster excen- 
tricus 

Urechis caupo 



Chaetopterus perga- 
mentaceus 



Nereis limbata 



Mactra sp. 



Delay just noticeable after about 
100 ergs/mm. 2 ; will develop even 
after 4,000; after 8,000 ergs/mm. 2 
become quite abnormal. 

Noticeable delay after 200 ergs/ 
mm. 2 but even after 2,000 ergs/ 
mm. 2 plutei, normal but smaller 
than controls, develop from eggs. 
After 4,000-8,000 ergs/mm. 2 eggs 
are quite abnormal. 

Slight delay after 1,600 ergs/mm. 2 ; 
strong after 6,400; quite abnormal 
after 25,000 ergs/mm. 2 

Some delay after 200 ergs/mm. 2 
Marked injury with abnormal 
cleavage after 5,000 ergs/mm. 2 

Slight delay only after about 4,000 
ergs/mm. 2 ; after 16,000 ergs/mm. 2 
still cleave but much delay and 
many cytolize. 

Even after 4,000 ergs/mm. 2 de- 
velop with little delay to the 
trochophore stage; after 8,000 ergs 
show delayed cleavage. 

Very slight delay after 500 ergs/ 
mm. 2 ; striking after 4,000-8,000. 



Noticeable delay 3 even after 10- 
20 ergs/mm. 2 Marked retardation 
as dosage above this is used. 

Noticeable delay even after less 
than 50 ergs/mm. 2 After 250 ergs 
still develop larvae but after 500 
quite abnormal. Even after 4,000 
ergs/mm. 2 sperm activate eggs. 

Slight delay after 200 ergs/mm. 2 ; 
abnormal after 800 ergs/mm 2 . 

Marked abnormalities after 200 
ergs/mm. 2 

Slight delay after 2,000 ergs/mm. 2 ; 
killed after about 8,000-16,000 
ergs/mm 2 . 

Slight delay between 4,000-8,000 
ergs/mm. 2 8,000 kills most sperm. 



Delay after 250 ergs/mm. 2 and 
progressive delay thereafter. 



2 The measurements with the Hanovia meter are accurate to about 10 per cent as checked by 
thermopile measurements in one instance. 

3 Amounting to 15-30 minutes delay at the third cleavage of eggs fertilized with such irradiated 
sperm. Marked delay means a delay of an hour or more. 



SENSITIVITY OF SPERM AND EGGS 



83 



sperm are much more sensitive than the eggs ; in the worms such as Urechis, 
Nereis, and Chaetopterus the sperm is slightly if at all more sensitive than the egg, 
as judged by cleavage delay. 

Such a lack of differences in susceptibility of the gametes might be more ap- 
parent than real. It is possible that when there is little or no cleavage delay follow- 
ing fertilization of an egg with an irradiated sperm, the sperm may be serving only 
to activate the egg to haploid parthenogenesis. Eggs of Arbacia and of Chaetop- 
terus were, therefore, fertilized with sperm treated either to a small dosage or to a 
medium dosage of radiations and at appropriate intervals samples were fixed in 
Benin's fluid and stained with iron hematoxylin. Although the preparations were 

k 

ACTION SPECTRUM FOR RETARDATION OF CLEAVAGE 



100 



80 

\ 
I 6 " 



40 



20 



SPERM^ 



A 

X. S 



EGGS D 

SETTING ACTION AT 2804 



N 



\ EQUAL TO 100 
\ 

\ 
\ 
\ 
\ 
\ 
\ 




P 



EGGS COMPARED TO SPERM 
ON AN ABSOLUTE BASIS 



2400 



2600 280O 

WAVELENGTH IN A 



3000 



320O 



FIGURE 1. Action spectra for retardation of cleavage of eggs fertilized with irradiated sperm 
at A and for irradiated eggs at C. At B the data for the eggs are compared on a relative basis 
setting the value at \ 2,804 A as 100 per cent efficient. See text below. 

not entirely satisfactory, evidence for pronuclear fusion was observed in both cases. 
No lagging or disintegrating sperm were observed in the cytoplasm of either egg. 
Since neither cytological nor physiological evidence suggests parthenogenesis, it 
seems likely that for the dosage ranges tested the delayed cleavage follows fusion 
of the gametic nuclei. The difference between the two types of sperm must lie in 
some other factor. Possible explanations will be considered in the discussion. 

The data in Table I show that the threshold for effects on cleavage is quite dif- 
ferent for eggs of different species. Thus Strongylocentrotus, Arbacia, Mactra, 
and Urechis eggs are retarded after brief exposures to ultraviolet as compared to 



84 



ARTHUR C. GIESE 



Nereis, Chaetopterus, and Dendraster. Whether this is due to mere physical 
screening by some inert materials in the egg or to differences in concentration of 
some light sensitive materials is not known. 

Action spectra for egg and sperm 

If irradiation of the nucleus alone causes retardation of division of the cell, the 
same action spectrum should be found for egg and sperm ; that is, there should be 
no qualitative difference in effectiveness of different wave-lengths even though the 
general susceptibility of the sperm is greater. If elements in the cell other than the 

ACTION SPECTRA FOR SPERM AND EGG AND PROTEIN ABSORPTION 



100 i- 



i 
I 



I 



i 



\ ABSORPTION BY 



SERUM ALBUMIN 



' SPERM 
ACTION 
SPECTRUM 



\ 
ABSORPTION 

BY NUCLEIC ACID 



EGG ACT/ON 
SPECTRUM 




20 - 



300 



2500 



270O 



2900 



3100 



WAVELENGTH IN ANGSTROM UNITS 



FIGURE 2. Comparison of the action spectra of Figure 1 with absorption spectra of nucleic 
acid and serum albumin. Data for nuclei acid from Caspersson (1938), for proteins from 
Smith (1929). Note that the action spectrum for the egg is quite different from the absorption 
spectrum for albumin at both ends. 

nucleus are involved the egg may show an action spectrum different from that of 
the sperm. 

The methods employed for the studies at different wave-lengths are similar to 
those already described elsewhere (Giese, 1938, 1939c), therefore, only the briefest 
mention need be made of them. The irradiated eggs are fertilized with normal 
sperm. The rate of division is then determined by observing for percentage of 
cleavage every 15 minutes. The times at which the eggs reach the 2, 4, 8, 16, and 
32 cell stages are recorded and the number of cleavages is plotted against the time 
after fertilization and compared with the control. The increase in time required to 
reach the third cleavage is taken as a measure of the retarding action of the radi- 



SENSITIVITY OF SPERM AND EGGS 85 

ations. The retardation is then plotted against dosage. From such curves for each 
of the wave-lengths the dosage required to bring about a given retardation can be 
determined. For Figures 1 and 2 the reciprocals of the relative amounts of energy 
at different wave-lengths required to produce a retardation of division by 1.5 hours 
were determined. In Figure 1 at A and C the sperm and egg are compared on 
this basis and a great difference in susceptibility between the gametes is evident. 
In B the data for the eggs are compared amongst themselves on a relative basis 
setting the action at A 2,804 A as 100 per cent efficient. 

The shape of the curves indicates that different materials are being affected in 
the two cases, since the action spectrum is considered to be a measure of the ab- 
sorption by the active constituent. To see if the absorbing materials can be identi- 
fied the absorption spectra for serum albumin and nucleic acid are given in Figure 
2. It is apparent that the action spectrum for sperm matches the absorption spec- 
trum for nucleic acid better than the absorption spectrum for albumin ; the reverse 
is true for the action spectrum of the egg. Since the simple proteins and nucleo- 
proteins are the major structural constituents of the cell and none of the other or- 
ganic or inorganic constituents have very specific absorption, the resemblances while 
imperfect are indicative of absorption by these two classes of compounds in the 
action of ultraviolet radiations on the gametes. 

DISCUSSION 

The occurrence of a differential susceptibility of gametes with the sperm more 
sensitive to ultraviolet light than the egg as first found in the sea urchin, Strongylo- 
centrotus pwpuratus, was verified on Arbacia and Dendraster and in preliminary 
trials on Pateria and 6\ franciscanus but not on Urechis, Chaetopterus, and Nereis. 
In the latter forms the sperm appears to be only slightly more sensitive than the egg 
(Table I). The former group of species belongs embryologically to the radially- 
cleaving, indeterminate egg type, the latter group to the spiral determinate type. 
In addition, the radial eggs used here are mature or nearly so at the time of shedding 
whereas the spiral eggs are generally immature. An illustration of the difference 
in response to ultraviolet light, depending on this difference in organization is seen 
in the local "burns" occurring in the spiral eggs. Thus a Nereis egg given a uni- 
lateral dosage of between 8,000-16,000 ergs/mm. 2 may develop apparently normally 
except on the burned surface which appears blistered. A Strongylocentrotus egg 
on the other hand unless given a large dosage of light will show general effects dis- 
tributed throughout the retarded egg. However, it is not possible to say which 
features of the organization account for the difference in sensitivity of the eggs and 
sperm of the two groups. 

One might envisage that in eggs the retarding effects of radiation on cleavage are 
due to the inactivation, by substances formed during irradiation, of some catalyst 
which is necessary for the reactions involved in cleavage. In one group of eggs 
perhaps the catalyst is present in excess of that necessary for a characteristic rate 
of cleavage, the rate being controlled by some other limiting factor, in the other it is 
present in just adequate concentration and itself constitutes the limiting factor. 
Even a considerable dosage of radiations will not reduce the concentration of cata- 
lyst below the critical level in the first case but will readily do so in the second. In 
the first case no cleavage delay would be expected until very large dosages of radi- 



86 ARTHUR C. GIESE 

ations had been administered, in the second the cleavage should be affected after 
very small dosages. One would have to assume that irradiated sperm on penetrat- 
ing unirradiated eggs introduce similar cleavage-inhibiting substances acting on the 
catalyst as those formed in the irradiated egg. In this case also the effect on cleav- 
age should depend upon the amount of catalyst present in the egg if in excess, the 
cleavage should not be easily inhibited, if limiting, the reverse should be true. \Ye 
should expect both sperm and egg to be relatively insensitive to the radiations in 
the former and this is found in most spirally cleaving eggs. 

Against the above postulation is the fact that the action spectrum for sperm re- 
sembles nucleoprotein absorption while for the egg it resembles simple protein ab- 
sorption indicating two different ultraviolet absorbing materials in the gametes by 
which the cleavage-retarding effect is produced. It is possible that absorption by 
both of these types of proteins leads to the formation of toxic photoproducts which 
inhibit the same catalyst. It is also possible that the toxic substance is much more 
rapidly formed by the nucleoproteins, but the necessary assumptions strain the 
imagination. 

It should be pointed out that the retardation of the early cleavage- is only the 
initial effect of the radiation. If the delayed effect could be studied we might find 
that the recovery from injury to the egg would resemble absorption by nucleo- 
protein, indicating a more lasting injury to the nucleus than to the cytoplasm, as is 
the case for division of Paramecium (Giese, 1945a). Because the number of cells 
cannot be satisfactorily determined in the later cleavages such experiments were not 
attempted with eggs. 

The action spectrum obtained for the egg is similar to that observed for "cyto- 
plasmic" effects such as increased time of ciliary reversal, retardation of excystment, 
immobilization of cilia, and prevention of hatching of eggs. The action spectrum 
for the sperm resembles that for "nuclear" effects such as recovery of paramecia 
from sublethal effects, bactericidal and fungicidal effects and the production of muta- 
tions (see Giese, 1945b, for references). It is interesting to note the difference be- 
tween the action spectrum for retardation of cleavage of the egg and for activation 
studied by Hollaender (1938). In the latter case no action was found until about 
A 2,650 A and the effectiveness of the light increased as the wave-length decreased. 
The mechanism of action of the ligbi must be different in these two instances. The 
action spectrum data thus lay the foundation for further analysis of the effect of 
these radiations upon gametes. 

SUMMARY 

1. The action spectrum for the retardation of division of eggs fertilized with ir- 
radiated sperm resembles the absorption of ultraviolet light by nucleoproteins. 

2. The action spectrum for retardation of division of irradiated eggs of the sea 
urchin resembles absorption by simple proteins like albumin except that at the short 
wave-length end there is no increase in action at A 2,483 A where absorption shows 
a definite upswing. 

3. The absolute amount of energy required to affect division to the same extent 
by affecting the sperm is very much less than that required to affect eggs. 

4. Other Echinoderms tested show a similar difference in susceptibility of eggs 
and sperm : 6\ jranciscanus, Arbacia punctnlata, Dendraster ex centric us, and Pa- 
ter la miniata 



SENSITIVITY OF SPERM AND EGGS 87 

5. Animals other than Echinoderms tested did not show as striking a difference 
between susceptibility of eggs and sperm : Urechis caupo, Mactra sp., Chactoptcrus 
pcrgamentaceus, and Nereis limb at a. 

6. In the eggs listed in paragraph 5, determinations are made more difficult by 
the tendency for the eggs to show irregular cleavage rather than retarded cleavage 
as the dosage increases. Such irregular cleavage occurs in Echinoderm eggs as well 
but the threshold is higher. 

7. If both eggs and sperm of the sea urchin are irradiated the effect on the rate 
of division is less than the sum of the effects which would be expected on each of the 
gametes alone. However, the percentage of abnormal cleavage greatly increases. 

LITERATURE CITED 

CASPKRSSON, T., 1936. Uher den chemischen Aufbau des Strukturen des Zellkernes. Skandinav. 

arch. f. physiol. Suppl, 8 to Vol. 73, 1-151. 
GIESE, A. C., 1938. The effects of ultraviolet radiations of various wavelengths upon cleavage 

of sea urchin eggs. Biol. Bull., 65 : 238-247. 
GIESE, A. C., 1939a. Retardation of early cleavage of Urechis by ultraviolet light. PhvsioJ. 

' Zool., 12 : 319-327. 
GIESE, A. C., 1939b. Ultraviolet light and cell division. Effects of x 2654 and 2804A upon 

Paramecium caudatum. /. Cell. Conip. Physiol., 13: 139-150. 

GIESE, A. C., 1939c. Ultraviolet radiation and cell division. Nuclear sensitivity : effect of ir- 
radiation of sea urchin sperm. /. Cell. Conip. Physiol., 14: 371-382. 
GIESE, A. C., 1945a. The ultraviolet action spectrum for retardation of division of Paramecium. 

/. Cell. Comp. Physiol., 26 : 47-55. 

GIESE, A. C., 1945b. Ultraviolet radiations and life. Physiol. Zool., 18 : 223-250. 
HINKICHS, M. A., 1927. Ultraviolet radiation and the fertilizing power of Arbacia sperm. 

Biol. Bull., 53: 416-437. 
HOI.LAENDER, A., 1938. Monochromatic ultraviolet radiation as an activating agent for the eggs 

of Arbacia punctulata. Biol. Bull., 75 : 248-257. 
SMITH, F. C., 1929. The ultraviolet absorption spectra of certain aromatic ainino acids and of 

proteins. Proc. Roy. Soc. London B, 104: 198-205. 



OBSERVATIONS ON THE FUNCTIONING OF THE ALIMENTARY 
SYSTEM OF THE SNAIL LYMNAEA STAGNALIS 

APPRESSA SAY 

MELBOURNE ROMAINE CARRIKER 
Zoological Laboratory of the University of Wisconsin, Madison 

INTRODUCTION 

Although records exist of functional studies on the alimentary system of Basom- 
matophora as far back as the early eighteen hundreds, the detailed story of the 
course and ultimate fate of food in the alimentary tract and the simultaneous move- 
ments of the tract is thinly scattered and far from complete. In the more recent 
emphasis placed on some gastropods because of their importance as vectors of para- 
sites of man, domestic animals, wild game, and fish, it is vitally important that the 
normal physiology of the system most frequented by these parasites be better known. 

It is the purpose of this paper to integrate the previous work on the physiology 
of the alimentary system of Lymnaea st agnails and allied forms (suborder Basom- 
matophora, order Pulmonata) with original research on the same system in L. s. 
apprcssa Say. The basic morphological (Carriker, 1945) and histological (Car- 
riker and Bilstad, 1946) studies on this system in L. s. apprcssa have been com- 
pleted and are in press. All terms used in this research have been described in these 
two papers. 

L. s. apprcssa has been selected for this research because it is a representative 
vector and because of its excellent response to laboratory culture, its relatively 
large size (maximum shell length, 62.5 mm.) as compared with other fresh water 
pulmonates, its short life cycle, and its relatively thin semitransparent shell and semi- 
transparent tissues. Snails used in the research were cultured entirely in the labo- 
ratory. They were grown through many generations in large battery jars and fed 
on lettuce and cooked "cream of wheat" cereal. The water in the jars was aerated 
by means of a small Marco air pump (Noland and Carriker, 1946). The original 
snails were collected in Fox Lake, Wisconsin, in 1939. Parasite-free cultures 
(especially of trematodes) from the original snails were obtained by the isolation 
of the egg mass soon after oviposition in separate aquaria. Each new culture was 
started in this way rendering transmission of infection very improbable. Detailed 
examination of succeeding generations has not disclosed parasites. 

This work was carried out at the University of Wisconsin (1939-1943) under 
the stimulating guidance of Prof. L. E. Noland, whose advice, encouragement, and 
friendly cooperation were much appreciated. 

HISTORICAL REVIEW 

Scanty observations on the function of the anterior part of the alimentary tract 
of Lymnaea were given by Semper (1857), Geddes (1879) and Moquin-Tandon 
(1885) ; more detailed information was given by Amaudrut (1898), Pieron (1908) 

88 



ALIMENTARY SYSTEM OF LYMNAEA 89 

and by Baecker (1932). The stomach region was investigated by Gartenauer 
(1875), Moquin-Tandon (1885), Colton (1908) and Heidermanns (1924). These 
experimental contributions of Colton and of Heidermanns, particularly of the 
latter, are noteworthy. The liver has been the object of most of the physiologi- 
cal work although the research has usually been incidental to that on the stylom- 
matophoran Helix: Barfurth (1880, 1881, 1883a and b), Frenzel (1886), Cuenot 
(1892), Enriques (1901, 1902), Faust (1920), Peczenik (1925) and Krijgsman 
(1928). Only the investigation of Peczenik is exclusively on L. stagnalis. 

EXPERIMENTAL METHODS AND RESULTS 
Lymnaea physiological salt solution 

The study of the living system has required the development of a physiological 
salt solution which will approximate the ionic and osmotic balances of the blood of 
Lymnaea more closely than do such commonly used solutions as Ringer's. On the 
basis of incomplete data given by Duval (1928) on Lymnaea and by Bernard and 
Bonnet (1930) on Helix on the molecular concentration of blood, the following 
solution was developed for L. s. appressa: 

NaCl 2.0 gms. per liter 

NaHCO., 2.0 " " " 

KH 2 PO 4 0.1 " " " 

MgCl 2 0.3 " " " 

CaCl 2 0.3 " " " 

This solution consists of 0.47 per cent salts and gives a pH of approximately 7.8. 
After about a week considerable precipitation of CaCO., occurs, although this 
seems to have no noticeable effect on the isolated organs. The vas deferens was 
used in testing the solution and was found superior to the heart for this purpose. 
The vas deferens, terminal preputium and prostate gland were removed under the 
physiological salt solution from the cephalic hemocoel without bruising. This por- 
tion of the reproductive tract is in part a strong muscular tube which is easily ex- 
cised and maintains a continuous squirming motion as long as the tissues are alive. 
It continued squirming for about 66 hours in the solution described above. A 
Ringer's solution of 0.7 per cent salts keeps it moving for about 12 hours, although 
at a much reduced rate. 

Hydrogen ion concentration 

The first work on the estimation of the pH of the alimentary tract of a fresh 
water snail seems to be that done by A. H. Rosenbloom on L. s. appressa in his 
bachelor's thesis in 1942 (unpublished) in this laboratory. He has kindly con- 
sented to the incorporation of his results in this paper. His method was essentially 
the colorimetric one employed by Yonge (1925) : fluids from the various lumina of 
the alimentary tract of the snails under variable feeding conditions were pressed 
out onto paraffined plates and thoroughly mixed with indicators (brom-thymol 
blue, neutral red, and methyl red). The colors were compared with those of indi- 
cators freshly prepared in buffered solutions checked on a Coleman pH electrome- 
ter. The results are given in Table I : 



90 



MELBOURNE ROMAINE CARRIKER 



TABLE I 

pH of the contents of various lumina of the alimentary tract of L. s. appressa 



Organ 


Average pH 


Maximum and 
minimum pH 


Number of tests 


Number of snails 


Buccal cavitv 




Same as water in 


external medium. 




Proesophagus 


6.9 


7.2-6.3 


10 


10 


Postesophagus 


7.2 


7.6-6.6 


10 


10 


Gizzard and crop . 


6.4 


7.2-6.3 


12 


12 


Pylorus .... 


6.6 


7.0-5.8 


10 


10 


Intestine 


7.1 


7.8-6.2 


35 


15 













Enzymes 

Preliminary tests were made for non-purified cathepsin, pepsin, trypsin, and 
amylase. The tests for the proteinases were made according to the methods of 
Anson (1938), Bradley (1938) and Folin and Ciocalteu (1927) ; those on amylase, 
by the iodine test of Hawk and Bergeim (1937). Semi-micro technics were ap- 
plied to large numbers of the excised organs. 

Maximum catheptic activity (at pH 3) over a ten-day period was found in the 
liver. That occurring in the buccal mass and gizzard and other portions of the 
alimentary system was not significant as compared to that in the liver. In an effort 
to determine to what extent cathepsin might be secreted from the liver, gut fluid 
from which the amebocytes had been centrifuged was tested. Under the conditions 
of the experiment, at least, no cathepsin was found in the gut juice. In some tests 
tryptic activity was found in the salivary glands. A very active amylase, optimum 
pH 7, was present in the salivary glands and in the liver. 

The only investigation of the hydrolytic enzymes of the alimentary system of the 
Basommatophora reported in the literature is that by Heidermanns (1924). He 
described a positive test for cellulase present in the digestive juice of the stomach 
organs (crop, gizzard, and pylorus) of L. stagnalis. 

Ciliatlon 

Ciliary currents were studied by the injection of fine carmine suspensions in 
Lymnaea physiological salt solution through various portions of the exposed tract, 
by application of carmine particles to the epithelium of the opened tract and by 
placing small bits of gut wall in a carmine suspension on an uncovered microscopic 
slide under high magnification. In some dissections the undisturbed food particles 
were seen passing through various portions of the excised gut on the natural ciliary 
currents. 

No work has been performed previously on the ciliation of the alimentary system 
of the Basommatophora. Merton (1923) in his research on the external ciliation 
of pulmonates included a brief study of the ciliation of the hepatic ducts of Helix. 

The entire alimentary system of L. s. apprcssa, with the exception of the gizzard 
and portions of the buccal cavity, is ciliated (see later in this paper), Figures 3, 9, 
and 11. 



ALIMENTARY SYSTEM OF LYMNAEA 91 

Muscular activity 

The activity of the alimentary system was observed under binoculars through the 
transparent walls of normal living young snails and in adult unanesthetized snails 
opened under Lymnaea physiological salt solution. The independent activity of 
the radula over the odontophore was clearly observed and conclusively verified by 
watching snails under the binocular under the following conditions : snails deprived 
of food for a day were placed in a finger bowl of well aerated water to which had 
been added strips of lettuce (1-2 mm. wide). A Petri dish was floated over the 
lettuce and the water. As the snails crawled upside down under the glass, feeding 
on the lettuce, the action of the radula and mouth parts was clearly visible under a 
strong beam of light. 

Sand in the gizzard 

In order to check the experiments of Heidermanns (1924) and to add additional 
information on the role of sand in the comminution of food by the gizzard of L. s. 
apprcssa, the following experiments were devised. 

Sixteen adult snails were placed in each of four aerated aquaria containing a 
one-half inch mesh wire platform over the bottom. By means of this contrivance 
the feces were removed from the vicinity of the snails soon after defecation. To 
three of the aquaria the following foods were added respectively : ( 1 ) cooked "cream 
of wheat," (2) filter paper, and (3) lettuce. (4) No food was added to the fourth 
tank. (5) A fifth tank was assembled as a control without the wire platform and 
with lettuce and sand. One snail from each aquarium was killed daily and opened 
immediately. After ten days the following was disclosed : eight of the forty-three 
experimental pulmonates contained no sand in the tract, thereby showing that it is 
possible to rid completely the tracts of a few of the snails of sand ; however, there 
was extensive variation in the ability of the different snails to retain sand. As the 
quantity of sand in the gut decreased, the snails consumed less food, until in the 
absence of sand in the tract, no food was ingested and the guts became void of food 
material and feces. The different diets indicated no significant difference in their 
respective values as sand eliminators. Sand was found most abundantly in the giz- 
zard lumen, then in decreasing amounts in the crop and retrocurrent passage of the 
pylorus '(anatomical terminology has been described elsewhere, Carriker, 1945). 
After the quantity of sand in the lumen of the gizzard reached a certain low level, 
it was retained with surprising tenacity for many days. The material in the fecal 
pellets of the control snails, particularly of the gizzard residues, was markedly 
brown and more thoroughly triturated than those of snails with sand-free diets. 

In a second set of experiments snails approximately 10 mm. in length were 
placed in a one-quarter inch mesh wire basket suspended in a large laboratory snail 
stock tank. The feces, propelled by the sluggish circulation of the water in the 
tank, passed out of the basket. All lettuce placed in the basket was carefully washed 
to remove sand. The experiment was continued for several months. In spite of 
precautions, small quantities of fine sand were always present in the tracts of some 
of the animals ; however, this did not seem to be enough for proper trituration as 
many of the snails died abnormally at an early age and none reached the normal 
adult size of the control snails in the tank outside the experimental basket. There 



MELBOURNE ROMAINE CARRIKER 

is unquestionably a vital need for the presence of at least a limited quantity of sand 
in the gizzard of these snails for sufficient breaking down of the food. 

These results are in agreement with the findings of Heidermanns (1924) and of 
Colton (1908). Heidermanns accidently discovered that the only way to entirely 
remove the sand from a live snail was to cause it to hibernate, in which state it 
emitted the total contents of the tract. Colton noted that in the presence of sand 
the plant food was cut to pieces by L. colnniclla, but that in the absence of sand it 
went unmolested. 

Digestive cell ingestiou 

By the use of a method patterned after that of Peczenik (1925) the ingestion of 
participate food by the digestive cells was investigated. White of egg was strained 
through cheese cloth. Carbon (lamp black) was ground into the egg albumen and 
the mixture was thoroughly beaten. This was steamed to a stiff mass and fed to 
snails starved for a few days. After feeding commenced, the snails were opened 
every other day. Indigestible residues within vacuoles in the digestive cells as well 
as similar residues in the fecal pellets showed the presence of very minute particles 
of carbon, particles not present in the control snails. The indigestible residues in 
the digestive cells appeared very similar to the albumen passing down the intestine 
in the gizzard residues. 

Fecal rhythms 

Some information was gathered on the rhythms of the liver and of the gizzard 
by a study of the rate and extent of passage of the various fecal strings. The fecal 
pellets of a 40 mm. snail were observed daily for twenty-four days. The animal 
was isolated in a two-liter glass jar over the bottom of which was placed a paraf- 
fined one-half inch mesh galvanized metal screen, so that all fecal pellets fell to the 
bottom of the jar and could not be reconsumed. The mollusc was fed lettuce on 
which was sufficient sand for the needs of the stomach region. Three egg masses 
were oviposited by the snail, and it added 2 mm. of shell during the twenty-four 
day period. Upon dissection at the end of the experiment the animal appeared nor- 
mal in all respects. For the first ten days the pellets were collected and examined 
microscopically every few hours during the day ; during the latter part of the ex- 
periment they were collected every twelve hours. Numerous examinations were 
made of fecal pellets from the stock snail tanks to corroborate the findings on the 
experimental snail. 

PHYSIOLOGY OF THE ALIMENTARY TRACT 
Bitccal mass and esophagus 

L. s. appressa is primarily an herbivore. In the laboratory it may complete its 
life cycle on lettuce alone and in its natural state feeds on the aquatic vegetation of 
its surroundings. Specialization of the alimentary system (Carriker, 1945) has 
been in keeping with a plant diet. However, animal food is also consumed as has 
been observed by Walter ( 1906) and by seven other authors cited by him. Repeat- 
edly in this laboratory L. s. appressa has been observed to eat the bodies out of the 



ALIMENTARY SYSTEM OF LYMNAEA 93 

shells of dead snails in the aquaria. Biochemical tests disclose the presence of some 
tryptic activity in the secretion of the salivary glands. 

Pieron (1908) has found in L. auricularia and L. stagnalis that there is a total 
absence of food discrimination in the buccal mass and that their feeding is a reflex 
which keeps the radula working most of the time. The only portion of the body 
showing any discrimination is the anterior surface of the foot which contains faintly 
sensitive chemoreceptors. In aquaria in this laboratory L. s. appressa rasps much 
of the time, whether on lettuce or over the newly cleaned glass surface of its tank. 
However it does also pass through regular "resting" periods in which no rasping 
occurs. In the rasping stroke the radula passes first to one side and then to the 
other describing a broad feeding track. 

Feeding can be followed clearly in normal immature "albino" L. s. appressa (a 
strain with very little dark pigment) feeding on a "cream of wheat" food mixture 
blackened with lamp black. This can lie seen to pass as far as the stomach region. 
On the protractor stroke the radula cups to an elongated spoon-shaped trowel about 
one-half the width of the upper mandible, and working against this, cuts out long 
narrow bits of food. Each denticle is sharp so the concerted action of the numerous 
denticles on the radula, sliding independently over the odontophore, provides an 
effective cutting-rasping apparatus. The food bits are pushed back through the 
dorsal food channel to the rear of the buccal cavity which dilates to receive them. 
The tip of the radula closely appresses to the dorsal wall of the buccal cavity in its 
rearward passage, as attested by the jagged pattern of the dorsal chitinous surface. 
The buccal aperture constricts strongly and rapidly after the receding radula. Some 
bits of food are dropped and remain in the dorsal food channel for the next rear- 
ward swing of the food-laden radula. Several food bits clump in the rear of the 
buccal cavity prior to being forced down the esophagus. The radula functions 
principally in cutting pieces of food of suitably small dimensions for convenient 
transport through the anterior portion of the alimentary tract ; it does not triturate 
the food to any considerable degree. 

Only the posterior third of the buccal cavity is ciliated. These cilia and those 
in the densely ciliated esophagus beat strongly posteriorly, bearing food bits from 
the rear of the buccal cavity to the crop. 

In connection with the functioning of the buccal mass, refer to a previous paper 
(Carriker, 1945) for the names, origin, and insertion and relations of the muscles 
and parts of the mass. The muscular activity of the buccal mass is divisible into 
four major synchronous movements: (1) opening and closing of the oral aperture 
and consequent spreading and approximation of the mandibles and lips, as well as 
dilation and contraction of the circular muscles about the anterior portion of the 
buccal cavity, (2) backward-forward and simultaneous elevator-depressor move- 
ments of the odontophore, with some slight turning of the odontophore on its longi- 
tudinal axis and some movement to the right and to the left, (3) movement of the 
radula and radular sac over the cartilage, and (4) backward -forward and simul- 
taneous elevator-depressor movements of the entire buccal mass. Consequently 
there exist in the buccal mass three intrinsic focal points about which the ma- 
jority of the muscles radiate: (1) the oral aperture, (2) the odontophoral cartilage, 
and (3) the radula and the radular sac. 

The activity of the odontophore with respect to the remainder of the buccal mass 
may be arbitrarily divided into four phases, and described as follows: (1) the quies- 



94 MELBOURNE ROMAINE CARRIKER 

cent stage in which the odontophore lies at rest in the rear of the huccal cavity with 
its longitudinal axis in a dorsoventral position. (2) The protracting stroke in 
which the proximal end of the odontophore swings in an arc of about 130 from its 
basal position to a point where it lies above the plane of the distal end, which then 
is in a position to pass partly out of the buccal cavity, bringing the radula against 
the substratum. At the beginning of this stroke the odontophore assumes a hori- 
zontal position as a result of the lowering of the distal end by contraction of the 
dorsal odontophoral flexor muscle, and a simultaneous raising of the proximal end 
by strong contraction of the posterior jugalis muscle. The oral aperture and the 
anterior portions of the buccal mass dilate to permit partial protrusion of the odon- 
tophore through the mouth ; the labial retractors, suboral dilators and dorsomandibu- 
lar dilators spread the mouth. The extrinsic postventral levators and posterior 
jugalis further raise the rear of the buccal mass so that the distal tip of the odon- 
tophore is directed towards the oral aperture, to which it seems to be guided prin- 
cipally by the action of the dorsal odontophoral flexor muscles. The inframedian 
radular tensors draw the radula over the distal end of the cartilage to the point 
where most of the radula outside the radular sac lies on the under side of the hori- 
zontally inclined cartilage, and the collostylar hood lies just behind the distal crest 
of the cartilage. The combined action of the radular sac and cartilage tensors holds 
the radula tautly drawn over the cartilage in readiness for the rasping stroke. Con- 
traction of the intracartilage tensors adds considerably to the rigidity of the cushion 
under the radula. As Woodward (1895) points out for Natalina caffra, the fibers 
of the cartilage act in much the same way as the intrinsic muscles of the human 
tongue and in contraction cause an elongation and consequent slight protrusion of 
the radula. The pressure of the blood in the odontophoral sinus probably provides 
further turgidity. Contraction of the extrinsic as well as of the intrinsic protractor 
muscles brings the odontophore to the substratum. (3) In the rasping stroke the 
distal tip of the odontophore is drawn over the substrate in a licking motion. The 
radula, independent of the principal motion of the cartilage under it, is itself simul- 
taneously slid quickly backward most of its length over the cartilage by the action 
of the heavy supralateral and supramedian radular tensor muscles. The odonto- 
phore is aided by contraction of the extrinsic preventral levator muscles which pull 
the anteroventral floor of the buccal cavity forward and upward. As the mouth 
opens during the previous stroke, the cutting distal margin of the dorsal mandible 
is turned partly forward by contraction of the dorsomandibular dilators and 
possibly the posterior jugals. Thus as the radula rasps forward it makes 
connection with and scrapes past the inner side of the dorsal mandible, much 
as two jaws would come together, so that the snail when feeding on thin por- 
tions of lettuce actually "bites" off pieces with each rasping stroke. It is only when 
feeding on thicker foods that true "rasping" comes into play. The dorsal mandible 
is governed by the dorsomandibular approximator muscle. The lateral mandibles 
afford mechanical protection to the sides of the mouth, and close in medially after 
the radula and under and behind the dorsal mandible. (4) The retractor stroke 
returns the odontophore to the resting condition, and completes the cycle, by action 
of the extrinsic retractor muscles and the supralateral and supramedian radular ten- 
sors and relaxation of the protractors. The oral aperture is closed after the reced- 
ing odontophore by action of the labial sphincter and the mandibular approximator 
muscles ; the buccal cavity, by a contraction of the buccal sphincter and related mus- 



ALIMENTARY SYSTEM OF LYMNAEA 95 

cles of the walls. In assuming the resting- position, the raclular sac is depressed 
behind the cartilage and the radula rests principally behind the vertically arranged 
cartilage so that the ventral tip of the sac projects slightly below the level of the 
buccal mass. As observed by Amaudrut (1898) for Lyiniiaca, the ventral wall of 
the buccal cavity between the esophageal ledge and the collostylar hood is also de- 
pressed, forming a slight dilation in front of the esophageal opening. As both the 
oral aperture and the proesophagus are closed during the retractive stroke of the 
radula, it is likely that this dilation is instrumental in creating a slight vacuum in 
front of the esophageal opening which aids in disengaging food particles from the 
radula. The dilation is caused principally by depression of the radular sac and 
possibly by contraction of the superior suspensor muscle of the radular sac and the 
hood tensor muscles. 

The proesophagus is limited in its muscular activity to slight peristaltic waves 
proceeding towards the postesophagus ; while the latter undergoes pronounced peri- 
staltic activity in either a forward or a backward direction, dilating broadly and 
contracting its entire length. In dilation it may become so large as to fill much of 
the cephalic hemocoel of the expanded mollusc. In expansion it is filled with a 
reddish fluid from the stomach region and food particles. 

In the buccal cavity the food receives generous quantities of fluid from the buccal 
gland cells, a fluid which is probably mostly mucoid in nature, judging from the 
positive mucicarmine stain and from negative tests for amylase and trypsin. This 
does not however preclude the possibility of the presence of other enzymes which 
were not tested for. As food passes under the openings of the salivary ducts it 
receives mucus, amylase, trypsin, and possibly other enzymes from the salivary 
glands. 

The proesophagus adds more secretion from buccal glands and mucous cells. 
The postesophagus functions as a temporary reservoir for the retention of food 
when the crop is full. Being capable of considerable distension, it may retain 
larger quantities of food than the crop. Digestion begins in the postesophagus 
because of enzymatic secretions received from the salivary glands. 

Stomach region 

Comminution of food particles is completed in the crop, gizzard, and anterior 
portions of the retrocurrent passage of the pylorus. These three organs act as a 
unit comparable to a grist-mill. The kneading motion of the anterior and posterior 
gizzard constrictor muscles and the gizzard lobes over the sand in the lumen pro- 
vides the grinding action. Food bits forced between the sand are soon crushed to 
minute particles upon which the digestive enzymes may act more efficiently. Two 
synchronized movements are present in the gizzard. In the first the anterior and 
posterior gizzard constrictor muscles alternate smoothly in mild contraction, thus 
mixing and forcing the contents of the gizzard slowly back and forth ; in the second, 
not as frequent as the first, the bulk of the gizzard compressor muscles contract 
suddenly and strongly, bringing pressure to bear on the contents of the gizzard. 
The presence of gritty material in the gizzard of the Lymnaeidae has been noted by 
many: Cuvier (1817), Wetherby (1879), Whitfield (1882), Moquin-Tandon 
(1885), Colton (1908), F. C. Baker (1900, 1911), and Heidermanns (1924). 

In the crop, all ciliary currents lead to the anterior margin of the right gizzard 



96 MELBOURNE ROMAINE CARRIKER 

pad, those on the left side beating ventrad and over to the right (Fig. 3). Thus 
fine food material accumulates on the right side of the crop at the anterior edge of 
the right gizzard lobe. The crop receives food from the postesophagus and forces 
it into the gizzard lumen. When ample sand is accessible to the animal, the crop 
and anterior portions of the retrocurrent pyloric passage are both filled with it. 
The walls of these organs act as mechanical obstructions to the open ends of the 
gizzard lumen and concentrate the pressure of the gizzard musculature upon the 
contents. They also cooperate in the muscular activity of the gizzard in a unified 
kneading and a slow rotation of the gritty contents. The retrocurrent passage re- 
turns to the crop those particles which have been dislodged from the gizzard con- 
tents by muscular movements of the stomach region. In this fashion the contents 
of the gizzard undergo thorough comminution and partial digestion before the resi- 
dues are shunted down the procurrent passage to the prointestine. 

The epithelium of the stomach region bears a complicated system of ciliary cur- 
rents (Figs. 1, 2, 3, 9). Cilia in the procurrent passage direct fine particles from 
the right ventral side of the gizzard cavity to the prointestine. Those in the retro- 
current passage are directed anteriad towards the left side of the gizzard cavity. 
The dorsal passage bears what in fixed sections appears to be nothing more than a 
brush border. Even in carmine suspensions under high magnification no distinct 
current could be detected in it. The cilia on the ventral fold are divided into two 
distinct functional areas : those on the right half of the fold beat obliquely posteriad 
and laterally in the direction of the currents in the procurrent passage ; those on the 
left half, obliquely anterolaterad in the direction of the gizzard and the currents in 
the retrocurrent passage. The currents on the minor fold whip obliquely antero- 
laterad ; those on the medial half of the major fold pass obliquely anterolaterad; 
while those on the lateral half of the major fold and those on the medial half of the 
fold adjacent the hepatic vestibule reach posterolaterad. The ciliary currents in the 
retrocurrent passage are noticeably faster than those in the procurrent passage. 
Currents on the atrial corrugations run into the incurrent tubule of the cecum. 
Thus the pylorus in cross section (Fig. 2) is composed of three channels, each with 
distinct ciliary currents and of three folds which almost meet centrally and whose 

EXPLANATION OF PLATE I 
(All figures concern L. s. appressa) 

FIGURE 1. Stereogram of the pylorus, hepatic vestibule, atrium, cecum, anterior portion of 
prointestine, and liver lobes. The vascularization is stressed. (Small arrows indicate the flow 
of blood in the arteries ; large arrows, the direction of movement of the contents of this part of 
the tract.) X 6. 

FIGURE 2. Stereogram of cross-section of the pylorus, taken midway between the gizzard 
and the hepatic vestibule. The stippled surfaces are heavily ciliated. (The small arrows in- 
dicate the direction of the ciliary beat; the large arrows, the direction of passage of material in 
the pylorus. The arrows with broken stems designate the direction of ciliary beat on surfaces 
behind the folds.) X 25. 

ABBREVIATIONS 

AT, atrial artery; CC, cecal artery; d.p.p., dorsal pyloric passage; GD, dorsogastric artery; 
HN, minor hepatic artery; HP, prohepatic artery; IP, prointestinal artery; ;./>./., major pyloric 
fold; n.p.j., minor pyloric fold; pc.p., procurrent pyloric passage; PM, major pyloric artery; 
PN, minor pyloric artery; PP, propyloric artery; PV , ventropyloric artery; re. p., retrocurrent 
pyloric passage ; v.p.j., ventropyloric fold ; VT, vestibular vascular arborescence. 



ALIMENTARY SYSTEM OF LYMNAEA 



97 



PLATE I 



PI/ 



or /ode of //t/er 



typhlosote 
-pm/f)fest/ne 




ftepot/c vestibule 
Aepafic ducts 



/ode 



c/orsa/ passage 
minor- 



major fo/d 




uentra/ fo/d 



ct/iatecf str/a 



ci/ia 



98 MELBOURNE ROMAINE CARRIKER 

ciliary currents pass out of the dorsal into both the procurrent and the retrocurrent 
passages. The major fold in addition bears a thin longitudinal strip of long cilia 
at its boundary with the dorsal passage. The major and minor folds in the living 
animal nearly always touch along their crests, so that the fluid contents of the dorsal 
passage may pass into the two ventral passages but coarse material from the ventral 
passage may not pass into the dorsal passage. The juxtaposition of the two folds 
is continued under the hepatic vestibule, where the folds provide a ventral floor to 
this chamber. At this point the cilia on the folds direct a powerful current out and 
away from the vestibule, again preventing the entrance of coarse material into the 
hepatic ducts and liver. 

As discovered for Helix by Merton (1923), the corrugations of the larger proxi- 
mal portions of the hepatic ducts of L. s. apprcssa bear two ciliary countercurrents 
(Fig. 11) : the cilia on the crests of the corrugations are long and beat into the 
liver, those in the grooves are shorter and pass particles in the direction of the he- 
patic vestibule and into the incurrent tubule of the cecum. The particles in the 
grooves are quickly entrapped in mucus secreted there and formed into delicate 
strings. The currents directed into the liver could be traced with certainty only in 
the large hepatic ducts, although cilia were observed as far as the peripheral folli- 
cles in isolated bits of living liver tissue. Yonge (1936) states that in Mollusca 
where food passes into the liver and waste material out, the ducts are ciliated in 
such a way that an inward passage exists on one side and an outward one, on the 
other. Such counter currents could not be determined in L. s. appressa. 

In the cecum the cilia on the cecal folds beat off the folds into the tubules (Fig. 
9) ; those in the incurrent tubule pass carmine particles directly to the distal end 
and around this into the excurrent tubule. Here the cilia beat circumferentially, 
rotating the contents of the tubule along the longitudinal axis. In the continuation 
of the excurrent tubule across the pyloric wall the ciliary stream is directed towards 
the prointestine. 

The crop, pylorus, liver, and hepatic ducts are as active as the postesophagus. 
Besides the usual peristaltic movements, they undergo a series of violent alternating 
pulsations, here designated pulsatory movements, in which the crop, pylorus, hepatic 
ducts, and liver pulsate successively, forcing the fluid contents slowly back and 
forth in swirling currents. In the pylorus the pulsations commence at a point be- 
tween the typhlosole and the atrium and pass towards the gizzard. They are of 
two types : ( 1 ) very strong pulsations in which the entire structure contracts and 
(2) minor pulsations running over restricted portions of the pylorus. In the liver 
the pulsations pass as far as the terminal follicles. This marked movement is most 
vividly observed in bits of living liver tissue under high magnification. Individual 
cells are seen to move against each other by contraction of the thin muscular con- 
nective sheet enveloping each follicle. The pylorus undergoes the most pronounced 
movements and appears to lead the other organs in activity. The incurrent tubule 
of the cecum is relatively thin-walled and does not appear to undergo peristaltic 
activity. The excurrent tubule is thicker-walled and has definite peristaltic move- 
ment in the direction of the outlet. 

It follows then that one of the important functions of the pylorus is that of a 
filter chamber, separating the digested and the fine, partly digested food particles 
from the gross material and sand. This is the conclusion which Heidermanns 
(1924) also reached when he stated that most of the time sand and gross material 



ALIMENTARY SYSTEM OF LYMNAEA 99 

are kept from passing into the liver by the pyloric folds. The major and minor 
folds remain in close approximation along their crests, leaving a narrow slit be- 
tween the dorsal and the ventral passages which may be called the pyloric filter. 
The cilia on the folds are well developed and beat away from the dorsal passage. 
During the pulsatory movements of the stomach region only the finest particles and 
fluid material are permitted ingress to the liver through this filter. The pulsatory 
currents, as these in the gut lumen may be named, are relatively strong and in their 
streaming between the sand particles and foot bits in the gizzard cavity dislodge 
large particles of food. Those which are carried into the pro- and retrocurrent 
passages and which are too large to pass through the pyloric filter, become entangled 
in the ciliary currents of the folds and are carried quickly back to the left side of 
the gizzard lumen by way of the retrocurrent passage. The particles carried into 
the crop on the forward streaming of the contents are soon entangled in the ciliary 
currents of the crop and conveyed to the right side of the gizzard lumen. Here, 
then, is a delicate adjustment by which the larger particles dislodged from the giz- 
zard contents are equally redistributed for further grinding within the gizzard. 

At certain intervals during the day the pulsatory movements appear to cease and 
a portion of the residual material and sand in the gizzard pass out through the pro- 
current passage to the prointestine. The propulsion of gizzard strings (Fig. 10), 
as these residues may be named, through the procurrent passage is very slow and 
mostly by cilia supplemented by slight peristalsis. Cilia were found active through- 
out all portions of the alimentary tract whenever opened ; no cessation of ciliary 
activity (as occurs in some lamellibranchs during increase of CO 2 concentration) 
or reversal of beating was observed. During emission of the gizzard string, the 
large portion of the ventral pyloric fold which partly occludes the gizzard lumen 
flattens to enlarge the opening. As suggested by Howells (1942) for Aplysia, it 
appears that the shape and position of the pyloric folds in L. s. appressa are partly 
maintained by blood pressure in the sinuses. 

To what extent digestion does occur in the postesophagus, crop, gizzard, and 
pylorus is questionable. As amylase from the liver and from the salivary glands, 
trypsin from the salivary glands and cellulase, at least, are present in the gut con- 
tents, some food may be partly hydrolized. Part of the remaining available food 
is reduced mechanically to particles small enough for ingestion by the digestive 
cells of the liver. The amebocytes of the gut also appear to aid in digestion. Ac- 
cording to Heidermanns (1924) fats and carbohydrates are absorbed in the pylorus 
by the ciliated cells. 

The pyloric filter permits only minute food particles to pass into the liver. Most 
of the radular teeth which are discarded continuously from the radula throughout the 
life of the snail (Carriker, 1943a) and grains of sand as large as 90^., by reason of 
the fact that they are considerably heavier than the lighter food particles of the 
same dimensions, are carried past the cilia by the force of the pulsatory currents. 
The larger free food particles, especially of lettuce, are very light and are readily 
barred by the cilia of the filter. In the proximal portions of the hepatic ducts, be- 
cause of counter ciliary currents, only the finer particles that fall into the grooves of 
the corrugations can be carried towards the cecum ; thus teeth and larger sand 
grains are held at this point by the ciliary currents of the crests of the corrugations 
until sufficient fecal material passes out of the liver to carry them with it. 

Ciliation of the crests of the corrugations may play a small role in the conduction 



100 MELBOURNE ROMAINE CARRIKER 

of food material into the follicles of the liver, but probably the principal conveyers 
are the pulsatory currents. Food in solution and in suspension is thus brought to 
all the internal surfaces of the liver follicles. Larger particles finding entrance 
through the filter and too large to remain readily in suspension appear to fall to 
the ductal epithelium. The smaller of these are soon propelled into the grooves of 
the corrugations. Liberal quantities of mucus are secreted there, trapping the par- 
ticles in mucous strings which pass towards the cecum, coalescing as they advance 
into the larger grooves (Fig. 11). From the incurrent cecal tubule the mucoid 
strings pass around the distal end of the cecum into the excurrent tubule. There 
the material receives a further transparent layer of mucoid and cementing material 
and is rotated into a smooth cylindrical continuous string, here designated the cccal 
string (Fig. 10). This, partly by ciliary action and partly by peristalsis, then passes 
on into the prointestine across the atrium. In snails feeding on green lettuce the 
strings are a vivid green because of a heavy accumulation of bits of chlorophyll 
bearing bodies which become entangled in mucous strings in the hepatic ducts. 
In gastropods fed on a food containing carbon, the cecal strings are a dense black. 
In animals on a starvation diet, the cecum continues to pass out cecal strings, just 
as in the feeding animal, but the strings are a mucoid, transparent, milky-white color 
and much reduced in diameter. It thus would seem that the function of the grooves 
in the hepatic corrugations and of the cecum is to collect and eliminate those fine 
particles which pass through the pyloric filter but which are too large to be engulfed 
by the digestive cells and which are thus mechanically eliminated by a "supple- 
mentary filter." Cecal strings pass out continuously, apparently at the same uni- 
form rate and without apparent interruption. They provide a kind of "time clock" 
by which the rate of passage of the gizzard strings and the residues from the liver 
can be compared (Fig. 10). 

EXPLANATION 01- PLATE 11 
(All figures concern L. s. appressa ) 

FIGURE 3. Ciliation currents of the postesophagus, crop, gizzard, pylorus, hepatic vestibule, 
atrium, and anterior portion of prointestine. The tract has been slit ventrally and spread. X 6. 

FIGURE 4. Irregular blue-green excretion bodies (in vacuoles) taken from the liver string. 
X500. 

FIGURE 5. Smooth blue-green, or brown, excretion bodies (in vacuoles) taken from the 
liver string. X 500. 

FIGURE 6. "Signet" excretion body (in vacuole) appearing in the liver strings. X 500. 

FIGURE 7. Clear nodules found in the liver strings which when pressed out under the cover 
slip display their crystalline nature. They dissolve in dilute HC1 and seem very similar to the 
calciferous concretions of the vesicular cells of the connective tissue. X 500. 

FIGURE 8. Indigestible residues from digestive cell (in vacuole), found abundantly in liver 
strings. X 500. 

FIGURE 9. Ciliation currents of the cecum, which has been opened along the incurrent cecal 
tubule and spread flat. X 6. 

FIGURE 10. Typical fecal pellet, showing the gizzard, liver and cecal strings, and the im- 
pression of the typhlosole in the pellet. X 6. 

FIGURE 11. Portion of the corrugated epithelium of the hepatic duct, taken at the opening of 
the duct into the hepatic vestibule. (Large arrows indicate the direction of the ciliary currents 
in the grooves ; the small arrows, that on the crests of the corrugations. ) X 50. 

ABBREVIATIONS 

c.s., cecal string; cxcur. tubule, excurrent tubule; g.s., gizzard string; inc. tubule, incurrent 
tubule; l.s., liver string; s., sand; t.i., impression of typhlosole in fecal pellet. 



PLATE II 



f-v, ::> !. ---\ :-! 
^itfY 



wnm^M'm 









procarren t 
pa-ssoge 



^wm/'-m^r^---- 
: 0:py' -mr< -*-f -/- 
Hl^f^Hii - x // . / . 



passage 
'or fo/d 
/ni/jor fo/d 



. fa date 
ceco/ fo/o? 
ezcur. fa bate 






w 



1 ^. ;V 

H^- &-':.' '. ' . .^ 

'-> sJ^'r.iX'.. : . 




102 MELBOURNE ROMAINE CARRIKER 

The excretory bodies and indigestible residues in the liver are voided periodi- 
cally. These are passed simultaneously in minute mucous strings from all parts of 
the liver towards the central hepatic ducts, there converging into larger strings 
which pass in the direction of the hepatic vestibule. At the proximal end of the 
hepatic ducts this material fills most of the main duct. The combined currents in 
the grooves of the corrugations appear to exert a stronger force than those on the 
crests, so forcing the waste material directly into the hepatic vestibule (Fig. 11). 
There it is caught by the outward flowing ciliary currents on the major and minor 
pyloric folds and passed rapidly into the prointestine. The excretory bodies and 
indigestible residues passing from both lobes of the liver are compressed in the 
hepatic vestibule into one bulky string which is distinct from the cecal and from 
the gizzard string and may be called the liver string (Fig. 10). It is drawn out 
of the liver at the same rate as the cecal string passes out of the ceum. Both strings 
are usually found parallel to each other and uncoiled in the fecal pellets. The giz- 
zard string, on the other hand, passes out much more slowly so that the cecal string 
occurs loosely and abundantly coiled therein (Fig. 10). A lapse of time seems to 
occur between the exit of the gizzard string and that of the liver string, as indicated 
by a conspicuous coiling of the cecal string between the last portion of the gizzard 
string and the forward end of the liver string. The gizzard string follows the liver 
string immediately, as indicated by no noticeable coiling of the cecal string between 
the two. There is also some evidence that, as the liver string is drawn from the 
liver, the pulsations of the stomach region cease. In animals opened for physiologi- 
cal observation of the tract, the stomach region was never pulsating when the liver 
strings were passing out of the liver. This is desirable to prevent the dismember- 
ment of the strings and their mixing with food material brought into the liver by 
the pulsatory currents. The merger of the strings in the prointestine produces the 
fecal pellets. 

The pylorus is composed of a complicated system of folds and passages, it is in- 
nervated by a pair of complex nerve plexuses and a nerve net, and all of the parts 
are exceptionally well vascularized. Functionally there is present in this portion 
of the tract an intricate system of counter ciliary currents and synchronized mus- 
cular movements, as well as partial vascular control of the folds. The pylorus is 
thus well equipped to convey digestive fluids from the liver to the gizzard and crop, 
to bear digested and semi-digested particles into the liver from the gizzard, to ex- 
clude large sand and other large particulate matter from the liver and transfer such 
residues to the prointestine, to receive waste material from the liver and transport 
it to the intestine, to act in conjunction with the cecum, liver, and hepatic ducts in 
shunting a continuous string of residual particles from the walls of these organs 
into the prointestine, to secrete fluids (of unknown nature) and finally to absorb 
fats and carbohydrates. 

Liver 

The liver is probably the most important organ of digestion in the alimentary 
system of the gastropods. Peczenik (1925) shows, as has been indicated in this 
work also in feeding experiments, that such proteins as egg albumen are engulfed 
and digested intracellularly in the digestive cells, and the indigestible residues are 
cast out in vacuoles. Krijgsman (1928) believes that digestive cells in Lymnaea 



ALIMENTARY SYSTEM OF LYMNAEA 103 

are also secretory as well as absorptive, as he has often observed numerous typical 
secretion granules in the liver cells of starved snails. Biochemical tests indicate 
that the greatest catheptic activity of the snail body is localized in the liver, yet none 
of this activity has been found in the fluid of the gut. This is in keeping with cath- 
eptic systems in other animals in which the enzyme has been shown to exist entirely 
as an intracellular protease. Hurst (1927) writes that in PJiysa fat and glycogen 
are stored in the digestive cells. Fat was also found in the lime cells of Helix by 
Griinbauin (1913). The problem of what size of food particle is engulfed through 
the distal membrane of the digestive cells is still an open question. It is likely, as 
indicated by the work of Krijgsman (1925. 1928) on Hcli.r. that the lime cells 
function in storing and in periodically secreting a buffering agent which adjusts 
the pH of the gut juice ; this point has not been investigated in L. s. appressa. The 
mucous cells of the liver provide the mucus utilized in the binding of the indi- 
gestible residues and the excretory bodies into the liver strings. 

Amebocytes were found in varying numbers in the contents of the lumina of the 
liver, postesophagus, gizzard, and pylorus. These were similar to those seen in 
the blood. In some instances those in the gut contained fecal vacuoles so large as 
to force the cell into a peripheral lobate ring. 

Rhythmic activity of the liver is suggested by inspection of sectioned liver tissue, 
of fecal pellets and of the living organ in various phases of its activity. Pulsatory 
movements of the stomach region are apparently interrupted only during the pas- 
sage of liver strings and of gizzard strings. This may explain why smaller hepatic 
excretory bodies occur in the upper pylorus, gizzard, crop, and postesophagus in 
such insignificant numbers. If the pulsatory currents persisted during the elimina- 
tion of the liver residues one would expect to find liver string detritus scattered 
over the gut in as great profusion as in the liver, along with the reddish colored se- 
cretions from the liver. 

The inclusion bodies of the digestive cells of L. s. appressa have been studied 
in detail in the living cells of normally feeding snails, starved snails, snails fed on 
special diets and in preserved tissue sections. The egested bodies have been fol- 
lowed in the fecal pellets over a period of weeks. The results of the study clearly 
indicate the presence in the digestive cells of excretion bodies, of indigestible resi- 
dues and of secretion in separate vacuoles. 

Figure 8 illustrates a vacuole from the digestive cells which is filled with indi- 
gestible particles. These vacuoles measure 12 to 25 /j. in diameter. In snails feed- 
ing on lettuce the contents are colored a greenish brown to dark brown and are 
composed of minute irregular particles, some of the larger ones of which measure 
about 3 ju, in diameter. In the digestive cells they occur one per cell and in varying 
stages of particulate concentration. These constitute the bulk of the liver strings 
and retain their identity in fecal pellets which have been voided for several days. 

The secretion granules are clearly evident in preserved histological sections 
stained with iron hematoxylin, especially grouped towards the distal area of the cell. 
Larger granules measure as much as 4 ^ in diameter. 

The excretion vacuoles (Figs. 4, 5, 6) when in the cells may measure as much 
as 25 p. in diameter, but in the fecal pellets have shrunk somewhat. In the living 
cells excretion bodies are found in variable form and color and are best observed 
when the cells are slowly pressed out under a cover slip as the fluids evaporate. 
The cell contents then pass rolling and turning from the ruptured cells, exposing the 



104 MELBOURNE ROMAINE CARRIKER 

different surfaces of the inclusions. There is one series in which the vacuoles range 
from small to large vacuoles containing variable numbers and sizes of minute blue- 
green, translucent, many-angled particles. The smaller particles are in constant 
Brownian movement, dancing around like a swarm of bees, and indicating the low 
viscosity of the fluids in the vacuoles (Fig. 4). In a second series the same vari- 
ation in size of the vacuole is encountered but the blue-green bodies are present in 
groups of only one to four per vacuole and are spherical and smooth ( Fig. 5 ) . In 
a third series the vacuoles and bodies are identical in form to the second series, but 
the color of the bodies varies from a light brown to a dark solid brown. The largest 
of these bodies are sometimes found free of the vacuoles. When compressed under 
a cover slip they spread with a flowing viscous movement, much as a drop of heavy 
molasses spreads when pressed between two smooth surfaces. In the fecal pellets 
these vacuoles are usually found varying in diameter from 3 to 15 ju, and the vacuole 
membrane presses closely around the excretion body. A fourth type of excretion 
body is found which varies in diameter from 12 to 18 /*, is colored a dark brown 
with a smooth center and possesses a periphery of irregular markings, such that the 
body resembles a signet ring (Fig. 6). The excretion bodies described above are 
present principally in the liver strings, and only in negligible numbers in the cecal 
strings. The "browns" and "signets," particularly, stain with methylene blue and 
neutral red and do not dissolve in strong HC1. The different types described are 
not all present in any liver string in equal abundance at any one time, but vary 
independently, in a sequence which did not seem significant. Because of the transi- 
tional stages between some of these excretion bodies it is probable that they are all 
different phases of the same type of metabolic excretion ; but the method of their 
formation is still a puzzle. 

Intestine and rectum 

Cilia on the typhlosole beat towards the lateral sides of the typhlosole (Fig. 3) ; 
those over the prointestine around the typhlosole beat circumferentially and some- 
what obliquely from the dorsal to the ventral sides in a symmetrical pattern. The 
division of the currents occurs along the dorsal line of the prointestine. Over the 
pellet-compressor the cilia beat transversely across the intestine. The raphe bears 
a strong current which streams directly posteriad. Thus in the pellet-forming re- 
gion, through ciliation and muscular movement, loose particles are gathered, rolled 
inward about the typhlosole and folded into a compact pellet. Strong ciliary cur- 
rents in the remainder of the intestine and rectum are limited almost entirely to 
the costae, raphe, and pseudoraphe ; cilia of the intercostal surfaces are relatively 
short and weak. Peristaltic activity is evident throughout the intestine and rectum, 
being noticeably strongest in the early portions of the prointestine, just behind the 
pellet-compressor. 

Abundant vascularization of the prointestine, in contrast to the relatively poor 
vascularization of the esophagus, suggests that this region of the intestine may also 
function in the absorption of food and water. 

Consolidation of the cecal and liver strings occurs at the hepatic vestibule ; of the 
gizzard residues and cecal string, in the pellet-forming region. The cecal string as 
it is moulded in the cecum is already a smooth well cemented string and undergoes 
no further change as it is forced continuously across the outer margin of the atrium. 



ALIMENTARY SYSTEM OF LYMNAEA 



105 



The liver string, characterized hy a fine dark brown mottling and almost as well 
concentrated as the cecal string, receives a final transparent envelope of cementing 
fluid which binds the cecal string to it (Fig. 10). 

The chief function of the pellet-forming region is that of consolidating and ce- 
menting the loose straggling gizzard residues which constitute by far the greatest 
bulk of the fecal pellet. The large numbers of mucous cells, basophilic flask cells 
and basal secreting cells about the pellet-forming region are indicative of the large 
quantities of cementing substance secreted during the moulding of the pellets. By 
means of ciliary streams and constriction of the tube at the pellet-forming region 
the gizzard residues are pressed into pellets, and the cecal strings, lying loosely 



40 



30 



20 



10 



length 

/engfn fiver str/fryi 

number t/cer strings 




10 



15 



20 



25 



Days 



FIGURE 1. Length in millimeters of the liver and gizzard strings and number of liver strings 
of the fecal pellets, calculated on a twenty-four hour basis. These were voided in a period of 
twenty-four days by a forty millimeter L. s. afiprcssa. The vertical arrows indicate the time 
at which egg masses were oviposited. 

coiled in these residues, are simultaneously incorporated in the pellets. These are 
then forced out of the pellet-forming region by ciliary activity and by strong peri- 
staltic movements which are noticeably stronger immediately behind the pellet- 
compressor. Peristaltic activity gradually diminishes in the direction of the anus. 
The conspicuous impression of the typhlosole remains in the fecal pellet, particu- 
larly in the gizzard string portion, as long after defecation as the pellet retains its 
form. Moore (1931) has found variable patterns in the fecal pellets of different 
Gastropoda and points out the importance of identification of animals by means of 
their pellets. A most striking fact about fecal pellet formation is the extreme com- 



106 MELBOURNE ROMAINE CARRIKER 

pleteness with which fecal material is compressed and cemented. This presumably 
prevents fouling of any portion of the tract. 

For any given snail the diameter of the gizzard string portion of the fecal pellet 
is constant, varying principally with the size of the snail. The liver string varies 
in diameter from that of the gizzard string to that of the fecal string. Figure 1 
indicates for a forty millimeter snail over a period of twenty-four days the rate and 
extent of voidance of fecal pellets. For the tabulation of this data the fecal pellets 
were collected daily and arranged end to end under the binoculars and measured to 
the nearest millimeter. The measurements given indicate only the lengths of the 
gizzard and liver strings, as the cecal string generally occurs embedded in the first 
two strings. The diameter of the gizzard string is reliably constant ; that of the 
liver, less so. 

Most conspicuous is the fact that the quantity of fecal pellets voided daily is 
quite variable from day to day. The quantity of gizzard strings fluctuates far more 
erratically than does that of the liver strings, indicating that the volume of material 
utilized by the liver is more constant than that which may pass through the gizzard. 
The number of liver strings is a more conservative indicator than the length of 
strings, and is probably not as accurate. Passage of food through the gizzard, and 
thus food consumption, seems to diminish during oviposition. 

As indicated by the following data, feces were voided in about equal quantity 
day and night, with just a slight daily increase, over a period of twenty days (9 P.M. 
to 9 A.M., and 9 A.M. to 9 P.M., respectively) : 

Pellets Night Day 

Total length of pellets, mm 1,987 2,134 

Total length of liver strings, mm 530 588 

Total number of liver strings 110 113 

The total length of fecal pellets passed in the twenty-four days was 5,645 mm. ; 
and the total length of liver strings, 1,491 mm., was passed in 289 liver strings, giv- 
ing an average length of 5.1 mm. per liver string. Actually the liver strings varied 
in length from one to 10 mm. The average calculated length of fecal pellets passed 
in twenty-four hours was 235 mm. ; of liver strings, 62 mm. In a normally feeding 
snail the sequence of the liver strings with the gizzard strings was always one of 
alternation. Liver strings do not generally mix with the gizzard strings. Gizzard 
strings as long as 52 mm. were found connecting liver strings. Three typical series 
of fecal pellets taken from days one, two, and three on Figure 1 are given below. 
The liver and gizzard strings are represented by the lengths in millimeters of the 
strings in the order of their elimination ; the figures for lengths of the gizzard strings 
are italicized. The total time for elimination of the pellets is given to the right in 
parenthesis : 

(1) 640 7 33 7 11 644 5 (5 hrs. 15 rnins.) 

(2) 48 7 52 8 13 4 50 5 38 6 (10 hrs. 15 mins.) 

(3) 207 87226299243 (10 hrs. 30 mins.) 

As indicated by the curve for total fecal pellets in Figure 1 and by the lengths of 
the gizzard strings in the series above, consumption of food appeared to follow an 
alternating heavy and light cycle. 



ALIMENTARY SYSTEM OF LYMNAEA 107 

In snails deprived of food the elimination of the gizzard strings ceased and liver 
strings then became connected only by slender lengths of cecal strings. When 
starvation had continued for ten or more days nothing but delicate white cecal 
strings and a few much reduced liver strings containing metabolic excretion bodies 
were found in the intestine. 

A. H. Rosenbloom (unpublished bachelor's thesis, 1942) by feeding colored 
food to L. s. appressa at different times through a period of a month found that in 
normally feeding snails of approximately forty millimeters shell length the minimum 
time for the passage of food from the mouth to the anus was two hours and twenty 
minutes ; in snails previously starved for a week, five hours and fifty minutes. He 
found also that previously starved snails feed for a longer consecutive time than 
do normally feeding snails. The present investigation shows clearly that the ali- 
mentary system becomes completely emptied of food a few days after starvation 
commences. Considerably more food and a longer time are required for a starved 
animal to fill the alimentary tract with food to the point where fecal material is 
voided than for a normally feeding snail. 

The rhythm of passage of liver strings is in keeping with the rhythm of the liver 
itself in which all digestive cells appear to assimilate food together and discharge 
indigestible residues simultaneously. This cycle, as indicated by the passage of 
liver strings, is not completely unvarying, because the number of liver strings dis- 
charged daily varied approximately from eight to nineteen. Thus the interval be- 
tween the discharge of liver residues, probably the time during which the liver was 
digesting food, varied in this experiment from seventy-five minutes to three hours. 
It is possible that oviposition ( Fig. 1 ) may account for some of the variability. 

There seems to be nothing in the literature concerning fecal cycles in the Gas- 
tropoda. Some few scattered observations are reported on the length of the fecal 
pellets. For example, Heidermanns (1924) writes that a 48 mm. L. stagnalis 
with a 90 mm. intestine, eliminated 120 mm. of feces in 24 hours. 

The long intestine is characteristic of the herbivorous snail nutrition of L. s. 
appressa. One of the most striking facts about the functioning of the alimentary 
system is the meticulous care with which all loose particles are collected and properly 
disposed of, in this way serving as a highly efficient sanitation system. The fecal 
pellets receive additional external layers of cementing material as they pass down 
the length of the intestine and rectum. The pH of the intestine is slightly more 
alkaline than that in the stomach region. As pointed out by Yonge (1935) mucus 
is an amphoteric protein whose viscosity is augmented by higher pH, thus more 
efficient consolidation of the feces occurs. Elimination of the fecal pellets through 
the anus is a fairly rapid and uniform process. The strong anal sphincter muscle 
remains tightly contracted except during defecation. Fecal pellets, being slightly 
heavier than water, settle slowly to the bottom of the aquaria. The marked effi- 
ciency of the mucoid coating over the feces is indicated by the extended period after 
defecation that pellets retain their identity. Thus it would seem that the alimentary 
system has not only become specialized in the maintenance of hygienic conditions 
within the system, but also in furthering a healthy external environment. 

Fecal pellets are ingested by snails even in the presence of fresh food and the 
animals appear to derive some nourishment from them. It is to be recalled that 
the gizzard is not a thoroughly efficient grinding mechanism and in many cases, 



108 MELBOURNE ROMAINE CARRIKER 

particularly in the absence of sufficient fine sand, considerable unused available 
food passes out in the gizzard strings. 

DISCUSSION 

The question as to whether the radula slides over the cartilage independent of 
cartilage activity has been a favorite point of academic controversy with certain 
malacologists for some time (in Lymnaeidae see Geddes, 1879; Amaudrut, 1898; 
and Pelseneer, 1935; in the Stenoglossa, a review: Carriker, 1943b). In L. s. 
appressa (and possibly in the majority of snails carefully investigated) there is no 
question but that the principal activity of the radula is that effected by the action 
of the cartilage and muscles under it, and a sliding of the total radula over the 
cartilage independent of the movement of the cartilage. 

A study of the movements of the gut in L. s. apprcssa suggests that rather than 
the presence of different pH in the different portions of the gut, the pH may vary 
with the rhythms and secretions of the liver, the secretions of the salivary glands, 
the secretions of the unicellular glands of the gut wall and with feeding. It is quite 
unlikely that with the constant mixing of the gut contents as a result of the pulsatory 
movements at certain periods, the pH would vary markedly in the different lumina 
of the tract at any time. The wide range obtained between the maximum and the 
minimum pH's and the insignificant variation of the maximum and of the minimum 
pH's is in keeping with this suggestion. The partial isolation of the intestine from 
the movements of the stomach region is in keeping with the slightly higher pH found 
in the intestinal lumen. 

The complexity and abundance of nervous tissues about the stomach region sug- 
gests a possible nervous control of the movements of the stomach region and of the 
liver. In its muscular structure there is no doubt that the buccal mass is the most 
complex organ in the alimentary system ; functionally it appears that the region in 
and about the pylorus is the most intricate. The dense ramifications of blood ves- 
sels, the presence of two nerve plexuses, the intricate series of folds and the compli- 
cated ciliary streams in this region lend credence to this postulation. 

Heidermanns (1924) has opened the question of the function of sand in the 
basommatophoran gizzard in his comparative study of Ancylus, Planorbis, Physa, 
Lymnaea and certain stylommatophorans. He points out that in land pulmonates 
the flaring portion of the esophagus is called the stomach, whereas in the aquatic 
pulmonates the esophagus is normal and the stomach has become differentiated into 
the crop, gizzard and pylorus. Thus the Stylommatophora have no organs that 
could properly be homologized with the stomach of the Basommatophora. The giz- 
zard and, with few exceptions, sand in the tract are absent from the land pulmo- 
nates. The gizzard, he states, reaches its peak of specialization in L. stagnalis and 
probably rose by reason of the ingestion of sand with food. He observed that in all 
Basommatophora the gizzard originates in front of the first flexure of the gut, appar- 
ently as a muscular band whose primitive function was to dispose of sand masses 
tending to congest there. This primitive type of. gizzard is exemplified by that of 
Ancylus and the intermediate type by that of Planorbis. Heidermanns in support 
of his theory of the origin of the gizzard through a specialization of a primordial 
portion of undifferentiated gut, attempted to show modification of the gizzard in 
one snail generation by the use of various diets. As might be anticipated, he got 
no significant structural changes. 



ALIMENTARY SYSTEM OF LYMNAEA 109 

The fact that Lymnaca possesses the gizzard grinding mill may explain the ob- 
servation stressed by Heidermanns that the cellulase of this snail is less active than 
that of Helix which has not developed a gizzard and consequently needs a strong 
cellulase for the hydrolysis of the cell walls of plant food consumed. 

There is striking similarity in the functioning of the alimentary tract of the 
herbivore Onchidella ccltica, ably presented by Fretter (1943) in a recent paper, 
and that of L. s. apprcssa. Perhaps this similarity is not to be wondered at when, 
as Fretter writes, "Many of the features which the Onchidiidae share with the pul- 
monates may be attributed to the close origin of the two groups, the similarity of 
their diet and their air-breathing habit." 

SUMMARY 

1. A balanced physiological salt solution was developed which maintains con- 
tractions of the vas deferens for approximately 66 hours. 

2. Cathepsin was found in greatest concentration in the liver and no activity 
could be ascertained in the gut fluids. Some trypsin was indicated in the salivary 
glands. Amylase showed greatest activity in the salivary glands and the liver. 

3. Muscular activity of the alimentary system involves the manipulation of the 
mouth parts in the buccal mass, peristalsis in the remainder of the tract, marked 
pulsatory movements of the postesophagus, crop, pylorus and liver, and a kneading 
motion of the gizzard. The radula is moved principally by the action of the odonto- 
phore but also operates independently of it. 

4. The entire alimentary system, with the exception of the gizzard and parts of 
the buccal cavity, is ciliated. The cilia show definite directional streams which 
function in propelling food particles, in sorting food and in consolidating fine refuse 
particles with the aid of mucoid substances. 

5. Sand is consumed normally by the snail and is necessary for the proper 
functioning of the gizzard in the crushing of food particles. Very little trituration 
is performed by the mouth parts. 

6. The pylorus is composed of a complicated system of folds and passages and 
counter ciliary currents and functions as a filter which permits only the soluble and 
the finer food particles to pass into the liver. It shunts the undigested residues 
from the gizzard into the prointestine. 

7. In the liver the digestive cells function in secretion, assimilation-, intracellular 
digestion and excretion. The indigestible foods and the excretory products, as vari- 
ably shaped and colored inclusion bodies, are eliminated in vacuoles. 

8. The cecum functions in collecting the finer residues from the liver and forces 
them in a continuous string into the prointestine. 

9. The residual material coming from the gizzard, liver and cecum is charac- 
teristic for each organ and is readily identified as distinct in the fecal pellet. 

10. The prointestine is specialized in the final consolidation of gizzard, liver and 
cecal strings with the aid of cementing substances secreted by the basophilic flask 
cells and the basal cells. 

11. The rhythmic nature of the liver is disclosed principally by a study of the 
fecal pellets. 

12. L. s. apprcssa is an herbivore. Food bits are cut away by the radula and 
swallowed. In the buccal cavity the food receives mucus from the buccal gland 



110 MELBOURNE ROMAINE CARRIKER 

cells, mucous cells and the salivaries and enzymes from the latter. Temporary stor- 
age and initial digestion occur in the postesophagus. Digestive fluids pass up from 
the liver in the pulsatory movements of the stomach region which keep the fluid gut 
contents in constant circulation. The crop, gizzard and anterior portion of the 
retrocurrent passage of the pylorus comminute the food. Amebocytes present in the 
gut contents appear to aid in digestion. Soluble and fine particles of food pass 
through the pyloric filter into the liver where it is assimilated by the digestive cells. 
Assimilation also occurs in the pylorus and absorption possibly in the intestine. 
There is some evidence that the pulsatory movements of the stomach region cease 
during the passage of the gizzard and the liver strings. 

LITERATURE CITED 

AMAUDRUT, A., 1898. La partie anterieure du tube digestif et la torsion chez les mollusques 

gasteropodes. Ann. sci. nat., 7 : 1-291. 
ANSON, M. L., 1938. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. 

Jour. Gen. PhysioL, 22 : 79. 
BAECKER, R., 1932. Die Mikromorphologie von Helix pomatia und einigen anderen Stylom- 

matophoren. Zs. Gcs. Anat. Abt. III. Eryeb. Anat. n. Entwicklungsgesch, 29: 449-585. 
BAKER, F. C, 1900. The gross anatomy of Limnaea emarginata variety mighelsi Binney. Bull. 

Chicago Acad. Sci., 2: 191-211. 
BAKER, F. C., 1911. The Lymnaeidae of North and Middle America, recent and fossil. Special 

Publ. 3, Chicago Acad. Sci. 
BARFURTH, D., 1880. Die "Leber" der Gastropoden, ein Hepatopancreas. Zoof. Ans., 3: 499- 

502. 
BARFURTH, D., 1881. Der Kalk in der Leber der Helicinen und seine biologische Bedeutung. 

Zool. Anz., .4 : 20-23. 
BARFURTH, D., 1883a. Der phosphorsaure Kalk der Gastropoden Leber. Biol. CentralbL, 3 : 

435-439. 
BARFURTH, D., 1883b. Ueber den Bau die Tatigkeit der Gastropodenleber. Arch. mikr. Anat., 

Anat.. 22: 473-524. 

BERNARD, A. AND V. BONNET, 1930. Composition minerale de I'hemolymphe et etude d'une solu- 
tion physiologique pour 1'escargot (Helix pomatia). C. R. soc. biol., 103: 1119-1120. 
BRADLEY, H. C., 1938. On toluene in autolysis. Physiol Rev., 18: 173. 
CARRIKER, M. R. 1943a. Variability, developmental changes and denticle-replacement in the radula 

of Lymnae stagnalis appressa Say. Nautilus, 57 : 52-59. 
CARRIKER, M. R., 1943b. On the structure and function of the proboscis in the common oyster 

drill, L T rosalpinx cinerea Say. Jour. Morph., 73 : 441-498. 
CARRIKER, M. R., 1945. Morphology of the alimentary system of the snail Lymnaea stagnalis 

appressa Say. Trans. Wis. Acad. Sci., 38. 
CARRIKER, M. R. AND N. M. BILSTAD, 1946. Histology of the alimentary system of the snail, 

Lymnaea stagnalis appressa Say. Trans. Micros. Soc., 65 (3). 
COLTON, H. S., 1908. Some effects of environment on the growth of Lymnaea columella Say. 

Proc. Acad. Nat. Sci. Philadelphia, 60: 410-448. 
CUENOT, M. L., 1892. fitudes physiologiques sur les Gasteropodes Pulmones. Arch. Biol., 12 : 

686-740. 
CUVIER, G., 1817. Memoircs pour servir a I'histoire ct a I'anatomie dcs mollusques. Paris 

(Unavailable). 
DUVAL, M., 1928. The molecular concentration of the blood of some fresh water molluscs. 

Role of Carbonates. Ann. physiol. physicochim. biol., 4: 27-43. 
ENRIQUES, P., 1901. II fegato di molluschi e le sue funzioni. Mittlg. Zool. Station Ncapel, 15: 

281-407. 

ENRIQUES, P., 1902. Le foie des mollusques et ses fonctions. Acch. ital. Biol., 37 : 177-199. 
FAUST, E. C., 1920. Pathological changes in the gastropod liver produced by fluke infection. 

Johns Hopkins Hasp. Bull., 31 : 79-84. 
FOLIN, O. AND V. CIOCALTEAU, 1927. On tyrosine and tryptophane determinations in proteins. 

Jour. Biol. Chem., 73 : 627-650. 



ALIMENTARY SYSTEM OF LYMNAEA 111 

FRENZEL, J., 1886. Mikrographie der Mitteldarmdruse (Leber) der Mollusken. I. Allgemeine 

Morphologic und Physiology des Drusenepithels. Nova Acta Acad. Caesareae Leop.- 

Carol. Germ. Nat. Cur., 48: 81-296. 
FRETTER, V., 1943. Studies on the functional morphology and embryology of Onchidella celtica 

(Forbes and Hanley) and their bearings on its relationships, jour. i\Iar. Biol. Assoc. 

U. K., 25 : 685-720." 
GARTENAUER, H. M., 1875. Uber den Darmkanal cinigcr cinheimischen Gasteropodcn. Inaug.- 

Disscrt. Strassburg (Unavailable). 
GEDDES, 1879. On the mechanism of the odontophore in certain Mollusca. Trans. Zool. Soc. 

London, 10: 485-491. 
GRUNBAUM, S., 1913. Sur la cellule calcigere et ses corpuscules dans le foie d'Helix. C. R. 

soc. bio!., 75 : 208-210. 

HAWK, P. B. AND O. BERGEIM, 1937. Practical physiological chemistry, llth. ed., Philadelphia. 
HEIDERMANNS, CURT, 1924. Uber den Muskelmagen der Siisswasserlungenschnecken. Zool. 

Jahrb. (Abt. Allg. Zool. Physiol.), 41 : 335-424. 
HOWELLS, H. H., 1942. The structure and function of the alimentary canal of Aplysia punctata. 

Q. J. Microsc. Sci., 83: 357-397. 
HURST, C. T., 1927. Structural and functional changes produced in the gastropod mollusc, 

Physa occidentalis, in the case of parasitism by the larvae of Echinostoma revolutum. 

Univ. Calif. Publ. Zool., 29 : 321-404. 
KRIJGSMAN, B. J., 1925. Arbeitsrhythmus der Verdauungsdriisen bei Helix pomatia L. Zs, 

vcrgl. Physiol., 2 : 264-296. 
KRIJGSMAN, B. J., 1928. Arbeitsrhythmus der Verdauungsdriisen be Helix pomatia L. II. 

Teil : Sekretion, Resorption und Phagocytose. Zs. vcrgl. Physiol.. 8 : 187-280. 
MERTON, H., 1923. Studien iiber Flimmerbewegung. Pfliigcrs Arch. Physiol., 198: 1-28. 
MOORE, H. B., 1931. The systematic value of a study of molluscan faeces. Proc. Malac. Soc. 

London, 19: 281-290. 
MOQUIN-TANDON, 1885. Histoirc natitrcllc dcs Molhisqucs tcrrcstrcs ct fluviatiles dc France. 

Paris. 
NOLAND, L. E. AND M. R. CARRIKER, 1936. Observations on the biology of the snail Lymnaea 

stagnalis appressa during twenty generations in laboratory culture. Amcr. Midland 

Nat. (In press). 
PECZENIK, O., 1925. Uber intracellulare Eiweissverdauung in der Mitteldarmdruse von Limnaea. 

Ztschr. zn'iss. Biol. Abt. C. Vgl. Physiol., 2: 215-225. 

PELSENEER, P., 1935. Essai d'ethologie zoologiqnc d'aprcs I'ctudc dcs Molhisqucs. Bruxelles. 
PIERON, H., 1908. La localisation du sens de discrimination alimentaire chez les Limnees 

C. R. Acad. Sci. Paris. 147 : 279-280. 
SEMPER, C., 1857. Beitrage zur Anatomic und Physiologic der Pulmonaten. Ztschr. zviss. Zool., 

8 : 340-399. 
WALTER, H., 1906. The behavior of the pond snail, Lymnaea elodes Say. Cold Spring Harbor 

Monog. No. 6, Inst. Arts Sci., Brooklyn, New York. 
WETHERBY, A. G., 1879. Notes on some new or little known North American Limnaeidae. 

Jour. Cin. Soc. Nat. Hist., 2: 93-100. 
WHITFIELD, R. P., 1882. Description of Lymnaea megasoma Say, with an account of changes 

produced in offspring by unfavorable conditions of life. Bull. Amcr. Mus. Nat. Hist., 

1 : 29-38. 
WOODWARD, M., 1895. On the anatomy of Natalina caffra Fer., with special reference to the 

buccal mass. Proc. Malac. Soc. London, 1 : 270-277. 
YONGE, C. M., 1925. The hydrogen ion concentration in the gut of certain lamellibranchs and 

gastropods. Jour. Mar. Biol. Assoc. Plymouth, NS 13 : 938-952. 
YONGE, C. M., 1935. On some aspects of digestion in ciliary feeding animals. Jour. Mar. Biol. 

Assoc. Plymouth, 20 : 341-346. 
YONGE, C. M., 1936. Evolution and adaptation in the digestive system of the Metazoa. Biol. 

Rev., 12: 87-115. 



TEMPORARY PAIR FORMATION IN PARAMECIUM 

BURSARIA 1 



TZE-TUAN CHEN 

Department of Zoology, University of California, Los Angeles 

In Parame'cium bursaria, the two members of a conjugating pair normally re- 
main united twenty to thirty-eight or more hours. During this time various nu- 
clear processes take place, including three pregamic divisions, exchange and fusion 
of pronuclei, and three post-zygotic divisions. Clones that are capable of under- 
going normal conjugation as described above are called "normal clones." 

But there are some clones of this species which are abnormal - in that when they 
are mixed with normal clones the pairs formed are not lasting but separate within 
a few hours. An examination was made of such temporary pairs in order to dis- 
cover what nuclear or other changes occur in them. 

Fourteen clones of Parameciion bursaria, all belonging to Variety I, have been 
used in the present study. These clones, all of which were collected in nature, are 
listed in Table I with data on each clone including (1) the mating type to which 
it belongs and (2) the locality where it was collected. 

TABLE I 

Clones of Paramecium bursaria employed in study of temporary pair formation 



Clone number 


Original designation 
of clone 


Mating type 


Locality collected 


1 


SAaS 


A 


Santa Ana River, Cal. 


2 


Or3 


A 


Vicinity of Capistrano, Cal. 


3 


SGa3 


A 


San Gabriel River, Cal. 


4 


BG35 


A 


Los Angeles, Cal. 


5 


La3 


B 


Laguna Canyon, Cal. 


6 


SAa7 


B 


Santa Ana River, Cal. 


7 


UC13 


B 


Los Angeles, Cal. 


8 


BH2 


B 


Beverly Hills, Cal. 


9 


SAa4 


D 


Santa Ana River, Cal. 


10 


LP10 


D 


Lone Pine, Cal. 


11 


UC14 


D 


Los Angeles, Cal. 


12 


SAal 


C 


Santa Ana River, Cal. 


13 


BH7 


C 


Beverly Hills, Cal. 


14 


BH101 


C 


Beverly Hills, Cal. 



Clones 1-11 are normal clones in that they are capable of undergoing normal conjugation. 
Clones 12-14 are abnormal clones in that when they are mixed with normal clones the pairs formed 
are not lasting but separate within a few hours. 

1 This work was aided by grants from the Committee for Research in Problems of Sex, 
National Research Council ; and from the Joseph Henry Fund of the National Academy of 
Sciences. 

- These clones are considered abnormal here only because they are incapable of taking part 
in the formation of lasting pairs when they are mixed with other, normal clones. 

112 



TEMPORARY PAIR FORMATION IN PARAMECIUM 113 

The animals were cultured in essentially the manner described by Jennings 
(1939). For cytological study the animals were fixed in Schaudinn's fluid con- 
taining glacial acetic acid, stained in iron hematoxylin, and destained in saturated 
aqueous solution of picric acid, following the technique the writer has described 
(Chen, 1944). 

EXPERIMENTAL STUDIES 

Most of the present work was done with the two abnormal clones (BH7, SAal), 
although some study was also made on a third abnormal clone (BH101). All of 
these three abnormal clones belong to mating type C of Variety I. These abnormal 
clones were mixed with normal clones. Eleven such normal clones (all belonging 
to Variety I) were used. Four of these normal clones belong to mating type A; 
four to type B ; and three to type D (see Table I). 

As an example of the phenomenon of temporary pair formation, the reaction be- 
tween the abnormal clone SAal and the normal clone UC13 will be described. On 
November 27. 1940, a large number of animals belonging to each of these two clones 
were mixed at about eleven o'clock in the morning. Strong agglutinative mating 
reaction occurred almost immediately. Half an hour after mixture, pairs were 
being formed. An hour after mixture (about noon) many pairs were formed. 
But in the early afternoon the pairs broke apart into single animals. By five o'clock 
all but a few pairs had separated. By evening all had separated. 

Such temporary pair formation was also observed when the abnormal clone 
SAal was mixed with the following normal clones: Or3, SGa3, SAaS, La3, SAa7, 
BH2, SAa4, LP10. and UC14; or when the abnormal clone BH7 was mixed with 
the normal clone LP10; or when the abnormal clone BH101 was mixed with the 
normal clone BG35. 

If such a mixture was placed in a moist chamber and kept from drying (with 
occasional replacement of the fluid that evaporated), the typical agglutinative mating 
reaction and temporary pair formation recurred the following day and almost daily 
over a period of many days. Some such mixtures were kept under daily observa- 
tion over a period of nineteen days. The following is the characteristic daily be- 
havior of the animals in such a mixture. The agglutinative mating reaction occurs 
in the late morning. By noon, many pairs are formed. These pairs persist for a 
few hours. Between four and six o'clock in the afternoon only a few pairs are 
found. In the early evening one or two pairs may remain ; none can be found after 
nine o'clock in the evening. 

CYTOLOGICAL STUDIES 
Nuclear conditions in the clones that jorm only temporary pairs 

The writer has made a cytological study of twenty-one abnormal clones, includ- 
ing the two clones BH7 and SAal, and nineteen of the twenty-two such clones de- 
scribed by Jennings (1944). It was found that fifteen of these clones possess 
micronuclei, while six appear to be amicronucleate. 

Thus the amicronucleate condition is not the general cause of the peculiar be- 
havior of these abnormal clones. It is probable that the persistence of the amicro- 
nucleate condition is a result of the inability to conjugate and acquire a micronucleus, 
rather than the cause of it. Apparently there are conjugating and non-conjugating 



114 TZE-TUAN CHEN 

races of amicronucleate ciliates. In nature those that can conjugate do so and ac- 
quire a micronucleus, leaving in the amicronucleate condition only those incapable 
of conjugation. In my experience with P. bursaria, which includes a study of the 
nuclei and chromosomes of many clones (collected from different parts of the United 
States. Canada, Russia, England, Ireland, and Czechoslovakia), the only amicro- 
nucleate animals found in nature are those which cannot conjugate. Since they can- 
not conjugate, it is likely that such clones will be permanently amicronucleate. In 
nature any amicronucleate animal that can conjugate would not remain amicro- 
nucleate for long, since it would become micronucleate after mating with a normal 
animal from whom it receives a pronucleus as a result of conjugation (Chen, 1940). 
Amicronucleate animals that can conjugate have been found in P. bnrsaria (Chen, 
1940) 3 and in Euplotcs patella (Kimball, 1941). They arose spontaneously in labo- 
ratory cultures. 

Nuclear changes in temporary pair jormation 

To determine whether nuclear changes occur in temporary pair formation, a series 
of preparations were made, in December, 1940, of temporary pairs (abnormal clone 
SAal X normal clone UC13) 4 and a number of separated animals belonging to the 
latter clone. The material included pairs 5 to 6 hours after onset of temporary 
mating, separated animals a few hours after separation, 13 to 17 hours after separa- 
tion, and 21 hours after separation. The micronuclei in these temporary pairs and 
separated animals were compared with the micronuclei of vegetative animals of 
clone UC13 (not mixed with any other clone). It was found that micronuclei in 
the majority of the temporary pairs and of the separated animals were slightly 
swollen. In some, no nuclear changes were apparent. 

In June, 1943, a series of preparations were made of temporary pairs (abnormal 
clone BH101 X normal clone BG35) 5 and a number of separated animals belonging 

3 The writer has recently found some additional cases of conjugation between amicronucleate 
and normal animals in Pafaincciinn bnrsaria, in Variety III. (Normal nuclear changes occur 
in the conjugants having the micronuclei.) These amicronucleate animals arose spontaneously 
in laboratory cultures. 

Schwartz (1939) in a brief preliminary paper reported "conjugation" in Paramecium 
bursaria between amicronucleate and normal animals and between two amicronucleate animals. 
In view of the lack of details in this report, it is impossible to tell whether temporary or lasting 
pair formation took place. 

4 Clone SAal appears to be amicronucleate; clone UC13 has a deeply staining micronucleus. 

5 Clone BH101 has a small, lightly staining micronucleus ; clone BG35 has a relatively large, 
deeply staining micronucleus. 

EXPLANATION OF FIGURES 

FIGURES 1-34. Micronuclei of animals belonging to clone UC13 before, during, and after 
temporary pairing with animals of clone BH7 (drawn by Mr. Earl Nielsen). All drawings 
were made with a camera lucida. X 3,300. 

FIGURES 1-5. Resting micronuclei of vegetative animals. 

FIGURES 6-10. Micronuclei in the members of temporary pairs 4 hours after onset of pair- 
ing. 

FIGURES 11-16. Micronuclei in the separated animals 18 hours after separation. 

FIGURES 17-22. Micronuclei in the separated animals 30 hours after separation. 

FIGURES 23-28. Micronuclei in the separated animals 42 hours after separation. 

FIGURES 29-34. Micronuclei in the separated animals 51 hours after separation. 



TEMPORARY PAIR FORMATION IN PARAMECIUM 



115 










6 






1 29 






17 




c 

& 

v- 



II 









12 



,' f 

' r 



1 



25 



31 

FIGURES 1-34. 



13 



J'i 



14 










116 TZE-TUAN CHEN 

to the latter clone. The material included pairs 3 to 4 hours after onset of temporary 
pairing, and separated animals 2 hours after separation, and a day after separation. 
It was found that the micronuclei in the majority or most of the temporary pairs 
and separated animals were slightly swollen. In others no nuclear changes were 
apparent. 

In October, 1944, a series of preparations were made of the temporary pairs (ab- 
normal clone BH7 X normal clone UC13) 6 and separated animals belonging to the 
latter clone. The material included pairs 4 hours after onset of temporary mating, 
and separated animals 7 hours after separation, 18 hours after separation, 30 hours 
after separation; 42 hours after separation, 51 hours after separation. A series of 
preparations of vegetative animals of clone UC13 (not mixed with any other clone) 
were used as controls (Figs. 1-5). It was found that the micronuclei in nearly all 
of the temporary pairs and separated animals were slightly but noticeably swollen 
(Figs. 6-34). This is true even of the separated animals 51 hours after separation 
(Figs. 29-34), indicating that the physiological effect of the contact between the 
animals in temporary pairing (as shown by the swelling of the micronucleus) is of 
long duration. 

GENERAL RELATIONS 

The temporary pair formation described in the present paper is similar to that 
reported by Sonneborn (1942) in P. aurclia and by Jennings (1944) in P. bursaria. 
Sonneborn (1942) concluded from his data that cell adhesion occurring in the initial 
stage of the mating reaction and cell fusion occurring during subsequent conjugation 
are due to two different mechanisms. 

SUMMARY AND CONCLUSIONS 

1. In normal conjugation of Paramecium bursaria, the two members of each pair 
remain united for 20 to 38 or more hours, during which time various nuclear proc- 
esses take place including three pregamic divisions, exchange and fusion of pro- 
nuclei, and three post-zygotic divisions. Clones that are capable of undergoing nor- 
mal conjugation as described above are called "normal clones." 

2. Some clones of this species are abnormal in that when they are mixed with 
normal clones the pairs formed are not lasting but separate within a few hours. 

3. In temporary pair formation, the animals of diverse mating types when mixed 
exhibit the typical agglutinative mating reaction. Within an hour many pairs are 
formed but in a few hours these pairs break apart into single animals. 

4. If such a mixture is placed in a moist chamber and kept from drying (with 
occasional replacement of fluid that evaporates) such agglutinative mating reaction 
and temporary pair formation will recur daily over a period of many days. 

5. Cytological study of 21 such abnormal clones shows that most of these clones 
have micronuclei ; some appear to be amicronucleate. Therefore, amicronucleate 
condition cannot explain the incapacity for taking part in the formation of lasting 
pairs. It is probable that the persistence of the amicronucleate condition is a result 
of the inability to conjugate and acquire a micronucleus rather than the cause of it. 

6. In temporary pair formation, there are no conspicuous nuclear changes either 
in the pairs or in the animals after their separation. In the majority of the tempo- 

G Clofie BH7 appears to be amicronucleate. 



TEMPORARY PAIR FORMATION IN PARAMECIUM 117 

rary pairs and separated animals, there is, however, a slight swelling of the micro- 
nuclei. This swelling persists for a considerable length of time after the separation 
of the animals, indicating that the physiological effect of the contact between the ani- 
mals in temporary mating (as shown by the swelling of the micronuclei) is of long 
duration. 

LITERATURE CITED 

CHEN, T. T., 1940. Conjugation in Paramecium bursaria between animals with diverse nuclear 

constitutions. Jour. Hercd., 31 : 185-196. 

CHEN, T. T., 1944. Staining nuclei and chromosomes in Protozoa. Stain Tcchn., 19 : 83-90. 
JENNINGS, H. S., 1939. Genetics of Paramecium bursaria. I. Mating types and groups, their 

interrelations and distribution ; mating behavior and self-sterility. Genetics, 24 : 202- 

233. 
JENNINGS, H. S., 1944. Paramecium bursaria: life history. I. Immaturity, maturity and age. 

Bio!. Bull., 86: 131-145. 
KIMBALL, R. F., 1941. Double animals and amicronucleate animals in Euplotes patella with 

particular reference to their conjugation. Jour. Exp. Zool., 86 : 1-32. 

SCHWARTZ, V., 1939. Konjugation micronucleusloser Paramaecien. Natumnss., 27: 724. 
SONNEBORN, T. M., 1942. Evidence for two distinct mechanisms in the mating reaction of 

Paramecium aurelia. Anat. Rec., 84 : 542-543. 



Vol. 91, No. 2 October, 1946 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



THE SPACE-TIME PATTERN OF SEGMENT FORMATION 

IN ARTEMIA SALINA 

PAUL B. WEISZ 

Department of Zoology, McGill University, Montreal, Canada 

i 

INTRODUCTION 

The present work was carried out in an attempt to arrive at a primary under- 
standing of the regularities and laws in the phenomenon of metameric segmentation, 
as related to the shape and size of animals. To date this phenomenon, although of 
widespread occurrence amongst the higher animal phyla and thus probably an in- 
tegral part in the more complex patterns of evolutionary organization, was never- 
theless surprisingly rarely, if at all, subjected to analytical inquiry. The reason 
for this can probably be found in an essential lack in the past of well-defined con- 
cepts about the interrelations between mass, shape, growth, and degree of develop- 
ment of living organisms. The problem of segment formation in relation to size 
and shape is primarily one involving a clear appreciation of the dynamic geometry 
of living matter, and initial insight into the problem can therefore only emerge from 
rigorous observation on a quantitative level, followed preferably by geometrical 
and mathematical analysis. Such a method has been employed in the present 
work, and the results gained are conclusive enough not only to point the way for 
further study of the problem at hand, but also to promise reasonable success in the 
application of the quantitative, geometrical method to questions of biological space- 
time pattern in general. 

The choice of Artemia has proven particularly fortunate for a study of meta- 
meric segmentation. The animal, held to be amongst the most primitive of living 
Crustaceans (Lockhead, 1941), develops few, if any, specialized structural features 
which would ordinarily tend to obscure the fundamental processes of morphogenesis. 
Moreover, the development of as many as nineteen body segments, a further primi- 
tive trait, is of obvious advantage in the investigation of the underlying principles 
of formation. Also, Artemia is easily obtained and can be reared in the laboratory 
without difficulty. 

METHODS AND MATERIALS 

Larvae of Artemia salina were obtained from commercial, air-dried egg cysts. 
Since excystment is retarded or inhibited in water above a certain salinity (Jennings 
and Whitaker, 1941), water of a specific gravity of 1.020 was used throughout as 
the initial medium. The egg shells cracked open usually 12 to 18 hours after 
contact with the water, and emergence of the larvae (Whitaker, 1940) took place 

119 



120 PAUL B. WEISZ 

between 18 and 24 hours. Portions of five stock solutions of brines with different 
salt concentration were employed as further media. The solutions were obtained 
from the original sea water by either diluting with doubly glass distilled water or 
concentrating with NaCl to specific gravities of 1.022, 1.033, 1.047, 1.066, and 
1.085, respectively. All solutions were vigorously aerated daily, and possible 
deviations from the proper specific gravity were adjusted in weekly intervals. All 
work was carried out at room temperature, corresponding to an average water 
temperature of 21-22 C. As soon as the embryos had emerged, still enclosed 
within the fine hatching membrane, they were transferred to water from either of 
the five stock solutions. The moment of hatching, occurring within 24 to 30 hours 
after the cysts had first made water contact, was taken as zero time for all further 
determinations. 

For observation the larvae were reared singly, in heavy crystal watch glasses. 
In the course of several observational series, a total of up to 100 individuals were 
observed, at least for certain periods of their development; of the 100, about 25 
individuals, evenly distributed among the solutions, were reared from hatching to 
the adult stage. The presence or absence of a molted shell, the time, the tempera- 
ture, the stage of development reached, and a series of measurements on bodily 
proportion were recorded for each individual twice daily in the earlier stages and 
daily for later stages. The animals were fed once every two days on a sea-water- 
yeast suspension. Each watch glass containing an animal was covered so that 
evaporation was nearly abolished, but a minimum of air circulation was always 
allowed for to equilibrize the CO 2 released by the yeast and the animal. The water 
was changed at two-day intervals for the younger stages and daily for older ones. 

Larval body measurements were taken under the microscope with the help -of 
a hemocytometer slide whose grid allows the direct reading off of lengths of 50 
micra and consistent estimations of lengths of 10, 20, and 30 micra. If the larva 
is placed on a coverslip with a minimum of water, the whole can be adjusted in 
relation to the grid ; evaporation is sufficiently slow to allow five or six measure- 
ments at a time. The error inherent in this method, viz., the parallax due to the 
thickness of the coverslip, is small enough to be negligible ; also, since all measure- 
ments were taken in this way, the relative values are consistent. 

PRELIMINARY OBSERVATIONS 

Barigozzi (1939) and Rugh (1941) observed the total developmental time from 
hatching to the adult stage of Artemia to be 3 to 4 weeks. This is true as a broad 
generalization, but with the egg cysts in the various salinity media here employed, 
certain statistically preferred tendencies become apparent, expressed empirically by 



r)_r). o 

*-> x k'O ^ 



-e.s 



where D x is the time, in days, for complete development from hatching in a solution 
of salt concentration x\ D , the (hypothetical) time, similarly, for development in 
distilled water (the algebraic value turned out to be 36.55) ; and S x , the specific 
gravity of the solution of concentration x. This relation holds good only in a 
statistical sense, within a specific gravity range of 1.020 and 1.1 ; it indicates that 
with higher salt concentrations the rate of development tends to be greater (Fig. 1). 
Irrespective of concentration a sigmoid curve of growth is always obtained. 



ARTEMIA SEGMENTATION PATTERN 



121 



Morphologically, different salinities have no differential effect on relative body 
proportions, a result to be expected in view of the conclusions of Bond (1932). 
An inverse relation between total size and salinity, observed by Bond, Heath 
(1924), and Warren (1938) for larvae from non-excysted eggs in the natural 
habitat, however, could not be observed for the excysted larvae here used; in the 
latter, total sizes are identical at equivalent stages of development, irrespective of 
salinity. 

The number of molts between hatching and sexual maturity is not constant. 
Even when reared in the same medium, slight differences in molting frequency 
between several larvae may occur. Moreover, there exists a rough statistical 
relation between salinity and the total number of molts, approximating closely the 



I/ S4 5 fi 7 8 9 10 II 12 13 14 15 IS 17 




05 



FIGURE 1. The effect of salinity on developmental time, from hatching to sexual maturity; 
absolute growth curves. Abscissa: total larval length; Ordinate: time in days. A-E, media 
of salt water, specific gravities from 1.022-1.085 respectively; numbers 1-19 above abscissa refer 
to number of body segments present. 

above relation between salinity and the time required for complete development; 
in general, however, the number of molts for a given salinity is somewhat lower than 
the number of days required for development. In larvae from excysted eggs and 
under artificial food conditions, a range of 12 to 16 molts was observed between 
hatching and maturity, at a specific gravity of 1 .085 ; this compares with 25 to 29 
molts at a specific gravity of 1.022, and gradually decreasing molting frequencies 
for the intermediate salinity ranges. A staging of larval development according 
to molts, as Heath has done for non-excysted individuals, would therefore not be 
possible in the present case. Heath's 13 stages would hold for excysted larvae 
only when reared in brine of a specific gravity of 1 .085 ; even then certain definite 
differences in the degree of development of equivalent molting stages can be 



122 PAUL B. WEISZ 

observed, as comparison of Heath's descriptions with those below makes apparent. 
With increasing developmental age the duration of instars increases; a 12 to 
24 hour interval between molts in the younger stages compares with 24 to 30 
hour intervals in older ones. The two factors of salinity and developmental age 
also determine the size increase between molts ; for higher salinities, as well as for 
older larvae, the size increase is greater. There is no observable relation however 
between the time at which a molt occurs and the size or the developmental stage 
attained, irrespective of whether test larvae are reared in the same or at different 
salinities. Molting is also greatly influenced by the food supply. Starving animals 
do not molt ; after 3-5 days an abortive attempt at molting is made which usually 
results in the death of the animal. Conversely, overfed larvae may molt twice in 
rapid succession without undue increase in size. 

ANALYSIS OF SEGMENT FORMATION 
Observations and definitions 

The larval development of Artemia can best be dealt with in terms of the 
number of body segments present. The first three segments become visible almost 
simultaneously at a total larval size of 0.745 mm. (stage 3), after the embryonic 
yolk has been digested away, and the termination of the hatching, nauplius, and 
metanauplius stages can therefore be represented as the termination of stages 0, 1, 
and 2, respectively ; at the end of any following stage the stage number will thus indi- 
cate directly the number of body segments present. It will be convenient to distin- 
guish between a thoracic period of development, comprising the first 11 stages, and 
an abdominal period, including stages 12 to 19; the latter can again be divided into 
a genital period (stages 12 and 13) and a post-abdominal 'one (stages 14 to 19). 

Except for the first three, each individual segment is initially recognizable as a 
transverse ring of thickened mesoderm, the segment rudiment, immediately under- 
neath the otherwise smooth epidermal layers (segmental stage a). Later, partial 
transverse constrictions appear externally in the epidermis and the chitin, in a 
plane just posterior to that of the segment rudiment (segmental stage b}. Even- 
tually, the constrictions become complete and deepen, with a concomitant bulging 
out of the body wall in the region of the segment rudiment (segmental stage c). 
At this stage, the segment can be considered "laid down," its shape resembling 
more or less a short cylinder. In thoracic segments, appendage buds appear in 
stage c ventro-laterally, on either side. The segments are considered mature when 
their pairs of swimming appendages first become independently motile. Stages a 
to c of the first and second, and stages a and b of the third segment can never be 
clearly seen; the first stages of these segments are attained prior to hatching and 
during the nauplius and metanauplius phases, when the presence of dense yolk 
conceals details of structure. As these segments become plainly visible in the 
third stage of the thoracic period, segment 3 is in stage c, but segments 1 and 2 
are already correspondingly ahead, both in size and the degree of their development. 

At the end of the thoracic period the llth segment has reached stage c and the 
first five segments have become mature. The llth segment attains maturity at 
the end of the abdominal phase of development (stage 19). Appendage buds 
similar to those on more anterior segments also develop on segments 12 and 13. 
But instead of developing into swimming appendages the buds on either side of 



ARTEMIA SEGMENTATION PATTERN 123 

both segments enlarge, and in the female fuse into a sac in stage 18, forming the 
left and right brood pouch; no male larvae were investigated. The remaining six 
segments develop similarly from segment rudiments, but no appendage buds are 
ever formed and stage c represents the first stage of maturity. At the end of the 
abdominal period the 19th and last segment has become mature. In the head, the 
ocellus becomes pigmented in stage 2 and the compound eyes in stage 4. The 
maxillae and maxillulae also form in stage 2. The end of stage 19 marks the time 
when the gnathobase and the setae have been lost entirely from the second antenna. 
After stage 19 an arbitrary number of non-segmental stages ensue before sexual 
maturity is reached. 

When individuals in identical stages of development from the same or from 
different salinity media are compared, it is strikingly apparent that total lengths 
and body proportions in general fall within well-defined size-classes ; the deviations 
from the underlying averages in no case exceed 3 per cent. In Table I the 
averages of a variety of body measurements are shown, from the 25 individuals 
watched throughout development, with the stage number as the basis of calculation ; 
these values, within 3 per cent, are true for individuals from any of the salinities 
here examined. A schematic diagram of an Artemia larva indicates, in Figure 2, 
how the various entities have been defined. Head length is understood to include 
maxillar and maxillular segments. The length of a segment refers to axial and 
the width to its lateral extent. Total abdominal length is the length of the seg- 
mental portion, whether actually cut up into segments or not, plus the length of a 
terminating anal piece; the segmental portion is the pygidium of annelid forms, 
and in Artemia is readily distinguished from the anal piece, or urosome, by a con- 
striction. During the abdominal period of development, the segmental abdomen 
contains a genital region composed of segments 12 and 13, as well as a post- 
abdomen (presumptive segments 14 to 19) with segmented and non-segmented 
portions. 

From observation and from examination of the data in Table I the following 
facts concerning the formation of segments in relation to larval shape and size are 
consistently found to occur : 

1. Every thoracic segment when newly formed (stage c) has a fixed length 
of 0.03 mm. and a fixed width of 0.144 mm. 

2. Every time a new thoracic segment is laid down in stage c, preceding seg- 
ments increase in length and in width. 

3. Throughout the thoracic period, the segmental part of the abdomen has a 
constant average length of 0.249 mm.; its anterior width, being slightly smaller 
than the width of the newest segment, is also constant (C = 0.142 mm.). 

4. During the thoracic period, the lateral contour-lines of the thorax are straight 
lines converging posteriorly; the lateral abdominal contour-lines are also straight, 
but generally they converge with a greater degree of taper than the thoracic contours. 

5. Appendages are longer the more anterior they are; the line joining the tips 
of the appendages on one side of the body is more or less a straight line. 

6. As the llth segment appears in stage c, the 5th segment has matured and 
the 19th segment has appeared in stage a. 

7. Between stage a and stage c of the thoracic segments, 4 stages of larval 
development intervene ; an interval of more than 4 developmental stages is neces- 
sary for an abdominal segment to reach stage c from stage a. 



124 



PAUL B. WEISZ 



TO. 
"t* 





W 
_1 

M 



^ 

"S 



S3 


o o 


o o 


00 


0- 


OO O 


<r. 


coc^ 




^^ 


^^ 


3cu > o |i 


^ 


O 


o o 


o o 


o o 


o o 





O 


o o 


o o 


o o 


*! bo M 


C/3 














to 10 


OO O 
** IO 


^^ -t*** 


S3 


J s J " 




















o o 


o o 


o o 


25-j^ 

IS co tf 


< 















to NO 


O to 

t~- OO 


o o 

ON 


o o 

CN CN 


- . - LH fe 

me" 

00 W ---C 


^ 














o o 


o o 





O 


=3 f t: *" 

^ ,- S -s 


ij 

f-H 


33 


%% 


%% 


33 


33 


3 










w) ^ g 03 

'5 S 5 - 




^ O 


o o 


o o 



















c c " 

.3 <u o js 


' 


03 


^^ l_O 
IO IO 


So 


s 


o^ ^-O 

^^ fV,J 

rxi CN 


cs 


CN CN 


CN CO 


CO co 


.0.- 


C T, *-" 

sip 




o o 


o o 





o o 





o 


O 


o o 


o o 


O 


= S^ *-s 

^ rt -5 ,- -S 


W 
















NO OO 

o o 


ON CN 


to oo 


(J ~ 

S 2 '* 


















o o 


o o 


o o 


HJSl 


co 


o o 


o o 


o o 








q 


o o 


o o 


o o 


IO IO 

o o 


1 6 1 

0/3 ^ -^ C 


H 





o o 


o o 


O 


o o 





o o 


o o 


o o 


O 


f^f |S 


(_i 


^^ ^^ 
<^5 ^) 


ss 


v^^ t^ 

C^ (^5 


; 


2g 


g 


> 


3 


S?K 


10 o 

t 00 


- M u 

j:- 8.2 

r/N 4-1 - u 




o o 


o o 


o o 


o o 


o o 


o 


o o 





o o 


o o 


l/J Mt/3 o ft) 

-^ ^ 'o c 




o o 


O ^H 


s 


5 CO 





X, 


f "N u~) 
t~~) ^.J 

C^l CN 


CN CN 


CN CN 


to*- 


c^i .- g 

i<~ju- 




o o 





o o 


o o 





c 


o o 


o o 


o o 


o o 


2^ < > 

a - S _ s 


a. 
















^o ^o 

o ~ 

o o' 


r- oo 

o' o" 


10 00 

r- 


to 13 " -- 

c ^-^ S 

1- l 1 ^ 1 


CH 














O to 
ON CN 
CN CO 


00 10 

fj ^d^ 


OO co 
to r^ 


s 2 


Slllf 
















o o 


o o 


o o 


O -H 


*O S - > " 

O ' ^ c 


J 














^t* ^^ 

O " 


CN-* 


IO to 

NO OO 


ON CO 


U ri M .5 

^S ..> s 

nS C -^ o 


O 




















o o 


o o 


JJ -'o 

3 cfl S tl J3 


< 


12 ?o 


ss 


els 


ss 


5 ^ 


s 










"o^S^ 

C/3 -D C uj 




o o 


o o 


o o 


o o 


o o 













<s^3lf5 


J 


o*> i o co 


33 





iS 


^D ^D 


5 


NO 


So 


o^ ^2 


IO IO 


2< g "-S 
2 .- a .I-S 


H 


o o o 


o o 


o o 


o o 


o o 


o 


o o 


o o 


O *-H 


1 1 


e-S"2 S3 


H 


^^ ^~^ 
""") ^^ 


ss 


is 


f^l ^^ 

^^ CO 


^^ ro 

^^ ^o 


i 


SS5 


So 


s$ 


o o 

IO I 


I ?. ^ 
i-S i?J 




O 


O 


o o 


o o 


o o 





o o 


O -H 






2 -a |3g 


J 
K 


^o o^ ^^ 

f^ ^^ ^t* 


s 


3 


s 


f^l t"** 

^^ ^^ 





s? 


5 


,, 


O 

o 10 

NO NO 


cj jy m I, 

^^ a^ 

JJ o o .. 




o o o 


o o 


o o 


o o 


o o 


o 


o o 


o o 


o o 


o o 


P ^ _ rt 4-> 

M^-5^ s 


o 


S!3 


^ s 


So 


JQ CO 


00 


^ 


IO ^^> 
IO ^^ 


IO N^^ 

IO ^^ 


s J 


O 

10 o 

** 


*H-f28 

^ .-^-5 w> 


H 


o o o 


o o 


O *-H 










CXI CN 


CN CO 


co ^ 


J= . M 
i- i C 


















^ IO 


\O t^ OO 


CO ON 


^ c<~'5 


g 






















i s^ x-- s 


CO 
















NO t- 


00 ON O 


CN *-( 


l^w^ E 

c ^ >- 


6 


^^ ^_H fS] 


co ^ 


.ONO 


t-00 


ONO 


,_, 


CN CO 


^10 


NOt-00 


ON 


n/|^fS 

w c S 
























. j J 6 

O HrH M U 


BJ 


^^ l-O 1 >O 


*-oO 


ONO 


> i rO 


UO J- 


OS 










^: ^ " u <* 

^ .. g UH 

J3 .5 o 


o 






















3 W) J3 
g C O rt " 

H, m -S >- "O 


a| 


f^l ^H pvl 


co * 


10 NO 


-00 


ON 


4 


CN CO 


^ 10 


NO*- 00 


ON 


oo i z o 'S 

o> J^ > 
J -3 5 - * 


g 
1 W 2 






















^ < 

JS S.JS "$ 



ARTEMIA SEGMENTATION PATTERN 



125 



8. Between stage c and maturity in the first 5 segments, 6 stages of develop- 
ment intervene; 8 stages intervene before maturity of segments 6 to 11. 

9. The anterior width of the non-segmented portion of the abdomen during the 
abdominal phase is of a fixed and constant magnitude and identical to the constant 
anterior width of the abdomen during the thoracic phase ; thus at the end of stage 




wu 



FIGURE 2. Schematic diagram of a larva of Artcmia salina. A, larva in the thoracic 
period ; B, larva in the post-abdominal period of development. A, length of segmental abdomen 
(pygidium) ; a, angle of thoracic taper; /3, angle of abdominal taper; C, Wo, WA , constant 
anterior width of segmental abdomen; GL, length of genital region; H, head length; NSPA, 
non-segmented post-abdomen ; P, length of post-abdomen ; PS, PS m , length of a post-abdominal 
segment; SPAL, length of segmented post-abdomen; SR, segment rudiment; T, T n , T m , length 
of thorax ; TAL, total abdominal length ; TS, TS n , length of first thoracic segment ; TSL, Ti, 
length of last (newest) thoracic segment: Tot.L, total larval length; U, urosomal length; W, 
W n , width of first thoracic segment; WTSL, Wi, width of last thoracic segment; WA, WA r , 
width of first abdominal (12th) segment; WASL, width of last (newest) abdominal segment; 
WU, anterior width of urosome. 



126 PAUL B. WEISZ 

19, the width of the urosome is the same as the posterior width of the head in 
stage when segmental development started. A constant width has seemingly 
travelled down the larva. 

10. At the beginning of stage 12, the segmental abdomen starts to grow in 
length at a fast rate, having retained a constant length in the thoracic period. 

.11. Segments 12 and 13 are of equal length at any time after their formation; 
they are individually always somewhat longer than the llth thoracic segment and 
become progressively shorter, relatively, than the 14th segment. At stage 18, 
6 developmental stages after segment 12 has reached stage c, segment 12 and 13 
fuse to form the brood pouch in the female and can then be considered matured. 
The interval for attainment of maturity is thus equal to the similar interval in the 
first 5 thoracic segments. 

12. Segments 14 to 19 are not of equal length when formed ; more posterior 
segments, when formed, are longer than more anterior ones when formed. Also, 
any one of these segments has always one-sixth of the length of the post abdomen, 
and at a given time post-abdominal segments are of equal length. 

13. When segment 19 reaches stage c, the llth segment attains maturity. 

14. During the abdominal phase, the thorax changes shape in the following 
way: the 2nd segment becomes longer and wider than the 1st, then the 3rd larger 
than the 2nd, etc., and the 5th becomes the largest, coincident with the end of 
stage 19. As a result, the lateral thoracic contours become curved, the widest part 
of the thorax being at segment 2 in stage 16, at segment 3 in stage 17, etc., and at 
segment 5 in stage 19. 

15. Similar differential increases take place in the appendages; at stage 19, 
the 5th pair of swimming appendages is longest and appendageal length regularly 
decreases towards the 1st and the llth pair. The line joining the appendage tips 
on one side of the thorax is now also curved. 

16. During the abdominal phase, and paralleling the differential increases in 
the thoracic segments, a progressive dorsal thoracic curvature develops, with an 
analogous shift backwards of the maximal flexure ; the latter arrives similarly at 
segment 5 at the end of stage 19. Due to this flexure the head now appears bent 
ventrad. 

17. The lateral contours of the abdomen remain straight lines throughout the 
abdominal phase, with a definite taper directed backwards. 

18. At the end of stage 19 segmental development is completed ; further devel- 
opment is still to take place in the head. The essential overall shape of the animal 
as now established, i.e., the possession of a barrel-shaped thorax and a straight 
tapering abdomen, is carried through to sexual maturity, although changes of detail 
do still occur. 

These observations are now to be interpreted and integrated analytically. 

The thoracic phase of development 

The lengths of the first thoracic segment, in successive stages, are 0.03, 0.04, 
0.05, 0.058, 0.065, 0.076 mm., etc. (Table I). The differences between these 
values, taken for all 11 thoracic stages, are very close to an average difference of 
0.0097 mm. The length of the first thoracic segment in successive stages can 



ARTEMIA SEGMENTATION PATTERN 127 

therefore be expressed as an arithmetical series 

TS n = (TSi -- TS ) + (n-- l)-Aj (1) 



where TS n refers to the length of the first thoracic segment at stage n; n, to the 
successive stage numbers from 1 to 11 (and thus to the number of segments 
present at the time) ; (TS\ TSo), to the initial length of the first segment at 
the end of stage 1 ; and As, to the increase of segmental length per stage (0.0097 
mm.). The expression would mean that the first segment grows in length by a 
constant amount As during each stage. 

Since every other thoracic segment is known to start off with an identical value 
for (TSi TSo), viz., 0.03 mm., it could be possible that other thoracic segments 
also increase a constant amount As during each stage. If that were true, then the 
newest segment, at any given stage, would have a length of (TSi - TS Q }, the 
segment immediately preceding it a length of (TSi -- TSo) + As, the third but 
last a length of (TSi - TS ) + 2As, . . . etc., and the first segment again a 
length of [(7\S"i -- TSo) + (n -- l)-As]. In other words the length of the entire 
thorax, being the sum of individual segments, should be the sum of an arithmetical 
series whose first term is (TSi - TS ) and whose last term is [(TSi T5" ) 
+ (n -- l)-As], This can be put as 



T n = n- (TS, - TS ) + -As (2) 

where T n is the total thoracic length at stage n; and (TSi - TS Q ), the constant 
length of the newest segment (or the length of the first segment when in stage c). 
Taking As as 0.0097 mm. and (TSi - TS ) as 0.03 mm., T n for successive values 
of n can be calculated. These calculated values are compared with the observed 
values for thoracic length in Table II ; the largest discrepancy is only approximately 
5 per cent, and the original suggestion is thus shown to be fact, i.e., every thoracic 
segment grows in length for a constant amount As, in each stage of the thoracic 
period. 

A similar approach can be employed to analyze thoracic changes in width. 
While in stage thoracic length T n is also 0, the anterior width of the presumptive 
thorax is already 0.142 mm. (C). In stage 1, the width of the first segment is 
0.144 mm. (Table I), and the initial increase (W\--C), analogous to (TS\ 
TSo) in equations 1 and 2, is therefore 0.002 mm. ; the anterior width of the 
presumptive second segment is again C = 0.142 mm. (each segment after stage c 
being regarded as a short cylinder). In succeeding stages, the width of the first 
segment increases 0.002, 0.004, 0.005 mm. . . . etc. (Table I). The increments 
per stage are then not constant, as they were for segmental length, but the figures 
suggest that the increases of the increments per stage might be constant. If the 
increment in stage 2 were 0.003 instead of 0.002 mm., the increase Azy over the 
initial increment (W\ C) would be 0.001, and (W\ -- C) + Aw would repre- 
sent the increase in width during stage 2. Similarly (W\ C) + 2Aw and 
(W\ C) + 3 Aw would indicate the increases during stages 3 and 4 respectively. 
In general, 



(W n - W n ^ = (Wi - C) + (n - 1) -Aw (3) 



128 



PAUL B. WEISZ 



would be true, where (W n ~ W n -i) represents the increase in width of the first 
segment during stage n. The total width increase of the first segment during the 
first n stages would then be the sum of an arithmetical series whose first term is 
(Wi C) and whose last term is [(Wi -- C) + (n -- 1) -Aw], for similar reasons 
as in thoracic length ; or 



and 



n(n -- 1) 



n(n - 1) 



Aw 



(4) 



(5) 



TABLE II 

Calculated and observed magnitudes of certain larval body regions, in millimeters 





Thoracic length 


Width of 1st thoracic segment 




Observed 


Calculated 


Observed 


Calculated 


. 1 


0.030 


0.030 


0.144 


0.144 


2 


0.070 


0.069 


0.146 


0.147 


3 


0.122 


0.119 


0.150 


0.151 


4 


0.185 


0.178 


0.155 


, 0.156 


5 


0.242 


0.247 


0.162 


0.162 


6 


0.311 


0.325 


0.167 


0.169 


7 


0.390 


0.413 


0.179 


0.177 


8 


0.480 


0.511 


0.191 


0.186 


9 


0.600 


0.619 


0.209 


0.196 


10 


0.733 


0.736 


0.225 


0.207 


11 


0.861 


0.863 


0.245 


0.220 




Length of post-abdomen 


Length of a post-abdominal 
segment 


Width of 12th segment 




Observed 


Calculated 


Observed 


Calculated 


Observed 


Calculated 


12 


(0.29) 


(0.28) 


(0.041) 


(0.04) 


0.150 


0.147 


13 


(0.325) 


(0.30) 


(0.054) 


(0.05) 


0.160 


0.154 


14 


0.38 


0.36 


0.06 


0.06 


0.170 


0.163 


15 


0.45 


0.45 


0.08 


0.075 


0.185 


0.174 


16 


0.58 


0.57 


0.09 


0.095 


0.190 


0.187 


17 


0.73 


0.72 


0.12 


0.12 


0.200 


0.202 


18 


0.91 


0.90 


0.15 


0.15 


0.210 


0.219 


19 


1.13 


1.1-1- 


0.18 


0.185 


0.230 


0.238 



Taking for (Wi C) and Aw the values 0.002 and 0.001 mm. respectively, W n 
has been calculated for successive values of n, and the comparison with the observed 
values is shown in Table II. The percentage discrepancies are greater than those 
observed for thoracic length, but nevertheless insignificant in view of the greater 
difficulty of taking accurate measurements of entities of so much smaller magni- 
tude. It is to be concluded that the width of the first thoracic segment grows 
similarly as the length of the thorax, i.e., by adding, in each stage, another term 



ARTEMIA SEGMENTATION PATTERN 129 

of an arithmetical series in which consecutive terms differ by a constant amount Aw. 
It must now be shown that other thoracic segments also increase in width 
according to equations 4 and 5 ; actual measurements for these segments have not 
been taken, but the proof can be arrived at indirectly. It is known from observa- 
tion that the lateral thoracic contours are straight lines converging posteriorly. The 
angle of taper a (Fig. 2) is always expressed by 

W n -C 

tan a = ?T (6) 

LL 

and this angle, on calculation, is seen to be very nearly constant for successive 
values of n. For n\ and n 11, tan a equals 0.033 and 0.045 respectively; 
the average from all eleven values is 0.039, corresponding to an angle of 2 18', 
15'. Since the contours are then straight lines, with a constant taper in all 
thoracic stages, the taper of individual segments must also be constant and identical, 
i.e., (IV n -- W n -\)/2TS n .', as the length TS V of a given segment in a given stage 
can be shown to be equal to the length, in the preceding stage, of the segment 
immediately anterior to it, an analogous equality must obtain for the width of a 
segment, for the taper in each case must be identical. In other words, when the 
width of the first segment is W n , the width of the succeeding segment is W n _\, 
in the same stage; this proves however, by extension, that all thoracic segments 
must increase in a manner identical to the first, since W n and W n -\ represent sums 
of the same arithmetical series as that in equation 5, W n containing one term more 
than Wn-i- 

The segmental abdomen during the thoracic phase maintains a constant length 
(A = 0.249 mm.) and a constant anterior width (C = 0.142 mm.). The posterior 
width W U, identical to the "width of the urosome," however increases (Table I). 
The angle of taper ft, therefore, expressed by 

C - 

~~ 



2A '- 

decreases. Stated in other words, the convergence of the abdominal contour-lines 
gradually diminishes. A stage will eventually be reached at which the thoracic 
and abdominal contours will form continuous straight lines, the thoracic contours 
having a constant taper (equation 6) ; at this time 



tan a = tan ft 

and 

W n -C C - WU n 



(8) 



2T n 2A 

from which WU n can be calculated, all other terms being known. WU n from 
equation (8) is 0.123 mm.; the value of WU n closest to this in Table I is 0.125 
mm. in stage 11. It follows therefore that the thoracic and abdominal contours 
become continuous straight lines as the end of the thoracic period of development 
is reached. 

For analytical purposes thoracic shape during the thoracic period can be re- 
garded as a regular cone from which the tip was cut off (frustrum of a cone). 
Dorso-ventral extent at any level would be very nearly equal to the lateral width 



130 PAUL B. WEISZ 

at that level. The diameters of the end faces of the frustrum can thus be assumed 
to be W n and W-L respectively, and since the length of the frustrum is always given 
by T n , the volume and the surface area of the thorax can be approximated by the 
use of known geometrical formulae. If the volume V\ of the first segment is 
known the total thoracic volume V n at any stage can also be calculated from a 
sum-of-a-series equation, of the general form 



which must obtain, since both length and width changes are governed by such 
equations. Furthermore, As and Aw are obviously related mathematically to 
Av. In sum, if the initial size and shape of the thorax (n 1), and the .values 
As and Aw are known, the size and shape of the thorax at any further thoracic 
stage can be predicted. 

The abdominal phase of development 

Abdominal growth. At the beginning of the abdominal period, the segmental 
abdomen starts to grow in length, having been constant before. During stages 12 
and 13 the abdominal increases are 0.08 mm. per stage, or almost exactly 8 X As 
(Table I) ; since the initial abdominal length at the beginning of stage 12 (or at 
the end of stage 11) is 0.249 mm. or approximately 8 X 0.03 m., it follows that during 
the genital period each 0.03 mm. portion of the segmental abdomen grows an 
amount As per stage. In other words, the segmental abdomen behaves as though 
it were already cut up into its eight segments, and each of these hypothetical seg- 
ments has the same antero-posterior growth potential as thoracic segments when 
first laid down, viz., increasing As per stage after having a length of 0.03 mm. If 
the 12th segment were laid down in the manner in which thoracic segments are 
formed, it would reach stage c at a length of 0.03 mm. But after stage 11 the 
entire segmental abdomen has already started to grow, at a rate of As per stage 
per 0.03 mm. Thus at the end of stage 12 when the 12th segment reaches stage c, 
it will be 0.03 + As, or 0.04 mm. instead of 0.03 mm. long ; the entire segmental 
abdomen should then be eight times 0.04, or 0.32 mm., and the post-abdomen 0.28 
mm. long. Analogously during stage 13, each 0.04 mm. portion of the segmental 
abdomen will now add an amount As, so that segment 13 when in stage c will be 
0.05 mm. and the entire segmental abdomen eight times 0.05, or 0.40 mm. long. 
At this point the genital region should be 0.10 (2 X 0.05) mm. and the post-abdomen 
0.30 mm. long. Actual figures in Table I support such an interpretation rather 
well, and the conclusion is justified that during the genital period the segmental 
tissue of the abdomen acquires the same growth potential in length as that of 
equivalent amounts of thoracic tissue during the thoracic period. 

The genital region continues to grow in length at the indicated rate, as the data 
in Table I tend to show. The post-abdomen would similarly do so, were it not 
for the fact that another change in the mode of growth occurred at the end of stage 
13. Successive post-abdominal lengths P m from stage 13 on are 0.325, 0.38, 0.45, 
0.58 mm. etc., in other words the increments are increasing. A sum-of-a-series 
expression, similar to that for thoracic width changes, fits these figures very 



ARTEMIA SEGMENTATION PATTERN 131 

closely, i.e., 

P m - Fo = (Pi - Fo) -m + m(m ~ 1} A/> ( (10) 

and 



where P m represents the total post-abdominal length for stages 14 to 19 ; PQ, the 
initial length at the end of stage 13; PI, the length at the end of stage 14; m, the 
successive integers from 1 to 6; and A/>, the increments per stage over the initial 
increase (Pi P ). The theoretical value for P was previously seen to be 0.30 
mm., and with 0.06 and 0.03 for (P x P ) and A/? respectively, the calculated 
values for P m compare well with the observed ones (Table II). 

If equation (11) is divided by six, the growth formula for individual segments 
is obtained, since each of these segments is one-sixth of the entire post-abdomen; 



+ (PS, - PS ) -m + A(/>) (12) 

PSo, PSi, and A(/>) are 0.05, 0.06 and 0.005 mm. respectively, and (PS l - PS ) 
is therefore 0.01, or very closely As; thus the initial increase of the presumptive 
segments 14 to 19, at the beginning of the post-abdominal period, is identical to 
the increase of these tissues during stages 12 and 13, and this increment is then 
augmented by a constant amount A(/>) in each subsequent stage. What is re- 
sponsible for this change in the mode of growth of post-abdominal segments? It 
is more than likely that non-formation of appendages is related to this, inasmuch 
as newly formed tissue will not be diverted for the establishment and subsequent 
growth of appendage buds ; augmented growth of the segments would therefore 
be facilitated. It can now be stated in general, that while body segments are 
formed, length increments per stage for all segments are constant, but the incre- 
ments may be added to an initial length as in thoracic and genital segments, or to 
an initial increase of length, as in post-abdominal segments. 

As in thoracic segmentation, the anterior width of the segmental abdomen has 
the constant value C = 0.142 mm., during the abdominal period. This value is 
the anterior abdominal width at the end of stage 11, and the anterior width of the 
presumptive 13th segment at the end of stage 12. The 12th segment, by this time, 
has attained a width of 0.15 mm. (Table I), and in succeeding stages this width 
increases to 0.16, 0.17, 0.185 mm. . . . etc. As for thoracic width the increases 
are found not to be uniformly constant, and a sum-of-a-series expression again 
approaches the data best, i.e., 



WA r -C --r-(WAi-C} + 2 "Awa (13) 

and 

J^ r = C + r(0Mi - C) + r(r 7 n -A7C'a (14) 



where WA r represents the width of the 12th segment at a stage r of the abdominal 
period; (WA\ C), the initial increase in width during stage 12; Awa, the in- 
crease in width, per stage, over the increment during the preceding stage; and r, 



132 PAUL B. WEISZ 



the successive integers from 1 to 8. If for (IVAi C) and Awa 0.005 and 
0.002 mm. respectively are taken, the calculated values for WA r compare well 
with the observed ones (Table II). 

Other abdominal segments can be shown to follow a similar mode of growth 
in width. The lateral contours being straight lines, the angle of taper /? is 
expressed by 

WA r - WU r 



where A r is the length of the entire segmental abdomen, i.e., genital plus post- 
abdominal lengths, and other values as before. Tan /?, when calculated from 
Table I for successive values of r, centers about the average of 0.036 0.004 ; in 
other words, the abdominal taper does not only remain constant during the ab- 
dominal period, but this taper is also practically identical with that reached by the 
segmental abdomen at the end of stage 11 (cf. above, equation 8). 

Unlike thoracic segments, which start development at stage c with the same 
length as that of more anterior segments at stage c, the abdominal segments begin 
development at a length identical with that of more anterior segments at the same 
time. In maintaining a constant taper, the initial increase of any presumptive 
thoracic segment over the width C is always expressed by the first term of the 
series applying to thoracic width (equations 3, 4, and 5), and the later a segment 
arises the fewer terms of the series can it add to its width during the thoracic period. 
Since abdominal segments have now also been shown to maintain a constant taper, 
and since their lengths at stage c are equal to those of more anterior segments 
already beyond stage c, an analogous relation must similarly exist for segmental 
width ; namely, the initial increase of a presumptive abdominal segment over the 
width C must be identical to the width increase experienced by other abdominal 
segments at the same time. If (WA]_ -- C) in equation (13) represents the initial 
increase of segment 12, then (WA 2 WA\) would do similarly for segment 13. 
In other words, the width of both segments follow the same series, but the second 
term for segment 12 becomes the first term for segment 13; the third term for 
segment 12, similarly, becomes the first term for segment 14, etc., and the eighth 
and last term for segment 12 is the first and last term for segment 19. Thus as 
with thoracic segments, the later an abdominal segment arises the fewer terms are 
added to its width, but while the width increases of thoracic segments start with 
the same and end with consecutive terms, those of abdominal segments start with 
consecutive and end with the same terms of the series. 

It should be observed parenthetically that equation (13) may have a slightly 
different constant Awa for the genital and post-abdominal segments respectively, 
reflecting the different modes of growth in length of these two groups of segments ; 
or, if the constant is actually identical the lateral abdominal contour would theo- 
retically not be an exact continuous straight line, but rather two straight lines with 
slightly different taper, joined between segments 13 and 14. In either case, the 
difference would be so small as to be unnoticeable in practice ; with the present 
techniques of observation and measurement, a single series relation holds for both 
groups of segments, and even if two separate series could be established with finer 
means, the principle of growth in width as outlined above would nevertheless hold. 

As for segmental growth in length, a general conclusion can now be stated for 



ARTEMIA SEGMENTATION PATTERN 133 

growth in width, viz., width increments per stage for all body segments are con- 
stant, and the increments are always added to an initial increase in width. The 
combined generalization is also true, that total segmental mass increments per stage 
are constant, and the increments are added either to initial masses or to initial 
increases of mass. 

Thoracic growth. One of four possible reasons could a priori be advanced 
in an attempt to account for the differential size changes in the thorax, such that 
the 5th segment ultimately becomes largest, during the abdominal period: i.e., 
either the segment rudiments in stage a differ in initial size but follow the same 
growth curves ; or the analogous converse ; or either the rudiments have both equal 
initial size and identical growth curves ; or the analogous negative. Since for all 
thoracic segments four stage-intervals elapse between stage a and stage c, length 
and width magnitudes at stage c are identical, and the increments per stage, no 
matter at which segmental stage, are identical (i.e., As), only the conclusion is 
admissible that thoracic segment rudiments have equal initial sizes and follow 
growth curves of the same shape. Under such conditions there are two factors 
which must be held responsible for the observed growth of thoracic segments, i.e., 
the time lag in the formation of consecutive segments, and segmental age. The 
time lag fully accounts for the regular gradation of segmental sizes at the end of 
the thoracic period and for the constant taper of the thorax ; as will be demon- 
strated below, the influence of this original time lag carries over importantly into 
the abdominal phase, and this, together with the factor of segmental age, can indeed 
be made the basis for a consistent interpretation of the manner of thoracic growth. 

Data in Table I show that thoracic length remains constant during the genital 
phase. Hereafter the values for length fit the equation 

r/-r-i / T-> T \ 1% \ 1H 1 / i f 1 S- \ 

m- : T m _! + (7\ - T ) m - -^- - As (16) 

where T and 7\ represent thoracic length at the end of stage 11 and stage 14 
respectively, and m, as before, the integers from 1 to 6. With 0.86 and 0.92 mm. 
for TO and 7\, and As as before, successive calculated values for T,,, are 0.92, 1.03, 
1.18, 1.36, 1.56, and 1.77 mm., significantly close to the observed data; the in- 
creases per stage are therefore 0.06, 0.11, 0.15, 0.18, 0.20, and 0.21 mm., and the 
differences between the increases are seen to diminish in a regular manner. 

The scheme in Table III will account for such a series of increases. The 
figures in this table represent multiples of As and they show the length increase 
of the indicated segment during the indicated stage. Sums of figures in vertical 
rows, multiplied by As, indicate the increases of the entire thorax during the given 
stages, and successive sums are seen to be equal to the values for the increases per 
stage as calculated from equation (16). Horizontal sums, multiplied by As, give 
the total increments of any thoracic segment during the post-abdominal period. 
This scheme is reproduced somewhat differently in Table IV, in which the figures, 
multiplied by As, indicate directly the size of any of the 19 body segments at any 
of the 19 developmental stages ; vertical sums have meanings analogous to equiva 
lent sums in Table III. 

It will be observed that all formulae previously deduced in connection with 
length increases are inherent in the figures in Table IV ; observational data are 



134 



PAUL B. WEISZ 



also incorporated. For example, the first segment when reaching maturity in 
stage 7 has a length of 0.09 mm. (cf. Tahle I). Succeeding thoracic segments 
must also mature at this size, in consequence to the equality of their growth curves ; 
thus segment 5 is shown to mature in stage 11 when the 19th segment appears in 
stage a, and segment 11 in stage 19, in conformity to the observational data in 
Table I. The scheme in Table IV also shows well the successive segmental pro- 
portions in the thorax during the abdominal phase. In stage 16, segments 1 and 2 
are longest, in stage 17 similarly segments 2 and 3, etc. ; maximal segmental length 
thus shifts caudad, fully corroborating observation. 

Segmental -growth of the thorax as indicated in the table can be interpreted 
provided two assumptions are postulated, i.e., (a) a segment can no longer grow 
by regularly increasing amounts after having passed through 14 segmental stages, 

TABLE III 

Scheme of segmental increments, in multiples of As, in the thorax during the post-abdominal 

phase of development 





Stage 


Total 




14 


15 


16 


17 


18 


19 


increases 


Segment 
















1 





1 


1 


1 


1 


1 


5 


2 





1 


2 


2 


2 


2 


9 


3 





1 


2 


3 


3 


3 


12 


4 





1 


2 


3 


4 


4 


14 


5 





1 


2 


3 


4 


5 


15 


6 


1 


1 


1 


1 


1 


1 


6 


7 


1 


1 


1 


1 


1 


1 


6 


8 


1 


1 


1 


1 


1 


1 


6 


9 


1 


1 


1 


1 


1 


1 


6 


10 


1 


1 


1 


1 


1 


1 


6 


11 


1 


1 


1 


1 


1 


1 


6 


Total 
















thoracic 
















increases 


6 


11 


15 


18 


20 


21 





counted from stage c, and (b) a segment, in order to grow by increasing amounts 
at all, must have matured within the first 6 segmental stages of its existence, 
counted from stage r. These two provisions constitute the limiting conditions of 
segmental age. 

Table IV reveals that only the first 5 segments fulfill the second condition ; 
segments 6 to 1 1 would also have matured in 6 stages of their individual existence, 
were it not for the fact that no thoracic growth takes place during the genital period, 
and maturation of the posterior thoracic segments is therefore delayed by two 
stages. Thus only the first five segments would be able to grow by increasing 
amounts, whenever such growth was made possible. It has been shown previously 
that at the beginning of the post-abdominal phase, the post-abdomen ceases to grow 
by constant increments and begins growth by increasing increments, with an initial 



ARTEMIA SEGMENTATION PATTERN 



135 



increase during stage 14 equal to that of stage 13. Apparently the phenomenon of 
increasing increments at this time is not confined to the post-abdomen but also 
affects thoracic segments, subject to the limiting provisions stated above. Thus 
the first five segments have an initial increase equal to the increment during stage 
13, viz., 0; segments 6 to 11, not fulfilling condition (b), simply continue at their 
former constant rates, viz., As per stage (cf. data in Table III, under increases 
during stage 14). From here on, the first five segments augment their increases 
by As in every stage, until their 14th segmental stage is passed ; then, by assump- 
tion, the increment of the 14th segmental stage can no longer be augmented, but 

TABLE IV 

Scheme of growth of body segments, in multiples of A.v 
(a refers to segmental stage a of any given segment) 













Stage 

















Thoracic phase 


Genital 
phase 


Post-abdominal 


phase 






1234567 


8 


9 


10 


11 12 13 


14 


15 


16 


17 


18 


19 


Seg- 
























ment 
























1 


3456789 


10 


11 


12 


13 


13 


14 


15 


16 


17 


18 


2 


345678 


9 


10 


11 


12 


12 


13 


15 


17 


19 


21 


3 


34567 


8 


9 


10 


11 


11 


12 


14 


17 


20 


23 


4 


3456 


7 


8 


9 


10 


10 


11 


13 


16 


20 


24 


5 


a 345 


6 


7 


8 


9 


9 


10 


12 


15 


19 


24 


6 


a 34 


5 


6 


7 


8 


9 


10 


11 


12 


13 


14 


7 


a 3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


13 


8 


a 


3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


9 


a 




3 


4. 5 


6 


7 


8 


9 


10 


11 


10 


a 






3 


4 


5 


6 


7 


8 


9 


10 


11 


a 








3 


4 


5 


6 


7 


8 


9 


12 


(a) 








4 5 


6 


7 


8 


9 


10 


11 


13 




a 






5 


6 


7 


8 


9 


10 


11 


14 






(a) 






6 


7.5 


9.5 


12 


15 


18.5 


15 






a 








7.5 


9.5 


12 


15 


18.5 


16 








(a) 








9.5 


12 


15 


18.5 


17 








a 










12 


15 


18.5 


18 










(a) 










15 


18.5 


19 










a 












18.5 



is retained as a constant increment till growth stops altogether. Thus segment 
one has increased its increment of zero by As at the end of stage 15 (Tables III 
and IV) ; but at this point its 14th segmental stage has already been passed and 
hereafter only a-constant increment of As per stage is possible. Segment 2 on the 
other hand is younger than segment one, being laid down in stage c with a time 
lag of one developmental stage. By the end of stage 15, therefore, when segment 
one has just passed its 14th segmental stage, segment 2 has only passed its 13th 
segmental stage and its increment of As during stage 15 can be augmented once 
more by As; when the 2nd segment has passed its 14th segmental stage, its in- 



136 PAUL B. WEISZ 

creases in subsequent stages will therefore be 2 As per stage. Similarly, segments 
3, 4, and 5, each being one stage younger than the preceding segment, are able to 
augment their increments by As 3, 4, and 5 times respectively, before they com- 
plete the 14th segmental stage. Segment 5 in consequence is as long as segment 
4 at the end of stage 19, but the former will continue to grow at a rate of 5 As 
per stage, while the rate of the latter can only be 4 As per stage ; at any time after 
stage 19 therefore the fifth segment will be longest. 

Analogous changes occur with regard to thoracic growth in width, and the 
thoracic cone-frustrum of stage 11 gradually assumes the shape of a barrel, with 
the "waist" at segment 5 after stage 19. The dorsal thoracic curvature of the 
animal, arising similarly after stage 11, can also be interpreted as a result of 
differential segmental increases in a dorsal direction, according to a scheme resem- 
bling that in Table III. 

The genital segments have been noted to mature, i.e., to form a broodpouch, in 
stage 18. Table IV reveals that at the end of this stage, segment 12 has just com- 
pleted its 6th, and segment 13, its 5th stage of segmental development, counted 
from stage c. Thus both segments fulfill one of the two age conditions assumed 
for thoracic segments ; the fulfillment of the other might be expected. Observa- 
tion proves that this is actually so. Genital segments of older larvae are known 
to bulge considerably beyond the general abdominal contour, giving them a knobby 
appearance. This could not be possible if the constant increases observed up to 
stage 18 were maintained any further; rather, after stage 18 the initial increment 
of an increasing rate will again be equal to the increase during the stage just 
passed, viz., As, and during a 20th stage this increment will be augmented by a 
given amount, during a 21st stage by twice this amount, etc., till the 14th seg- 
mental stage is passed. 

The post-abdominal segments have previously been shown to grow by regu- 
larly augmented increases as soon as they afe laid down. But since these segments 
bear no appendages, stage c for them is equivalent to attainment of maturity, as 
already observed above. Maturity thus proves to be an important temporal 
threshold for all body segments, and the statement that augmented growth will 
occur in any mature segment, provided maturity was reached in a definite time, 
has general application ; the concept of segmental maturity is apparently not only 
a working hypothesis, as has been assumed at the start, but seems to have real 
biological meaning. 

There is no doubt that the scheme of growth here presented describes correctly 
the actual events of later thoracic development ; but the assumptions, while justified 
by the interpretations they allow, still remain to be explained. Only tissue culture 
studies will be able to reveal why segments not matured in the first 6 stages of 
existence are at too early a stage of development, and why segments after 14 stages 
of existence are at too advanced a stage to do more than keep up a constant rate. 

Growth of appendages; integration oj segmental development 

In the preceding section it has been reasoned that segment rudiments in the 
thorax have equal initial size and identical growth curves; observation tends to 
confirm not only this but also that equal-sized rudiments develop for all body 
segments. It can be assumed that in these rudiments certain tissue masses (ap- 



ARTEMIA SEGMENTATION PATTERN 137 

pendage rudiments), initially also of equal size and of equal growth capacity in 
equal times, differentiate independently towards the establishment of appendage 
buds. Such buds however never appear in the post-abdomen, and when they 
appear in other regions they may develop into swimming appendages or into a 
broodpouch. In the evidence presented in Tables I and IV, an important clue 
can be found to at least one of the factors preventing serial analogy despite the 
observed serial homology in appendageal development. 

Every thoracic appendage rudiment reaches the bud stage after an interval of 
four developmental stages. The segment as a whole is at stage c at this point, 
and the appendage buds of any thoracic segment must be of identical size, due to 
the identity of initial size and of growth capacity for all appendage rudiments, and 
of identical shape, since every thoracic segment at stage c has identical propor- 
tions. Enough appendageal tissue has apparently been manufactured, during the 
four preceding stages, to initiate the development of a swimming appendage. 

When a genital segment reaches stage c, 4 + and 5 developmental stages have 
elapsed since stage a. The appendage rudiments therefore have time to manu- 
facture proportionately more appendageal tissue, at the same intensity as that of 
thoracic rudiments. If the genital segments in stage c had larger sizes, propor- 
tionate to the longer time interval available, the appendage buds of genital segments 
would have the same size and shape as those of thoracic segments. However, both 
the length and the width of genital segments are greater in stage c than the size 
which would be proportionate to the longer time of formation. The length of 
any thoracic segment when laid down is 0.03 mm., for example, and four develop- 
mental stages have elapsed since stage a ; the length/time ratio is thus 0.03/4. In 
genital segments this ratio is larger, viz., 0.04/4 + and 0.05/5, and analogously for 
width. Appendageal tissue in genital segments can therefore not be developed in 
sufficient quantity, in proportion to segmental size, to produce appendage buds of 
dimensions equal to those of thoracic buds, even though more time is available. 
Genital buds will thus be relatively smaller and flatter, and the amount of appenda- 
geal tissue manufactured will be spread more thinly over the presumptive appen- 
dage region ; the quantity of tissue present per unit area is apparently already 
below the threshold necessary for the formation of comparatively specialized 
swimming appendages, and only enough tissue is available to initiate the formation 
of a relatively simple sac. 

In post-abdominal segments at stage c the size/time ratio becomes progressively 
larger still, and appendageal tissue consequently cannot even accumulate in quanti- 
ties sufficient to form a bud. 

After the appendage buds are laid down, an appendage retains a definite size- 
proportionality to the segment bearing it. When, for example, the thoracic con- 
tour is a straight line, during the thoracic phase of development, the line joining 
the tips of the appendages on one side is also a straight line, and, as with the 
segments themselves, the time lag in bud formation accounts for the taper. Simi- 
larly, as the thorax gradually becomes barrel-shaped in the abdominal phase, the 
transformation is reflected in differential length increases in the appendages, and 
when the appendageal tips on one side of the body are joined by a line, the result 
is an analogously barrel-shaped contour. 

From the above analyses, the following integrated sequence of events becomes 
apparent with regard to segmental development. 



138 PAUL B. WEISZ 

Shortly before hatching segmental rudiments of equal size begin to be formed, 
at a rate of one per developmental stage ; with a time lag of four stages, segments 
are constricted off in posterior succession, all with constant initial sizes and in- 
creasing by a constant amount during each stage. As the first segment reaches 
maturity, the rate of segment rudiment formation increases to two per stage. 
Rudiments are laid down at this rate till the newest rudiment appears at the 
posterior end of the segmental abdomen which latter had so 'far maintained a 
constant length. The last formed rudiment happens to be the 19th and by this 
time, 11 segments have been constricted off, five of which have already matured. 

The process of rudiment deposition and segment constriction could be assumed 
to go on at length, were it not for the fact that the "end" of the animal has been 
reached. This is apparently the cue for a general change in the mode of growth. 
The entire segmental abdomen begins growth, increasing as yet equal amounts 
per stage, and the thorax ceases to grow. After two genital segments of equal 
size are formed another general change occurs* to the effect that hereafter any 
segment maturing within a definite time may grow by augmented increases, as 
described in detail above. This type of growth is maintained, in each segment in 
which it takes place until the 14th segmental stage is passed, whereupon the total 
increment of the 14th stage is reproduced without further increase in each suc- 
ceeding stage. Segments not matured within the required time continue to grow 
by constant increments. The eventual result of this varied manner of growth, 
maintained up to sexual maturity, is the barrel-shape of the thorax, the presence 
of a dorsal thoracic curvature, the knobby appearance of the broodpouch seg- 
ments, etc. 

DISCUSSION 

Throughout the present analysis of metamerism in Artemia salina, the time 
scale employed was that of developmental stages, defined as the number of body 
segments present. It must be eminently realized that this is a scale of relative, 
biological time. Events in nature take place in a space-time continuum, and to 
Artemia equivalent happenings in space, i.e., the establishment of segments, must 
be correlated to the passage of equivalent units of (relative) time, i.e., what here 
had been called "stages." In hours and minutes, segment formation occurs of 
course not in equivalent times, since the phenomenon is dependent on the environ- 
ment on the one hand, and on changes in growth rates with age on the other. 
Artemia and other similarly primitive forms are particularly suited for a ready 
identification of relative time, but in segmented animals of greater complexity, as 
well as in non-segmented groups, "equivalent happenings in space" cannot be picked 
out with comparative ease, and it will be more difficult to tell what the relative time 
scale actually is ; but that it is intrinsically present in biological phenomena has 
already been acknowledged by others. Thus Needham (1942), after briefly re- 
viewing the pertinent literature, states : 

"Mouse time must bear the same, or a similar, relation to elephant time as 
mouse spatial magnitudes to elephant spatial magnitudes. Indeed, unless the 
time factor is brought into account, we may understand morphological similarity, 
but we can never hope to understand physiological, still less embryological, 
similarity." 



ARTEMIA SEGMENTATION PATTERN 139 

t 

Measurements on Artemia in absolute time would never have brought to light 
the truly amazing simplicity of the laws of segment formation, as given by the series 
and the sum-of-series formulae, and in terms of relative time these formulae as- 
sume a simple biological meaning, viz., (a) that equivalent spatial events take 
place during equivalent relative times, and also (b) that equivalent spatial events 
take place in tissues of equivalent relative age. For illustration, the thorax during 
the thoracic period of development may be considered, where the increments per 
stage of (TSi -- TS ) and (W\ -- C) (equations 1 and 3) are indeed equivalent 
and constant, and where every other segment grows similarly in this same manner ; 
summation of the increments must then result in the sum-of-series expression. 
Analogous interpretations, based on the idea of spatial and temporal equivalence 
can be adduced in every other case in which the formulae hold, i.e., virtually for 
the entire period of segmental development. Before and after this period, relative 
time is of course still operative, but its expression is latent, inasmuch as its passage 
is not paralleled by morphological events clearly identified as equivalent. The 
same would be true for the majority of living organisms, but it can be asserted 
with a fair amount of logical conviction, that if and when it will be possible to 
make explicit the relative time scales of living organisms as a whole, size incre- 
ments in relative time units will be found to be equivalent, and series formulae of 
linear, quadratic, and perhaps even of higher degree will be found to hold. 

It should in general be useful to have a specific term to distinguish relative 
biological time from absolute duration ; the concept as a whole might be called 
"biochronism," and the relative time scale could be said to have one "biochron" 
as its unit. Also, in order to transcend the usual connotations of "growth rate," 
"biochronal rate" could be substituted. Whenever in the text above "increase per 
developmental stage" was mentioned, "increase per biochron" was really implied. 
In this connection, the type of analysis in the present report is clearly different 
from "allometric," "heterauxetic," or "heterogonic" inquiries. The term "mor- 
phometry" is suggested to indicate generally any quantitative appreciation of or- 
ganic size, shape, and time as an integrated dynamic pattern. While it is realized 
that apologies are in order, more or less categorically, for the introduction of any 
new term into present-day biology, it should be kept in mind that new terms become 
unavoidable as different methods of inquiry and fresh fields of study appear. 

Two immediate issues have not been touched on at all in the present analysis. 
First, what determines the changes in the mode of segmental growth at the end 
of both the thoracic and the genital periods? That the changes occur is fairly 
definitely established, and this would support the view that division of segmental 
development into periods is real, i.e., physiological as well as morphological. But 
beyond that, speculation into the nature and history of the changes would be futile, 
for lack of direct evidence. Secondly, and this is the fundamental question in the 
study of metamerism, why are segments formed at all? It will readily be ad- 
mitted that even an attempt to answer this problem can only be made after a great 
deal more is known about segmentation phenomena as a whole. 

Excepting these two questions however, the final size and shape of Artemia 
nevertheless has here been accounted for in terms of initial body proportions much 
as Berrill (1941) has done for the ascidian, Botryllus. When copepods, crayfish, 
and other diverse crustacean forms oE higher evolutionary rank are considered, 
similarly in possession of a barrel-shaped thorax and a straight tapering abdomen, 



140 PAUL B. WEISZ 

it is perhaps justifiable to reflect that Crustacea as a group might have evolved 
with a single and basic geometrical pattern of growth. 

SUMMARY 

1. Growth and the dynamic pattern of segment formation in excysted larvae of 
Artemia salina have been quantitatively studied. The final shape of Artemia at 
sexual maturity can be accounted for in terms of initial shape at hatching. 

2. In analyzing the pattern of metamerism, the stages of development are 
gauged by the number of body segments present. Growth during the entire period 
of segment formation is found to be governed by arithmetical series and sum-of- 
series relations, implying that growth increments per stage over either initial sizes 
or initial increases are constant and identical for thoracic, genital, and abdominal 
segments, respectively. Later transformations of larval shape, resulting in the 
barrel-shape of the thorax, the presence of a dorsal thoracic curvature, the knobby 
appearance of the genital segments, and the presence of a straight tapering ab- 
domen, are accounted for analytically on the basis of concepts concerning the age 
of segments and the time lag involved in segment formation. 

3. The presence, absence, and the difference of structure of appendages are 
shown to be determined, at least in part, by the size of segments when first laid 
down, and by the time available for appendage rudiments to form appendageal 
tissues.. 

4. The time scale employed in the analysis of the segmentation pattern in 
Artemia is interpreted to be a relative, biological one, and the meaning of the series 
formulae with regard to this relative scale is illustrated. The notion of "bio- 
chronism" is introduced, as a general concept applying to biological events in 
relative time. 

LITERATURE CITED 

BARIGOZZI, C, 1939. La biologia di Artemia salina Leach studiata aquario. Atti Soc. Ital. 

Sci. Nat., 78 : 137-160. 

BERRILL, N. J., 1941. Size and morphogenesis in the bud of Botryllus. Biol. Bull, 80: 185-193. 
BOND, R. M., 1932. Observations on Artemia "franciscana" Kellogg, especially on the relation 

of environment to morphology. Int. Rev. dcr gcs. Hydrobiol. u. Hydrogr., 28: 117-125. 
HEATH, H., 1924. The external development of certain phyllopods. Jour. Morph., 38 : 453-483. 
JENNINGS, R. H. AND D. M. WHITAKER, 1941. The effect of salinity upon the rate of excyst- 

ment of Artemia. Biol. Bull, 80: 194-201. 

LOCKHEAD, J. H., 1941. Artemia, the brine shrimp. Turtox 'News, 19: 4145. 
NEEDHAM, J., 1942. Biochemistry and morphogenesis. Cambr. Univ. Press, Cambridge, 1942 

(p. 561). 

RUGH, R., 1941. Experimental embryology. New York Univ. Press, N. Y., 1941 (p. 206). 
WARREN, H. S., 1938. The segmental excretory glands of Artemia salina Linn. var. principalis 

Simon (the brine shrimp). Jour. Morph., 62: 263-289. 
WHITAKER, D. M., 1940. The tolerance of Artemia cysts for cold and high vacuum. Jour. 

E.rp. Zoo/. 83 : 391-399. 



ELECTRON MICROSCOPE OBSERVATIONS OF THE 
TRICHOCYSTS AND CILIA OF PARAMECIUM 

M. A. JAKUS AND C. E. HALL 

/ V/></r///;<'/ /if Binliiiiy. Massachusetts Institute of Tcclui/>!o//y, Cambridge, .Massachusetts 

In previous publications, electron micrographs have been shown of trichocysts 
(Jakus, 1945) and of cilia (Schmitt, Hall, and Jakus, 1943). Recently we have 
re-examined both these organelles using the shadow-casting technique of Williams 
and Wyckoff (1945). The new technique shows structural detail with improved 
clarity and reveals some features not previously visible in specimens prepared in 
the conventional manner. 

TUNGSTEN FILAMENT 
WITH METAL 




METAL DEPOSIT 

^ COLLODION FILM 
"SHADOW" 

FIGUKE 1. Diagram uf shadow-casting technique. 

The shadow-casting technique is illustrated diagrammatically in Figure 1. A 
specimen is placed in a vacuum bell- jar containing a conical tungsten filament in 
which are placed some small pieces of a suitable metal such as chromium. When 
the filament is raised to a high temperature by the passage of an electric current 
the metal evaporates, travelling in straight lines and depositing on the specimen as 
indicated. Structures projecting above the surface of the supporting film cast per- 
manent shadows to the "leeward" and intercept metal to the "windward." Speci- 
mens are then examined in the electron microscope in the usual manner. In posi- 
tive prints the shadows appear bright because they represent relatively transparent 
regions in the object. It is customary, therefore, to prepare micrographs as nega- 
tive prints so that the shadows will appear darker than the background. 

TRICHOCYSTS 

The structure and properties of the trichocysts of Paramecium have been de- 
scribed in a previous paper (Jakus, 1945). In electron micrographs, the discharged 
trichocyst consists of a sharply-pointed tip and an elongated, cross-striated shaft 
with a periodicity of about 550 A. The cross-striated structure appears to be a 

141 



142 M. A. JAKUS AND C. E. HALL 

thin membrane formed by the lateral aggregation of fine fibrils. The tip, in con- 
trast to the shaft, is quite opaque. The reason for this opacity was not obvious. 

Further information about the morphology of the dried extruded trichocyst is 
obtained from electron micrographs of shadowed specimens (Fig. 2). The tip is seen 
to be a compact structure which stands up from the film and is not flattened to any 
great extent as a result of dehydration. The contour of its shadow indicates that 
it is shaped somewhat like a golf tee. In contrast to the tip, the dried shaft is very 
flat, as is evident from the short shadow it casts. The cross striations previously 
observed are enhanced by the metal, indicating that the surface has a regularly cor- 
rugated contour. The elevated regions correspond to the darker bands in both un- 
treated trichocysts and those stained with phosphotungstic acid. Other details of 
structure observed previously may also be found in some shadowed trichocysts. 
These are the fine longitudinal striations of the shaft membrane and the larger 
periodicity (2.200 A) frequently noted along the shaft. The latter may appear 
simply as a slight further intensification of every fourth dark band, suggesting that 
these ridges have a somewhat higher elevation than do the others. 

In some specimens the pointed tip appears regularly cross-striated, if the amount 
of metal deposited has not been excessive and the orientation of the tip is approxi- 
mately parallel to the direction of deposition. This banding has not been seen in 
either stained or unstained specimens and, while it is readily visible in the original 
micrographs of shadowed tips, it is not considered to be of sufficient clarity for re- 
production. Although relatively constant in any one tip, the spacing varied from 
280 to 365 A in the different tips measured and had an average value of about 300 A. 
This is to be compared with the average period of about 550 A in the trichocyst 
shaft. 

>- 

CILIA 

The cilia of Paramecium are shed quite readily if the cell is injured and both 
intact cilia and fragments are observed frequently in preparations of trichocysts. 
Each cilium consists of a bundle of fibrils (about eleven in number), extending the 
full length of the cilium (Fig. 3). The diameter of the dried fibrils lies between 
300 and 500 A. It may be of significance that both the number of fibrils and their 
diameter are in close agreement with the corresponding values observed in the sperm 
tails of numerous animal forms ( Schmitt, Hall, and Jakus. 1943). 

In fixed preparations (for example, with OsO 4 ), the component fibrils usually 
adhere to form a compact bundle, while in unfixed cilia they separate to a greater 
or lesser extent. They are clearly defined in shadowed specimens. Usually the 
separation of fibrils is not complete and they remain in close contact near the end of 
the cilium which was attached to the cell. Here they appear sometimes to be joined 
into two closely adjacent bundles. 

It is not evident what holds the fibrils together in the living cilium. No spiral 
sheath similar to that observed in mammalian sperm tails (Schmitt. Hall, and Jakus, 
1943) or in Euglena flagella (Brown. 1945) has been seen. If a sheath does exist, 
it must be very fragile and easily ruptured. In some cilia, a rather poorly-defined 
cross-striation has been noted, particularly in two or more adjacent fibrils. This 
striation appears to be unlike that of clearly cross-striated proteins and, if it is not 
an inherent periodicity in the fibril, it may represent the remnants of some binding 
or enveloping structure. 



TRICHOCYSTS AND CILIA 



143 




FIGURE 2. Trichocysts from Paramecium, shadow-cast with chromium. X 16,000. 
FIGURE 3. Ciliuni from Paramecium, shadow-cast with chromium. X 11,000. 



144 M. A. JAKUS AND C. E. tJALL 

SUMMARY 

Electron micrographs of shadow-cast trichocysts of Paramecium show that the 
dried trichocyst shaft is flattened on the supporting film, while the pointed tip is 
apparently more resistant to collapse on dehydration. Accentuation, by the metal, 
of the cross striation previously observed in the shaft indicates that the periodicity 
is accompanied by corrugation of the dried surface. A cross striation in the tip is 
also visible in some micrographs of shadow-cast specimens. In the few cases where 
the periodicity could be measured, the average spacing was about 300 A, as com- 
pared to about 550 A for the well-defined shaft striation. 

In electron micrographs of shadow-cast specimens of Paramecium cilia, the 
component fibrils are seen with greatly increased clarity. 

LITERATURE CITED 

BROWN, H. P., 1945. On the structure and mechanics of the protozoan flagellum. Ohio Jour. 
Science. 45: 247-301. 

JAKUS, M. A., 1945. The structure and properties of the trichocysts of Paramecium. Jour. 
E.r/>. ZooL. 100: 457-485. 

SCHMITT, F. O., C. E. HALL, AND M. A. JAKUS, 1943. The ultrastructure of protoplasmic 
fibrils. Biol. Symp.. 10: 261-276. 

WILLIAMS, R. C., AND R. W. G. WYCKOFF, 1945. Electron shadow-micrography of virus par- 
ticles. Proc. Soc. E.\-p. Hiol. Mcd., 58: 265-270. 



HYDROSTATIC PRESSURE EFFECTS UPON THE SPINDLE FIGURE 

AND CHROMOSOME MOVEMENT. 11. EXPERIMENTS ON 

THE MEIOTIC DIVISIONS OF TRADESCANTIA 

POLLEN MOTHER CELLS 

DANIEL C. PEASE 

Department of Anatomy, the Medical School, the University of Southern California, 

Los Angeles, California 

INTRODUCTION 

Hydrostatic pressure increments are known to reduce progressively the rigidity 
of plasmagels and the viscosity of plasmasols. Eventually complete solation results. 
Marsland (1939 and 1942) has been able to formulate what appears to be a gen- 
eral quantitative law on the basis of a considerable volume of work with very di- 
verse material. He has found that with each increment of 1,000 lbs./in. 2 hydro- 
static pressure, the relative rigidity or viscosity decreased to 76 per cent of the 
initial value. This applied no matter whether the cytoplasm of amoebae, Arbacia 
eggs, or Elodca was being studied. Furthermore, these direct effects have always 
proved very rapidly reversible when the pressure was released. The subsequent 
pattern of cell events, however, has sometimes been found to have been changed by 
new reorganization patterns (cf., Pease, 1940, 1941). 

In the first paper of this series (Pease, 1941), experiments w 7 ere reported in 
which advantage was taken of these known effects of hydrostatic pressure to study 
the first cleavage division spindle apparatus in Urechis eggs. The material was not 
well suited for this sort of work, and some interpretations were open to question. 
However, the following facts were clear and significant. 1 ) Pressure could so 
affect the cell that no trace of the spindle figure appeared in the fixed preparations, 
and presumably the spindle had been completely liquified. 2) The pressures de- 
stroying the spindle blocked all anaphase movement. 3) The chromosomes ag- 
gregated in clumps (originally thought to be vesicles) under lower pressures than 
were required to block anaphase movement. 4) Numerous cytasters appeared in 
material given a brief recovery period before fixation. 5) Peculiar "half-spindles" 
developed de novo within cytasters whenever the latter came in contact with nuclear 
material. 6) By their very nature, the half-spindles lacked "continuous fibers" 
since only one pole was involved, and also there were no "interzonal fibers." 7) 
Yet there was ample evidence that such half-spindles were functional in moving 
chromosomes, and even recently-formed nuclei with membranes were at least de- 
formed, and* probably moved, by them. The role of cytoplasmic components in the 
spindle was stressed (perhaps unduly), and the role of the "traction fibers" mini- 
mized (perhaps incorrectly as will be seen later). 

To find out whether or not nuclear gels behaved in the same manner as cyto- 
plasmic gels when hydrostatic pressures were applied, the extraordinary equational 
meiotic division in Stcatococcus spermatocytes has been studied in unpublished work 
by the author. In these cells the spindle is formed inside the nuclear membrane, 

145 



146 DANIEL C. PEASE 

and the anaphase movement nearly completed, before the nuclear membrane dis- 
integrates. In this case, there can be no question but that the whole spindle appa- 
ratus is of nuclear derivation. It was found that sufficiently high pressures de- 
stroyed it by liquefaction, and anaphase movement was blocked. The spindle 
re-formed once more when the pressure was removed and the cells allowed a short 
recovery period. Thus the physiological action of hydrostatic pressure appears to 
be qualitatively identical in gels of nuclear and cytoplasmic origin. 

For the present work, Tradcscentia pollen mother cells (PMC) were selected 
as material for several reasons. The spindle is characterized by relatively enor- 
mous "traction fibers" going to the poles from comparatively large and easily visible 
kinetochores. The cells have the advantage of a small number of chromosomes 
which are relatively large. The only important disadvantages are the impossibility 
of getting controls which necessarily divide at the same time as the experimental 
material, and the extreme difficulties (which proved insuperable with pressure tech- 
niques) of actually observing the divisions in vivo (cf., Shimakura, 1934). 

The material was collected and prepared at Stanford University, and the author 
is indebted to Dr. Reed Rollins of that institution's botany department for technical 
advice on handling procedures and for the plants which were used. The material 
was studied mostly at Columbia University before the war interrupted the work. 
Dr. F. Schrader, Dr. S. Hughes-Schrader, and Dr. H. Ris followed its course with 
interest, enthusiasm, and valuable suggestions. Dr. C. W. Metz of the University 
of Pennsylvania also contributed excellent comments on an early draft of the 
manuscript. 

MATERIAL AND METHODS 

The half dozen Tradcscantia paludosa plants used in these experiments pos- 
sessed six pairs of chromosomes. They were derived from a common stock. The 
anthers were prepared by separating the connective which joins the two lobes. One 
lobe was then fixed as a control just at the time of pressure application to the other 
lobe. The bisection of the anthers with a small lance could be accomplished easily 
without rupturing the anther lobe walls. The lobes were handled and finally 
mounted in a 7.4 gm./lOO ml. saccharose (Merck, C. P.) solution which Shimakura 
(1934) has found to be isotonic with Tradcscantia pollen mother cells. 

The pressure bomb used in these experiments held a half dram homeopathic vial, 
and was so designed that it could be opened very rapidly. After filling with sugar 
solution and a few anther lobes, the vial was sealed with "Parafilm" wax sheet held 
in place with a rubber band. The experimental material was always kept under the 
desired hydrostatic pressure for a one hour period. In a few experiments the mate- 
rial was fixed 30 minutes after the release of pressure which allowed time for some 
recovery. But in most of the experiments, the pressure was released, the bomb 
opened, and the fixative added within one minute. Preliminary experiments had 
shown that there was no appreciable reorganization within that short time limit. 

Experiments were performed using 1,000 Ib. pressure increments from 1,000 to 
6,000 lbs./in. 2 , and with 8,000, 10,000, and 15,000 lbs./in. 2 Control experiments 
were performed giving identical treatment, but at atmospheric pressure, and at the 
relatively low pressure of 150 lbs./in. 2 

Bouin's fixative, to which 3 per cent urea was added, was used throughout. 
For study, eight micra sections were prepared, and stained by Heidenhain's hema- 



ANAPHASE MOVEMENT UNDER PRESSURE. II 147 

toxylin method. Both mordanting and staining were prolonged (never less than 
5 hours each), and the sections were destained in saturated picric acid in such a 
fashion that considerable stain remained in the cytoplasm. There was a good deal 
of shrinkage, but the cytoplasmic differentiation (particularly of the spindle) was 
good. 

RESULTS 
Effects upon the first division spindle 

The first division spindle was particularly sensitive to a critical hydrostatic pres- 
sure that was found to be between 4,000 and 5,000 lbs./in. 2 Even after 4,000 Ibs. 
had been applied, the spindle figures looked essentially normal. There was no re- 
duction in the length or diameter of "traction fibers" (compare Fig. 28 with Figs. 
25 and 26). However, many of the "continuous fibers" had apparently been lost 
for the net effect was a more diffuse looking spindle mass with fewer and less con- 
spicuous continuous fibers. The abnormalities of chromosome movement under 
even the lower pressures prevented any adequate study of "interzonal connections," 
but occasional examples that looked normal have been found after 4,000 lbs./in. 2 

In striking contrast were the results after 5,000 Ibs. had been applied. The 
traction fibers were then reduced in length and in diameter so that they appeared 
as delicate structures (Fig. 30). Small numbers of faint and very thin continuous 
fibers were usually visible, although not always. Ordinarily 6,000 Ibs. pressure 
obliterated the spindle completely, but in a small fraction of the cells a fine residual 
fiber structure remained visible. Figure 31 is a photograph of the heaviest and 
most extensive fibers which have been observed in material fixed after an exposure 
to this pressure. It must be emphasized that this is an entirely atypical cell. No 
sign of continuous fibers has been seen after exposures to 8,000 Ibs., and it was the 
very rare cell which showed indications of traction fibers. When visible, as in 
Figure 33 (arrows), they were thin and short. No oriented fiber structure of any 
sort was ever observed after exposures to 10,000 or 15,000 lbs./in. 2 

In summary, it can be said that the first division spindle looked essentially nor- 
mal after treatments with 4,000 lbs./in. 2 pressure, but was profoundly affected by 
5,000 Ibs. This demarkation was really very sharp ! 

Effects upon the second division spindle 

The spindle of the second meiotic division was considerably more resistant to 
hydrostatic pressure than that of the first division. The spindles appeared nearly 
normal after 4,000 lbs./in. 2 pressure, and after 6,000 Ibs. the spindles of some cells did 
not seem to be greatly affected. After 6,000 Ibs. pressure there was a considerable 
individual variability in different cells, even within the same anther lobe. The best 
spindles were somewhat fainter than normal, and the fibers seemed generally thin- 
ner, but they sometimes extended from one pole to the other. After 8,000 Ibs. pres- 
sure there were occasionally evidences of traction and continuous fibers, although 
they were always thin and faint if present. No fiber structure was ever visible 
after pressures of 10,000 Ibs. or more. 

It thus appears that the second division spindle withstood nearly 2,000 lbs./in. 2 
more pressure than the spindle of the first division. It will appear later that the 
pressure required to block anaphase movement was similarly proportional. 



148 DANIEL C. PEASE 

It may also be noted here that there was a little evidence that the spindles of the 
somatic cells in the connective were even more resistant to pressure, and were not 
entirely destroyed unless pressures in excess of 8,000 Ibs. were applied. 

i 

Effects upon the chromosomes fusion 

Increasing hydrostatic pressures made the chromosomes progressively more 
"sticky" and "soft." Chromosomes tended to aggregate in fused masses. In Fig- 
ure 27 a metaphase plate is shown, fixed just after the release of 2,000 Ibs. pressure. 
It will be noted that there are stained "bridges" connecting all of the chromosomes. 
At this low pressure, the bridges were, on the average, only slightly heavier than 
comparable bridges which could be found in controls of the proper stage. However, 
they persisted much longer than normally, well into the anaphase stages. 

When pressures of 3,000 Ibs. or more were applied, the inter-chromosomal 
bridges tended to become much thicker, and entirely out of the range of normal 
variation. Figure 32 shows such connections in a cell fixed just after the release of 
6,000 Ibs. pressure. With progressively higher pressures, there was an increasing 
tendency for the fusion of chromosomes into a single mass. This can be seen in 
Figures 33 and 34. The extreme condition was reached at 15,000 Ibs. /in. 2 when 
it was nearly always quite impossible to recognize individual chromosomes. This 
is well shown in Figure 36. 

It must be emphasized that the preceding description and the photographs are 
typical of cells to which the pressure was applied in late metaphase stages. When 
the pressure was applied to early metaphases, the chromosomes showed a much 
greater degree of fusion for corresponding pressures. Of considerable importance 
must have been the proximity of chromosomes, and probably also the initial pres- 
ence of thin connections. The existence of some movement in the low pressure 
range may have aided the process. 

Not only were metaphase chromosomes fused together by treatment with hydro- 
static pressures, but a comparable effect was observed with late diakinesis chro- 
mosomes before the nuclear membrane broke down. Here the chromosomes are 
apparently normally kept separate from one another by gel structure within the nu- 
cleus, for nucleoplasm strands showed clearly enough in fixed preparations. These 
strands continued to be visible until pressures of 6,000 or 8,000 Ibs./in. 2 were ap- 
plied. As long as they were present the chromosomes kept apart and did not fuse. 
After the higher pressures the strands were no longer visible, and the chromosomes 
were all in a single clump together. But, as with the metaphase chromosomes, the 
individual chromosomes did not lose their visible identity until pressures of 15.000 
Ibs. were applied. 

At metaphase, the chromosomes were not only found fused laterally in the plane 
of the equatorial plate, but the homologous chromosomes were also fused together 
so that their separation was greatly complicated. This was very obvious when first 
diyision anaphases fixed just after the release of 3,000 or 4,000 Ibs. pressure were 
studied. Practically every cell showed evidences of fusion with bridges that were 
often long and massive (cf.. Figs. 1-12). Such bridges always stained just as the 
chromosome proper with hematoxylin (Fig. 39), and the larger ones, at least, were 
stained by the Feulgen reaction. These bridges were frequently between homolo- 
gous chromosomes, but also commonly involved lateral fusion with non-homologous 
chromosomes. 



ANAPHASE MOVEMENT UNDER PRESSURE. II 149 

Even more massive bridges were found in the second meiotic division material 
subjected to the higher pressures which still allowed a good spindle to exist. Then, 
after 6,000 lbs./in. 2 pressure, most or all of the chromosomes were frequently so 
fused together that they nearly lost their visible identity. However, the mass of 
chromosomes often would be strung out from one end of the cell to the other (Fig. 
15). 

It should be noted that the chromosomes of somatic cells showed the same type 
of fusion. These have occasionally been seen in the tissue of the connective, and 
Figure 41 shows one bridge out of a total of three present in such a cell fixed just 
after the release of 4,000 Ibs. pressure. 

Effects upon the chromosomes rounding 

It should be emphasized that all of the fusion bridges between chromosomes had 
rounded outlines. This shows well in Figures 27 and 32, and suggests a consider- 
able plasticity. 

In addition, the chromosomes as a whole tended to round up under the higher 
pressures. This was most obvious in the second division chromatids which were 
V-shaped with relatively long and thin arms. After 3,000 Ibs. pressure there was 
very little noticeable change in shape even though there might be some fusion (Fig. 
13). However, after 4,000 Ibs. there was a striking alteration. The chromatids 
were then decidedly thickened and shortened (Fig. 14). This tendency became 
more pronounced with increased pressures (Fig. 15, 6,000 Ibs.). 

The short and thick chromosomes of the first meiotic division were not as suited 
for study, but the same tendency was obviously present. Particularly after 10,000 
Ibs., when the identity of individual chromosomes could still be seen, they were de- 
cidedly shortened and rounded except at the kinetochore region (Fig. 34). 

Effects upon the chromosomes the spindle attachment region 

The first meiotic division material gave the impression that 1,000-3,000 lbs./in. 2 
pressure allowed a greater extension of the attachment region of the chromosomes 
than was normal (compare Fig. 26 with 25). More particularly, this region of 
some chromosomes was extended far beyond what could be found in the controls. 
The attachment region gave the impression of being unduly short in the material 
exposed to 4,000 Ibs. pressure. An attempt to measure statistical samples was de- 
cided upon. 

In Table I the mean extensions of the attachment regions of first division chro- 
mosomes are given for pressures up to 4,000 lbs./in. 2 There were, of course, real 
difficulties in measuring such small distances, but errors should have cancelled out 
in the averages. While no great reliance should be placed on the absolute values, 
they certainly indicate the general trend. 

The measurements were made with a filar micrometer. In each group, 50 meas- 
urements were made at random, excepting that only cells in anaphase were selected, 
and individual chromosomes that had not yet separated and left the metaphase plate 
were measured. The micrometer hair was moved up to a chromosome until it just 
touched the distal tip of the kinetochore (indicated by the arrows in Figs. 25 and 
26), and a reading made. Then the hair was swung across the field, and moved 
back in the other direction until the hair just touched the base of the attachment 



150 



DANIEL C. PEASE 



stalk which was ordinarily rather well defined from the body of the chromosome by 
its relative translucency. Then a second reading was made. The difference meas- 
ured the length of the stalk plus the width of the hair in the micrometer. The hair 
width was measured in the same way in relation to a fixed point, and this value was 
subtracted from all of the measurements. The figures were then converted to micra. 
The control measurements actually used for comparison were combined from data 
upon the control anther lobes of the 1,000 and 3,000 Ib. experimental material, and 
a control anther which was left mounted in the bomb for one hour before fixation, 
but without pressure. 

It is to be- concluded that the mean length of the attachment stalk was definitely 
increased by pressures from 1,000 to 3,000 lbs./in. 2 , and it has also been found that 
there is no overlap in the extreme extensions between control cells and experimental 
cells exposed to this pressure range. With 4,000 Ibs. pressure the mean extension 
was significantly less than in the controls, and the greatest extensions found after 
this treatment did not even approach the maxima found in the controls. 



TABLE I 



Pounds pressure 


Mean extension 
in micra 


Percentage increase 
in length 


Percentage overlap with 
control mean 


control 


0.85 






1,000 


1.4 


59.4 


2 


2,000 


1.2 


38.6 


6 


3,000 


1.2 


42.9 


4 


4,000 


0.68 


-19.9 


22 



The distance between the tip of the kinetochore and the base of its stalk is given in the 
second column. In the experimental series, 50 measurements were made at random, excepting 
only that early anaphase cells were selected. The control average, however, is a combined average 
of three sets of measurements upon different material. The mean percentage increases in length 
are based upon figures carried to the third decimal place. The last column gives the percentage 
of measurements which overlapped the mean of the control. 

Effects upon the chromosomes chromoneinata 

We have already seen that late prophase and metaphase chromosomes fused to- 
gether and rounded up under the influence of hydrostatic pressure. This, however, 
only applied to condensed chromosomes. Uncondensed early prophase chromo- 
somes did not seem to be affected by even the highest pressures employed. This 
agrees with the findings of Pease and Regnery (1941) who were unable to detect 
any effect of 15,000 lbs./in. 2 pressure upon Drosophila salivary chromosomes which 
are similarly uncondensed. It must be admitted that no detailed study has been 
made of the early prophase chromosomes. While there was certainly no general 
clumping, it is possible that very local fusions could have been overlooked, but there 
was no indication of shortening or thickening. 

An "accidental experiment" gave further information, and additional reason for 
believing that the chromonemata were not affected by hydrostatic pressure. An 
anther lobe which had been pricked was exposed to 15,000 lbs./in. 2 pressure for one 
hour and was then rapidly fixed in the usual fashion. The surrounding sugar solu- 



ANAPHASE MOVEMENT UNDER PRESSURE. II 



151 



tion had entered the anther, and apparently was somewhat hypertonic. All of the 
cells were slightly plasmolized and had more or less swollen chromosomes. In one 
small section of the anther, conditions were such that the spiral structure was visible. 
Figures 37a and b are photographs of one of these early anaphase cells, and it is 
obvious that the spiral structure was unaffected. Oddly enough there was no tend- 
ency for the chromosomes to fuse under these circumstances. 




First division cells. Figures 1-10 are of sections from material which was fixed just after 
the release of 4,000 lbs./in. 2 pressure. Figures 11 and 12 are of sections fixed just after the re- 
lease of 3,000 Ihs. pressure. The broken lines represent traction fibers except in Figure 7 where 
they represent the pathways of "continuous fibers." All of the chromosomes visible were not 
necessarily included. 

Abnormalities of chromosome movement under pressure 

Because of the fusion of metaphase chromosomes, even by relatively low pres- 
sures, their ultimate distribution to the two spindle poles was usually very abnormal 
whenever anaphase movement took place during the pressure treatment. The par- 
ticular pattern which resulted apparently depended upon the balance between ana- 



152 



DANIEL C. PEASE 



phase forces and the local resistances of whatever fused bridges happened to be pres- 
ent. Greater or lesser fusions might occur between homologous chromosomes and, 
laterally, between non-homologous chromosomes. Almost any conceivable vari- 
ation in the resulting pattern could be found in all degrees. Some of the more 
interesting variations which have been seen are included in Figures 1-15, which 
are also perfectly typical of material exposed to 3,000 or 4,000 Ibs. pressure. 

Homologous chromosomes might be so extensively fused that separation could 
not occur. Such pairs of chromosomes, fused as in Figure 2 in the metaphase plate 




Second division cells. Figure 13 is from material fixed just after the release of 3,000 
lbs./in. 2 pressure; Figure 14, after 4,000 Ibs. pressure; and Figure 15, after 6,000 Ibs. pressure. 
Figure 16 is from recovery material, fixed 30 minutes after the release of 10,000 Ibs. pressure. 
The broken lines indicate traction fibers except in the upper cell of Figure 15 in which they 
indicate the pathways of the "continuous fibers." Not all visible chromatids were necessarily 
included. 




Figures 17-24 are all from first division recovery material which was fixed 30 minutes after 
the release of 10,000 Ibs. /in. 2 pressure. The broken lines indicate traction fibers. Not all visible 
chromosomes were included except in the last three figures. 



ANAPHASE MOVEMENT UNDER PRESSURE. II 



153 









27 




28 






Figure 25 is a first division early anaphase control exposed in the bomb for an hour (but 
without pressure) before fixing. Figure 26 is of a cell fixed just after the release of 2,000 
lbs./in. 2 pressure. Figure 27 is a metaphase plate of the same material. Figure 28 is of a cell 
fixed just after the release of 4,000 Ibs. pressure, and note the anaphase separation of the homolo- 
gous chromosomes a' and a". The small arrows indicate the distal ends of the kinetochores. 
The magnification of these and the following photographs is approximately X 3,000. 



154 DANIEL C. PEASE 

region, would presumably have remained there, and eventually formed micronuclei 
(Figs. 9 and 10). 

Even though there was no lateral fusion with other chromosomes, there might 
be slight differences in the forces directed towards the two poles, or possibly in the 
strength of the traction fibers going to opposite poles. An extensively fused pair 
of chromosomes might then go as a unit to one pole (Fig. 5). Then there would 
always be an abnormally long, but otherwise normal looking traction fiber (with 
full thickness) going most of the way across the cell to the other pole. 

Figures 4 and 5 show very extensive lateral fusion between non-homologous 
chromosomes. -Such anaphase cells would probably have given rise to extensive 
bridges in telophase, and between daughter nuclei, such as are shown in Figures 8, 
10, and 12. 

In Figure 6 the lower member of a pair of homologous chromosomes, indicated 
by an arow, was laterally fused with a non-homologous chromosome going to the 
upper pole. Seemingly it was being carried to that pole in spite of its traction fiber 
to the other pole. 

We have already spoken of the massive bridges which characterized the second 
meiotic division material exposed to 6,000 Ibs. pressure, and which often involved 
all of the chromatids (Fig. 15). There was less fusion with lower pressures, and 
the abnormalities more nearly resembled what has just been described for the first 
division. 

The critical pressure blocking anapliasc movement 

The best evidence for chromosome movement under pressure is certainly the 
presence of extensive bridging. The author sees no rational way of accounting 
for the bridges other than to suppose that anaphase movement occurred after the 
chromosomes established fusions in the metaphase plate and then pulled out the 
bridging connections. 

With this as a criterion of movement, it is possible to state that anaphase move- 
ment continued at 4,000 Ibs. /in. 2 hydrostatic pressure in the first meiotic division, 
but was blocked by 5.000 Ibs. pressure. No extended bridge has been seen in any 
cell of this division exposed to 5,000 or more pounds pressure. Nor were there 
ever signs of asynchrony, or of directionally atypical movements. 

It must also be emphasized that abnormal division resulting from fusion charac- 
terized practically ez>cry anaphase cell exposed to 4,000 Ibs. pressure. It was also 
extremely common after 3,000 Ib. treatments. Similar abnormalities appeared on 
a lesser scale after 1,000 or 2,000 Ibs., but then the separation was more frequently 
fairly normal, and characterized only by loss of division synchrony. 

In the second meiotic division very abnormal anaphase movement involving 
massive fusions took place in some cells exposed to 6,000 Ibs. /in. 2 pressure (Fig. 
15), but none w r as possible at 8,000 Ibs. 

Bridging has been found even after 8,000 Ibs. pressure in the somatic cells of 
the connective. Figure 42 is from a somatic cell forming daughter nuclei at this 
pressure, and two out of a total of five bridges are visible in the plane of the 
photograph. 

In the meiotic divisions, at least, the presence of a good visible spindle was corre- 
lated with anaphase movement. When the spindle was obviously considerably af- 



ANAPHASE MOVEMENT UNDER PRESSURE. II 



155 







. 



30. 





31 



32. 



Figure 29 is a late anaphase cell from the same material as Figure 28 (exposed to 4,000 
lbs./in. 2 pressure). Figure 30 is a cell fixed just after the release of 5,000 Ibs. pressure. Fig- 
ures 31 and 32 are from material fixed just after the release of 6,000 Ibs. pressure. 



156 DANIEL C. PEASE 

fected there were no longer evidences of anaphase movement. This was also prob- 
ably true of the somatic cells, but they have not been carefully studied. It is clear 
that movement is most sensitive to hydrostatic pressure during the first meiotic divi- 
sion, withstands nearly 2,000 Ibs. more pressure in the second division, and seem- 
ingly about 2,000 Ibs. more in the somatic cells. This, in turn, appears due to dif- 
ferent characteristics of the spindle gels, rather than being due to differential pres- 
sure effects upon the chromosomes. For in the first and second meiotic divisions, 
and probably also in the somatic divisions, the chromosomes seemed affected equally 
by equal pressures. 

Spindle recovery after pressure release 

At the time of making these experiments the importance of the recovery stages 
was largely unsuspected, and relatively little material was gathered. But after one 
hour exposures to 10,000 and 15,000 Ibs. /in.- pressures, some experimental material 
was removed from the bomb and given a 30 minute recovery period before fixing. 
Many of these cells showed excellent spindles with massive traction fibers (Fig. 38). 

Of particular interest is the fact that the traction fibers of these recovery spindles 
were de novo formations. Conclusive evidence of this was afforded by paired ho- 
mologous chromosomes (still fused as a result of the pressure treatment) which 
formed traction fibers from both kinetochores that went to the same pole. Figure 
39 is a photograph of such a condition. Figure 40a is a drawing of another ex- 
ample. Figure 40fr seems further complicated for apparently one traction fiber had 
to curve around a blocking chromosome before its direction to the "wrong" pole 
could become definitive. In Figure 40r each traction fiber can probably be con- 
sidered as having gone to the "wrong" pole so that the original polarity of each 
chromosome was entirely reversed. 

Figures such as those described in the last paragraph were not rare, although 
out of the ordinary. They were never seen in the controls, nor is the author aware 
of similar accounts in the literature. 

Most commonly the spindle appeared to re-form nearly along its original axis 
if it is assumed that the metaphase plate was not displaced, and remained as an 
index of that polarity. The pattern thus usually seemed very nearly normal. 
However, the long axis of the new spindle was sometimes very oblique to the plate, 
and presumably to the original spindle axis. In extreme cases a 90 shift was 
indicated. 

Also, not infrequently multipolar spindles were found which were very rare in 
the control material. Three-pole spindles such as Figure 24 were fairly common, 
and a few four-pole spindles have been seen. All possible variants were seen with 
equal or very unequal poles, spaced equidistant from one another, or barely 
separated. 

These several lines of evidence all imply that the spindle was re-formed de novo, 
and was not rebuilt upon residual structure which had survived the pressure treat- 
ment and persisted to give a framework. New patterns appeared, and whatever 
molecules were involved, they were at least rearranged. 

The development of the recovery spindle 

One can select a series of cells which apparently show the different steps of 
spindle re-formation after the release of pressure. In some cells fiber structure con- 



ANAPHASE MOVEMENT UNDER PRESSURE. II 



157 





J 









- 



35. 






36. 



Figure 33 is of a cell fixed just after the release of 8,000 lbs./in. : pressure (the arrows 
indicate very faintly visible traction fibers), and Figure 34 after 10,000 Ibs. pressure. Figure 35 
is of a cell fixed just after the release of 15,000 Ibs. pressure, and the orientation is thought to 
be in the plane of the original spindle axis. Figure 36 is from the same material, but sectioned 
in the plane of the metaphase plate. 



158 DANIEL C. PEASE 

sisted of thin fibrils tangled around the clumped chromosomes of the equatorial 
plate, and without any polar orientation. The fiber direction was roughly circum- 
ferential to the enclosed mass of chromosomes (as in a cocoon. Fig. 44). This 
could be regarded as the first recovery stage. 

Many cells showed polarized fibers as in Figure 45a. The section of Figure 45 
is oblique to the spindle axis. The focus of Figure 45a is tangent to the slant height 
of the cone which makes up one-half of the entire spindle (the "surface" of the 
spindle, so to say). The visible fibers are the continuous fibers of the new spindle. 
Figure 45b is a lower focus of the same cell. It should be observed that there are 
no continuous fibers in the center of the cone. Instead, there are only slight indi- 
cations of traction fibers. The continuous fibers were thus largely peripheral, but 
the extensive lateral fusion of the chromosomes to make a practically solid meta- 
phase plate probably had much to do with this morphological pattern which was 
typical of recovery material. 

Traction fibers were not seen in cells without polarized continuous fibers. But 
when the latter had formed, traction fibers could usually be found. In some cells 
they would be thin and short, in others longer and more massive. Thus the trac- 
tion fibers appeared to "grow" outward directly away from the kinetochore region, 
and full thickness was not achieved until they practically reached the poles. 

It was possible to find many minor irregularities in the developmental pattern 
of traction fibers. These resulted whenever the kinetochore pointed in some other 
direction than directly towards a pole. A graded series could be found, the ex- 
treme examples being when kinetochores pointed more or less to "wrong" poles. 
Invariably the base of the traction fiber extended directly away from the kinetochore, 
and it did not bend towards a pole until it became associated with continuous fibers. 
The bend would then be towards the pole less than 90 away from the initial growth 
direction even if this happened to be the "wrong" pole. It thus looked as though 
the growth direction was unimpeded until the traction fiber became associated with 
continuous fibers, and then the further extension of the traction fiber followed the 
path of least" resistance in the pattern expressed by the continuous fibers. Thus the 
traction fiber' even developed around obstructions as in Figure 40b. 

The fusion of traction fibers 

A very rare situation casts further light on the formation of traction fibers if 
the interpretation is correct. It was possible to find non-homologous chromosomes 
in the recovery material which appeared to be bridged across the kinetochore re- 
gions. A photograph of such a bridge is shown in Figure 43. These bridges dif- 
fered from all the other ordinary bridges which have been seen in that they were 
achromatic. Although they were short, they had exactly the appearance in the 
fixed and stained preparations that traction fibers had. They certainly gave the 
impression that they represented fused traction fibers, traction fibers which started 
to develop from each separate kinetochore in opposite directions, and which grew 
terminally into each other to fuse end to end. 

The author hesitates to emphasize these structures. The material has been 
thoroughly searched and only two good examples have been seen, plus another which 
was more questionable because overlying material partially obscured it. There 
may be good reason for their rarity, for it is obviously an exceptional situation to 
have two kinetochores pointed directly towards each other. If we accept their 



ANAPHASE MOVEMENT UNDER PRESSURE. II 



159 






37 



a. 



b. 38. 









39. 



A 40. 




Figure 37 is from a slightly plasmolized cell fixed just after the release of 15,000 lbs./in. 2 
pressure (a and b are different focal levels). Figures 38-40 are from recovery material fixed 
30 minutes after the release of 10,000 Ibs. pressure. In Figure 38 note the bridge, br. In Fig- 
ures 39 and 40 de novo recovery traction fibers of fused homologous chromosomes go to the 
"wrong" pole. The direction of a pole is indicated by arrows in Figure 40. 



160 



DANIEL C. PEASE 





42. 



ijte 



a. 








. 






44. 



b. 




Figure 41 is of a somatic anaphase cell fixed just after the release of 4,000 Ibs. /in. 2 pressure. 
Figure 42 is of a somatic cell forming daughter nuclei, fixed just after the release of 8,000 Ibs. 
pressure. Figure 43 is from recovery material fixed 30 minutes after the release of 10,000 Ibs. 
pressure, and shows achromatic bridging between non-homologous chromosomes (fused trac- 
tion fibers?). Figure 44 shows an early stage of spindle recovery in material fixed 30 minutes 
after the release of 10,000 Ibs. pressure. Figure 45 is from the same material, but spindle re- 
covery is more advanced (a and /> are different focal levels of the same cell). 



ANAPHASE MOVEMENT UNDER PRESSURE. II 161 

reality and the above interpretation, however, the implications are of considerable 
interest, for it means that developing fibers can mutually terminalize each other. 
Yet there is no effect as far as lateral growth is concerned, and the fibers thicken 
as normally. There is simply no growing end left. We can say that fibers extend 
by terminal additions rather than from the kinetochore, or by elongation from 
within their length. 

Having gone this far, we can make another deduction as to the role of the 
kinetochore in traction fiber formation. We can regard it as an "organizing center" 
which initiates linear extension and controls fiber diameter. The linear gowth is 
self perpetuating once started until the fiber reaches a pole, or is terminalized as 
above. The fiber thickens by further organization at the kinetochore, and additional 
linear growth parallel to the initially thin fiber, thus adding enclosing layers. The 
final fiber has a thickness equal to the diameter of the organizing center. The au- 
thor reiterates that this hypothesis has a slender experimental basis, and depends 
upon a correct interpretation of three figures. 

Chromosome movement in recovery material 

There were obvious indications of chromosome movement in recovery material 
after the spindles re-formed. The movement was abnormal because of strong and 
persistent fusion bridges,, and in many 'ways resembled the anaphase movement 
which occurred under low pressures (3,000 and 4,000 Ibs.). 

Frequently fused pairs of homologous chromosomes were found going to, or 
after they had reached, a single pole as in Figures 38 and 17. In such cases one 
traction fiber extended all the way across the cell to the other pole but seemed to 
be of normal thickness. This type of movement often seemed to be aided by lateral 
fusion with non-homologous chromosomes as in Figures 18 and 21. Less fre- 
quently the fusion between homologous chromosomes was relatively slight, and there 
would be a partial separation with the formation of more or less long and thin bridges 
(Figs. 19, 20, and 38, /-.). Quite frequently very massive bridges were formed 
involving most if not all of the chromosomes which would be fused together (Figs. 
22 and 23). There were no important differences between first and second meiotic 
division cells (note Fig. 16). 

None of the material was allowed a sufficient recovery period so that daughter 
nuclei formed in cells which began their anaphase movement after the application 
of pressure. It can be presumed, however, that many of the cells would form only 
a single nucleus because of an inability on the part of the chromosomes to separate. 
Other cells would be expected to form bridged nuclei, and probably multiple 
micronuclei. 1 

Chromosome structure in recovery material 

The persistence of chromosome fusion in the recovery material would seem to 
suggest just one possibility that the initial fusion under high pressure must have 
been due to at least a partial liquefaction of some chromosomal element, and that 
the fusion bridges then gelled when the pressure was released. In the recovery 
material the chromosomes were thus stuck together by very viscous bridges.' After 
examining a great deal of material, the author is of the opinion that it is very doubt- 

1 Pease (1941) definitely found this to be the case in Urechis eggs. 



162 DANIEL C. PEASE 

ful that fused chromosomes were ever able to separate completely before the forma- 
tion of daughter nuclei. Most commonly there were few signs of any separation, 
but even in extreme cases, thin and very long bridges persisted as in Figures 19 
and 20. The moderately thick bridges, at least, stained with Feulgen. 

There is another, and much more puzzling, aspect of chromosome structure 
which is brought to light by a study of the recovery material. Even after the re- 
lease of 15,000 lbs./in. 2 pressure (which resulted in the very complete fusion of the 
chromosomes as in Figure 36) the chromosomes regained their visible identity and 
their approximately normal shape. This tendency can be seen (in 10,000 Ibs. mate- 
rial) by comparing Figure 38 with Figure 34, but it is best seen by comparing the 
long chromatids of the second meiotic division (compare Fig. 16 with Figs. 14 and 
15). In regaining the normal shape, the fusion areas must necessarily have been 
reduced in cross-section, and it is likely that some fusion bridges were lost entirely 
during this change. The effects of this change were best demonstrated by the sepa- 
ration of the second division chromatids in material recovering from 10,000 Ibs. 
pressure. Extensive separation sometimes occurred, thus differing in degree from 
the first division. Figure 16 gives an indication of typical difficulties which were 
essentially the same as in the first division. 

Absolute pressure and recovery rate 

In Urechis egg material Pease (1941) found that the rate of recovery was 
roughly proportional to the absolute pressure which had been applied. In the 
Tradescantia PMC material we can only compare the effects of 10,000 and 15,000 
lbs./in. 2 pressures. Comparison is subjective, but there was not the slightest doubt 
but that the cells subjected to 10,000 Ibs. pressure showed a much greater amount 
of recovery of the spindle elements in 30 minutes than the cells exposed to 15,000 
Ibs. showed in the same length of time. Fully developed new spindles were only 
rarely found in the 15,000 Ib. material, but were common in the 10,000 Ib. mate- 
rial. In both, however, the chromosomes had regained their visible identity and 
approximately normal shapes. 

CONCLUSIONS 

A single hypothesis readily accounts for most of the manifold effects of hydro- 
static pressure upon spindle, chromosomes, and anaphase movement. This sup- 
poses that increasing hydrostatic pressures progressively reduce gel rigidity, with 
liquefaction as the end result. Conversely, after the release of pressure, conditions 
return to a state such that gel structures can be re-formed once more. There is, 
of course, an excellent experimental background for this thesis, particularly in so 
far as it applies to cytoplasmic systems. This has been indicated in the introduction, 
and has been outlined at greater length in the first paper of this series (Pease, 1941 ). 

It is, however, unfortunate that this work depends upon an interpretation of 
fixed material. However, we have every reason for believing that the presence of 
good fiber structures in such material is a good index of oriented gel structure in 
life. It is only on that assumption that a comprehensive pattern appears, consistent 
throughout its details. It is true that whenever we have contributory evidence of 
liquefaction (such as a block of anaphase movement), we do not find fiber struc- 
tures in the cytological material. Apparently extensive fiber structures are only 



ANAPHASE MOVEMENT UNDER PRESSURE. II 163 

precipitated by fixation agents when molecules are at least organized into an oriented 
pattern and probably also concentrated in a gel. 

Spindle structure and formation 

In view of the above considerations, it is not surprising to find that the spindle 
no longer appears in cytological preparations after a critical pressure has been ap- 
plied before fixation. This is to be interpreted as indicating a liquefaction of pre- 
existing gel structures, with a consequent loss of molecular organization. 

It has been demonstrated that the pattern of the recovery spindle can be very 
different from that of the original spindle. High hydrostatic pressure seems able 
to break down the oriented structure of the original spindle so completely that it 
re-forms de novo, and sometimes with a new polarity. In the re-formation of the 
spindle much the same protoplasmic material may well be used, but the unit mole- 
cules or micells are rearranged in a different manner, just as a pile of second-hand 
bricks might be rearranged to build a new house. This conclusion can probably be 
accepted as a generalization for it agrees with the findings in Urechis eggs which, 
in their formation of "half spindles," were even more striking (Pease, 1941), with 
certain other observations on cytoplasmic systems (cf., Pease, 1940), and with gen- 
eral theory. 

It is not clear just what does orient the new spindle axis in Tradcscantia PMC. 
Cytasters accomplished this end in Urechis eggs, and obviously played the impor- 
tant role. These were never observed in the PMC material. Instead, we find a 
strong tendency for the new axis to coincide more or less with the original. The 
recovery spindle encountered one unusual difficulty in its organization in that the 
chromosomes were no longer completely separate entities. After the higher pres- 
sures there was usually a continuous plate of fused chromosomes in the equatorial 
region. Continuous fibers did not, indeed could not, penetrate this obstruction. 
However, note that homologues were not even found as half spindle components. 
Continuous fibers were only found sweeping around the blocking mass leaving the 
core of the spindle devoid of visible oriented structure except for traction fibers. 
Apparently, therefore, the continuous fibers are entirely a product of the cytoplasm, 
and are not directly related to the chromosomes. The latter, in fact, are obstacles 
to be by-passed. This does not, however, preclude the possibility of a generalized 
interaction between chromosomes and cytoplasm in that the former may "activate" 
the latter to form gel structures. Such an "activation" was quite definitely shown 
by Urechis eggs recovering from the effects of hydrostatic pressure (Pease, 1941). 
A more accurate interpretation might be not to stress the continuous fibers as such, 
but to consider them simply as an index of a more fundamental structural organiza- 
tion of molecules. They thus may signify nothing more than the basic pattern of 
an extensive gel framework. 

On the other hand, the kinetochore apparently quite specifically "organizes" the 
protoplasm to form the attached traction fiber. This process is partially separable 
from the development of continuous fibers. We have good reason for believing 
that developing traction fibers simply follow the path of least resistance in the struc- 
tural pattern of the bulk of the spindle, which, in turn, is expressed by the distribu- 
tion of the continuous fibers. Thus the structural pattern of the body of the spindle 
limits the course taken by the traction fibers as they develop outwards away from 
the kinetochores. It seems likely that this is a progressive wave of molecular or- 



164 DANIEL C. PEASE 

ganization. This view is quite similar to that of Schrader (1932), although based 
upon different evidence. However, it is fundamentally distinct from that of Belar 
(1929) who supposed a very different relationship between traction and continuous 
fiber. Further tentative conclusions on the growth of traction fibers have already 
been given in describing the experimental results. 

The extension of the attachment region in chromosomes subjected to relatively 
low pressures indicates a real pull by or through the traction fibers. It is almost 
impossible to imagine that it could be due to "repulsive forces" between the kineto- 
chores for, if that was so, the extension should continue to increase with progres- 
sively higher, pressures which further soften the chromosomes. Instead, we find the 
extension to be subnormal while we still have evidence of traction fibers and ana- 
phase movement (at 4,000 Ibs. in the first meiotic division). Our conclusion, then, 
is that the traction fiber is a reasonably stiff gel. No doubt it progressively loses 
rigidity with increasing pressure, but it has a margin of strength, and there is no 
important weakness until a pressure threshold is passed. The extension of the 
attachment stalk is therefore thought due to a pressure effect upon the chromosome 
itself so that it is softened, and can be unduly pulled out. The subnormal exten- 
sion at 4,000 Ibs. indicates a significant weakness of either the traction fiber or 
available force. It is interesting for comparison that the centrifuging experiments 
of Shimamura (1940) with comparable material (Liliwn PMC) also lead to the 
conclusion that the traction fiber is a fairly stiff gelled structure. The latter's 
work seems to the author to be quite conclusive. 

Chromosome structure 

It seems obvious that some portion of the condensed chromosome tends to be 
softened, and finally liquefied, by hydrostatic pressures. Since there was no ap- 
parent effect upon uncondensed chromosomes, or upon the spirally coiled chro- 
monemata, the portion affected would seem to be the "matrix" (no morphologically 
separable "sheath" is visible, and presumably more than a sheath would be involved 
when the attachment region is extended). 2 

A critical analysis of the data, however, discloses some relationships that cannot 
yet be interpreted with any assurance of certainty. The normal presence of an at- 
tachment stalk, and its further extension under relatively low pressures, suggests 
that the rigidity of the matrix is normally low, but is further reduced by pressure. 
One might suppose it to be viscous rather than a stiff gel. While the spindle gels 
are liquefied by moderate pressures, the matrix is not entirely liquefied until pres- 
sures of about 15,000 Ibs. /in. 2 are applied when the chromosomes so fuse that they 
lose their visible identity. Thus a structural viscosity appears to persist and with- 
stand very considerable pressures. 

It is a fair assumption that the spindle gels obey Marsland's (1939) law, so that 
their rigidity is reduced 24 per cent by each pressure increment of 1,000 lbs./in.- 
Liquefaction then occurs at a critical pressure, when gel linkages tend to break more 

2 In the first paper of this series (Pease, 1941) chromosome aggregation was described in 
Urechis eggs subjected to hydrostatic pressure. The cytological appearance suggested that a 
"sheath" was involved in this fusion rather than the matrix. The Urechis chromosomes were 
so small, though; that the details were not visible. In view of the present work it seems more 
likely that the matrix as a whole was involved. 



ANAPHASE MOVEMENT UNDER PRESSURE. II 165 

rapidly than they can be formed. Whereas we can probably apply Mainland's law 
to the spindle gels, it does not seem applicable to the chromosome matrix, unless we 
assume that the matrix material has a much lower pressure/rigidity constant than 
cytoplasmic or spindle gels, i.e., much less than 24 per cent per 1,000 lbs./in. 2 
That other different gels in vitro do, in fact, have different constants has been dem- 
onstrated by Marsland and Brown (1942). 

There is yet another aspect of chromosome structure to be considered. Why is 
it that with increasing pressures we find chromosomes rounding up and tending to 
fuse into a single mass? This looks like an interfacial phenomenon to be exp' lined 
on the basis of surface tension laws. We do not observe this with uncondensed 
chromosomes. The author does not see how these and related observations can 
be explained except by the assumption that a true interface does exist between con- 
densed chromosome and surrounding protoplasm (cf., Hirschler, 1942). Many 
workers do not believe that there is an osmotically active membrane separating 
chromosome from protoplasm, although this could explain many of the observa- 
tions of chromosome swelling. However, a real interfacial boundary would not 
necessarily imply an osmotically active system. 

In any case, it can be presumed safely that any intracellular interface would exert 
only a very low tension, certainly not more than a fraction of a dyne, or the very 
few dynes, that have invariably been recorded for water/cell interfaces, or intra- 
cellular oil/protoplasm interfaces (cf., Harvey and Shapiro, 1934 and Harvey and 
Schoepfle, 1939). The presence and properties of dissolved proteins would always 
prevent high values. Thus any interfacial tension at the surface of a chromosome 
would be so low that complete rounding of the aspherical shape would occur only 
when both chromosome and surrounding protoplasm were essentially fluid, and 
practically without structural viscosity. It is only at a pressure of about 15,000 
Ibs./in. 2 that the observed effect indicates these conditions as being nearly fulfilled. 

The spindle in chromosome movement 

It has already been pointed out that there is a direct and definite correlation be- 
tween anaphase movement and the presence of a good visible spindle. Hence, our 
outstanding conclusion is that the presence of gel structure in a spindle is essential 
for anaphase movement. When the gel rigidity is sufficiently reduced, the move- 
ment necessarily ceases. Other types of experimentation have less directly led to 
the same conclusion (cf., particularly the work of von Mollendorff, 1938 and 1939, 
on the specific effects of chemical agents). On the other hand, hypotheses involving 
attractive or repulsive forces are well nigh incompatible with the results. It is 
hard to imagine hydrostatic pressure affecting such forces, particularly in the low 
pressure range. Under pressure, with conditions of reduced viscosity, the chro- 
mosomes should move apart all the more rapidly and easily if such forces were in- 
volved. Furthermore, since Marsland's law relating pressure and viscosity ex- 
presses a logarithmic relationship, the effect should be most noticeable in the low 
pressure range. Obviously this is in direct disagreement with the present findings. 

But what is the role of gel structure in anaphase movement? Certainly there 
are at least two separable structures to be considered the traction fibers and the 
spindle mass. 

Considering the traction fibers first, Cornman (1944) in a thought-provoking 
review comes to the conclusion that they are contractile structures and supply the 



166 DANIEL C. PEASE 

force for movement. However. Cornman ignores one major difficulty in his other- 
wise excellent analysis. No one has yet been able to demonstrate that traction 
fibers thicken as they shorten, although this would be expected if we were dealing 
with contractile bodies. The author has certainly seen no evidence of this in his 
own preparations, nor has he been able to observe the converse of any visible thin- 
ning when a traction fiber was extended all the way across the cell from one pole 
to the other. We, therefore, seem to require a different explanation. 

It is the author's thought that Schrader (1932) was correct in regarding trac- 
tion fibers as being no more than passively semi-elastic structures. . This has been 
given excellent experimental foundation by Ris (1943) who has been able to meas- 
ure directly anaphase movement in living cells (insect spermatogonia and spermato- 
cytes). In some cases he has demonstrated that anaphase movement is very defi- 
nitely a two step process. The first, relatively rapid movement can be explained 
as due to the release of elastic tension so that the traction fibers do actually shorten. 
The remaining movement is then due to the spindle mass, with the traction fibers 
serving simply as passive connections to the chromosomes. Lewis '(1939) pro- 
duced an accelerated motion picture of dividing fibroblasts in vitro which beauti- 
fully showed the same phenomenon, although he has not commented upon it. 

A general hypothesis of anaphase movement can be advanced on the assumption 
that the traction fiber is anchored at one end to the chromosome, and along some of 
its length to the larger gelled mass of the spindle which, in turn, is in motion. Thus 
it is simply a more or less elastic connection from the spindle body to the chromo- 
some a rope, so to say, between the machine and the load. This interpretation 
forces our attention to the body of the spindle. 

The analysis of anaphase movement by Belar (1929) does much to delimit the 
problem, even though we cannot accept his general hypothesis. He demonstrated 
that it was impossible to account for the total movement on the basis of simple 
swelling or elongation of the main spindle mass (or, more specifically, the Stemm- 
korper). There is, however, an obvious way to avoid the difficulties outlined by 
Belar (other than his own solution), and still be consistent with his findings and 
other knowledge. 

It is proposed that motion and force may be imparted to the spindle mass by 
means of two phase transformations. The postulate supposes that gel material is 
added either in the interzonal region 3 or along the greater part of the spindle, 
while a proportional solation occurs at the poles. Thus a material circulation is 
established, but a circulation by means of sol-gel-sol transformations rather than 
within a single phase. Actually a somewhat comparable idea has been proposed 
by Wassermann (1929 and 1939). Such an idea would be regarded by many as 
entirely too speculative, and not subject to either proof or disproof. The author, 
however, wishes to point out some comparable effects which are not likely to be 
known to most cytologists. 

Dan ct al. (1938 and 1940) discovered a remarkable phenomenon in dividing 
sea urchin eggs. After the furrow completes its intrusion, an entirely new region 
of gelled cortex is added in the center of the furrow region as the original cortical 

3 Note that Schmidt (1939) did not find birefringence with polarized light in the mid-region 
of sea urchin egg spindles, and that Shimamura (1940) found this to be the "weak" region in 
centrifuging experiments upon Lilium PMC. 



ANAPHASE MOVEMENT UNDER PRESSURE. II 167 

material backs out. Pease (1943) calculated that this de novo cortex came to cover 
about 11 per cent of the cell surface. This gel growth is obviously analogous to a 
system that could very well work within a spindle. 

Since the advent of hydrostatic pressure techniques, it has also become clear 
that all sorts of other cell processes involving movement are dependent upon gel 
structure. Thus amoeboid movement, cyclosis, streaming, cytoplasmic division, 
the movement of pigment granules, and the pole cell nuclei of Drosophila eggs, and 
even sperm penetration both through the egg surface and also to their final central 
position all cease (reversibly) when the gel is liquefied. All of these movements 
depend upon the rather unexpected, and admittedly little understood, properties of 
protoplasmic gels. Obviously the gel rearranges itself, and is itself in motion (cf., 
the review of Marsland, 1942). No doubt gel-sol transformations are usually if 
not always involved along with the rearrangement. Thus we do find empirically 
a common denominator for all movements other than such specialized activities as 
muscle contraction and ciliary motion. The author believes that a general theory 
of anaphase movement is in sight, and that it will come from a better physico- 
chemical understanding of protoplasmic gel-sol systems. 

SUMMARY 

Hydrostatic pressures have been applied to Tradescantia pollen mother cells as 
a technique for studying the structure of division spindles and chromosomes and 
the mechanics of anaphase movement. The procedure has given pertinent informa- 
tion by virtue of the fact that increasing pressures progressively reduce gel rigidity. 
Sufficiently high pressure results in liquefaction. Yet the effects are reversible. 

The spindle of the first meiotic division was but slightly affected by 4,000 
lbs./in. 2 pressure, yet was mostly liquefied by 5,000 Ibs. The spindle of the second 
meiotic division withstood about 2,000 Ibs. more pressure. The somatic cells were 
even more resistant. 

Condensed chromosomes were significantly softened by even 1,000 lbs./in. 2 
pressure as indicated by an undue elongation of the kinetochore stalk. Fusion 
bridges became particularly obvious when 3,000 Ibs. was applied. Significant short- 
ening and rounding occurred at 4,000 Ibs. Total fusion and rounding, indicating 
complete liquefaction of the matrix, did not occur until pressures of 15,000 lbs./in. 2 
were applied. The fusion and rounding appeared to be a surface tension effect, and 
suggested the existence of a true interfacial membrane between condensed chromo- 
some and cytoplasm. Not even these highest pressures, however, affected the un- 
condensed prophase chromosomes so that the effect of pressure was thought to be 
only upon the matrix material. 

Chromosome movement was limited to those pressures which did not liquefy 
the spindle. The presence of fusion bridges, however, resulted in very abnormal 
movement. 

After the release of high pressures, spindles re-formed. That these were de 
novo structures was indicated by their sometimes abnormal orientation, by the fre- 
quency of multipolar spindles, and by abnormalities in the course of traction fibers. 
Thus, the traction fibers of two homologous chromosomes might go to a single 
pole. Abnormalities made it seem likely that the growth of traction fibers was in 
a large measure independent of the growth of the body of the spindle. The direc- 



168 DANIEL C. PEASE 

tion of growth of the traction fiber was not specifically oriented until it reached the 
oriented bulk of the spindle. 

Chromosome movement in recovery material was abnormal in that the fusion 
bridges persisted. Thus the chromosome matrix which had been liquefied, had 
become highly viscous once more. Under such circumstances, homologous chromo- 
somes frequently went to a single pole, and the traction fiber to the other pole ex- 
tended all the way across the cell. However, such traction fibers were not thinner 
than normal. 

The outstanding conclusion is that a gel structure in the spindle is essential for 
anaphase movement. The traction fiber apparently serves as nothing more than 
a semi-elastic connection between the chromosome and the main mass of the spindle 
which, in turn, is in motion. It is suggested that motion and force is imparted by 
means of sol-gel-sol transformations, with gel being added to the central bulk of the 
spindle while a proportional solation goes on at the poles. 

LITERATURE CITED 

BEI.AR, K., 1929. Beitrage zur Kausalanalyse der Mitose. II. Untersuchungen an der Sperma- 

tocyten von Chorthippus (Stenobothrus) lineatus Panz. Arch. f. Entivickmcch., 118: 

359-484. 
CORNMAN, I., 1944. A summary of evidence in favor of the traction fiber in mitosis. Amer. 

Nat., 78 : 41(M22. 
DAN, K., AND J. C. DAN, 1940. Behavior of the cell surface during cleavage. III. On the 

formation of new surface in the eggs of Strongylocentrotus pulcherrimus. Biol. Bull., 

78: 486-501. 
DAN, K., J. C. DAN, AND T. YANAGITA, 1938. Behavior of the cell surface during cleavage. II. 

Cytologia, 8: 521-531. 
HARVEY, E. N., AND G. SCHOEPFLE, 1939. The interfacial tension of intracellular oil drops in 

the eggs of Daphnia pulex and in Amoeba proteus. Jour. Cell. Comp. Physiol., 13: 

383-389. 
HARVEY, E. N., AND H. SHAPIRO, 1934. The interfacial tension between oil and protoplasm 

within living cells. Jour. Cell. Coinp. Physiol., 5 : 255-267. 
HIRSCHLER, J., 1942. Osmiumschwarzung perichromosomaler Membranen in der Spermato- 

cyten der Rhynchoten-Art Palomena viridissima Poder. Naturw., 30: 105. 
LEWIS, W. H., 1939. Dividing normal adult rat fibroblasts in vitro. (A 16 mm. motion pic- 
ture film distributed by the Wistar Institute of Anatomy and Biology, Philadelphia, 

Pa.) Anat. Rcc., 80: 396, 1941 (review). 
MARSLAND, D. A., 1939. The effects of high hydrostatic pressure upon the mechanics of cell 

division. Arch. f. cxp. Zellforsch., 22: 268-269. 
MARSLAND, D. A., 1942. Protoplasmic streaming in relation to gel structure in the cytoplasm. 

From The Structure of Protoplasm, a mongraph published by the Iowa State College 

Press, Ames, Iowa, p. 127-161. 
MARSLAND, D. A., AND D. E. S. BROWN, 1942. The effects of pressure on sol-gel equilibria, 

with special reference to myosin and other protoplasmic gels. Jour. Cell. Cornp. 

Physiol., 20 : 295-305. 

VON MOLLENDORF, W. V., 1938. Zur Kenntis der Mitose. I. Arch. f. c.rp. Zellforsch., 12 : 1-66. 
VON MOLLENDORF, W. V., 1939. Zur Kenntis der Mitose. VIII. Arch. f. e.rp. Zellforsch., 

29 : 706. 

PEASE, D. C., 1940. The effects of hydrostatic pressure upon the polar lobe and cleavage pat- 
tern in the Chaetopterus egg. Biol. Bull., 78: 103-110. 
PEASE, D. C., 1941. Hydrostatic pressure effects upon the spindle figure and chromosome 

movement. I. Experiments on the first mitotic division of Urechis eggs. Jour. 

Morph., 69: 405-441. 
PEASE, D. C., 1943. Surface movements during the cleavage of echinoderm eggs. Anat. Rec.. 

87: 36 (suppl). 



ANAPHASE MOVEMENT UNDER PRESSURE. II 169 

PEASE, D. C. Hydrostatic pressure effects upon the spindle figure and chromosome movement. 

III. Experiments on the intranuclear meiotic division of Steatococcus spermatocytes 

(in preparation). 
PEASE, D. C., AND D. REGNERY, 1941. Drosophila salivary chromosomes subjected to high 

hydrostatic pressure. Jour. Cell. Comp. Physiol., 17 : 397-398. 
Ris, H., 1943. A quantitative study of anaphase movement in the aphid Tamalia. Biol. Bull., 

85 : 164-178. 
SCHMIDT, W. J., 1939. Doppelbrechung der Kernspindel und Zugfasertheorie der Chromo- 

. somenbewegung. Chromosoma, 1 : 253-264. 

SCHRADER, F., 1932. Recent hypotheses on the structure of spindles in the light of certain ob- 
servations in Hemiptera. Zcitschr. f. zviss. Zool., 142: 520-539. 
SCHRADER, F., 1944. Mitosis. Columbia Univ. Press, New York, N. Y. 
SHIMAKURA, K., 1934. The capability of continuing divisions of the Tradescantia pollen 

mother-cell in saccharose solution. Cytologia, 5 : 363-372. 
SHIMAMURA, T., 1940. Studies on the effect of centrifugal force upon nuclear division. (K. 

Fujii et al. On the mechanism of nuclear division and chromosome arrangement. IV.) 

Cytologia 11: 186-216. 
WASSERMANN, F., 1929. Wachstum und Vermehrung der lebendigen Masse. From Handbuch 

der mikroskopischen Anatomic des Menschen. Springer, Berlin. 
WASSERMANN, F., 1939. Mechanismus der Mitose. Arch. f. exp. Zelljorsch., 22: 238-251. 




THE COMPARATIVE DISTRIBUTION OF TWO CHROMA- 

TOPHOROTROPIC HORMONES (CDH AND CBLH) 

IN CRUSTACEAN NERVOUS SYSTEMS 

FRANK A. BROWN, JR., AND LORRAINE M. SAIGH 

Department of Zoology, N or thivc stern University, Evanston, 111., and 
Marine Biological Laboratory, Woods Hole, Mass. 

INTRODUCTION 

It was demonstrated by Brown (1933) that sea-water extracts of the crustacean 
central nervous organs contained material having a definite and characteristic effect 
upon certain chromatophores of the body. The nervous organs were the only tis- 
sues of the body other than the eyestalks, with their included sinus glands, that 
yielded such a chromatophorotropically active substance, thus suggesting that the 
former possibly contained a source or sources of normal, color-changing hormonal 
material. In the shrimp, Palaeinonetes, injection of extracts of the nervous system 
were shown to bring about a rapid blanching of dark-colored specimens through 
concentration of the red and yellow pigments within the chromatophores, an action 
similar to that which could be induced by extracts of the sinus gland of the eyestalk. 

Similar activity of the nervous system was described by Hosoi (1934) for 
Penacus japonicus and by Hanstrom (1937) for Penacus brasilicnsis. Knowles 
(1939) found that extracts of the central nervous system of Lcander adspersus 
caused concentration of the white pigment within that species. Concentration of 
white pigment by extracts of central nervous system was also reported for Cambarus 
by Brown and Meglitsch (1940) who worked with the chromatophores in isolated 
pieces of integument. Sinus gland extracts had an antagonistic action upon this 
pigment, thus proving that the sinus glands and nervous system did not yield exclu- 
sively identical chromatophorotropic substances. 

Evidence that the central nervous organs contained sources of hormones nor- 
mally involved in the adaptive color-changes of Palacmonetcs was presented by 
Brown (1935) who found that any vigorous stimulation of the cut ends of the optic 
nerves in darkened eyestalkless specimens would induce a blanching characteristic 
of that following injection of extracts of central nervous organs. Roller (1930) 
had also observed comparable responses of eyestalkless Crago but did not at that 
time consider the central nervous organs to be a source of the active material. 

More convincing evidence for the production of a normal chromatophorotropic 
hormone in the crustacean nervous system was presented by Brown and Ederstrom 
(1940). Their observations concerned the reactions of the particularly sensitive 
melanophores in the telson and uropods of the shrimp, Crago. Amputation of the 
eyestalks of a white-adapted animal brought about, within 3-6 minutes, a complete 
dispersion of black pigment in the melanophores giving the animal a "black-tailed" 
appearance. The condition persisted for about an hour whereupon the pigment re- 
turned to its former concentrated state, the latter condition typically lasting for 
several days. Brown and Ederstrom found that the black pigment could be caused 

170 



HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 171 

to disperse again by stimulation of the eyestubs or by the injection of extracts of 
the circumoesophageal connectives. Upon more extensive experimentation they 
concluded that the mid-region of the connectives, including the connective ganglia, 
contained the origin of the Crago tail-darkening hormone (CDH) involved here. 
The results of these investigators were confirmed and extended when Brown and 
Wulff (1941) gave evidence for a second chromatophorotropic principle within the 
central nervous system, namely a Crago body-lightening hormone (CBLH) by de- 
scribing that strong stimulation of the eyestubs simultaneously darkened the telson 
and uropods and lightened the remainder of the body, an action duplicated by in- 
jection of extracts of the central nervous system as a whole. It was shown that 
these two actions were due to two separable principles in that injection of ethyl - 
alcohol extracts of the nervous system gave only body-lightening action, the tail- 
darkening principle remaining in the alcohol-insoluble residue, and, that mild stimu- 
lation of the eyestubs of eyestalkless animals produced both tail-darkening and 
body-darkening. Brown and Wulff speculated that CDH was. in the absence of 
CBLH, a general body-darkening principle. This hypothesis was more specifically 
set forth and given experimental support by Brown (1946) who clearly demon- 
strated the source of this darkening principle to lie, not in the circumoesophageal 
connectives proper, but in the minute tritocerebral commissure interconnecting the 
connectives immediately posterior to the oesophagus. Injection of sea-water ex- 
tract of this commissure in various experiments produced in every case tail- 
darkening but various degrees of either body-lightening or body-darkening. The 
variable effects upon the body seemed reasonably explained in terms of varying 
concentrations of an antagonistic body-lightening principle. 

In the following experiments a survey was made of the effects of sea- water ex- 
tracts of the central nervous systems of thirteen species of higher crustaceans repre- 
senting the Isopoda, Natantia, Ashicura, Anomura, and Brachyura upon Crago 
color-change. The distribution of both the Crago tail-darkening hormone, CDH, 
and the Crago body-lightening hormone, CBLH, was considered. We have con- 
cerned ourselves primarily with the presence or absence of each substance within 
the centra] nervous systems and, when the hormones are present in a particular 
species, with a survey of the relative concentrations of the principles within the 
parts containing the hormone in question. 

EXPERIMENTS AND RESULTS 

The experiments to determine the distribution of CDH and CBLH were con- 
ducted in the following manner. Animals for use in assaying the concentration 
of active principles in extracts of nervous tissue were first prepared. The eyestalks 
of a number of Crago septevnspinosus, ranging from 3-6 cm. in length, were ampu- 
tated by means of a sharp scalpel and the eyestubs cauterized with an electric cautery 
needle. No animals were used for assay purposes until at least twelve hours fol- 
lowing this operation, at which time they could best be described as possessing 
mottled black and white bodies and light telson and uropods (see Fig. \A, control). 

A relatively simple but effective method was used in the preparation of central- 
nervous-system extracts. The donor of the nervous tissue first had eyestalks re- 
moved and stubs cauterized in the same manner as described above for Crago. The 
dorsal portion of the exoskeleton was then cut away. After removing surrounding 



172 



FRANK A. BROWN, JR., AND LORRAINE M. SAIGH 



viscera and muscles the nervous organs were removed under a dissecting micro- 
scope by carefully severing the nerves about the brain, thoracic and abdominal cords 
and gently lifting the entire system out of the animal. Particular caution was ob- 
served in the removal of the circumoesophageal connectives so as to prevent any 
damage to the tritocerebral commissure. The nervous system was then placed in 
a watchglass containing a small amount of sea-water and divided by means of a 
sharp scalpel into the desired portions which usually comprised brain, connectives, 
thoracic cord, and abdominal cord. 





A 







B 

FIGURE 1. ^.Darkening of eyestalkless Crago following injection of a sea-water extract 
of the abdominal nerve cord of Homarns (cone. = 1 cord/0.5 ml. sea-water). The two speci- 
mens on the left are two uninjected ones used for a control. The injections for the animals on 
the right were made 15 min. before the photographs were made. B. Lightening of eyestalkless 
Crago following injection of a sea-water extract of the circumoesophageal connectives of fY<; 
(cone. = 3 pr. conn, to 0.2 ml. sea-water). The two specimens on the right were injected 8 
minutes before the photographs were made. 



HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 



173 



Following this procedure the organs were transferred to individual glass mortars 
where excess sea-water was removed and the tissues allowed to dry partially. The 
tissue was then triturated with a measured amount of sea-water varying in quantity 
with the different species from 0.1-0.5 cc. per portion depending upon the size of 
the nervous system as a whole. In some cases, such as that of Idothca, it was 
necessary to use the parts of several nervous systems in the preparation of each 
extract in order to obtain adequate concentration and amount for assay. All ex- 
tracts were centrifuged for three minutes at approximately 3,500 R.P.M. and the 
supernatant liquid of each injected into the dorsal musculature of the abdomen of 
at least two test-animals prepared as described above. The amount of extract in- 
jected into each varied with the size of the test-animal, but was normally between 

TABLE I 

Responses of eyestalkless Crago to injection of extracts of various portions of the central nervous system 
of other crustaceans. No. of cases signifies the number of donors 

Body-lightening or 

Tail-darkening darkening 

Time (min.) Time (min.) 



Species 


Organ 


No. 
cases 





5 


10 


15 


30 


45 


60 





5 


10 


15 


30 


45 


60 


Homarus 


Brain 
Connectives 
Thoracic cord 
Abdominal cord 


7 
8 
8 
2 


0.0 
0.0 
0.0 
0.0 


3.3 
1.9 
1.6 
1.0 


3.6 
2.3 
2.1 
1.5 


3.9 
2.3 
2.2 
2.5 


3.7 
2.3 
2.8 
3.0 


3.7 
2.1 
2.7 
2.5 


1.6 
1.4 

2.5 
1.5 


0.0 
0.0 
0.0 
0.0 


0.0 
-1.4 
-0.8 
+3.0 


+0.7 
-1.0 
-0.3 
+3.5 


+2.0 
- 0.0 
+0.4 
+4.0 


+2.6 
+0.6 
+2.8 
+4.0 


+ 1.9 
+0.4 
+2.8 
+4.0 


+0.5 
0.0 

+4.0 


Cambarus 


Brain 
Connectives 
Thoracic cord 
Abdominal cord 


10 
10 
10 
10 


0.0 
0.0 
0.0 
0.0 


3.4 
2.5 
2.7 
2.6 


3.4 
2.9 
3.1 
2.8 


3.4 
3.2 
3.4 
2.8 


2.6 
2.2 
2.9 
2.5 


1.3 
0.9 
2.1 
1.4 


0.4 
0.3 
1.3 
1.0 


0.0 
0.0 
0.0 
0.0 


+0.8 
-1.7 
-1.9 
+0.8 


+ 1.2 
-0.9 
-1.7 

+ 1.2 


+ 1.7 
-0.6 
-0.9 

+ 1.4 


+0.9 
-0.1 
+0.1 
+ 1.1 


+0.4 
+0.1 
+0.3 
+0.6 


+0.2 
+0.3 
0.0 
+0.2 


Upogebia 


Brain 
Connectives 
Thoracic cord 
Abdominal cord 


7 
6 

7 
7 


().() 

0.0 
0.0 
0.0 


1.3 
0.2 
1.5 
1.0 


2.3 
0.6 
2.1 
1.4 


2.3 
0.0 
2.7 
1.4 


2.2 
0.0 
2.5 
1.2 


0.8 
0.0 
2.0 
0.9 


0.0 
0.0 
1.4 
0.4 


0.0 
0.0 
0.0 
0.0 


-2.0 
-2.3 
-0.2 
-0.6 


-1.7 
-2.6 
+0.7 
-0.6 


-1.5 
-2.0 
+ 1.2 
-0.3 


-0.4 
-1.7 
+0.8 
0.0 


-0.2 
-0.4 
+0.5 
0.0 


0.0 
-0.2 
+0.2 
0.0 


Pagurus 


Brain 
Connectives 
Thoracic cord 


8 
8 
8 


0.0 
0.0 
0.0 


0.4 
0.0 
1.9 


0.5 
0.0 
2.9 


0.4 
0.0 
3.1 


0.2 
0.0 

2.4 


0.2 
0.0 
1.4 


0.0 
0.0 
0.2 


0.0 
0.0 
0.0 


- .6 
- .8 
- .3 


-1.6 
-1.6 
-1.3 


-1.1 
-1.0 
-1.3 


-0.5 
0.0 
-0.5 


-0.1 
+0.1 
+0.2 


0.0 
+0.1 
0.0 


Emerita 


Brain 
Connectives 
Thoracic cord 


8 
8 
8 


0.0 
0.0 
0.0 


0.0 
0.0 
1.0 


0.0 
0.0 
1.6 


0.0 
0.0 
1.6 


0.0 
0.0 
1.0 


0.0 
0.0 
0.5 


0.0 
0.0 
0.1 


0.0 
0.0 
0.0 


- .5 
- .4 
-0.9 


-0.6 
-0.6 
-0.6 


-0.5 
-0.2 
-0.1 


-0.2 
0.0 
-0.1 


0.0 
0.0 
0.0 


0.0 
0.0 
0.0 


Libinia 


Brain 
Connectives 
Thoracic cord 


7 
7 
7 


0.0 
0.0 
0.0 


0.0 

0.0 
0.0 


0.0 
0.0 
0.0 


0.0 
0.0 
0.0 


0.0 
0.0 
0.0 


0.0 
0.0 
0.0 


0.0 
0.0 
0.0 


0.0 
0.0 
0.0 


-2.6 
-1.7 

-1.7 


-2.8 
-1.8 
-1.7 


-2.0 
-1.7 
-1.8 


-1.0 
-0.4 
-0.9 


-0.3 
-0.1 
-0.4 


0.0 
0.0 
0.0 



0.025 and 0.04 cc. Sea-water injected or uninjected controls were observed simul- 
taneously with all test-animals. 

Observations of the color changes in both body and tail were taken at five- 
minute intervals up to fifteen minutes and at fifteen-minute intervals thereafter. 
The degree of darkness of the tail or body was described within the range, + 1 to 
+ 4, the number + 4 representing the maximum extent of darkening and the num- 
ber + 1, the minimum observable one. In a similar manner body-lightening was 
indicated by the range, - - 1 to -- 4, with -- 4 denoting the greatest extent of body- 
lightening. Final results for a number of experiments were averaged and are pre- 
sented in tabular -form in Table I. These results have been further analyzed so as 
to present the distribution of CDH and CBLH within the central nervous system 



174 



FRANK A. BROWN, JR., AND LORRAINE M. SAIGH 



of each of the species considered (see Tables II and III). In these tables, the 
relative distribution of activity of the hormones is calculated for the various portions 
of the nervous system for each species. 

This was done as follows. The average values of the chromatophores at 5, 10, 
15, and 30 minutes following extract-injection were of themselves averaged. Then 
for Table II the portion of the nervous system producing maximum darkening was 

TABLE II 

The quantitative distribution of CDH activity within the central nervous systems of a number of 
crustaceans. The region of maximum activity is arbitrarily given the value 1.00. It is important to 
note that each portion of the nervous system, regardless of size, is extracted in an equal volume of sea- 
water, and the relative concentrations of the principles investigated are expressed solely in terms of their 
activities. This note applies equally to Table III. 



Classification 


Species or genus 


Brain 


Connec- 
tives 


Thoracic 
cord 


Abdominal 
cord 


Isopoda 

Decapoda 
Natantia 

Reptantia 
Astacura 

Anomura 

Brachyura 
Oxyrhyncha 

Brachyrhyncha 


Idothea baltica 

Crago scptemspinosus 
Palaemonetes vulgaris 

Homarus americanus 
Cambarus virilis 
Upogebia affinis 
Pagurus sp. 
Emerita talpoidea 

Libinia sp. 
Cancer irroratus 
Carcinides maenas 
Ovalipes ocellatus 
Uca pugilator 


1.00 


1.00 


1.00 


1.00 


some 
0.06 


1.00 


0.22 


0.21 


0.85 


1.00 


0.97 


0.98 


1.00 


0.61 


0.61 


0.53 


1.00 


0.84 


0.94 


0.84 


1.00 


0.10 


0.80 


0.65 


0.17 





1.00 











1.00 


































































arbitrarily given the value 1.00, the activity of the other parts being expressed in 
terms of simple proportions of this. For Table III the part showing maximum 
lightening was given the value -- 1.00 with the activity of other parts similarly ex- 
pressed proportionately. The positive values in the latter table obviously indicate 
darkening rather than lightening. 

Within the single species of Isopoda investigated, Idothea baltica, there appears 
to be roughly a uniform distribution of CDH throughout the central nervous sys- 
tem, all organs darkening the telson and uropods of Crago to approximately the 



HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 



175 



same degree. Great variations in distribution of the hormones occur among the 
decapods. The Natantian, Crago apparently possesses significant CDH activity only 
in the regions of the circumoesophageal connectives. CDH is differentially distrib- 
uted throughout the central nervous system of the anomurans with highest quantity 
usually in the posterior region of the thoracic cord, is relatively uniformly distributed 
within the central nervous system of the astacurans and Palaemonetes, and is en- 
tirely absent within that of the brachyurans. 

The quantitative distribution of CBLH was considered here solely within the 
reptantian nervous system, although it is known to be present throughout the cen- 
tral nervous system of the natantians (Brown and Wulff, 1941). Both the anomu- 
rans and brachyurans show wide distribution of this principle throughout brain. 



TABLE III 

The quantitative distribution of CBLH activity within the central nervous systems of a number 
of crustaceans. The region of maximum body -lightening is arbitrarily assigned the value 1.00. 
The + values indicate body-darkening. 



Classification 


Species or genus 


Brain 


Connec- 
tives 


Thoracic 
cord 


Abdominal 
cord 


Isopoda 


Idothea baltica 


pres. 


pres. 


pres. 


pres. 


Decapoda 












Natantia 


Crago septemspinosus 


pres. 


pres. 


pres. 


pres. 




Palaemonetes vulgaris 


pres. 


pres. 


pres. 


pres. 


Reptantia 












Astacura 


Homarus americanus 


+ 2.40 


-1.00 


+ 2.20 


+ 7.20 




Cambarus virilis 


+ 1.09 


-0.73 


-1.00 


+ 1.00 


Anomura 


Upogebia affinis 


-0.64 


-1.00 


+0.27 


-0.18 




Pagurus sp. 


-0.92 


-0.85 


-1.00 






Emerita talpoidea 


-1.00 


-0.86 


-0.57 




Brachyura 












Oxyrhyncha 


Libinia sp. 


-1.00 


-0.67 


-0.71 




Brachyrhyncha 


Cancer irroratus 


pres. 


pres. 


pres. 






Carcinides maenas 


pres. 


pres. 


pres. 






Ovalipes ocellatus 


pres. 


pres. 


pres. 






Uca pugilator 


pres. 


pres. 


pres. 





connectives, and thoracic cord. However, a striking feature is noted in the asta- 
curans and the natantian, Palaemonetes, in which a darkening (see Fig. I A), as well 
as a lightening, of the body occurs. 

The two species of astacurans with which we have concerned ourselves more or 
less parallel one another with respect to the distribution of CDH. In Homarus and 
Cambarus the region of greatest quantity of this principle is the brain, and is fol- 
lowed by an apparent gradual diminution of the substance from anterior to posterior 
within the nervous system. The problem of CBLH distribution seems somewhat 
more complex since, as has been previously mentioned, certain of these nervous- 
system extracts appear to produce body-darkening preceded by a body-lightening. 
The abdominal-cord extract is particularly active in body-darkening and only the 



176 



FRANK A. BROWN, JR., AND LORRAINE M. SAIGH 



connectives and thoracic cords of Homarus and Cambarus show any body-lightening 
activity at all. In these cases where body-lightening is indicated, the lightening 
persists for only a short time and is followed by a definite darkening. These ob- 
servations suggest that the body-darkening activity observable for extracts of the 
astacuran central nervous system is explainable in terms of CDH. It is significant 
that in no case is body-darkening ever obtained from a portion of the nervous 
system lacking tail-darkening activity. However, since there is no essential direct 
correlation between the degree of tail-darkening and the degree of body-darkening 
even within a single species, the observed results must be the consequences of vary- 
ing proportions of the two principles within the extracts, with the degree of influ- 
ence of either one being a function of its relative concentration at any given instant. 
There are significant differences in the distribution of CDH within the group 
of anomurans. Pagurus and Emcrita exhibit similar tail-darkening activities and 
these are shown chiefly by thoracic cord extracts. On the other hand, extracts of 



TABLE IV 

The responses of eyestalkless Crago to injections of extracts of parts of the thoracic cord of some 
anomurans, showing the differing distributions of CBLH and CDH activity. No. of cases signifies 
number of donors. 

Body-lightening or 

Tail-darkening darkening 

Time (min.) Time (min.) 



Species 


Part of thor. 


No. 





5 


10 


15 


30 


45 


60 





5 


10 


15 


30 


45 


60 






cases 






























Pagurus pollicaris 


Anterior J 


8 


0.0 


0.3 


0.3 


0.4 


0.3 


0.0 


0.0 


0.0 


-2.3 


-2.2 


-2.0 


-0.6 


-0.3 


0.0 




Second J4 


8 


0.0 


0.6 


0.7 


0.7 


0.2 


0.0 


0.0 


0.0 


-0.7 


-0.6 


-0.3 


-0.3 


0.0 


0.0 




Third y 


8 


0.0 


1.5 


1.6 


1.6 


0.5 


0.5 


0.4 


0.0 


-0.4 


-0.4 


-0.3 


0.0 


0.0 


0.0 




Posterior J 


8 


0.0 


2.6 


2.6 


2.4 


0.9 


0.5 


0.0 


0.0 


-0.5 


-0.5 


-0.3 


-0.1 


0.0 


0.0 


Pagurus 


Anterior J 


6 


0.0 


0.5 


0.5 


0.5 


0.0 


0.0 


0.0 


0.0 


-1.7 


-1.6 


-1.2 


-0.4 


-0.2 


0.0 


longicarpus 


Second J4 


6 


0.0 


0.2 


0.2 


0.2 


0.0 


0.0 


0.0 


0.0 


-1.0 


-1.0 


-0.3 


0.0 


0.0 


0.0 




Third M 


6 


0.0 


1.3 


1.4 


1.5 


0.8 


0.5 


0.2 


0.0 


-1.2 


-0.8 


-0.2 


0.0 


+0.2 


+0.2 




Posterior J 


6 


0.0 


2.0 


2.0 


2.0 


1.2 


0.5 


0.0 


0.0 


-0.5 


-0.4 


-0.2 


-0.2 


0.0 


0.0 


Emerila talpoidea 


Anterior J^ 


4 


0.0 


0.8 


0.8 


0.8 


0.3 


0.0 


0.0 


0.0 


-2.3 


-2.3 


-1.8 


-0.5 


0.0 


0.0 




Second }- 


4 


0.0 


0.0 


0.0 


0.0 


0.0 


().() 


0.0 


().() 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 




Third H 


4 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


-0.3 


-0.3 


0.0 


0.0 


0.0 


0.0 




Fourth Ji 


4 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


-0.5 


-0.5 


0.0 


0.0 


0.0 


0.0 




Posterior J 


4 


0.0 


0.3 


1.8 


1.8 


0.3 


0.1 


0.0 


0.0 


-0.3 


+0.8 


+0.8 


0.0 


0.0 


0.0 



brain, thoracic and abdominal cords of Upogebia all contain notable amounts of 
CDH. Another similarity between extracts from Pagurus and Emerita is seen in 
the distribution of CBLH. CBLH is found in considerable amounts in brain, con- 
nectives, and thoracic cord of both genera. However, the thoracic cord extracts 
of Upogebia show almost a complete absence of CBLH activity while the extracts 
of the remaining parts of the central nervous system produce a definite body- 
lightening, the connectives being most active in this respect. 

The absence of CDH within the brachyurans investigated as well as the restric- 
tion of this principle to the connective region of the natantians studied confirms the 
results of Brown and Ederstrom (1940). Experimental data show that moderate 
amounts of CBLH are found in brain, thoracic cord, and connectives. Although 
results for CBLH distribution for brachyurans are shown only for Libinia, it has 
been found that they are qualitatively the same for Uca, Cancer, Carcinides, and 



HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 177 

Oval i pcs. The striking body -lightening effect of a strong extract of Uca connec- 
tives and commissures is illustrated in Figure IB. 

An attempt was made to analyze further the localization of CDH and CBLH 
within the thoracic cords of Emcrita and two species of Pagnnis: pollicaris and 
longicarpns (Table IV). The procedure consisted of dividing the thoracic cords 
into a number of approximately equal portions, four in the case of Pagunis and 
five in that of Emerita. It was observed that the concentration of CDH within 
the thoracic cord of both P. pollicaris and P. longicarpus is greatest in the posterior 
fourth of the cord and decreases gradually along the cord as one proceeds anteriorly. 
In Emcrita the highest region of CDH concentration is also the posterior portion 
of the thoracic cord. However, there is a lack of CDH in any of the central por- 
tions of the thoracic cord in Emcrita. It would seem then that the distribution of 
CDH in the thoracic cord of Emcrita is more restricted than in Pagunis. 

The distribution of CBLH in the thoracic cord of P. pollicaris and P. longicarpus 
is similar. The most intense body-lightening effect is brought about by extracts 
of the anterior fourth of the cord while less intense reactions are produced by ex- 
tracts of the remaining portions. Experiments with extracts of Emcrita thoracic 
cord indicate a higher concentration of CBLH in the anterior portion of the cord, 
and apparent absence of CBLH in the second portion and only slight amounts of 
the principle in the third, fourth and fifth divisions of the cord. In summarizing 
the distribution of CDH and CBLH within the thoracic cords of Pagnnis and 
Emcrita we can say that CDH is relatively more concentrated posteriorly in the 
thoracic cord while CBLH appears more concentrated anteriorly. 

DISCUSSION OF RESULTS 

The effect of the extracts of the central nervous system upon the dark pigments 
of the telson and uropods of Crago possesses a characteristic pattern in each of the 
major groups of the order Decapoda. In the Xatantian, Crago, we have observed 
the restriction of CDH activity to the circumoesophageal connectives, whereas the 
Astacnra and Palacmonctcs exhibit a more generalized occurrence of the hormone 
within the organs of the central nervous system. However, as one proceeds to the 
Anoinnra. these contain changes from the widespread condition in the astacurans to 
a more specialized one as evidenced by the restriction of CDH in the thoracic cord of 
two of the three genera examined. Finallv there is an entire lack of CDH among the 

O J 

brachyurans. 

Experimental data concerning the distribution of CBLH in the reptantians pre- 
sent an interesting problem. Although both the anomurans and brachyurans pos- 
sess the body-lightening hormone in varying amounts throughout the entire central 
nervous system, the astacurans appear to limit the hormone to connectives and 
thoracic cord. The simplest explanation for the body-darkening activity of the 
astacuran central-nervous-system extracts involves action of the tail-darkening prin- 
ciple. It is thought that CDH produces body-darkening after CBLH has been ex- 
hausted or in the absence of CBLH. This is indicated in Figure 2 in which selected 
portions of the central nervous system of Libinia, Cainbanis, and Homarus are 
shown to produce a graded series of differential effects upon the coloration of the 
body of eyestalkless Crago. These range all the \vay from maximum body-lightening 
and no trace of darkening (Libinia brain) through initial lightening followed by 



178 



FRANK A. BROWN, JR., AND LORRA1NK M. SAIGH 



darkening, to immediate and extensive body-darkening (Hoinanis abdominal cord). 
These results are believed to be explained in terms of different relative amounts of 
CDH and CBLH. The former is known to be absent in the case of Libinia, and 
it is assumed that the latter is absent or nearly so in the case of Honianis abdominal 
cord. In the case of the extracts of Honuinis thoracic cord and connectives and 
those of Cainbarus thoracic cord, CBLH is present in small amounts and lightens 
the body for a short time, thereby delaying the darkening influence of CDH on the 
body. 

A comparison of tail-darkening and body-darkening within Craf/o injected with 
nervous system extracts from numerous sources suggests a rough positive correla- 
tion between the two (Fig. 3). Generally speaking, we may infer from these data 
that the tendency towards body-darkening is greater in those animals showing a 
high degree of tail-darkening. This gives further support for an active role of 
CDH in body-darkening. 

Unlike the Dccapoda the Isof>oda apparently exhibit a uniform distribution of 
CDH within the central nervous system. However, since only a single species was 
considered, further experimentation is deemed necessary before any decisive state- 
ment is made concerning CDH distribution within this group. 




10 



20 



TIME 



30 

IN MINUTES 



FIGURE 2. The influences of extracts of selected portions of the central nervous system of 
some crustaceans upon the body coloration of eyestalkless Crago. 

From most positive to most negative at the end of 10 minutes are shown, respectively, Huniants 
abdominal cord, Homarus brain, Hoiuanis thoracic cord, Huniants circumoesophageal connectives, 
Cainbarus thoracic cord, and J.ibinia brain. Concentration in each experiment was: organs of 
one specimen/0.5 ml. sea-water. 



HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 



179 



20 



15 



O 



10 



o- 



o o 







o 



o 
o 



00 



o o o oo 



-IO -5 O +5 

BODY LIGHTENING (-) OR DARKENING 



HO 



FIGURE 3. The general relationship between the degree of darkening or lightening of the 
body proper of eyestalkless Crago and the degree of darkening of the telson and uropods. 
Darkening of the tail is expressed as the algebraic sum of the intensities of the reactions at 5, 
10, 15, 30, 45, and 60 min. following extract injection, thereby including a measure of both in- 
tensity and duration of the effect. Body-lightening, being more rapidly transitory, is expressed 
as the algebraic sum of the values at 5, 10, 15, and 30 min. 



SUMMARY 

1. A survey was made of the effects upon Crago color-change of sea-water ex- 
tracts of various parts of the central nervous system of 'thirteen species of higher 
crustaceans. The crustaceans represented the groups Isopoda, Natantia, Astacura, 
Anomura, and Brachyura. 

2. Extracts of various portions of the nervous system among the various groups 
showed wide differences in their total chromatophorotropic activities, producing 
various degrees of telson and uropod darkening and of body-lightening and 
darkening. 

3. An analysis of the results gave support to the hypothesis that most crustacean 
nervous systems possess at least two principles, a) a Crago body-lightening prin- 
ciple, CBLH, lightening all portions of the body except telson and uropods, and b} 
a CVa<70-darkening hormone, CDH, darkening the telson and uropods, and, in the 
absence of CBLH, the body as well. 



180 FRANK A. BROWN, JR., AND LORRAINE M. SAIGtt 

4. CBLH is more or less uniformly distributed throughout the nervous systems 
of all the species examined except the astacurans in which it is demonstrated only 
for the circumoesophageal connectives and thoracic cord. 

5. CDH is restricted to the circumoesophageal connective region of the Natantia, 
is differentially distributed throughout the nervous systems of anomurans, with 
highest concentration in the posterior region of the thoracic cord, and is distributed 
throughout the nervous systems of the other species except the brachyurans in 
w r hich it is absent. 

LITERATURE CITED 

BROWN, F. A., JR., 1933. The controlling mechanism of chromatophores in Palaemonetes. 
Proc. Nat. Acad. Sci, Washington, 19: 327-329. 

BROWN, F. A., JR., 1935. Control of pigment migration within the chromatophores of Palaemo- 
netes vulgaris. Jour. Exp. Zool., 71 : 1-15. 

BROWN, F. A., JR., 1946. The source and activity of Crago-darkening hormone (CDH). 
Physiol. Zool., 19: 215-223. 

BROWN, F. A., JR., AND H. E. EDERSTROM, 1940. Dual control of certain black chromatophores 
of Crago. Jour. Exp. Zool., 85 : 53-69. 

BROWN, F. A., JR., AND A. MEGLITSCH, 1940. Comparison of the chromatophorotropic activity 
of insect corpora cardiaca with that of crustacean sinus glands. Biol. Bull., 79 : 409- 
418. 

BROWN, F. A., JR., AND V. J. WULFF, 1941. Chromatophore types in Crago and their endo- 
crine control. Jour. Cell. Comp. Physiol., 18 : 339-353. 

HANSTROM, B., 1937. Die Sinusdruse und der hormonal bedingte Farbwechsel der Crustaceen. 
Kungl. Svenska Vetenskap. Handl, 16: Nr. 3, 1-99. 

Hosoi, T., 1934. Chromatophore activating substance in the shrimps. Jour. Fac. Sci. Imp. 
Univ. Tokyo, 3 : 265-270. 

KNOWLES, F. G. W., 1939. The control of white-reflecting chromatophores in Crustacea. Pub- 
bli. Stas. Napoli, 17: 174-182. 

ROLLER, G., 1930. Weitere Untersuchungen iiber FaYw'echsel und Farbwechsel-hormonen. 
Biol. Centralbl, 50: 759-768. 



PHYSIOLOGICAL OBSERVATIONS ON WATER LOSS AND 
OXYGEN CONSUMPTION IN PERIPATUS x 

PETER R. MORRISON 

Biological Laboratories, Harvard University, Cambridge, Massachusetts 

The small group of species which comprises the Onychophora have long been 
of interest because of their unique combination of arthropod and annelid characters 
which places them in a phylogenetic position intermediate to those two extensive 
groups (Snodgrass, 1938). They are further of interest because of their close 
homogeneity despite a sporadic distribution that encompasses a large portion of the 
world and points to an ancient separation of some of the genera (Clark, 1915 ; Brues, 
1923). This homogeneity appears to be physiological and ecological as well as 
morphological, 2 since Peripatus is restricted everywhere to a moist but terres'rial 
environment. Further, their sporadic and fluctuating local distribution suggests 
that environmental variation, presumably in moisture, is actively limiting their oc- 
currence even in the regions where they are found. 

Physiological observations on members of this group, then, are of interest, and 
it is of particular interest to examine the process of water loss and to contrast Perip- 
atus in this respect to comparable annelids and arthropods. Manton and Ramsay 
(1937) have reported a value for water loss in Peripatus (Pcripatopsis) at 30 
and with wind velocity of 7.0 m./sec. (16 m.p.h.). These conditions, however, 
seem rather severe for a species which is uncomfortable at temperatures above 20 
(Manton, 1938) and which, living in crevices, must have little exposure to wind. 
The experiments reported here were made under conditions which more nearly ap- 
proximate those encountered by the animal in nature. 

In this connection reports in the literature suggest that there may have been 
some temperature adaptation in the Onychophora. Thus the two species studied 
here, both from Panama (lat. 9 N.), stayed in good condition at a temperature of 
25 =t. In contrast as already noted, Peripatus from near Cape Town (lat. 34 S.) 
became uneasy at temperatures above 20 although low temperatures, even down to 
freezing, did not bother them. They survived very well in England (Manton, 
1938; Sedgwick, 1885) as have specimens from New Zealand (lat. 40 S.). The 
latter were only successfully transported through the intervening tropical regions 
with the aid of refrigeration (Sedgwick, 1887). Peripatus from New Zealand 
(Hutton, 1876) and from Australia (Steel, 1896) are reported to become torpid 
during the winter but with no subsequent ill effects. On the other hand Sclater 
(1887) reported that his specimens from British Guiana (lat. 7 N.) successfully 

1 These observations are by no means complete, but because the literature contains little data on 
living Onychophora, particularly on New World species, and because there was no immediate 
prospect of obtaining a further supply of these unusual animals, it seemed advisable to present 
them at this time. 

2 It should be noted, however, that for such a small group of Onychophora show remarkable 
diversity in their embryological development and their mode of reproduction. 

181 



182 PETER R. MORRISON 

survived the trip to England, "but unfortunately were much affected by the cold, 
and were therefore killed." 

MATERIAL 

These Peripatus were secured on Barro Colorado Island, Canal Zone, through 
the great kindness of Mr. James Zetek. Two species (note Clark and Zetek, 1946) 
were obtained, the larger of which (Epipcripatus brasiliensis varians) had a con- 
tracted length of 50 mm. and was uniformly colored a rich red-brown. The smaller 
species (Oroperipatus corradi) had a contracted length of 25 mm. and was a choco- 
late color with lighter underside and with darker legs and a dark, median, dorsal 
stripe 0.3 mm. in width. The animals were taken in -early September and these 
observations were made in Cambridge about a month later. During the interim 
they were kept in moist forest debris but were not given suitable food other than the 
supply of termites initially in the debris. The animals survived the trip well and 
apparently stayed in good health until just before death which presumably occurred 
through starvation. 

The general behavior of these individuals corresponded to that described for 
other species (Manton, 1938; Holliday, 1942; Andrews, 1933; Steel, 1896; Sedg- 
wick, 1885; etc.). They were retiring and preferred to remain inactive in some 
dark crevice. They are sensitive to light but react even more sharply to dryness 
which stimulates them to constant activity. The smaller species were definitely 
more sensitive in this respect and could not be held still even for a moment. 

An occurrence involving an individual of the larger species may be of particular 
interest. On the occasion of mechanical injury to one of its antennae that member 
was placed in the mouth and the injured portion, about half the length, was removed. 
The stump healed and the individual did not appear to be inconvenienced by the 
loss. Parturition as observed in these specimens has been described elsewhere 
(see Morrison, 1946). 

The rate of oxygen consumption and water loss in Peripatus was compared 
to several arthropods and annelids of fairly similar size, habitat and body form : 
centipeds (Lithobius) ; millipeds (Julits} ; sow bugs (Onlscus) ; and earthworms. 
These were all collected locally with the exception of one small tropical earthworm 
found among the debris. 

OBSERVATIONS 

Sensory responses 

With the exception of the antennae the animals showed equal tactile sensitivity 
all over the body, on the dorsal and ventral surfaces and on the legs. A very light 
stimulation could be applied with no response, a light one produced a local with- 
drawal of a leg or small section of the body, while a strong stimulus led to a general 
withdrawal. Holliday (1942) noted that fairly large wood lice and centipeds could 
crawl over the body of a Peripatus without evoking any response. The antennae 
are much more sensitive and the lightest touch here results in the retraction of one 
or both. With stronger or repeated stimulation the animal will completely contract 
and change its direction of progression ; further irritation provoked the well known 
ejection from the slime glands. These responses are in accord with the histological 
findings of Manton (1937) that while a single well ensconced sense capsule was 



OBSERVATIONS ON PERIPATUS 183 

found in each primary body papilla, each antennal papilla bore at least three much 
more exposed capsules with much heavier innervation. 

The animals usually walked forwards but when startled would often reverse 
their direction, apparently walking backwards with equal ease. Occasionally they 
would half turn backwards and then move in the form of a "U" with the legs of 
the anterior half walking forward and those of the posterior half walking backwards. 
This mode of progression must impose an interesting problem in coordination. 

The response of the animals to a single point source of light (a two-cell flashlight 
with reflector and glass removed, at a distance of 0.5 to 1 m.) was recorded by trac- 
ing their path on a large underlying sheet of paper. A number of records were 
made both with the light fixed and with it moved through 90 or 180 halfway 
through the record. Examination of the records showed no oriented negative 
phototropism ; indeed, the animals actually travelled towards the light more often 
than away from it. Thus these animals would appear to be unable to localize light 
but only to be aware of it. This corresponds to the observations of Manton (1938) 
that the movement of objects near Peripatus elicited a response only when accom- 
panied by air movement. These experiments were not carried out in a saturated 
environment, however, and it is possible that with the very strong stimulus of dry- 
ness removed, some phototropic pattern might be observed. 

Water balance 

In measuring water loss the animals were placed in large (D = 5 cm.) flat, 
weighing bottles containing a layer of calcium chloride covered by a floor of brass 
gauze. Measurements were made at 24 which is within the range normally en- 
countered by these species (Kenoyer, 1929), and for periods of 30 minutes. No 
circulation was supplied, the movement of the animals themselves providing for 
convection. The Peripatus were particularly uneasy in this very dry atmosphere 
and kept in constant and vigorous motion. 

The values obtained for the two species of Peripatus and for several other ani- 
mals are summarized in Table I. Water loss has been computed on the basis of 
both body weight, and the two-thirds power of the body weight. 3 The latter is 
perhaps a more reasonable basis for comparing animals of different size. The two 
values for Peripatus agree well and lie between those found for the annelids and 
arthropods. They indicate that Peripatus has a twofold advantage over the earth- 
worm 4 in the conservation of water ; and that it is at a twofold disadvantage as 
compared to the centiped, the most xerosensitive arthropod studied. Other arthro- 
pods showed values ranging down to one-twentieth that observed in Peripatus. 
These data are presented graphically in Figure 1. 

Manton and Ramsay (1937) reported on water loss in Peripatus under the 
much more rigorous conditions of 30 with a 7 m./sec. (16 m.p.h.) wind and a rela- 

3 This quantity is proportional to the surface area in animals of similar body form. In 
Peripatus and the arthropods where the actual body surface is increased by appendages and 
papillae, loss of water very probably takes place largely through the trachae (note Mellanby, 
1935). Water loss will therefore be related to respiration which is also roughly proportional 
to the two-thirds power of the body weight in animals of different size (Krogh, 1916). 

4 This will be a minimum figure since the body weight of the earthworm includes a con- 
siderable amount of dirt in the gut. These earthworms were kept in clean wet containers for 
l 1 /^ days before use, during which time they evacuated up to 15 per cent of their weight, but 
more undoubtedly remained. 



184 



PETER R. MORRISON 



tive humidity of 27.5 per cent. They found a value of 13.0 mg./g. min. or 2 to 3 
times our value. A similarly measured value for an earthworm was about half as 
large on a weight basis or of equal magnitude on the basis of surface area. How- 
ever, the advisability of making measurements under physiological conditions should 
be stressed since under abnormal circumstances quite different relations may hold. 
Thus, for example, Ramsay (1935) showed that in the cockroach water was lost 



O 



UJ 
Q_ 

i 



25 



20 



* 15 



c/) 
O 

.J 

CL 
UJ 



10 




0.2 



0.4 



0.6 



0.8 



BODY WEIGHT IN GRAMS 

FIGURE 1. Water loss in Peripatus and other animals at 24 over calcium chloride as a 
function of the body weight. Open circles, earthworms ; crossed circles, Peripatus ; half-closed 
circles, centipeds ; lined circles, sow bugs ; closed circles, millipeds. The curves represent 
}" -= K (X) 2/3 , where the values for K are the average values given in Table I. 

much more rapidly at temperatures above 30 with an apparent breakdown of the 
hydrophobic character of the body surface. 

In considering this function it is of interest to note that Clark (1915) concluded 
on the basis of distributional and taxonomic considerations that the Onychophora 
had originally evolved in a cooler rather than a warmer environment. Thus, the 
more primitive groups are found on mountains or in the "temperate" regions while 



OBSERVATIONS ON PERIPATUS 



185 



the more recent forms are tropical. This is, of course, entirely in accord with the 
physiological considerations since the xerotic stress would be reduced at a lower 
temperature and such an environment would be more favorable for evolution from 
an aquatic to a terrestrial mode of life. 

TABLE I 

Water loss in Peripatus and other animals at 24 over calcium chloride 









Water loss 


. . . 


Number and weight 


Duration of experi- 






in mg. 


ment in min. 












mg./g.min. 


mg./g. 2/3 min. 


Earthworm 


884 


15 


7.4 


7.1 




703 


15 


12.0 


10.4 




360 


15 


13.4 


9.6 




208 


10 


17.3 


; 10.4 




105 


8 


22.6 


10.7 


Peripatus 










Epiperipatus 


788 


30 


5.2 


4.7 


Oroperipatiis 


423 


30 


6.6 


5.0 


Centiped 


150 


60 


5.6 


3.0 




135 


20 


5.9 


3.1 




4X95 


60 


4.1 


1.9 




4X63 


30 


7.2 


2.8 


Sow bug 


158 


25 


3.0 


1.6 




97 


40 


2.5 


1.2 




6X49 


60 


3.0 


1.1 




48 


20 


4.5 


1.6 


Milliped 


3X76 


120 


0.56 


0.24 




3X98 


600 


0.44 


0.21 


A verages 








Earthworm 


15 Experiments 




9.9 


Peripatus 


2 Experiments 




4.9 


Centiped 


4 Experiments 




2.5 


Sow bug 


5 Experiments 




1.3 


Milliped 


2 Experiments 




0.22 



Respiration 

The oxygen consumption of the larger species of Peripatus and of various other 
animals was measured in a Warburg apparatus. 5 Carbon dioxide was absorbed in 
sodium hydroxide in a small cup fused to the bottom of the chamber. The animals 
were placed directly in the chamber and were kept from the lye by a small screen 
shield. Measurements were made at 25.0 C. over a period of 60 minutes. 

The results on Peripatus are shown in Figure 2. After a restless initial period 
(10 minutes) it settled down to a very uniform rate of oxygen consumption. The 
centipeds, also shown in Figure 2, were less regular. The results for the various 

5 I am indebted to Dr. William Carroll for the use of his calibrated Warburg assembly. 



186 



PETER R. MORRISON 



animals are summarized in Table II. The exact significance of the "resting" or 
"basal" oxygen consumption is not known but some correlation between it and the 
"intensity" of the organism has been observed. Compared on a weight basis Perip- 
atus consumes oxygen at the same rate as the earthworm and at about half the rate 
of the arthropods. It has been observed, however, that within a given group, the 
metabolism per unit of weight varies with the size of the animal (note Edwards, 
1946, for example), and that the metabolism is more nearly proportional to some 



16 



ID 
O 



Q. 

2 
ID 
(/) 
Z 
O 

o 



LL) 
O 

X 

o 



8 




20 



40 



60 



TIME IN MINUTES 



FIGURE 2. Oxygen consumption in Peripatus and the centiped as a function of time. Open 
circles, Peripatus (Epiperipatus), 0.68 g. ; closed circles, 3 centipeds, total weight 0.33 g. ; 
temperature, 25. 

lower power of the weight. As a first approximation this may be taken as the 
two-thirds power (Krogh, 1916). When the oxygen consumption is compared on 
this basis, Peripatus agrees more closely with the arthropods and has a higher value 
than the earthworm. 

The hydrophobic character of the body surface has been noted by many ob- 
servers. It is particularly evident when the animal is submerged since the body 
papillae hold the water away from the body surface and leave the animal entirely 



OBSERVATIONS ON PERIPATUS 



187 



surrounded by a sheath of air. It would seem entirely possible that this air sheath 
may function as a respiratory surface under water. Such a mechanism has been 
demonstrated in certain aquatic insects which carry down an air supply by means 
of hydrophobic hairs and which, by this means, greatly extend their periods under 
water (Krogh, 1941 ; Wigglesworth, 1931). Since Peripatus must be often covered 
by water in rainstorms, particularly as its lack of resistance to dessication forces it 
to frequent wet places, this mechanism could be of real utility and have a consider- 
able survival value. This would provide a functional explanation for the papilla- 
covered body surface which is characteristic and unique in the Onychophora. 

TABLE II 

Oxygen consumption in Peripatus and other animals at 25 C. 







Oxygen consumption 


Animal 


Weight in mg. 












cc./g.hr. 


cc./g. 2 hr. 


Earthworm 


96 


0.22 


0.10 ' 


Peripatus 








(Epiperipatus) 


680 


0.23 


0.20 


Millipeds 


3X111 


0.46 


0.22 


Centipeds 


2X69 


0.56 


0.22 


Pill bugs 


5X61 


0.35 2 


0.14 



1 Lesser (1908) reported values of 0.4 cc. per g. 2/3 hr. at 19 at which temperature the oxygen 
consumption should be about half that measured at 25 (Vernon, 1897). 

2 Edwards (1946) reports a similar value but at a temperature of 17. 

SUMMARY 

The Onychophora represent a morphological transition between the annelids and 
the arthropods. They also represent a physiological transition between the aquatic 
and the terrestrial environment. In the latter transition the most important adapta- 
tions are those involving the functions of water conservation and respiration. 

The ability of Peripatus to conserve water has been compared to that of com- 
parable annelids and arthropods. Peripatus is shown to be intermediate to those 
two groups in this function, losing twice as much water as the centiped, but only 
one-half as much as the earthworm. This corresponds to its taxonomic and ecologi- 
cal positions. 

The "resting" rate of oxygen consumption has also been compared to other ani- 
mals. The rate in Peripatus is comparable to that in the arthropods and larger than 
that in the earthworm. 

It is suggested that the unique papilla-covered body surface may represent an 
adaptation for underwater respiration to meet the environmental restriction imposed 
by the inadequate regulation of water loss. 

LITERATURE CITED 

ANDREWS, E. A., 1933. Peripatus in Jamaica. Quart. Rev. Biol., 8 : 155. 

BRUES, C. T., 1923. The geographical distribution of the Onychophora. Amcr. Nat., 57: 210. 
CLARK, A. H., 1915. The present distribution of the Onychophora, a group of terrestrial in- 
vertebrates. Smithsonian Misc. Coll., 65 : 1-25. 



188 PETER R. MORRISON 

CLARK, A. H., AND J. ZETEK, 1946. The Onychophores of Panama and the Canal Zone. Proc. 
U. S. Nat. Mus., 96 : 205. 

EDWARDS, G. A., 1946. The influence of the temperature upon the oxygen consumption of sev- 
eral arthropods. Jour. Cell. Comp. Physiol., 27 : 53. 

HOLLIDAY, R. A., 1942. Some observations on Natal Onychophora. Ann. Natal. Mus., 10: 237. 

HUTTON, F. W., 1876. On Peripatus Novae-Zealandiae. Ann. Mag. Nat. Hist. (4) 18: 361. 

KENOYER, L. A., 1929. General and successional ecology of the lower tropical rain forest at 
Barro Colorado Island, Panama. Ecology, 10: 210. 

KROGH, A., 1916. The respiratory exchange of animals and man. London, Longmans, Green 
and Co., 173 pp. 

KROGH, A., 1941. The comparative physiology of respiratory mechanisms. Philadelphia, The 
University of Pennsylvania Press, 172 pp. 

LESSER, E. J., 1908. Chemische Prozesse bei Regenwiirmern. Zeit, f. Biol., 50: 421. 

MANTON, S. M., 1937. Studies on the Onychophora. II. The feeding, digestion, excretion and 
food storage of Peripatopsis. Proc. Roy. Soc. London, B, 227 : 411. 

MANTON, S. M., 1938. Studies on the Onychophora. VI. The life history of Peripatopsis. 
Ann. Mag. Nat. Hist. (11), 1 : 515. 

MANTON, S. M., AND J. A. RAMSAY, 1937. Studies on the Onychophora. III. The control of 
water loss in Peripatopsis. Jour. Exp. Bio!., 14: 470. 

MELLANBY, K., 1935. The evaporation of water from insects. Biol. Rev., 10: 317. 

MORRISON, P. R., 1946. Parturition in Peripatus. Psyche (In press). 

RAMSAY, J. A., 1935. The evaporation of water from the cockroach. /. Exp. Biol., 12 : 373. 

SCLATER, W. L., 1887. Notes on the Peripatus of British Guiana. Proc. Zool. Soc. London, 
130. 

SEDGWICK, A., 1885. A monograph on the development of Peripatus capensis. Quart. Jour. 
Micr. Sci., 25 : 449. 

SEDGWICK, A., 1887. A monograph of the genus Peripatus. Quart. Jour. Micr. Sci., 28: 431. 

SNODGRASS, R. E., 1938. Evolution of the Annelida, Onychophora, and Arthropoda. Smith- 
sonian Misc. Coll., 97 : 6. 

STEEL, T., 1896. Observations on Peripatus. Proc. Linn can Soc. New South Wales, 21 : 94. 

VERNON, H. M., 1897. The relation of the respiratory exchange of cold blooded animals to 
temperature. Jour. Physiol., 21 : 443. 

WIGGLESWORTH, V. B., 1931. The respiration of insects. Biol. Rev., 6: 181. 

J 



STUDIES ON CILIATES OF THE FAMILY ANCISTROCOMIDAE 

CHATTON AND LWOFF (ORDER HOLOTRICHA, 

SUBORDER THIGMOTRICHA) 

III. ANCISTROCOMA PELSENEERI CHATTON AND LWOFF, 

ANCISTROCOMA DISSIMILIS SP. NOV., AND 

HYPOCOMAGALMA PHOLADIDIS SP. NOV. 

EUGENE N. KOZLOFF 
Lewis and Clark College, Portland, Oregon 

INTRODUCTION 

Chatton and Lwoff described in 1926 two ciliates for which they created the 
genus Ancistrocorna: A. pelseneeri, from the gills and palps of Macoma balthica 
(L.) ; and A. pholadis, from Barnea (Pholas) Candida (L.). Their descriptions of 
these two species are of a preliminary nature and are not accompanied by illustra- 
tions. More detailed descriptions of A. pelseneeri, together with illustrations, are 
given in two papers of Raabe (1934, 1938). 

Kofoid and Bush (1936) described as Parachaenia myae a ciliate from the peri- 
cardial cavity and excurrent siphon of My a arenaria L. which Kirby (1941) noted 
was in several respects apparently identical with A. pelseneeri. Kudo (1946), 
however, listed Parachaenia myae as a valid species in the suborder Gymnostomata. 
Kofoid and Bush stated that they did not find P. myae in any other molluscs which 
were present in the same localities as the host species. I have studied the ciliate 
associated with Mya arenaria in San Francisco Bay and have compared it with 
similar forms from Cryptomya calif ornica (Conrad), Macoma inconspicua Broderip 
and Sowerby, 1 Macoma nasuta (Conrad), and Macoma ims (Hanley) from San 
Francisco Bay, and from Macoma sect a (Conrad) from Tomales Bay, California. 
I have concluded that the ciliate described by Kofoid and Bush as Parachaenia myae 
is not specific in Mya arenaria and that P. myae is identical with Ancistrbcoma 
pelseneeri Chatton and Lwoff. 

On the gills and palps of the rock-boring piddock Pholadidea penita (Conrad) 
there occurs a species of Ancistrocoma which is clearly distinct from A. pelseneeri 
and which I will describe in this paper as Ancistrocoma dissimilis sp. nov. Another 
ciliate I have studied from P. penita is referable to the genus Hypocomagalma, cre- 
ated by Jarocki and Raabe (1932) for H. dreissenae, from the fresh water mussel 
Drcissena polyrnorpha (Pall.). It will be described herein as Hypocomagalma 
pholadidis sp. nov. 

1 By some malacologists the small species of Macoma referred to in this paper as M. incon- 
spicua is considered to be conspecific with M. balthica; by others it is considered to be a sub- 
species of M. balthica. No conclusive evidence has been presented in the literature in recent 
years either to support or refute these contentions. 

189 



190 



EUGENE N. KOZLOFF 



ANCISTROCOMA PELSENEERI CHATTON AND LWOFF 
(Figure 1 ; Plate I, Figs. 1, 2) 

The body is elongated and somewhat flattened dorso-ventrally. 2 As seen in 
lateral view, the ciliate is banana-shaped, the ventral surface being incurved. The 
anterior end is more or less attenuated. The body is usually wjdest and thickest 
in its posterior third. Forty living individuals taken at random from Mya arenaria 
ranged in length from 50^ to 83 p., in width from 14 /A to 20 /*, and in thickness 
from 11 /A to 16^,, averaging about 62 ,11 by 16 /x by 12.5 //. Twenty individuals 
from Macoma inconspiaia ranged in length from 52 /u. to 78 /*, in width from 14 ju, 
to 19 p., and in thickness from 11 p. to 15 /JL. 





FIGURE 1. Ancistrocoma pelsenceri Chatton and Lwoff. Distribution of ciliary rows, somewhat 
diagrammatic. 3 A, dorsal aspect ; B, ventral aspect. 

The anterior end is provided with a contractile suctorial tentacle which enables 
the ciliate to attach itself to the epithelial cells of the gills and palps of the host and 
to suck out their contents. The internal tubular canal continuous with the tentacle 
is directed at first dorsally and then ventrally and obliquely toward the right side 
of the body. It can usually be traced in fixed individuals stained with iron hema- 
toxylin for about two-thirds the length of the body. Kofoid and Bush suggested 
only in the title of their paper that the form which they named Parachaenia myae 
was parasitic in Mya arenaria, but did not describe attachment of the ciliate to the 
epithelium. They found the ciliate in the pericardial cavity and excurrent siphon 

2 Kofoid and Bush stated in their description of "Parachaenia myae" that the body of this 
ciliate is bilaterally compressed, the transverse diameter being about two-thirds the dorso-ventral 
diameter. Obviously their orientation of the form in question is not in agreement with the 
orientation assigned to it by Chatton and Lwoff, Raabe, and myself. 

3 All text-figures illustrating this paper are based on camera lucida drawings of individuals 
fixed in Schaudinn's fluid and impregnated with activated silver albumose (protargol). 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 191 

oi the clams and apparently believed it to be unattached and to feed as a gymno- 
stome, by producing a current in the medium by means of vigorous ciliary activity 
which carries food particles to the mouth. They stated that they observed a few 
instances of food taking, in which "debris containing bacteria enters the mouth and 
moves along the cytopharynx, forming little globules which continue back and aggre- 
gate in the large food vacuoles which distend the posterior part of the body." They 
stated further that "stained specimens show some vacuoles containing broken-up 
nuclear material similar to that of the epithelial cells which are removed when the 
fluid is taken from the clam." I have not observed any instances of ingestion of 
food such as that described by Kofoid and Bush, and although I admit it is perhaps 
possible for the ciliates to ingest food in this manner, I believe that they are pri- 
marily branchial parasites which feed by means of the suctorial tentacle. 

The cilia of A. pelseneeri are disposed on the ventral, lateral, and dorso-lateral 
surfaces of the body in longitudinal rows originating at the anterior end. In all 
individuals which I examined carefully the number of ciliary rows was fourteen, 
but Raabe stated that in some specimens there are but thirteen rows. According to 
Raabe the ciliary system is composed of three separate complexes, the first consist- 
ing of eight or nine rows spiralling from the left side of the body toward the right 
and terminating progressively more posteriorly on the ventral surface, the second 
consisting of two approximately meridional rows situated on the central part of the 
ventral surface, and the third consisting of three rows spiralling from the right side 
of the body toward the left and terminating on the ventral surface. After studying 
a large number of the ciliates from Mya arenaria and Macoma inconspicua I cannot 
agree with Raabe on this matter. The ciliary rows appear collectively to form a 
single complex. There are usually five approximately equal row r s about two-thirds 
the length of the body occupying the central portion of the ventral surface ; these are 
bounded on the right by three progressively longer and more widely-spaced rows 
and on the left by six progressively longer and more widely-spaced rows. In some 
specimens the number of longer rows on the left side is greater than six, in which 
case the number of approximately equal and more or less meridional rows is pro- 
portionately decreased. Some of the outer rows on either side of the body, which 
originate on the lateral margins or on the dorsal surface, curve ventrally as they ex- 
tend posteriorly, but the last two rows on the left side and the last row on the right 
side are typically dorso-lateral in position over their entire length. The outermost 
row on either side extends almost to the posterior tip of the body. Kofoid and 
Bush stated that the ciliary rows of "Parachacnia myac" may unite with one an- 
other, but I have never observed this to be the case, although in some seriously 
shrunken fixed individuals a few of the rows converge in such a way that they ap- 
pear to be united. 

In one of the illustrations accompanying the first of Raabe's papers in which 
there is a detailed discussion of A. pelseneeri (1934) the, outermost ciliary row on 
the right side of the body is shown to originate as far anteriorly as the more cen- 
tral rows, while the outer three or four rows on the left side are shown to originate 
progressively more posteriorly. According to my own observations, however, the 
outermost row on the right side originates at about the same level as the last row 
on the left side. In all suitably impregnated individuals which I have studied the 
eighth row from the right side originates a little posterior to the level of origin of 
the adjacent ventral rows. 



192 EUGENE N. KOZLOFF 

The cilia of A. pelseneeri are 8 ^ to 10 p. in length. Those at the anterior end 
of the body are usually the more active and may be employed for thigmotactic at- 
tachment. Kofoid and Bush stated that the cilia of the "dorso-bilateral region" of 
"Parachaenia myae" are about 20 /x long near the anterior end, becoming somewhat 
shorter posteriorly ; the cilia of the ventral surface, on the other hand, were said 
by them to be about one-half the length of those of the dorso-bilateral area. I have 
noted, however, no significant disparity between the lengths of the cilia of various 
parts of the ciliary system. When dissociated from the host the ciliate swims ener- 
getically, rotating on its longitudinal axis or swaying from side to side. 

In the original description of A. pelseneeri given by Chatton and Lwoff refer- 
ence is made to a "frange peristomienne" which they supposed corresponded to the 
peristomal fringe of cilia in species of Ancistrmna. In his paper of 1934, Raabe 
described a short (approximately 13 /x long) row of basal granules lying in a dorsal 
anterior depression just above the anterior part of the internal tubular canal which 
he thought may represent the "frange peristomienne" described by Chatton and 
Lwoff. In his paper of 1938, however, Raabe stated that on certain of his prepara- 
tions of this ciliate he could distinguish a row of basal granules such as he described 
in 1934, but did not refer to it as the peristomal fringe, and suggested that Chatton 
and Lwoff may have mistaken the stained outline of the internal tubular canal for 
a row of basal granules homologous with those of the peristomal fringe of ancis- 
trumid ciliates. In my study of living, stained, and impregnated individuals of the 
ciliate I believe to be A. pelseneeri I have found no evidence whatever of a dorsal 
anterior depression or a row of basal granules such as that described by Raabe. 

Kofoid and Bush described internal fibrillar structures, w r hich they believed to 
represent elements of the neuromotor system, extending for a short distance poste- 
riorly from an annular commissure ("cytostomal ring") around the "cytostome." 
One of the fibrils was said by them to pass along the internal tubular canal ("cyto- 
pharynx") to a slight thickening on the surface of the canal, then "towards the 
dorsal surface where it joins a relatively large granule which is closely associated 
with the mid-dorsal ciliary fibril." They stated further that "from points of the 
cytostomal ring on the ventral side, two fibrils are given off which soon unite and 
continue as a slender thread along the ventral surface of the cytopharynx." I have 
been unable to detect any structures in A. pelseneeri which might be construed as 
elements of a neuromotor system, but perhaps it is a siderophilic fibril-like structure 
of the type that Kofoid and Bush described that Raabe may have thought to repre- 
sent a series of basal granules. The "cytostomal ring" around the "cytopharynx" 
was stated by Kofoid and Bush to be connected with the longitudinal ciliary rows, 

EXPLANATION OF PLATE I 

FIGURE 1. Ancistrocoma pelseneeri Chatton and Lwoff (from Mya arenaria). Ventral 
aspect. Heidenhain's "susa" fixative-iron hematoxylin. X 1,680. 

FIGURE 2. Ancistrocoma pelseneeri Chatton and Lwoff (from Macoma inconspicua) . 
Lateral aspect from left side, from life. 

FIGURE 3. Ancistrocoma dissimilis sp. nov. Ventral aspect. Schaudinn's fixative-iron 
hematoxylin. X 1,680. 

FIGURE 4. Hypocomagalma pholadidis sp. nov. Dorsal aspect. Schaudinn's fixative-iron 
hematoxylin. X 1,260. 

FIGURE 5. Hypocomagalma pholadidis sp. nov. Ventral aspect. Schaudinn's fixative-iron 
hematoxylin. X 1,260. 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 



193 



ii\\ 




- 



ii 

. 

r 






JK ; 





PLATE I 



194 EUGENE N. KOZLOFF 

but I have not observed this to be the case in A. pclscnccri. As has been pointed 
out above, some of the rows do not originate as close to the base of the suctorial 
tentacle as others. It is possible that the structure referred to by Kofoid and Bush 
as the "cytostomal ring" represents the siderophilic anterior edge of the contracted 
suctorial tentacle. 

The cytoplasm is colorless and contains numerous small refractile granules of a 
lipoid substance. In the posterior part of the body there are in addition to typical 
food vacuoles containing ingested fragments of epithelial cells one or more large 
vacuoles containing globular masses usually of a dense, homogeneous character. 
Raabe referred to this type of vacuole as "Konkrementenvacuole" and suggested 
that since he observed the internal tubular canal to terminate very near the 
"Konkrementenvacuole" the material within the vacuole may represent an accumula- 
tion of waste material which was not digested and absorbed as the ingested food 
material passed backward down the canal. It is quite true that these concrement 
vacuoles do not resemble the typical food vacuoles of most other ancistrocomid 
ciliates which I have studied. It would be interesting to determine whether or not 
digestion and absorption take place in the internal tubular canal, and how the mate- 
rial in the concrement vacuole, if it represents undigested wastes, is gotten rid of 
by the ciliate. 

The macronucleus is usually sausage-shaped, rarely ovoid, and typically is situ- 
ated dorsally near the middle of the body. In some fixed specimens stained with 
iron hematoxylin the chromatin appears to be distributed in irregular masses scat- 
tered through the macronuclear material ; in other iron hematoxylin preparations 
and in most specimens stained by the Feulgen reaction the chromatin is aggregated 
into a dense reticulum enclosing vacuole-like clear spaces. In twenty individuals 
from Mya arcnaria fixed in Schaudinn's fluid and stained by the Feulgen reaction 
the macronucleus ranged in length from 11 ^ to 16 p. and in width from 4 p. to 7 p. 

The micronucleus is ovoid, fusiform, or sausage-shaped, and usually is seen to 
lie to the right of the macronucleus. In fixed and stained specimens the chromatin 
is ordinarily aggregated into granules. In twenty individuals from Mya arcnaria 
fixed in Schaudinn's fluid and stained by the Feulgen reaction the micronucleus 
ranged in size from 1.2 /x by 3 ^ to 2.1 /j, by 3.2 j u. 

Ancistrocoina pclscnccri is very common in M\a arcnaria in all localities in San 
Francisco Bay where I have collected this mollusc. I have found it to be present, 
although usually in smaller numbers, also in Cryptoniya calljornlca, Maconia incon- 
splcua, M. nasuta, and M. irus from several localities in San Francisco Bay, and in 
Macoma sccta from Tomales Bay. It is peculiar that this ciliate was not recorded 
by Raabe from Mya arenaria at the marine biological station at Hel. Raabe listed 
Sphcnophyra doslniac Chatton and LwofF. Hypocomidium grainun Raabe, and a 
species of Ancistruma which he provisionally referred to A. cyclidioides (Issel), 
from M. arcnaria. I have found 6". doslniac in a small percentage of M. arcnaria 
and in a fairly large percentage of Cryptomya calijornica from San Francisco Bay. 
I have also found in M. arcnaria the ciliate thought by Raabe to be A. cyclidioides, 
but not Hypocomidium graimin. 

Ancistrocoina pclscnccri Chatton and Lzvoff (= Parachacnia myac Kofoid and 
Bush} 

Diagnosis: Length 50 /*-83 /JL (according to Kofoid and Bush 40/x-100/i), aver- 
age about 62 p.; width 14//.-20/*, average about 16^; thickness 11 ^-16 /x, average 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 195 

about 12.5 JJL. The ciliary rows are fourteen (according to Raabe thirteen or four- 
teen) in number and are distributed on the ventral, lateral, and dorso-lateral sur- 
faces of the body. There are usually five approximately equal rows about two- 
thirds the length of the body on the ventral surface, bounded on the right by three 
progressively longer and more widely-spaced rows and on the left by six progres- 
sively longer and more widely-spaced row r s. The outermost row on either side 
extends almost to the posterior tip of the body. The more central rows originate 
close to the base of the suctorial tentacle, while the several outer rows on either side 
originate progressively more posteriorly on the lateral margins and the dorsal sur- 
face. Some of these rows curve ventrally as they extend posteriorly, but the two 
outer rows on the left side and the outermost row on the right side are typically 
dorso-lateral in position over their entire length. The macronucleus is usually 
sausage-shaped. The micronucleus is ovoid, fusiform, or sausage-shaped. Para- 
sitic on the epithelium of the gills and palps of Maconia balthica (L.) (Wimereux 
[Chatton and Lw r off] ; Hel [Raabe] ) ; J\Iacoina inconspicua Broderip and Sowerby, 
Macoma uasuta (Conrad), Maconia inis (Hanley), Cryptomya calijornica (Con- 
rad) (San Francisco Bay, California) ; Maconia sccta (Conrad) (Tomales Bay, 
California) ; My a arcnaria L. (Tomales Bay [Kofoid and Bush] ; San Francisco 
Bay). 

ANCISTROCOMA DISSIMILIS SP. NOV. 
(Figure 2; Plate I, Fig. 3) 

The body is elongated, attenuated anteriorly, and somewhat flattened dorso- 
ventrally. The ciliary system, to be described presently, is disposed for the most 
part on the incurved and slightly concave ventral surface. The body is widest and 
thickest in its posterior third and rounded posteriorly. Twenty living individuals 
taken at random ranged in length from 33 /x to 51 /x, in width from 10 ^ to 14.5 /x, 
and in thickness from 8 /x to 12 /x, averaging about 44 ^ by 13 ^ by 10 /x. 

The anterior end is provided with a contractile suctorial tentacle continuous 
with an internal tubular canal. The canal is directed at first dorsally and then 
ventrally and obliquely toward the right side of the body. In fixed specimens 
stained with iron hematoxylin it can usually be traced posteriorly for about one-half 
the length of the body. 

The cilia of A. disshnilis are 7 it to 8 /x in length and are disposed in longitudinal 
rows originating at the anterior end. The typical number of ciliary rows is eleven, 
but specimens with twelve rows are not uncommon, and I have seen some with four- 
teen rows. There are usually five approximately equal rows about three-fifths the 
length of the body occupying the central portion of the ventral surface ; these are 
bounded on either side by three progressively longer rows, the outermost rows being 
three-fourths to four-fifths the length of the body. In specimens having twelve 
ciliary rows there are four longer ro\vs on the left side instead of three ; in speci- 
mens having fourteen rows there are four longer rows on the right side and five 
longer rows on the left. In some cases, particularly if the number of ciliary rows 
exceeds eleven, the five central rows are of unequal length, becoming progressively 
longer from right to left. One or two of the outer rows on either side originate 
on the lateral margin or the dorsal surface, usually a short distance posterior to the 
level of origin of the other rows. These rows curve ventrally and inward as they 
extend posteriorly, so that at least their distal portions are visible in ventral view. 



196 



EUGENE N. KOZLOFF 



The cytoplasm is colorless and contains numerous small refractile granules of a 
lipoid substance in addition to food inclusions. One or more larger food vacuoles 
are usually present in the posterior part of the body. The contractile vacuole lies 
near the middle of the body and opens to the exterior on the ventral surface. 

The macronucleus is ovoid and situated dorsally near the middle of the body. 
In fixed and stained preparations the outline of the macronucleus is nearly always 
very irregular and the chromatin appears to be aggregated into a dense reticulum 
enclosing vacuole-like clear spaces of varying size. In twenty individuals fixed in 
Schaudinn's fluid and stained with iron hematoxylin the macronucleus ranged in 
length from 6.8 ^ to 13.7 /n and in width from 5.4 ^ to 7.2 /A. 

The micronucleus is typically ovoid, rarely spherical, and commonly is situated 
a short distance anterior to or to one side of the macronucleus. In fixed and stained 





FIGURE 2. Ancistrocoma dissimilis sp. nov. Distribution of ciliary rows, somewhat 
diagrammatic. A, dorsal aspect ; B, ventral aspect. 

preparations the chromatin appears to be dispersed in granules of varying size. In 
twenty individuals fixed in Schaudinn's fluid and stained with iron hematoxylin 
the micronucleus ranged in size from 2.2 ^ by 2.4 ,u to 2.2 ^ by 3.2 p.. 

I found Ancistrocoma dissimilis to be present on the gills and palps of twenty- 
one of thirty-six specimens of Pholadidca pcnita which I examined from localities 
near Moss Beach, California. It is sometimes found in association with Hypo- 
comagalma pholadidis. In some individuals of P. penita I have encountered a ciliate 
of the genus Sphenophrya which I hope to describe in a later paper and a species 
of Boveria which may also be new. 

Ancistrocoma disshnilis sp. nov. 

Diagnosis: Length 33^-51^, average about 44^; width lO/x-14.5^, average 
about 13 ju,; thickness 8 fi-12 /JL, average about 10 /x. The ciliary rows are eleven to 
fourteen (typically eleven) in number and are distributed for the most part on the 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 197 

ventral surface and lateral margins of the body. Most of the rows originate on the 
ventral surface close to the base of the suctorial tentacle, while one or two outer 
rows on either side originate on the lateral margin or the dorsal surface and curve 
ventrally and inward as they extend posteriorly. There are usually five approxi- 
mately equal rows about three-fifths the length of the body bounded on the right 
by three progressively longer rows and on the left by four progressively longer 
rows. The outermost row on either side is three-fourths to four-fifths the length of 
the body. The macronucleus is ovoid. The micronucleus is typically ovoid. Para- 
sitic on the gills and palps of Pholadidea penita (Conrad) (Moss Beach, California). 
Syntypes are in the collection of the author. 

HYPOCOMAGALMA PHOLADIDIS SP. NOV. 
(Figure 3; Plate I, Figs. 4, 5) 

The body is elongated, strongly attenuated anteriorly, and markedly asymmetri- 
cal. The anterior end is deflected toward the left and bent ventrally. The dorso- 
ventral flattening characteristic of most ancistrocomid ciliates is not conspicuous in 
this species. As viewed from the posterior end the body appears in its middle and 
posterior portions to be almost as thick as wide. In its anterior third the body is 
nearly round in cross section. Most fixed specimens are considerably distorted and 
compressed in such a way that they appear to be widest near the middle. Twenty 
living individuals taken at random ranged in length from 63 /* to 89 ju, in width from 
18 ju to 25 JJL, and in thickness from 16 /z to 21 /*, averaging about 76 ^, by 22 /x by 19 //.. 

The anterior end is provided with a contractile suctorial tentacle continuous 
with an internal tubular canal. The canal can usually be traced in fixed specimens 
stained with iron hematoxylin down the middle of the attenuated anterior part of 
the body and then obliquely toward the right side. I have not succeeded in demon- 
strating the course of the canal beyond the anterior one-third of the body. 

The cilia of Hypocomagalma pJwladidis are approximately 9/x to 10 ^ in length. 
The ciliary system consists of twenty-four or twenty-five longitudinal rows. The 
body is almost completely invested by cilia except for a cilia-free "cap" at the pos- 
terior end. Two rows on the right side of the body usually appear to be set apart 
from the others, but in some specimens the spacing between these rows and the adja- 
cent rows on either side is not significantly wider than the spacing between some 
of the other rows. Perhaps these two rows are homologous with the one or two 
rows constituting the right ciliary complex of Crebricoma carinata (Raabe), Insig- 
nicoma venusta Kozloff, and species of Hypocomides. They originate near the base 
of the suctorial tentacle on the right margin or the dorsal surface close to the right 
margin and curve ventrally and to the left as they extend backward. The outer 
row, as seen in ventral view, is the longer and extends almost to the posterior end 
of the body. The inner row terminates a short distance more anteriorly than the 
outer row, but is conspicuously longer than the first of the next series of rows, 
which usually is about two-thirds the length of the body. The first eight to ten 
rows to the left of the two longer rows all originate at about the same level on the 
ventral surface close to the base of the suctorial tentacle. The remaining rows, 
which are disposed along the left margin of the body and on the dorsal surface, 
originate progressively more posteriorly. The tenth or eleventh row of this com- 
plex is usually the longest, although some of the shorter rows on the dorsal surface 



198 



EUGENE N. KOZLOFF 



may terminate more posteriorly. The last ciliary row on the right side of the 
dorsal surface is always the shortest row, originating at a point about one-third the 
distance from the anterior end of the body to the posterior end and terminating at 
a point about three-fourths or four-fifths the distance from the anterior end to the 
posterior end. 

The cytoplasm is colorless and contains numerous small refractile granules of a 
lipoid substance in addition to food inclusions. One or more larger food vacuoles 
containing fragments of cells from the epithelial tissues of the gills or palps of the 
host are usually evident in the posterior part of the body. The contractile vacuole, 
when single, is located near the middle of the body and opens to the exterior on 
the ventral surface. In a larger percentage of the living specimens of H. pholadidis 
which I examined there were two or more contractile vacuoles scattered through 






FIGURE 3. Hypocomagaluia pholadidis sp. nov. Distribution of ciliary rows, somewhat 
diagrammatic. A, dorsal aspect; B, ventral aspect. 

the body which emptied their contents to the exterior on the ventral surface. In a 
large percentage of the living specimens of H. pholadidis which I examined there 
were two or more contractile vacuoles scattered through the body which emptied 
their contents to the exterior independently of one another. Jarocki and Raabe 
(1932) reported that in H. dreissenae the contractile vacuole was sometimes single, 
but that in some specimens there were several smaller ones. 

The macronucleus typically is sausage-shaped and lies in the posterior third of 
the body, its longitudinal axis placed obliquely to the longitudinal axis of the body. 
In light iron hematoxylin preparations and in specimens stained by the Feulgen 
reaction the chromatin of the macronucleus appears to be aggregated into a dense 
reticulum enclosing vacuole-like spaces which frequently contain globular masses 
of deeply-staining material. In ten . individuals fixed in Schaudinn's fluid and 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 199 

stained by the Feulgen reaction the macronucleus ranged in length from 12.5 ju, to 
20 p. and in width from 5 /* to 8.9 /*. 

The micronucleus is spherical and usually is situated a short distance anterior 
to or to one side of the macronucleus. In most fixed and stained preparations the 
chromatin appears to be homogeneous, although in some the chromatin appears to 
be in part aggregated into granules or peripheral strands. In ten individuals fixed 
in Schaudinn's fluid and stained by the Feulgen reaction the diameter of the micro- 
nucleus ranged from 2.4 p. to 3.3 p.. 

I found Hypocomagalma pholadidis to be present on the gills and palps of 
twenty-eight of thirty-six specimens of Pholadidea penita which I examined from 
localities near Moss Beach, California. When the ciliate is dissociated from the 
host it swims erratically, usually rotating on its longitudinal axis and tracing wide 
arcs with its attenuated anterior end. The cilia of the anterior half of the body are 
more active than those of the posterior half and are sometimes observed to beat 
metachronously. The ventral cilia near the base of the suctorial tentacle are mark- 
edly thigmotactic. 

Hypocomagalma pholadidis sp. nov. 

Diagnosis: Length 63 /*-S9 /u, average about 76 /*; width 18^-25^, average 
about 22 p; thickness 16,u-21 /x, average about 19ft. The anterior end of the body 
is attenuated, conspicuously deflected toward the left, and bent ventrally. The 
ciliary system consists of twenty-four or twenty-five rows. Two long rows on the 
right side of the body appear in most specimens to be set apart from the remaining 
rows ; these two rows originate near the base of the suctorial tentacle and extend 
almost to the posterior end of the body. The first eight to ten rows to the left of 
these two longer rows originate at about the same level on the ventral surface, while 
the remaining rows, disposed along the left lateral margin and the dorsal surface, 
originate progressively more posteriorly. The contractile vacuole may be single or 
represented by several independent vacuoles opening to the exterior on the ventral 
surface. The macronucleus is sausage-shaped. The micronucleus is spherical. 
Parasitic on the epithelium of the gills and palps of Pholadidea penita (Conrad) 
(Moss Beach, California). Syntypes are in the collection of the author. 

LITERATURE CITED 

CHATTON, E., AND A. LWOFF, 1926. Diagnoses de cilies thigmotriches nouveaux. Bull. Soc. 

Zool. France, 51 : 345. 
JAROCKI, J., AND Z. RAABE, 1932. t)ber drei neue Infusorien-Genera der Familie Hypocomidae 

(Ciliata Thigmotricha), Parasiten in Susswassernmuscheln. Bull. int. Acad. Cracovie, 

Cl. Sci. math, not., B(II), 1932: 29. 
KIRBY, H., 1941. Relationships between certain Protozoa and other animals. In Calkins, G., 

and F. Summers (editors) : Protozoa in Biological Research. Columbia University 

Press, New York. 
KOFOID, C., AND M. BUSH, 1936. The life cycle of Parachaenia myae gen. nov., sp. nov., a ciliate 

parasitic in Mya arenaria Linn, from San Francisco Bay, California. Bull. Mus. Hist. 

not. Belgique, 12, No. 22. 

KUDO, R., 1946. Protozoology. 3rd ed. Charles C. Thomas, Springfield. 
RAABE, Z., 1945. tiber einige an den Kiemen von Mytilus edulis L. und Macoma balthica (L.) 

parasitierende Ciliaten-Arten. Ann. Mus. sool. polon., 10: 289. 
RAABE, Z., 1938. Weitere Untersuchungen an parasitischen Ciliaten aus dem polnischen Teil 

der Ostsee. II. Ciliata Thigmotricha aus den Familien: Hypocomidae Biitschli und 

Sphaenophryidae Ch. & Lw. Ann. Mus. zo.ol. polon., 13: 41. 



STUDIES ON CILIATES OF THE FAMILY ANCISTROCOMIDAE 

CHATTON AND LWOFF (ORDER HOLOTRICHA, 

SUBORDER THIGMOTRICHA) 

IV. HETEROCINETA JANICKII JAROCKI, HETEROCINETA 

GONIOBASIDIS SP. NOV., HETEROCINETA FLUMINI- 

COLAE SP. NOV., AND ENERTHECOMA 

PROPERANS JAROCKI 

EUGENE N. KOZLOFF 
Lewis and Clark College, Portland, Oregon 

INTRODUCTION 

The genus Heterocineta was established by Mavrodiadi (1923) for a ciliate 
which he named Heterocincta anodontae, and which he had formerly believed to 
represent a gregariniform stage in the development of Conchaphthirus anodontae 
(Ehrenberg). Unaware of the fact that Mavrodiadi had abandoned his earlier con- 
ception and applied the name Heterocincta anodontae to this ciliate, Jarocki and 
Raabe (1932) described the same species, from Anodonta cygnca (L.) and Unio 
pictorum L., as Hypocomatophora unionidarum. Jarocki later (1934) pointed out 
that Hypocomatophora unionidarum was a synonym of Hcterocineta anodontae. 

In his papers of 1934 and 1935 Jarocki described seven additional species of 
the genus Heterocincta ectoparasitic on fresh water gastropods : H. janickii, from 
Physa fontinalis (L.) ; H. Iwoffi, from Viviparus jasciatus Miiller ; H. chattoni, from 
Radix ovata (Drap.) ; H. krsysiki, from Bithynia tcntaculata (L.) ; H. maziarskii, 
from Coretus corncus (L.) ; H. turi, from Tropidiscus planorbis (L.) and Spiralina 
vortex (L.) ; and H. siedlcckii, from Acroloxus lacustris (L. ). In 1945 I de- 
scribed as Heterocineta phoronopsidis a ciliate from the tentacles of Phoronopsis 
viridis Hilton. This species is the only representative of the genus thus far de- 
scribed which is not a parasite of fresh water molluscs or of the annelid commensal 
Chaetogastcr limnaci von Baer when the latter is associated with infected snails. 

On the fresh water prosobranch snails Goniobasis plicijera silicula (Gould) and 
Fluminicola virens (Lea) I have found two new species of Heterocincta which will 
be described herein as H. goniobasidis sp. nov. and H. fluminicolae sp. nov. I have 
also studied a species of Heterocineta from Physa cooperi Tryon which agrees with 
the original description of H. janickii. It seems advisable, for comparative pur- 
poses, and in view of the fact that Jarocki's description of H. janickii is not accom- 
panied by illustrations, to include an account of the morphology of this form in the 
present paper. 

The genus Encrthccoina was proposed by Jarocki (1935) for a single species, 
E. propcrans, parasitic on the gills of Viviparus jasciatus. Although the original 
description of this species is quite adequate, it is not supplemented by illustrations, 
and the second installment of Jarocki's "Studies on ciliates from fresh-water mol- 
luscs," in which figures of E. properans and several other ciliates were to be pub- 

200 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV 



201 



lished, has not come to my attention. A ciliate which I have found to infest Vivi- 
parus malleatus (Reeve) apparently is identical with E. properans. This ciliate 
will be described and illustrated here. 

HETEROCINETA JANICKII JAROCKI 
(Figure 1; Plate I, Fig. 1) 

The body is elongated and flattened dorso-ventrally. The anterior end is attenu- 
ated, bent ventrally, and deflected slightly toward the left. The anterior one-half 
of the left margin is not quite so rounded as the right margin and typically is nearly 
straight or weakly indented. The body is widest a short distance behind the mid- 
dle and rounded posteriorly. The ciliary system, to be described presently, is dis- 
posed on a shallow concavity occupying the anterior two-thirds of the ventral sur- 
face ; the dorsal surface and that part of the ventral surface posterior to the ciliary 
area are convex. Twenty living individuals from Pliysa cooperi ranged in length 
from 25 fj. to 32 //., in width from 12 /x to 15^,, and in thickness from 10 /x to 12 /j., 




FIGURE 1. 



^> X- 

Hcterocineta janickii Jarocki. Distribution of ciliary rows, somewhat diagram- 
matic. 1 Ventral aspect. 



averaging about 30^ by 14 ^ by 11 /x. The specimens of H. janickii from Pliysa 
joiitinalis which were studied by Jarocki ranged in length from 23^ to 32 (*,, in 
width from 12^ to 17 /*, and in thickness from 10^, to 13^. 

The anterior end of the body is provided with a short contractile suctorial ten- 
tacle which enables the ciliate to attach itself to the epithelial cells of the host and 
to feed upon their contents. When fully extended the tentacle is about 3 /* to 4 ju, 
(according to Jarocki about 4.5 p.) in length. The internal tubular canal continuous 
with the tentacle is directed at first dorsally and then ventrally and obliquely toward 
the right side, and in specimens stained with iron hematoxylin can usually be traced 
for about one-half the length of the body. 

The ciliary system consists of eight longitudinal rows originating close to the 
base of the suctorial tentacle. The first four rows from the right side are approxi- 
mately one-half the length of the body. The remaining four rows become increas- 
ingly longer and terminate one behind the other a little to the left of the midline. 

1 The text figures illustrating this paper are based on camera lucida drawings of specimens 
impregnated with silver nitrate by Klein's method. 



202 EUGENE N. KOZLOFF 

The longest row is about two-thirds the length of the body. The cilia are about 
6 p. to 7 fji (according to Jarocki about 5 p. to 7 /x) in length. While attached to the 
skin of the host the parasites are as a rule almost immobile, their cilia exhibiting 
only a feeble motion. When dissociated from the host Hetcrocineta janickii swims 
sluggishly, usually rotating on its longitudinal axis and tracing wide arcs with its 
attenuated anterior end. 

The cytoplasm is colorless and contains numerous small refractile granules in 
addition to food inclusions. One or more large food vacuoles are present in the 
posteriorpart of the body behind the macronucleus. The contractile vacuole is situ- 
ated near the middle of the body and opens to the exterior on the ventral surface. 
I have observed no permanent opening in the pellicle. 

The macronucleus is typically sausage-shaped and is located near the middle of 
the body or somewhat posterior to the middle. As seen in dorsal or ventral view 
the longitudinal axis of the macronucleus is placed obliquely to the longitudinal axis 
of the body. As seen in lateral view, the anterior end of the macronucleus is di- 
rected dorsally, while the posterior end is directed ventrally. In fixed and stained 
preparations the chrbmatin appears to be more or less homogeneous. In ten indi- 
viduals fixed in Schaudinn's fluid and stained by the Feulgen reaction the macro- 
nucleus ranged in length from 7 //. to 1 1 ju, and in width from 4 /* to 5 /JL. 

The micronucleus is ovoid or spherical and is situated near the dorsal surface 
anterior to or to one side of the macronucleus. In most fixed and stained specimens 
the chromatin is homogeneous, although in some it appears to be concentrated in 
peripheral granules. In ten individuals fixed in Schaudinn's fluid and stained with 
iron hematoxylin the size of the micronucleus ranged from 1 .4 p. by 1 .4 ^ to A .6 /A 
by 2 p.. 

Hetcrocineta janickii was present in very small numbers on the tentacles, mantle, 
and margins of the foot of most of the specimens of Physa coopcri which I col- 
lected in a stream near Mt. Eden, California. The degree of infestation increased 
rapidly on snails kept in laboratory aquaria for a period of six weeks. 

Heterocineta janickii Jarocki 

Diagnosis: Length 25 ju-32 p. (according to Jarocki 23/x.-32/x), average about 
30 p; width 12/^-15 p. (according to Jarocki 12/x-17/t), average about 14/x; thick- 
ness 10/x-12/x (according to Jarocki 10/x-13/x), average about 11 p.. The ciliary 
system consists of eight rows originating close to the base of the suctorial tentacle. 
The first four rows from the right are about one-half the length of the body, while 
the remaining four rows become progressively longer and terminate one behind the 
other a little to the left of the midline. The longest row is about two-thirds the 
length of the body. Parasitic on the epithelium of the tentacles, mantle, and foot 
of Physa jontinalis (L.) (Warsaw [Jarocki])' and Physa coopcri Tryon (Mt. 
Eden, California). 

HETEROCINETA GONIOBASIDIS SP. NOV. 
(Figure 2; Plate I, Figs. 2, 3) 

The body is elongated and flattened dorso-ventrally. The anterior end is at- 
tenuated, bent ventrally, and deflected slightly toward the left. The anterior one- 
half of the left margin is not so rounded as the right margin and typically is nearly 



CILIATES OF THE FAMILY ANC1STROCOMIDAE. IV 



203 



straight or weakly indented. The body is widest at the middle or a short distance 
anterior to the middle. The ciliary system is disposed on a shallow concavity oc- 
cupying the anterior two-thirds of the ventral surface ; the dorsal surface and that 
part of the ventral surface posterior to the ciliary area are convex. Twenty-five 
living specimens taken at random ranged in length from 36 /j. to 48 /JL, in width from 
15,u, to 20 fj., and in thickness from 11 /A to 14 /JL, averaging about 43 /x by 18 /A 
by 13 fjL. 

The anterior end is provided with a contractile suctorial tentacle continuous with 
an internal tubular canal. The nature of the canal is very similar to that of other 
members of the genus. It is directed at first dorsally and then ventrally and ob- 
liquely toward the right side of the body. It can be traced in most fixed specimens 
stained with iron hematoxylin for about one-half to two-thirds of the length of the 
body. 




FIGURE 2. 



Hetcrocineta goniobasidis sp. nov. Distribution of ciliary rows, somewhat 
diagrammatic. Ventral aspect. 



The cilia of H. goniobasidis are about 9 /A long. Those of the anterior part of 
the ciliary system are markedly thigmotactic. The ciliary system consists of ten 
longitudinal rows. The first six rows are approximately the same length, being 
about one-half the length of the body, although on careful examination the first 
row is seen to originate some distance posterior to the level of origin of the other 
five rows. The seventh, eighth, ninth, and tenth rows originate progressively more 
posteriorly and become increasingly longer, terminating one behind the other a little 
to the left of the midline. The longest row is two-thirds to three-fourths the length 
of the body. The last one or two rows usually originate on the left margin and 
curve ventrally as they extend backward. The cilia of the distal portions of the 
longer rows are nearly always practically motionless and directed posteriorly. When 
dissociated from the host the ciliate swims sluggishly and erratically, rotating on its 
longitudinal axis. 

The cytoplasm is colorless and contains numerous refractile granules of a lipoid 
substance in addition to food inclusions. There are usually one or two large food 
vacuoles in the posterior part of the body behind the macronucleus. The contractile 
vacuole is central and opens to the exterior on the ventral surface. 



204 EUGENE N. KOZLOFF 

The macronucleus is situated in the middle portion of the body. It is elongated 
and typically somewhat narrower at its anterior end than at its posterior end. As 
seen in dorsal or ventral aspect, the longitudinal axis of the macronucleus is placed 
obliquely to the longitudinal axis of the body. As seen in lateral view, the anterior 
end of the macronucleus is directed dorsally, while the posterior end is directed 
ventrally. In ten individuals fixed in Schaudinn's fluid and stained with iron 
hematoxylin the macronucleus ranged in length from 10^ to 13. 5 /A and in width 
from 4 ^ to 5.5 /x. 

The spherical or ovoid micronucleus is very difficult to distinguish in the living 
ciliates. It is usually situated near the dorsal surface a short distance anterior to 
the macronucleus. In fixed and stained preparations the micronucleus is vesicular, 
the chromatin being concentrated along the periphery. In ten individuals fixed in 
Schaudinn's fluid and stained with iron hematoxylin the micronucleus ranged in 
size from 1 .2 ^ by 1 .5 ;u, to 1 .5 p, by 1.7 /i. 

Heterocineta goniobasidis was found to be present on the epithelium of the gills 
and mantle of a small percentage of the specimens of Goniobasis plicifcra silicula 
which I collected in Crystal Springs Creek, in Portland, Oregon. The degree of 
infestation on freshly collected snails was very low, but increased during the four 
weeks the specimens were kept in laboratory aquaria. 

Heterocineta goniobasidis sp. nov. 

Diagnosis: Length 36^ 1-8//., average about 43 yu.; width 15^-20/x, average 
about 18/x; thickness ll/x-14/x, average about 13/x. The ciliary system h com- 
posed of ten rows. The first six rows from the right side are about one-half the 
length of the body and, with the exception of the first row, originate close to the 
base of the suctorial tentacle. The remaining rows originate progressively more 
posteriorly and become increasingly longer, terminating one behind the other a little 
to the left of the midline. The longest row is two-thirds to three-fourths the length 
of the body. Parasitic on the gills and mantle of Goniobasis plicifera silicula 
(Gould) (Portland, Oregon). Syntypes are in the collection of the author. 

HETEROCINETA FLUMINICOLAE SP. NOV. 
(Figure 3; Plate I, Fig. 4) 

The body is elongated and flattened dorso-ventrally. The anterior end is at- 
tenuated, bent ventrally, and deflected slightly toward the left. The anterior part 



EXPLANATION OF PLATE I 
All figures except Figure 2 have been prepared with the aid of a camera lucida. 

FIGURE 1. Heterocineta janickii Jarocki. Ventral aspect. Schaudinn's fixative-iron hema- 
toxylin. X 1,720. 

FIGURE 2. 'Heterocineta goniobasidis sp. nov. Lateral aspect from left side, from life. 

FIGURE 3. Heterocineta goniobasidis sp. nov. Ventral aspect. Schaudinn's fixative-iron 
hematoxylin. X 1,720. 

FIGURE 4. Heterocineta fluniinicolae sp. nov. Ventral aspect. Schaudinn's fixative-iron 
hematoxylin. X 1,720. 

FIGURE 5. Encrthccoma properans Jarocki. Macro- and micronuclei from three specimens. 
Schaudinn's fixative-Feulgen reaction. X 1,720. 

FIGURE 6. Enerthecoma properans Jarocki. Ventral aspect. Schaudinn's fixative-iron 
hematoxylin. X 1,720. 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV 



205 


















ff. 













PLATE I 



206 



EUGENE N. KOZLOFF 



of the left margin is not so rounded as the right margin and typically is weakly 
indented. The body is widest a short distance behind the middle and rounded 
posteriorly. The ciliary system is disposed on a shallow concavity occupying the 
major portion of the ventral surface ; the dorsal surface and that part of the ventral 
surface posterior to the ciliary area are convex. Twenty-five living individuals 
taken at random ranged in length from 30^ to 36 /x, in width from 13 /x to 17 /M, 
and in thickness from 10 /A to 12 //,, averaging about 33 /A by 15/u by 11 /*. 

The anterior end is provided with a contractile suctorial tentacle continuous 
with an 'internal tubular canal. The canal is directed at first ventrally and then 
obliquely toward the right side of the body. It can be traced in most fixed speci- 
mens stained with iron hematoxylin for about one-half the length of the body. 

The cilia of H. fluminicolae are about 6/x or 7 p. long. Those of the anterior 
part of the ciliary system are strongly thigmotactic. The ciliary system consists of 
ten longitudinal rows. The first row on the right side of the ciliary complex origi- 




FIGURE 3. Hctcrochicla flnininicolac sp. nov. Distribution of ciliary rows, somewhat 

diagrammatic. Ventral aspect. 

nates close to the base of the suctorial tentacle ; each of the remaining rows origi- 
nates progressively more posteriorly. The first six rows from the right side are 
approximately the same length, being about two-thirds the length of the body. The 
last four rows become increasingly longer and incurved in such a \vay that they 
terminate one behind the other not far to the left of the midline. The longest row 
usually extends almost to the posterior end of the body. The cilia of the distal 
portions of these longer rows are usually directed posteriorly. When the ciliate is 
dissociated from the host it swims erratically, rotating on its longitudinal axis and 
tracing wide arcs with its anterior end. 

The cytoplasm is colorless and contains numerous small refractile granules of 
a lipoid substance in addition to food inclusions. One or more large food vacuoles 
are usually present in the posterior part of the body behind the macronucleus. The 
contractile vacuole is central and opens to the exterior on the ventral surface. I 
have not observed a permanent opening in the pellicle. 

The sausage-shaped macronucleus is situated dorsally a short distance behind 
the middle of the body with its longitudinal axis placed obliquely to the longitudinal 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV 207 

axis of the body. In fixed and stained preparations the chromatin appears to be 
more or less homogeneous. In ten individuals fixed in Schaudinn's fluid and 
stained with iron hematoxylin the macronucleus ranged in length from 7.4 p, to 10 p. 
and in width from 3.9 p, to 4.4 /JL. 

The micronucleus is round, fusiform, or ovoid, and .is usually placed dorsally 
near the middle of the body anterior to or to one side of the macronucleus. In 
fixed and stained specimens the chromatin is seen to be concentrated primarily along 
the periphery. In ten individuals fixed in Schaudinn's fluid and stained with iron 
hematoxylin the micronucleus ranged in size from 1.5^ by 1.2 /JL to 1.7 p, by 1.5 /A. 

Heterocineta fluminicolae was present in small numbers on the epithelium of the 
gills and the edge of the mantle of nearly all specimens of Fluminicola virens which 
I collected in Crystal Springs Creek in Portland, Oregon. 

Heterocineta fluminicolae sp. nov. 

Diagnosis : Length 30 /A-36 /JL, average about 33 p. ; width 13 p.-\7 p., average about 
15 p.; thickness lO/t-12//,, average about 11 p. The ciliary system is composed of 
ten rows originating progressively more posteriorly from the right side to the left. 
The first six rows from the right side are about two-thirds the length of the body. 
The remaining four rows become increasingly longer and terminate one behind the 
other a little to the left of the midline. The longest row extends almost to the pos- 
terior end of the body. Parasitic on the gills and mantle of Fluminicola virens 
(Lea) (Portland, Oregon). Syntypes are in the collection of the author. 

ENERTHECOMA PROPERANS JAROCKI 
(Figure 4; Plate I, Figs. 5, 6) 

The body is elongated, nearly symmetrical as seen in dorsal or ventral view, at- 
tenuated anteriorly, and flattened dorso-ventrally. The anterior end is bent ven- 
trally and deflected inconspicuously toward the left. The ciliary system is disposed 
on a narrow, relatively flat area occupying the anterior two-thirds of the ventral 
surface ; the dorsal surface and that part of the ventral surface posterior to the 
ciliary area are convex. The body is widest at a point about two-thirds the dis- 
tance from the anterior end to the posterior end. Twenty-five living individuals 
taken at random from Viviparus malleatus ranged in length from 32 p, to 56 p, in 
width from 13 ^ to 21 p., aiid in thickness from 10 p. to 13 /x, averaging about 44 p. 
by 18 p. by 11.5^. Specimens from Viviparus fasciatus which were measured by 
Jarocki ranged in length from 33 p. to 60 p., in \vidth from 15 p. to 22 p, and in thick- 
ness from 10 p. to 13 p.. 

The contractile suctorial tentacle is continuous with an internal tubular canal 
which is directed at first dorsally and then ventrally and obliquely toward the right 
side of the body. In specimens stained with iron hematoxylin the canal can usually 
be traced for about two-thirds or three-fourths the length of the body. 

The ciliary system is composed of eight approximately equal rows about two- 
thirds the length of the body. These rows originate close to the base of the suc- 
torial tentacle. The first five rows from the right side are usually a little more 
widely spaced than the last three rows. This was noted also by Jarocki. who stated 
that the ciliary system was separated into two complexes by an "inconsiderable 
eminence stretching from the base of the tentacle to the end of the system," which 



208 



EUGENE N. KOZLOFF 



segregated the five rows on the right from the three rows on the left. This eminence 
was evident on many of the living specimens which I examined but is never con- 
spicuous. The cilia of E. proper ans are about 9 ^ in length and exhibit a feeble 
undulatory motion while the parasites are attached to the epithelium of the gills of 
the host. When dissociated from the host the ciliates swim slow and erratically, 
usually rotating on their longitudinal axes. 

The cytoplasm is colorless and contains numerous small refractile granules ot 
a lipoid substance in addition to food inclusions. One or more larger food vacuoles 
are usually present in the posterior part of the body. The contractile vacuole is 
situated a short distance behind the middle of the body and opens to the exterior 
on the ventral surface. I have not detected a permanent opening in the pellicle. 

The macronucleus is typically sausage-shaped and is situated in the posterior 
half of the body with its longitudinal axis placed obliquely to the longitudinal axis 




FIGURE 4. Enerthccoma propcrans Jarocki. Distribution of ciliary rows, somewhat 

diagrammatic. Ventral aspect. 

of the body. In specimens stained with iron hematoxylin the chromatin appears 
to be more or less homogeneous, but in preparations stained by the Feulgen re- 
action it appears to be organized into a dense reticulum enclosing vacuole-like clear 
spaces of varying size. In ten individuals fixed in Schaudinn's fluid and stained 
by the Feulgen reaction the macronucleus ranged in length from 10 /A to 19 ^ and 
in width from 4 ^ to 7 p. 

The micronucleus is situated anterior to or to one side of the macronucleus. 
In most of the individuals of E. properans which I examined, the micronucleus is 
elongated and more or less fusiform. I have observed very few specimens to have 
a round micronucleus such as that described by Jarocki. The micronucleus does 
not stain readily with iron hematoxylin and it is possible that Jarocki may have 
mistaken food inclusions for micronuclei. In specimens stained by the Feulgen 
reaction the chromatin of the micronucleus appears to be concentrated in peripheral 
granules or strands. In ten individuals fixed in Schaudinn's solution and stained 



CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV 209 

by the Feulgen reaction the micronucleus ranged in size from 0.8 p. by 2.3 /* to 1 /* 
by 3.8 n. 

Enerthecoma properans was abundant on the gills of nearly all specimens of 
Viviparus mallcatus which I collected in Stow Lake, San Francisco, California, 
and in Evans Lake, Riverside, California. It is undoubtedly a common parasite 
of this introduced snail wherever the latter has become established. 

Enerthecoma properans Jarocki 

Diagnosis: Length 32/^-56^ (according to Jarocki 33^-60^), average about 
44/.I. ; width 13 /x-21 p. (according to Jarocki 15/x-22/x), average about 18 /x; thick- 
ness 10fi-13/x, average about 11.5/x. The ciliary system is composed of eight ap- 
proximately equal rows about two-thirds the length of the body which originate 
close to the base of the suctorial tentacle and occupy a narrow, relatively flat area 
on the ventral surface. The first five row's from the right are more widely-spaced 
than the remaining three rows, and in living specimens appear to be segregated 
from the latter by an inconspicuous longitudinal eminence. The macronucleus is 
elongated ; the micronucleus is typically elongated and more or less fusiform (ac- 
cording to Jarocki, spherical). Parasitic on the gills of Viviparus jasciatus Miiller 
(Warsaw [Jarocki]) and Viviparus mallcatus (Reeve) (San Francisco, California; 
Riverside, California). 

LITERATURE CITED 

JAROCKI, J., 1934. Two new hypocomid ciliates, Heterocineta janickii sp. n. and H. Iwoffi 
sp. n., ectoparasites of Physa fontinalis (L.) and Viviparus fasciatus Miiller. Mem. 
Acad. Cracovic, Cl. Sci. math, not., B, 1934: 167. 

JAROCKI, J., 1935. Studies on ciliates from fresh-water molluscs. I. General remarks on pro- 
tozoan parasites of Pulmonata. Transfer experiments with species of Heterocineta 
and Chaetogaster limnaei, their additional host. Some new hypocomid ciliates. Bull, 
int. Acad. Cracovic, Cl. Sci. math, nat., B (II), 1935: 201. 

JAROCKI, J., AND Z. RAABE, 1932. Uber drei neue Infusorien-Genera der Familie Hypocomidae 
(Ciliata Thigmotricha), Parasiten in Siisswassermuscheln. Bull. int. Acad. Cracovie, 
Cl. Sci. math, nat., B (II), 1932: 29. 

KOZLOFF, E., 1945. Heterocineta phoronopsidis sp. nov., a ciliate from the tentacles of Phoro- 
nopsis viridis Hilton. Biol. Bull., 89 : 180. 

MAVRODIADI, P., 1923. "Kosoe" delenie u infuzoril. Pratsy Bclaruskaga dziarshafinaga univer- 
sytctu H Mcuskn, 4-5 : 166. 



PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED 
AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1946 

JULY 9 
DR. J. E. KINDRED. No abstract submitted. 

The cyanide sensitivity of the unfertilized sea urchin egg. W. A. ROBBIE. 

Reinvestigation of the cyanide sensitivity of unfertilized eggs of Arbacia punctulata, using 
recently devised methods for the control of HCN concentration in manometric experiments, 
showed that there was a definite inhibition of respiration. The respiration is depressed by 
concentrations of HCN as low as 10~ 5 M., and for a four-hour period in KT 4 M. it is only 40 
per cent of the control value. There is complete inhibition for the first hour or more. In 4 per 
cent CX-96 per cent Nj mixture there is no depression of the respiration of the control egg, but 
on the addition of 10~ l M. HCN the oxygen consumption is reduced, for a four-hour exposure, 
to 20 per cent of the control level. 

At concentrations of cyanide higher than 10 4 M. there is apparently a stimulation in oxygen 
uptake. This is increased with high and reduced with low oxygen tensions. It is possibly 
associated with oxidations proceeding through a cyanide-hemin system, or with the metabolism 
of a carbohydrate intermediate catalyzed by HCN. 

Inhibition of fertilisation in sea urchins by means of univalent antibodies vs. 
antifertilisin. ALBERT TYLER. 

In order to obtain further information as to the role of the specific interacting substance 
of eggs and sperm in fertilization, antisera were prepared against them by immunization of 
rabbits, and the antibodies tested for their ability to interfere with fertilization. The present 
report concerns tests with antibodies prepared against purified anifertilizin derived from sperm 
of the sea urchin Lytcchinus pictus and the gephyrean worm Urcchis caupo. The antisera ag- 
glutinate the species sperm to high titer, but cannot be used directly to test for specific action on 
fertilization, since the mechanical effect of tying up the sperm would itself constitute a block to 
fertilization. However, by a previously described method, namely photo-oxidation, antibodies 
can be converted into a non-agglutinating form, termed "univalent." This treatment was, 
therefore, applied to the anti-antifertilizin sera and the "univalent" antibodies thus obtained 
were tested for possible action on the ability of the sperm to fertilize eggs of the same species. 
The results showed a considerable reduction in fertilizing power of the sperm, ranging in dif- 
ferent tests from 32-fold to greater than 128-fold. At the same time, the motility of the treated 
sperm was found to be quite as high as the controls. 

JULY 16 
Intermediate steps in the visual cycle. A. F. BLISS. 

The primary functions of a visual pigment are absorption of radiant energy and its transfer 
to the stimulatory mechanism of the visual cell. At present four such pigments are known : 
rhodopsin and porphyropsin, the photosensitive pigments of vertebrate night vision; iodopsin, 
the corresponding pigment of daylight vision; and cephalopsin, the photostable red pigment of 
cephalopods and probably other invertebrates (/. Gen. Physiol, 1943). Instability in the light 
has generally been accepted as a diagnostic test for a visual pigment. The existence of a light- 
stable visual pigment in the squid however throws doubt on the validity of this assumption. 

The bleaching by light of vertebrate visual pigments is nevertheless an interesting and 
complex process which has not been adequately analyzed into its component steps. The first 
known product of bleaching visual purple is a thermally unstable complex lipid, called Tran- 

210 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 211 

sient Orange by Lythgoe and Provisual Red by Krause. Its sluggish reaction to base and tem- 
perature near C. suggest that it is the acid tautomer of the first relatively stable product of 
bleaching, appropriately named Indicator Yellow by Lythgoe. Acid Indicator Yellow is a red 
lipid, becoming reversibly decolorized in base, and irreversibly converted in chloroform to the 
greenish yellow carotenoid retinene, extracted by Wald from freshly bleached frog retinas. If 
bleached retinas are allowed to stand an hour before extraction, retinene is no longer found. 
Instead an equivalent amount of vitamin A is extracted. Retinene, however, is not the precursor 
to vitamin A, but is due to the irreversible side reaction described above. In the normal retina 
and in fresh neutral solution Indicator Yellow forms vitamin A under the influence of a labile 
protein, the reaction being presumbaly enzymatic in nature. In the dark, dissolved rhodopsin 
is reformed in part from Indicator Yellow. In the living animal the vitamin A released by 
bleaching is reincorporated into rhodopsin by unknown means. 

MR. W. H. PRICE. No abstract submitted. 

The dependence of the resting potential of nerve on potassium, calcium, and 
hydrogen ions.* ABRAHAM M. SHANES. 

On the basis of new as well as recent -experimental results it now appears possible to de- 
scribe a specific mechanism necessary and sufficient to account for the relationships between the 
resting potential and metabolic processes in frog nerve. Cellular hydrogen ion production ac- 
companied by an exchange with extracellular potassium ions is apparently involved ; under the 
conditions of study this process contributes about 50 per cent of the total resting potential. 
Calcium reduces the rate of ionic exchange, an effect of possible importance in the energy ex- 
penditure necessary to maintain concentration gradients and associated potentials. 

The evidence consists of demonstrating first that hydrogen ions from (1) CO, produced 
by nerve, (2) CO 2 applied to nerve, and (3) lactic acid and possibly other sources of acid within 
the fibers are directly concerned with the production and maintenance of the potentials. This 
has been possible chiefly with the aid of inhibitors of carbonic anhydrase sulfanilamide and 
thiophene-2-sulfonamide by means of which the role of hydrogen ions can be followed during 
anoxia, upon return to oxygen following anoxia, and to some extent during relatively normal 
aerobic conditions. The changes of potential associated with the application of CQ..-O 2 mix- 
tures and related experiments show that the effectiveness of the hydrogen ions is dependent on 
the ionic gradients established. 

The involvement of extracellular potassium is demonstrated by the suppression in its ab- 
sence of the changes in potential normally produced by CO 2 . This effect is used to show, fur- 
ther, that the potassium in the extracellular spaces is reduced by the rapid large increase in 
potential induced by the return of the anoxic nerves to oxygen. A small secondary decline of 
potential which follows the rise in CO 2 and which is independent of extracellular buffering is 
also dependent on extracellular potassium, which suggests that the dense connective tissue or 
the other sheathing materials of nerve are interfering with the potassium exchange between the 
extracellular space immediately adjacent to the fibers and that more remote. 

Calcium slows the rate of potential rise upon application of CO 2 and this effect is directly 
related to calcium concentration ; in view of the above results and available evidence, this is 
interpreted as an effect on ionic exchange. At lower concentrations calcium depresses to almost 
the same degree the potential changes in response to oxygen following anoxia and to CO 2 ; 
higher calcium concentrations, known to suppress the metabolic processes, exert a more marked 
effect on the former. 

These results therefore focus attention on factors important in the production and modifica- 
tion of the resting potential. The action of any agent on the potential must be considered from 
several possible standpoints: (1) inhibition or activation of metabolism or of carbonic anhy- 
drase, (2) production of hydrogen ions, (3) production of a membrane diffusion potential, (4) 
modification of equilibrium or membrane diffusion potentials. The experimental procedures 
which have been applied provide means of distinguishing these possibilities. In view of the 
conclusions reached, these methods should also prove useful in studies of the "potassium pump" 
and of the biochemical processes concerned with CO 2 production and fixation in relatively intact 
cells, both are problems of considerable interest at the present time. 

* Aided by a grant from the Penrose Fund of the American Philosophical Society. 



212 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

DR. J T. BONNER. No abstract submitted. 

JULY 23 

Oxidation-reduction studies as a clue to the mechanism of fertilisation of marine 
eggs. MATILDA M. BROOKS. 

Eggs, sperm, and larvae at stages up to pluteus of three marine animals (Arbacia punctu- 
lata, Asterias Forbesii and Chactopterus pcrgcmentaceus) were measured for E h and Ph. The 
egg, sperm or larvae were centrifuged and 1 cc. of the mass used for measurement in a glass 
vessel in the Coleman electrometer. It was found that there is a definite correlation between 
the rate of Q.. consumption and the redox potential of these cells. It was also found that the 
redox potential of sea water, as diluted by hypertonic NaCl, CaCU, MgCl,., butyric acid or su- 
crose, became more negative than that of sea water alone. These facts were used as a basis 
for the hypothesis that a proper redox potential or ratio of oxidants to reductants of the respira- 
tory enzymes was necessary for producing fertilization of the egg. The hypothesis as presented 
states that the redox potential of the external solution or sperm as compared with that of the 
exterior of the egg itself is an important factor in producing fertilization of the egg. 

From the results with KCN in sea water which produced fertilization membranes but not 
cleavage, it was concluded that the formation of the fertilization membrane is not associated 
with oxidations, and appears rather to be due to change in the physical aggregation of some 
proteins at the surface of the egg or to a denaturation process occurring as the redox potentials 
is changing. 

DR. C. L. YNTEMA. No abstract submitted. 

JULY 30 

The action oj napthoquinone antimalarials on respiratory systems. CHRISTIAN 
B. ANFISEN AND ERIC G. BALL. 

In confirmation of the findings of Wendel (unpublished reports) a series of 2-hydroxy-3- 
alkyl-naphthoquinones have been found to exert a powerful inhibitory effect on the respiratory 
metabolism of the malarial parasite. The most powerful tested to date is the compound 2- 
hydroxy-3(2-methyl-octyl)-naphthoquinone-l,4 (M-285) which, at a level of 1 mg./liter in- 
hibits Plasmodium kiwzvlcsi respiration 60 per cent. In experiments to localize the site of ac- 
tion of M-285 in the main respiratory chain of enzymes it was found that p-phenylene-diamine 
oxidation, requiring only the cytochrome system, was not inhibited by the drug, while the oxida- 
tion of succinate to fumarate by succinic oxidase prepared from beef heart was completely in- 
hibited at about 1 mg./liter. The drug, therefore, appears to inhibit at an oxidation-reduction 
potention level below that of cytochrome C. Succinic dehydrogenase activity, as measured by the 
Thunberg methylene blue technique, was only very slightly diminished even at high drug con- 
centrations. Similarly, the respiration of both fertilized and unfertilized eggs of Arbacia punc- 
tulata, neither presumably containing succinic dehydrogenase, was inhibited strongly at levels 
as low as 0.1 mg./liter (2 X 10" 7 M.).This enzyme, therefore, does not seem to be the inhibited 
system. A number of flavoproteins including d-amino acid oxidase and xanthine oxidase, as 
well as several systems involving the mediation of the pyridine nucleotides, showed no decrease 
in activity in the presence of the drug. 

It appears possible that the naphthoquinones under study are inhibiting a hitherto un- 
detected enzyme or enzyme group in the main chain of oxidative metabolism having an E below 
that of cytochrome C and above that of the flavoproteins. 

Chemical sense and taste in the Sea Robin, Prionotus. ERNST SCHARRER. 

The differentiation of taste and chemical sense is partly based on the concept that chemical 
sensitivity can evoke only negative or defensive reactions ; positive reactions to food are medi- 
ated by the sense of taste (Kappers, Huber, Crosby, 1936, p. 347). Observations in the sea 
robin, Prionotus, do not support this conclusion. Prionotus possesses three free fin rays. Their 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 213 

epithelium is innervated by spinal nerves ; taste buds are absent. The afferent fibers end in 
accessory lobes on the dorsal surface of the cephalic end of the spinal cord. Secondary fibers 
from these lobes which represent the greatly enlarged dorsal horns, ascend cephalad to the 
funicular nucleus from which fibers pass ventromedially, crossing in the ventral commissure, 
and ending in the contralateral ventral horn. When the free fin rays of blinded and sufficiently 
hungry sea robins are stimulated with extracts of clams or crabs the animals react positively by 
turning to and snapping in the direction from where the juice comes. Positive reactions to 
chemical stimuli are mediated in this case by spinal nerves and in the absence of taste buds. 
The differentiation between chemical sense and taste can, therefore, be based only on the inner- 
vation and the presence or absence of taste buds. The reaction of the animal cannot be used 
as a criterion. 

Studies o\ the respiration of the iniaginal discs of Drosophila using the Cartesian 
diver ultramicrorespiromctcr. CLAUDE A. VILLEE. 

A determination of the effects of a mutant gene on the metabolic activity of a particular 
group of cells provides a basic approach to the analysis of gene action in development. In 
most animals it is impossible to locate exactly the cells which will give rise to a particular 
structure, but in Drosophila each organ develops from a discrete group of cells, an imaginal 
disc, which can be dissected out of the larva. The rate of respiration of wing and leg 
discs from wild, "miniature" wing, and "vestigial" wing stocks were determined by the 
Cartesian diver ultramicrorespirometer and their weights measured by the quartz fiber balance 
of Lowry. The legs of the adults of all three stocks are normal, the wings of adult "miniature" 
flies are about two-thirds normal size but of normal shape, and the wings of adult "vestigial" 
flies are small misshapen stumps, less than one-quarter the size of the normal wing. At each 
of several stages studied, before, at and after pupation, the Qo 2 of wild type discs and the leg 
discs of all stocks used varied only slightly from 20 cu. mm. O 2 per hour per milligram of 
tissue. The Qo 2 of "miniature" wing discs was 18 and of "vestigial" wing discs 9 cu. mm. O,. 
per hour per milligram of tissue. The weights of "vestigial," "miniature" and wild type wing 
discs are the same at corresponding developmental stages in the larvae and early (1-2 hour) 
pupae. The discs contain considerable reserves of substrate and will respire in the divers 
twelve hours or more. The mutant genes "vestigial" and "miniature" produce their effects 
by altering the rate of some chemical reaction in the wing disc of the larva which is reflected 
by a lowered rate of oxygen consumption. These results do not mean that the "miniature" 
and "vestigial" genes affect the same chemical reaction in development to a different extent 
but rather that in affecting different processes they each lower the overall metabolic rate of the 
disc. The metabolism of the leg discs, and probably of the other discs as well, is not changed, 
although the cells contain the mutant gene. The "vestigial" and "miniature" genes therefore 
produce their physiological as well as their morphological effects only in certain cells of the 
body, presumably due to the interaction of the gene or gene products with specific components 
of the cytoplasm of those cells. 

AUGUST 6 

The specificity of chlorine est erase. PHILIP B. ARMSTRONG. 

The relative rates of hydrolysis of choline esters acting at the nerve terminations in the 
sphincter pupillae of the turtle could be inferred by determining the relative potentiations by 
eserine of threshold concentrations for pupillary constriction of the choline esters. A com- 
parison of the potentiations in vivo with the relative hydrolysis rates of the choline esters 
by the purified specific choline esterase in vitro indicates that the enzyme as it functions in vivo 
is as specific if not more so than in vitro. The choline ester substrate concentrations in vivo 
at which eserine was effective were much lower than those for effective substrate hydrolysis 
in vitro. 

DR. T. H. BULLOCK. No abstract submitted. 



214 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

The endocrine role of the corpora allata in insects. BERTA SCHARRER. 

In Lcncophaca wadcrac (Orthoptera) extirpation of the corpora allata at nymphal stages 
earlier than the last causes an abbreviation of development (suppression of molts) which results 
in animals with adult-like characters ("adultoids"). In operated seventh instars the following 
nymphal molt is suppressed, and the animals emerge as adultoids, resembling normal adults 
except for their smaller size and comparatively shorter wings. Allatectomized sixth or fifth 
instars result in "pre-dultoid" stages which show less adultoid differentiation and require an 
additional molt before becoming adultoids. In the adult insect the corpora allata are necessary 
for the development of the eggs. In females allatectomized shortly after the beginning of a 
reproductive cycle the eggs do not develop appreciably beyond the stage typical of the ovary 
at the time of operation. The accessory sex glands in these operated females show little or no 
sign of secretion in contrast to normal control glands. Reimplantation of the corpora allata 
into allatectomized females causes the eggs and the nymphs hatching from them to develop 
as normally as those of unoperated animals. In a series of experiments in which the time of 
allatectomy is varied it can be demonstrated that the corpora allata are necessary throughout 
the period of growth and yolk deposition which constitutes about the first third of the total 
period required for the development of the eggs. The corpora allata are apparently not essential 
for the reproductive activity of male Leucophaea. Allatectomized males when mated with 
normal virgin females are capable of fertilizing the eggs. 

Contrasts between visible and dominant lethal mutation rates in x-rayed Habro- 
bracon eggs. ANNA R. WHITING AND H. C. GEORGE. 

Senior author has previously reported that eggs x-rayed in late metaphase I have lethal dose 
about 2,000 r and one-hit dose-hatchability curve. Death appears to be due to terminal deletions. 
Eggs x-rayed in prophase I have lethal dose about 45,000 r and complex dose-hatchability curve. 
Death appears to be due to several factors, including translocations and inversions. Majority of 
lethal effects in both stages are dominant. Recently, two groups of females were treated with 
doses giving about 90 per cent mortality, one with 1,120 r for metaphase I and the other with 
28,000 r for prophase I. They were then crossed with untreated males and their daughters were 

F, 5? heterozygous 

tested for heterozygosis for visible mutations. -p ~T - was 2.11 per cent lor eggs 

.b, Y tested 

treated in metaphase I, 12.69 per cent for eggs treated in prophase I. By x 2 test there is less 
than one chance in one hundred that these stages belong to same class in respect to visible muta- 
tion rate although they have same dominant lethality rate. This strengthens theory of terminal 
deletions (which would not produce visibles) as most common response to x-rays of metaphase 
I. Visibles produced in metaphase I are probably genie and their low percentage is what 
would be expected at low doses tolerated by this stage. Most visibles from treated prophase 
I are probably also genie although a few may be due to position effects of translocations or 
inversions. Their high percentage is possible because of high doses tolerated. 

AUGUST 13 

A new factor from the adrenal influencing fat deposition in the liver. KATHERINE 
A. BROW NELL. 

Starvation in the normal mouse leads to a large deposition of fat in the liver. This fails 
to occur after adrenalectomy. With these facts as a basis we have developed a test for a fat 
factor in various fractions prepared from ox adrenals. 

The method is briefly as follows : Adrenalectomized mice are fed for 24 hours then fasted 
for 24 hours. During this 48-hour period they are injected every 6 hours with 0.2 cc. of the 
preparation to be tested. Two to 3 hours after the final injection the livers are removed and 
the total lipid determined gravimetrically. 

Over 30 fractions from the adrenal gland including crystalline compounds have been tested 
by this method. The table shows results on adrenalectomized untreated animals; two fractions, 
a whole extract from which these fractions were taken and three crystalline compounds already 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 



215 



proven to have glyconeogenic potency. Both fractions are crude, being specific in only one 
respect namely, that the carbohydrate factor fraction has no electrolyte potency and the sodium 
factor fraction no glyconeogenic potency. The only fraction that gave a highly significant 
response was that containing the carbohydrate factor. The low response given by whole 
extract, we attribute to inhibiting substances, three of which have been tested. 

Since the liver fat response was given almost exclusively by the carbohydrate factor 
fraction, some of the crystalline compounds having glyconeogenic properties were tried to de- 
termine whether or not they were responsible. The table shows that the only one used which 
gave a significant response was dehydrocorticosterone ; a 25 per cent increase over the control 
level and in order to obtain this response two and one half times as much pure substance (0.96 
mgm.) was used as that estimated to be present in our carbohydrate factor fraction (0.35 
mgin.). The other two compounds, corticosterone and 17-hydroxy- 11 -dehydrocorticosterone, 
gave liver fat responses only on the borderline of significance and to obtain even these small 
responses two to two and one half times as much material was used as that estimated to be 
present in the carbohydrate factor fraction. The fourth known glyconeogenic compound, 
hydroxycorticosterone, we were unable to test on account of lack of material. 

There remain two possibilties : (1) that hydroxycorticosterone is the fat factor. If so, 
the effect on fat metabolism is a new property. (2) There is in the carbohydrate factor fraction 
a new factor regulating fat deposition in the liver. 

Effect of adrenal fractions on deposition of fat in the liver 



Treatment 


No. of animals 


Total lipid per cent 


Increase per cent 


Adect. untreated 


29 


6.31 





Carbo. factor fraction * 


15 


8.42 


33 


No factor fraction * 


7 


6.74 


9 


Whole extract * 


7 


7.13 


13 


Dehydrocorticosterone f 


8 


7.87 


25 


Corticosterone f 


8 


7.11 


13 


1 7-hydroxy- 1 1 -dehydrocorticosterone t 


7 


6.87 


9 



* The extracts represent 300 gm. of tissue per cc. 

t The solutions of crystals represent 0.6 mgm. solid per cc. 

Hyper activity of the adrenal cortex. FRANK A. HARTMAN. 



At rest or under conditions of minimal activity there is a basal secretion of adrenal cortical 
hormones. In response to various stresses such as exercise, exposure to cold, trauma, anoxia, 
and poisons, there is an increase in output of the hormones which subsides after the stimulus dis- 
appears. After removal of a large proportion of both adrenals by enucleation, in the mouse, a 
considerable rise in the basal secretion occurs. This higher level of secretion is maintained for 
months. The following table illustrates these changes. Fat and glycogen (as sugar) in the 
liver were determined after 24 hours' starvation. 



Values indicating changes in hormone production after 
enucleation of both adrenals 



Normal 

Adrenalectomized 
Enucleated 2 days 
Enucleated 7 days 
Enucleated 15 days 
Enucleated 29 days 
Enucleated 99 days 



Total lipid 
per cent 

8.5 
6.3 
6.6 

11.8 

10.0 

10.0 



Glycogen 
per cent 

0.12 

0.04 
0.24 
0.58 



216 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

The wide difference in time at which the peaks for the production of the fat factor and car- 
bohydrate factor occur, is evidence that the two factors are not identical. 

By enucleation we removed an average of 75 per cent of the adrenal tissue. Less than 25 
per cent of the active tissue remained since the circulation was disturbed and this 25 included the 
capsule. Thirteen days after enucleation the adrenals averaged 0.69 per cent of the body weight 
which is one-half the normal weight. Removal of cortical tissue probably reduces the in- 
hibitory effect on the adrenotrophic hormone production by the pituitary so that after a lag 
of three or four days there is sufficient recovery of the remaining cortices to respond to the in- 
creased output of adrenotrophic hormone. However, the new level of cortical hormone pro- 
duction .does not return the adrenotrophic output to the old level. Thus a higher basal level is 
established. The performance of a relatively small number of cortical cells indicates a large fac- 
tor of safety. This capacity of cortical cells for sustained activity in disease where a large 
proportion of cortical tissue is destroyed is important in prolonging life. 

There is now evidence for three mother hormones secreted by the adrenal cortex ; the fat 
factor, the carbohydrate factor, and the sodium factor. 

Studies on the mechanism of allo.nin action. ARNOLD LAZAROW AND STANLEY 
LEVEY. 

A number of compounds related to alloxan were synthesized and tested for their diabetogenic 
effect. These compounds were injected intraperitoneally into rats in high doses and the blood 
sugar was determined at 0. 1. 3, 8, 24, 48, and 72 hours after injection. Alloxan, N-methyl 
alloxan, and alloxantin which dissociates into alloxan all produced diabetes. N-N-dimethyl 
alloxan was toxic and, therefore, could not be injected in doses equivalent to that required for 
the production of diabetes with alloxan. Since alloxan is a ureid of mesoxalic acid, some deriva- 
tives were prepared in which the urea or mesoxalic acid portions of the molecule were intact. 
None of these (mesoxalamide, mesoxalic acid, dimethyl mesoxylate, or diacetyl urea) produced 
diabetes in the doses used. Freshly prepared dialuric acid, alloxanic acid, and barbituric acid 
did not produce diabetes ; whereas, dialuric acid which was allowed to stand overnight was di- 
abetogenic. (This is interpreted as oxidation of dialuric acid to alloxan by molecular oxygen.) 
Slight alterations in the structure of alloxan abolish its diabetogenic effect. 

It has been reported by other investigators that alloxan combines with sulfhydryl groups of 
proteins and that on injection it produces a rapid drop in the blood and tissue glutathione. Since 
one of us has shown that injection of glutathione or cysteine immediately preceeding a diabetogenic 
dose of alloxan completely protected the animals from diabetes ; and since others have shown that 
pancreas contains less glutathione than do other tissues ; it was suggested that variations in 
tissue glutathione may determine the selectivity of alloxan. Studies are now being carried out 
to determine the glutathione content of the beta cells of the pancreas which are selectively de- 
stroyed by alloxan. 

Biological specificity and the synthesis of native proteins. DOROTHY WRINCH. 

A common starting point for the discussion of biological specificity today is the assumption 
that biological function is an outward and visible sign of atomic pattern. Furthermore indications 
from many fields reinforce the old assumption that the native protein is the dominant structure 
type in all living systems. A vast number of physiological problems turn upon questions of 
atomic pattern, particularly such matters as (1) local stereochemical features and (2) the pres- 
ence of internal OH...O, NH...O and NH...N bridges and of linkages dependent upon the 
presence of a foreign ion. 

Of all these problems, the most fundamental is the synthesis of native proteins. We must 
presume that the power of native proteins to produce replicas of themselves depends in some 
basic way upon their structure, and that it is intimately related to the presence on native protein 
surfaces of 'active patches' to use Warburg's term, each of which functioning as a template or 
mold permits the laying down on itself of a complementary constellation. 

It is useful to notice that the associations of simple molecules within crystals offer many ex- 
amples of such complementary constellations, e.g., (1) hexamethylene tetramine, with pairs 
which are not identical associated about tetrahedrally related planes with a common three-fold 
axis and (2) the phosphotungstic acid 29-hydrate with identical (i.e., self complementary) con- 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 217 

stellations associated about such planes with a common three-fold axis and, in addition, self-com- 
plementary constellations associated about cube planes with a common two-fold axis. 

Visualizing the formation of new 'active patches' on the surfaces of an already existing 
species of native protein molecules, we see that such new constellations comprise the material re- 
quired for the formation of a new and identical molecule if (1) the species carries complementary 
patches (which may be but need not be individually self-complementary) and (2) the molecule 
is wholly made up of such patches, i.e., is a surface structure. 

In order to have a mechanism whereby these isolated constellations on several different 
molecules may be integrated so as to interlock in the same spatial pattern as in the original mole- 
cule, something has to be postulated as to the capacity of the original molecules to form a crystal. 
Thus for example, let us visualize a body-centered cubic lattice with molecules placed a: the 8 
body-centers and the 6 cube corners nearest the origin, with the molecule at the origin missing. 
With the complementary constellations in position on each of the 8+6 faces of the molecules 
turned to the origin, we have a situation in which interlocking, possibly in a number of distinct 
steps, could take place, the resulting molecule being a replica of the original molecule. This is 
but one example of a number of such possibilities, with the original molecules characterized by 
antipodal pairs of complementary patches. All, however, have in common the dependence upon 
the capacity of the original molecules to crystallize, an outstanding characteristic and most re- 
markable property of unnumbered native proteins. Similarly, all theories as to the formation 
of new native protein molecules by autocatalysis must, it would seem, have in common the picture 
of such molecules as surface structures, i.e., atomic fabric cages. 

AUGUST 20 

Naturally occurring polyploidy in the development of Allium cepa L. Dr. C. A. 
BERGER. 

One of the factors in the developmental pattern of Allium cepa is the formation of some 
tetraploid cells and their division as tetraploids. These cells are found throughout the cortex of 
the cotyledon and of the intermediate region between root and shoot. They are found in seed- 
lings between 20 and 40 mm. in length. They are never found in the root. During prophase of 
mitosis in tetraploid cells the chromosomes are closely paired and relationally coiled. The two 
members of each pair are united at a single undivided SA-region. These cytological details 
show that the chromosomes have not separated since the time of x their formation. Since the pair- 
ing and relational coiling is present from earliest prophase the double chromosome reduplica- 
tion must have taken place during the resting stage immediately preceding the 4n division. At 
metaphase the tetrachromosomes undergo two successive divisions of the SA-region and ana- 
phase is normal. Since no tetraploid division figures were found with unpaired chromosomes it 
was concluded that only one division of these tetraploid cells occurs. 

Chick embryology at the medical schools of Ancient Greece. TAGE U. H. EL- 
LINGER. 

Of the seventy titles comprising the Hippocratic Corpus, the most significant work, from 
a biological standpoint, is the lecture on embryology represented by the two texts On Semen and 
On the Development of the Child. It deals with human embryology from the formation of the 
semen to the birth of the child. The author is unknown, but he was not Hippocrates nor one 
of his followers. His work reflects the teachings of the medical school at Cnidos and of that 
of Empedocles whose influence is evident in doctrine as well as in scientific method and in the 
choice of vocabulary. This pre-Aristotelian author, who wrote in the last quarter of the fifth 
century B.C., at the time of Socrates, was indeed a very great scientist and a great teacher as well. 

To the modern reader perhaps the most amazing revelation is the use made of observations 
on chick embryology in explaining to the students the development of the human embryo. The 
following quotations are in the author's translation. 

In chapter 13, the Greek physician after describing a "semen which had stayed six days in 
the womb and which fell out," adds "A little later I will describe another test in addition to this 
one, that will enable anyone who seeks knowledge to see this for himself, as well as a proof that 
my whole discourse is correct, as far as that is possible for a mortal discussing such a matter." 



218 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

He returns to this topic in chapter 29 : "Now I shall recount the crucial test, that I promised 
a little while ago to make known, which is as clear as possible to a human intelligence and makes 
plain to anyone who wants to be informed about it, that the semen is in a membrane and that the 
navel is in the middle of it, and that it first draws air in and expels it outward," (according to 
the Empedocles* pneuma theory of differentiation) "and that there are membranes from the navel. 
You will also find the further growth of the child, as I have described it, to be from beginning 
to end, such as it is in my account, if you will apply the method of inquiry that I am about to 
describe. Take twenty eggs or more and give them to hatch to two hens or more ; then on every 
day from the second to the last, that of hatching, remove an egg, break it and examine it. You 
will find that everything in it conforms with my statements, in so far as one can compare the 
growth of a bird with that of man. That there are membranes extending from the navel, and 
all my other statements about the child, you will find illustrated from beginning to end in the 
hen's egg ; and he who has not yet made these observations will be surprised that there is a 
navel in a hen's egg. Such are the facts, and such is my account of them." 

Again in chapter 30, the Greek author advances chick observations to illustrate and explain 
conditions in man. He states : "Now in proof of my theory, that it is the lack of nourishment 
that causes the child to come forth, provided it suffers no violence, I offer the following evidence. 
The bird develops from the yolk of the egg in the following way. Under the brooding mother 
the egg is heated and the content of matter inside receives the impulse to development from the 
mother. When the content of the egg is heated, it forms air and attracts other cold air from 
the atmosphere through the egg; for the egg is porous enough to admit the attracted air in 
sufficient quantity to the matter inside. The bird grows in the egg and is differentiated in the 
same or in a similar way to the child, as I have already said above. It develops from the yolk, 
but it receives its nourishment and material for growth from the white that is in the egg. This 
was at once apparent to all those who have given attention to it. Whenever nourishment from the 
egg is insufficient for the chick, then, not having sufficient nourishment to live on, it moves vio- 
lently in the egg seeking more nourishment, and the membranes about it burst. When the mother 
notices that the chick has moved violently, she pecks and removes the shell. And this happens 
in twenty days. And it is evident that this is so, for, when the mother pecks the shell of the egg, 
there remains in it no liquid worth mentioning, since it has been expended on the chick." 

Reproductive economy in closccrossed species zvith haploid males. P. W. WHIT- 
ING AND RUDOLPH G. SCHMEIDER. 

According to the multiple-allele theory of sex determination, proved true for the wasp 
Habrobracon, every mating must involve either three or two sex alleles. The three-allele 
matings produce only females (sex heterozygotes) and normal (haploid) males (azygotes) ; but 
the two-allele matings produce also sex-homozygotes which either develop into sterile (diploid) 
males or are inviable. Outcrossing reduces the chance for two-allele crosses with their attend- 
ant reproductive wastage. The Habrobracon theory has been tentatively applied to the six or 
seven invertebrate groups characterized by male haploidy. Since many species, however, re- 
produce with much inbreeding, this theory would imply loss approximating half of the fertilized 
eggs. It has now been shown that in the wasp Melittobia over 90 per cent of the eggs from close- 
crosses, including selfcrosses (mother X haploid son), may develop into females. If Melittobia 
females are sex-heterozygotes, some method must therefore have been evolved other than multiple 
alleles for avoiding production of sex-homozygotes equal in number to the females. Although 
the method of sex determination in Melittobia is not yet understood, it has now for the first time 
been shown that reproductive economy is high in a closecrossed species with haploid males. 

A comparative study of the lipids in some marine annelidcs. CHARLES G. WILBER. 

Studies on the metabolism of lipids have been in the past confined to observations made on 
vertebrates. Very few studies have been made on the lipids in the invertebrates; consequently 
a detailed investigation seems justified. 

The following marine annelides were studied: Nereis pelagica, Amphitrite ornata, Arcnicola 
marina, Phascolosoma gouldii, Lepidonotus squamatus, Glycera americana, and Chactoptcrns 
variopedatus. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 219 

Whole worms or individual tissues were prepared by grinding or in the Waring-blendor. 
Lipids were extracted with boiling alcohol. Phospholipids were precipitated with acetone and 
magnesium chloride and estimated by oxidation-titration method of Bloor. Fatty acids were 
estimated by oxidation-titration and cholesterol colorimetrically using the acetic anhydride-sul- 
furic acid reagent. The ratios, cholesterol/fatty acid (lipocytic index) and cholesterol/phos- 
pholipid, were calculated. 

It was found that the absolute values of the various lipids in the same species and in different 
species were not always the same. On the other hand, the lipocytic index and the relation, 
cholesterol/phospholipid, were constant for a given species and tissue. If the lipocytic index of 
each worm were plotted against the phospholipid of the same worm the points representing the 
various species fell along a straight line ; a similar straight line was obtained when the cholesterol 
was plotted against phospholipid. 

There is, therefore, an apparent relationship between cholesterol and phospholipid and be- 
tween phospholipid and lipocytic index in marine annelides. Tissues with a high lipocytic index 
or high cholesterol content have a high phospholipid content. These results indicate that in 
the marine annelides, just as Bloor found in the vertebrates, since cholesterol is associated with 
and in constant relation to phospholipids, it is probably a normal protoplasmic constituent. These 
results confirm in part the results of analyses made on vertebrate tissues and agree with the con- 
tention of Mayer and Schaeffer that the lipocytic index is characteristic of the organ of an animal 
in a given species. 

GENERAL SCIENTIFIC MEETINGS 
AUGUST 23 

Vascular reactions to ergonovine maleate * as seen directly with the microscope 
in the living mammal. 1 RICHARD G. ABELL. 

Erognovine was injected intravenously in amounts varying from 0.005 mgm. to 0.2 mgm., 
and its effect upon the arterioles, capillaries and venules observed directly with the microscope 
in transparent 'moat' chambers (Abell and Clark, '32) in rabbits' ears. The clinical intravenous 
dose of ergonovine is 0.1 mgm. The equivalent dose in the rabbit is approximately 0.005 mgm. 
Injections of 0.005 mgra. caused constriction of arterioles to approximately 0.7 to 0.9 of their con- 
trol diameters, and a slight reduction in velocity of flow. The arterioles returned to their con- 
trol diameters and the flow to its control rate within 3 to 5 minutes. Daily injections of 0.005 
mgm. made for a period of 2 weeks caused similar results. One hundredth mgm. (twice the 
clinical dose) caused constriction of the arterioles to approximately 0.6 to 0.8 of their control 
diameters, and a slightly greater reduction in rate of flow than 0.005 mgm. One tenth mgm. 
(20 times the clinical dose) caused arterioles 15 to 30 microns in diameter to constrict to the point 
of obliterating their lumens and stopping the blood flow for approximately 30 seconds to one 
minute. The vessels relaxed to their control diameters within approximately 12 minutes. Two 
tenths mgm. (40 times the clinical dose) caused more vigorous and prolonged arteriolar con- 
striction, which lasted for from 1 to 1% minutes, and stopped all of the blood flow within the 
chamber. The venules constricted to approximately 0.6 to 0.7 of their control diameters. The 
arterioles returned to their control diameters in approximately 15 to 20 minutes. Four injections 
of 0.2 mgm. at 15 minute intervals made the small arterioles (15 to 30 microns in diameter) un- 
responsive to further injections, but not the larger arterioles (80 to 90 microns). Intravenous 
injections of 0.025 mgm. of epinephrine while the small arterioles were still unresponsive to 
ergonovine, caused them to constrict to the point of obliterating their lumens, which is the typical 
response to this amount of epinephrine. 

None of the above injections caused any sign of injury to the blood vessels, or any abnormali- 
ties in appearance and distribution of the red blood cells, the white blood cells, or the platelets. 
Thus it is clear that ergonovine maleate, which is used widely to prevent post partum hemorrhage 
and to give symptomatic relief of migraine headache, does not cause any observable injury to the 
blood vessels and associated structures even when given in amounts of 40 times the clinical dose. 

*"Ergotrate" (Ergonovine Maleate, U.S. P., Lilly). 

1 This work was aided by a grant made by Eli Lilly and Company to the Department of 
Anatomy of the University of Pennsylvania Medical School. 



220 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

The effect of halogcnated alkyl amines on the respiration of Arbacia eggs and 
sperm. E. S. GUZMAN BARRON, E. G. MENDES AND H. T. NARAHARA. 

Halogenated alkyl amines at 0.001 M concentration produce an inhibition of the respiration 
of animal tissues, and complete inhibition of pyruvate and choline oxidation (Barron et al. 1 ). In 
smaller concentrations the early cleavage of the fertilized sea urchin egg is inhibited or retarded 
(Cannan et al. 1 ). There is also inhibition of mitosis in the corneal epithelium of mammals 
(Friedenwald and Scholz 1 ) and a high incidence of sex-linked lethals as well as a significant 
number of translocations and inversions in the chromosomes of Drosophila inelanogastcr ( Auer- 
bach et al. 1 ). 

Dichloroethylmethylamine HC1, and trichloroethylamine HC1 at a concentration of 0.001 M. 
and dissolved in sea water, produced a definite increase in the respiration of sea urchin sperm 
(from 170 to 50 per cent). The increase of respiration could be noticed even with 1 X 10" 5 M. 
The respiration of sea urchin eggs, fertilized or unfertilized, was slightly inhibited by this con- 
centration of alkyl amine (14 to 17 per cent). Higher concentrations produced inhibition of 
respiration probably due to a decrease in pH as a result of the hydrolysis of these compounds. 
When the alkyl amines were previously neutralized and the sperm and eggs suspended in 0.05 M 
citrate buffer, pH 6.8, the effect of the alkyl amines was erratic. It is quite possible that 
penetration of the alkyl amines into the cell occurs only in an acid milieu. 

The experiments of Cannan et al. 1 on retardation of the rate of cleavage of fertilized Ar- 
bacia eggs were confirmed. Eggs treated with 0.001 M dichloroethylmethylamine HCL (dis- 
solved in sea water) for 15 minutes prior to insemination, and fertilized eggs treated at the time 
of the first cleavage showed a definite retardation in the rate of cleavage. Furthermore none 
of the treated eggs reached the pluteus stage. 

The effect of uranyl nitrate on the respiration of Arbacia- sperm. D. BENEDICT 
AND E. S. G. BARRON. 

Uranium, like other heavy metals, is quite toxic and it has been extensively used for the 
production and study of experimental nephritis. Uranyl nitrate in concentrations varying from 
10~ 2 to 5 X 10" 5 M. inhibited the respiration of Arbacia sperm. The inhibition was complete at 
5 X 10~ 4 M. (92 per cent inhibition). W 4 M. UO 2 (NO S ) 2 produced partial inhibition (from 53 
to 15 per cent), 5 X 10" 5 M. inhibited 15 per cent, and 10" 5 M. had no effect at all. This inhibi- 
tion must be due to combination of respiratory enzymes with uranium, a combination which can be 
reversed completely on addition of a citrate at a ratio of U : citrate of 1 : 2. Addition of phosphate 
at a ratio of 1 :100 brought only partial release (25 per cent). The experiments were performed 
in acetate-sea water buffer at pH 6.4 to avoid precipitation of the uranyl salt. Dry weights of 
sperm were obtained after centrifugation of the sperm at 16,000 g. There was in the control 
experiments a rise in the pH value of about 0.6 units at the end of one hour, probably due to the 
formation of NH :! . 

Some properties of purified squid visual pigment. ALFRED F. BLISS. 

The photostable red visual pigment of the squid (Bliss, 1943, Jour. Gen. Pliysiol.) was 
found to become reversibly light sensitive in the presence of formalin. A method was devised 
for the extraction of this pigment in a state of purity approximating that of the best prepara- 
tions of vertebrate rhodopsin. The principal impurity of previous extracts, melanoprotein, was 
rendered insoluble by the following procedure. Retinas were rinsed in distilled water and kept 
frozen until use. They were then homogenizd with 0.2 M Na L . HPO 4 and centrifuged. The resi- 
due was washed with pH 4.5 buffer and distilled water. The visual pigment was extracted with 
3 per cent digitonin at 6 C. for 2 minutes and centrifuged 5 minutes. The absorption spectrum 
of the extracted pigment did not differ significantly from that of rhodopsin. In its chemical prop- 
erties it differed significantly from rhodopsin, since it was rapidly destroyed by digitonin even at 
6 C. The primary breakdown product in cold acetone was, like that of rhodopsin (Bliss, 1946, 
Biol. Bull.), the acid tautomer of the lipid "Indicator Yellow." Because of the distinctive prop- 
erties of the squid rhodopsin, a differentiating name, cephalopsin, is suggested. 

1 All quoted from Gilman, A., and Philips, F., Science 103: 409 (1946). 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 221 

Studies on the viscosity and elasticity of striated muscle. MANFRED BRUST. 

By the use of a spring vibrating against the resistance of frogs' (Rana pipicns) sartorius 
muscles as described by Gasser and Hill (Proc. Roy. Soc. B. 96: 398, 1924) the effects of 
urea and iodoacetic acid (IAA) on the viscosity and elasticity of these muscles were studied. 
All initial slack was removed from the system by stretching the muscles 17.5 per cent beyond 
their resting length and putting them under 3.5 gm. tension. 

Thirty minutes immersion in solutions of 2.5 M urea in Ringer's shortens the muscles on 
the average by 26.1 per cent. When extended to their original length they still exert the ten- 
sion originally exerted at that length. They will not return to their urea induced length when re- 
leased from stretch. Their viscosity is reduced on the average of 53.6 + 13.0 per cent of that 
in the untreated muscles, while the elasticity is similarly reduced to 60.4 + 18.6 per cent. 

Sixty minutes immersion of Ringer's equilibrated muscles in 1-80000 IAA (6.72 X 10" 5 M) 
in Ringer's sometimes causes a rise in viscosity and elasticity even without activity by the poisoned 
muscles. Summer frogs show this response less often than winter frogs. Measurements made 
during 30 second rest periods between 5 second isometric tetani show a short initial decrease fol- 
lowed by a gradual increase in both viscosity and elasticity. Average maximum rigor values of 
181 per cent and 258 per cent respectively of the untreated muscle values are attained. 

The urea results would agree with the findings by other authors that this agent disrupts myosin 
and other protein molecules thus transforming them into disconnected less asymmetric entities. 
Collagen is not believed to be markedly affected since muscle shape is maintained while tension 
remains the same as before treatment at the same lengths. The IAA results would agree with 
the progressively diminishing solubility and increase in hardness of actomyosin in gradually de- 
creasing concentrations of adenosine triphosphate reported by the Szent-Gyorgyi group (Acta 
I'liysiol. Scand. 9: Snppl. xxv, 1945). 

Arterial anastomoses. ELIOT R. CLARK AND ELEANOR LINTON CLARK. 

This study represents an attempt to discover factors responsible for the presence or absence 
of arterial anastomoses, which vary so greatly in different organs. 

The governing factor appears to be the histo-mechanical principle established by R. Thoma 
in 1892, corroborated by E. R. Clark in 1918 in studies on living vessels in the tadpole's tail, that 
the size of the lumen of an artery is regulated by the amount of blood flow. In the absence of 
flow, the lumen is reduced to zero and the artery obliterated. In order, then, for arterial an^to- 
moses to survive, conditions must be such as to provide a fldw of blood through the .terminal 
connecting portion. 

In most cases this requires the presence of factors which force the blood to flow part of the 
time in one and part of the time in the reverse direction. Such factors are present in the periph- 
eral parts of the body in the form of varying outside pressures that are exerted irregularly upon 
large supplying arteries or small distributing arterioles. 

A study, with the aid of artificial chambers, of the living circulation in the rabbit's ear, where 
anastomeses are abundant, reveals frequent reversals of flow in connecting portions of anasto- 
moses, but controlled by an unsuspected factor, namely, the irregular contraction of the arteries or 
arterioles themselves, described in an earlier paper. 

In types of artificial chambers, installed in rabbits' ears, which are invaded by new tissue, 
there are often arterioles that, for weeks, are unprovided with nerves and hence contract little, 
if at all. In many such chambers no arterial anastomoses survive. However, in this type of 
chamber, occasionally arterioles receive a nerve supply, and in such cases arterial anastomoses 
may survive. In every case in which such anastomoses have persisted in newly-formed tissue, 
there have been frequent reversals of flow in the connecting portion. 

The effects of the ultra-violet radiations on Styela eggs. A. M. DALCQ. 

The M.D.L. installation for microphotography with U.V. rays (2537 A) may be used for 
irradiating part of the Ascidian egg or certain of the various blastomeres up to the Vlll-cell 
stage. The method was worked out with the aid of Dr. G. I. Lavin. The egg is placed in a 
drop of sea water near the edge of a thin quartz coverslip, which is itself put on the transverse 
arm of the mechanical stage. The coverslip is adjusted under the microscope in such a way 
that the part of the egg to be irradiated protrudes over the edge of the metallic stage arm which 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 

acts as a protection screen for the rest of the egg. Attention should be paid to two sources of 
error: (1) the effect of hypertony due to evaporation of the drop and (2) to the reflexion of 
the rays by the objective lens of the microscope, which is easily eliminated by interposing some 
black paper during the irradiation. In exploring a considerable range of exposure no favorable 
effect of the irradiation could be found. If feeble, it produces a delayed disorganization of the 
embryonic layers. If stronger, it stops the cleavage with rapidity varying according to the 
dosage. In order to obtain stopping of the next cleavage, exposures of at least 10 minutes are 
necessary. When a division is suppressed, the cell may manifest a delayed attempt at cleavage, 
but this is always abortive. After exposure of the unsegmented eggs, deviations of the first 
cleavage plane may be observed. Observations of the movements of yolk and yellow pigment 
and the elongation of the cell-body indicate that the effect of the radiation is not primarily on 
the nuclear activity. That the influence of the rays is exerted on the surface protoplasm is 
shown by the transitory appearance of alterations of the surface film (small protuberances, 
"blisters") in coincidence with attempts at cleavage. 

By means of this method, the division of one or more blastomeres of the II, IV, and VIII 
cell stages has been inhibited. The non-irradiated cells exhibit normal development with respect 
to mitotic rhythm and arrangement. Their capacity for differentiation, which seems rather poor 
when large blastomeres remain undivided in the germ, must still be studied in sections. 

A correlation betzveen gill surface and activity in marine fishes. I. E. GRAY. 

The units of respiration in the gills of fishes are the numerous microscopic secondary lamel- 
lae which appear as thin, leaf-like plates set at right angles to the main axes of the primary 
lamellae. Within each plate lies a capillary network through which the interchange of gases 
takes place. Among fishes there are species differences, not only in the number of gills, but 
also in the number and length of the gill filaments (primary lamellae) and in the number of 
respiratory units (secondary lamellae). By determining the number of respiratory units per 
gram of body weight it is possible to obtain an estimate of the relative respiratory ability of 
different fishes. 

There is a marked contrast in the number of respiratory units per gram of body weight 
between the active, surface, migratory fishes (mackerel, 2550; butterfish, 1725; menhaden, 
1685) and the sluggish bottom fishes (flounder, 265; toadfish, 135; goosefish, 50). The number 
of respiratory units of fishes of medium activity fall between these two extremes (scup, 1325; 
sea 'trout, 1250; sea bass, 1110; eel, 900; sea robin, 800: puffer, 505; tautog, 440). A four 
hundred gram mackerel has a total of nearly three-fourths million respiratory units while a 
toadfish of the same weight has only fifty thousand. The number of respiratory units is also 
directly correlated with the amounts of sugar and hemoglobin in the blood. 

The distribution of lipid between the light and heavy halves of the Arbacia egg. 
F. R. HUNTER AND A. K. PARPART. 

Unfertilized Arbacia eggs were centrifuged for 10-20 minutes in an air turbine at ap- 
proximately 16,000 X G. in a medium of graded density obtained by mixing sea water and 0.95 
1 molal sucrose. The light and heavy halves which resulted were collected, packed in an air 
turbine, frozen, dried in a vacuum desiccator and weighed. This dried material was then ex- 
tracted with ether, dried and again weighed. The loss in weight was taken as a measure of 
the amount of free fats and sterols. This material was then extracted with alcohol-ether and 
again dried and weighed. This was considered to give a value for the bound lipid. In order 
to relate the amount of lipid to the number of halves, counts were made on suspensions of halves 
prior to drying. The following values expressed as nigs, of lipid per million halves were' ob- 
tained : heavy halves 6.6 (ether fraction), 12.2 (alcohol-ether fraction) ; light halves 2.2 
(ether fraction), 9.6 (alcohol-ether fraction). Thus, 75.0', per cent of the free fats and sterols, 
56.0 of the bound lifids and 61.6 per cent of the total lipids are in the heavy halves. The sum 
of the total lipids in the two halves is equal to 30.6 mgs. per 10"' cells which compares favorably 
with the value 34.1 mgs. per 10" cells calculated from the data given by Parpart (Biol. Bull., 
81: 296, 1941) for unfertilized, whole eggs. Similarly a comparison can be made between the 
sum of the bound lipids of the two halves and of the whole egg. Their values are 71.3 per 
cent and 77 per cent, respectively. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 

Evidence for enzymatic participation in the penetration of the human erythrocyte 
by glyccrol. PAUL G. LEFEVRE. 

Jacobs and his associates have reported that an amount of copper sufficient to cover only 
a very small fraction of the surface of the cells involved markedly inhibits hemolysis of human 
red cells in isotonic glycerol. This report concerns the extension of this finding to the effects 
of other substances which inhibit the same types of enzymes affected by traces of copper. 

Following the pattern prescribed by Barren and Singer for identification of sulfhydryl 
activity, iodine, mercuric ion, the arsenical Mapharsen, and p-chloromercuribenzoate were shown 
to inhibit hemolysis by glycerol, buffered at pH 7.1. This inhibition failed in the presence of 
cysteine, glutathione, or thioglycolic acid ; and could be reversed by later addition of these sub- 
stances at 2-3 times the concentration of the inhibitor, except with Mapharsen. These relations 
indicate strongly that active - SH groups are involved in carrying glycerol into the cell. 
Though sensitive to the inhibitors mentioned, the hemolytic process was not affected by iodoace- 
tate ; this indicates that the sulfhydryl groups involved are of the difficultly available type, not 
inactivated by the alkylating agents. 

Since phosphorylation is apparently essential in transfer of sugars and other substances 
across the membranes of the kidney tubule and the intestinal cell, it is proposed tentatively that 
the enzymatic step involved in the present studies is the phosphorylation of glycerol. Adenosine 
triphosphatase, capable of this step, is present in the erythrocyte, and shows the same pattern 
of sensitivity to inhibitors as found in the present instance, as well as similar relations of ac- 
tivity to pH. Further, more decisive tests of the proposed identity of the enzymatic factor are 
planned. 

"Accommodation" and opening excitation in nerve and muscle. PAUL G. LE- 
FEVRE. 

In his mathematical analysis of electrical excitation in 1936, Hill pointed out that the accom- 
modative process (recession of threshold under the influence of a stimulus) itself accounted for 
the phenomenon of excitation at the anode at the "break" of a constant current. There seems 
to have been no attempt to test this neglected implication of Hill's theory: that "accommodation" 
is an essential prerequisite for "opening excitation" at the anode. This report concerns the 
occurrence of opening excitation in tissues showing no accommodation. 

Following Solandt's practise, frog sciatic nerves were treated with citrate until they no 
longer showed any accommodation : their threshold was independent of the rate of increase of 
the excitatory current (delivered with Solandt's condenser-charge arrangement). In such 
preparations, in spite of the absence of accommodation, there was no difficulty in eliciting an 
anodal response at the cessation of a steady current. The same result was readily obtained with 
exposed sciatic nerves of anesthetized rats treated with citrate. 

Frog sartorii, or the pharyngeal retractors of Thyone, if stimulated in the nerve-free re- 
gions, or following neural degeneration, also showed no accommodation. But all attempts to 
demonstrate any response at "break" in these muscles failed ; this is in accordance with the 
predictions of Hill's theory. Only in the case of citrated nerves was seen the troublesome occur- 
rence of opening excitation in the absence of any accommodation. 

A further analysis of this matter is planned, to determine whether the results may be ex- 
plained on the basis of a postulated fundamental difference between accommodation at the 
cathode and that at the anode ; the latter persisting in the absence of the calcium ion required 
by the former. 

A photometric study of the kinetics of fibrin formation. JOSEPH LEIN. 

The clotting of fibrinogen solutions by thrombin was studied by measuring the optical 
density and light scatter as the process occurred. The results can be analyzed kinetically only 
when purified preparations were used. If other plasma proteins are present the degree of light 
scatter also depends on their concentrations. This is believed due to a trapping effect of non- 
clottable proteins by the fibrin as it is formed. The light scatter studies were particularly useful 
in the kinetic analysis of the clotting process. 



224 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

The reaction was considered from a polymerization viewpoint, the fibrin representing the 
polymer formed through the action of thrombin on the monomer fibrinogen. First order re- 
action kinetics were employed. The following assumptions were made. (1) With constant 
thrombin concentrations the rate of increase of the polymer size is proportional to the fibrinogen 
concentration (dN/dT K,F~). (2) The rate of decrease of the fibrinogen is proportional to 
the fibrinogen concentration ( dF/dT -= K~F). (3) The light scatter under the conditions 
of the experiment is proportional to the increase in particle size once it reaches the critical size 
(N ) which first scatters light. (LS K S (N N ). From these formulas a relationship 
was derived that included light scatter (LS}, the initial concentration of fibrinogen (F n ), time 
(T), and time (T a ) for the polymer to reach a size (AM that would scatter light. The for- 
mula may be expressed as : 

log (K.KJK, F.e-K^T, - LS) = - 0.434 K,T + log K t KJK, F n 

The relationship was tested on a series of experiments in which the initial concentration 
of fibrinogen was altered, the thrombin being kept constant. The calculated values of the con- 
stants agreed with the experimental values within a 6 per cent average deviation. It thus ap- 
pears that the course of the reaction may be considered to be molecular and that thrombin acts 
as a true catalyst, not forming part of the final fibrin product. 

The effect of iodacetate on the changes in muscular latency induced by activity. 
A. SANDOW. No abstract submitted. 

Formation of the nuclear membrane and other mitotic events in Chaos chaos Linn 
and Chaos ncos (nczv species). A. A. SCHAEFFER. 

The mitotic stages of the amebas mentioned are easily followed in the living animal. The 
principal stages are the following: 1. the nucleus about to divide swells up to about 6 times its 
former volume. 2. The chromatin grains (300 to 600 in number) gradually disappear, as if 
going into solution. Some of these grains coalesce before going into solution. 3. A new mass 
of small grains (about 2500) appear, before all the larger grains of the so-called resting nucleus 
have disappeared. These small grains arrange themselves first as a lens-shaped cloud, then as 
a plate of about 2 grains thickness. At this stage the plate of grains is occasionally seen to be 
indistinctly divided into at least 4, possibly as many as 8 or 12, smaller groups of equal size. 
4. This plate of grains then separates into 2 plates which rapidly move apart. 5. Fibers analo- 
gous to, if not identical with, spindle fibers, appear between the plates, as the plates separate. 
Fibers also appear on the other face of the plates. All fibers are at first horizontal and parallel. 
6. The plates separate and the inter-plate fibers lengthen until the plates are separated to about 
2 or 3 times their diameter when, because of the streaming of the protoplasm, the plates are 
torn apart. During this time the nuclear membrane breaks into pieces which eventually com- 
pletely disappear. 7. The separated plates, still granular, become bent like a concavo-convex 
lens, with polar fibers still attached. The granules soon disappear, leaving a very thin, per- 
fectly homogeneous flat disk that shows a brilliant blue green color when seen on edge. No 
refractory edge can be made out. This stage is difficult to see. 8. After a few minutes the 
disk shrinks in diameter and is thrown into rope-like folds around the periphery. 9. Very soon 
thereafter a refractory edge begins to appear as the folds disappear. 10. Very fine grains pres- 
ently begin to appear until about 1,400 are formed. Many of these coalesce to form larger 
grains until only about 600 to 700 remain in the newly formed daughter nucleus. (Further 
reduction in number may occur during the next few hours.) While the small grains are ap- 
pearing, the edge of the nucleus becomes more and more refractory until in the new nucleus it 
is seen as the new nuclear membrane. The steps outlined here require about 28 minutes. 

The mitotic events of Chaos difflucns, as far as they have been observed, are practically 
identical with those of the above-mentioned species, except for size. 

Correlated histories of individual sense organs and their nerves, as seen in living 
frog tadpoles. CARL CASKEY SPEIDEL. 

In the living frog tadpole it is possible to make daily observations on the same individual 
nerves and sense organs of the lateral-line for many weeks or months. By suitable operations 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 225 

some sense organs may be deprived of their nerve supply (nerveless organs), and conversely, 
some aberrant lateral-line branches may be induced to grow without reaching any sense organ 
(organless nerves). 

Prolonged observations of nerveless organs (1 to 21 months) reveal the following: (1) 
During the first two months in regenerating or growing zones the sense organs are largely 
independent of their nerve supply. They grow and divide readily. (2) During later months, 
however, regressive changes of atrophy and degeneration take place. The organs become 
smaller. Some degenerate and disappear. Occasionally, however, a nerveless organ may per- 
sist for more than a year. 

Prolonged observations of organless nerves reveal the following: (1) During the first two 
months they are largely independent of the sense organs. They grow and become provided 
with both neurilemma and myelin sheaths. (2) During later months, however, regressive 
changes ensue. The myelin sheath is not maintained on any functionless fiber. It becomes 
thinner and ultimately disappears. The untnyelinated fiber resulting may then itself degenerate, 
leaving only a collapsed neurilemma tube. 

Thus, the structural integrity of both lateral-line sense organ and nerve fiber is definitely 
correlated with the successful establishment of a functional relationship between the two. 

Sense hairs and orange granules are specialized structures of lateral-line organs. The be- 
havior of both of these under various experimental conditions indicates their relative inde- 
pendence of nerve influence. 

Many other histories involving nerve and sense organ relations in wound zones have also 
been recorded. Illustrative cine-photomicrographs have been made. 

The effect of prolonged starvation on the lipids in Phascolosoma gonldii. CHARLES 
G. WILBER. 

It is known that the muscle in vertebrates serves as a storehouse of fats and that during 
starvation the fats in muscle decrease whereas the fat in various internal organs is not changed. 
Whether this is true for invertebrates is not known. 

In order to throw light on the problem, worms (Phascolosoma c/onldii) were starved for 
one month and then the whole worm, the muscle, and the perivisceral fluid respectively were 
analyzed for phospholipid, for cholesterol, and for fatty acid. These results were compared 
with the results of similar analyses made on control worms. 

It was found that in the whole worm there was a loss of all lipid constituents. In the peri- 
visceral fluid, phospholipid and fatty acid decreased greatly, but cholesterol did not decrease. 
In the muscle there was an apparent increase in lipid material which can be explained on the 
basis of absorption of some of the tissue. In muscle the fatty acid is decreased, as is clear from 
the larger lipocytic coefficient of the muscle of starved worms. 

It is concluded that the perivisceral fluid serves as a storehouse of lipid in Phascolosoma 
and that the muscle does not. Moreover, it seems that phospholipid and fatty acid are used 
during starvation. Whether cholesterol is also used is not certain. Phascolosoma differs, 
therefore, from the vertebrates in the use of phospholipid during starvation and in the fact that 
muscle is not the important storehouse of fat. 

Protoplasmic clotting in isolated muscle fibers. ARTHUR A. WOODWARD. 

Isolated muscle fibers provide a material favorably adapted to the quantitative study of 
protoplasmic clotting. The cut ends form clots which pass in waves over the length of the fiber. 
The rate of the clotting reaction can be measured and is expressed as mm. of fiber converted into 
clot per minute. Single fibers are teased from the adductor magnus of Rana pipicus; all solutions 
used are kept at pH 7.1 7.4 with glycine buffer. 

In Ringer's solution, used as a standard for comparison, the rate of clot formation is constant 
for a given fiber and varies only moderately from fiber to fiber within a muscle. The normal 
rate is about 0.050 mm./min. 

Ca ion causes a very rapid clot formation ; in this case it is shown that the rate is largely 
a function of the speed with which Ca diffuses into the end of the fiber, the protoplasm clotting 
with great rapidity once it is exposed to free Ca ion. 



226 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

The clotting process is relatively insensitive to pH changes in the region from pH 5 to pH 9 ; 
above and below this the rate increases very rapidly. Near the regions in which thrombin is 
inactivated liquefaction has been observed under certain conditions. 

Solutions of crystalline trypsin cause clot formation at a rate averaging about 50 times that 
of the control. In the absence of Ca, trypsin produces only a slight increase over the control. 
Crystalline chymotrypsin is much less active than trypsin and also has very little effect in the 
absence of Ca. Preparations of crude papain cause clot formation at a moderately high rate ; 
addition of glutathione increases the effect to the magnitude of that produced by trypsin. Ab- 
sence of Ca has no effect on the action of papain. 

AUGUST 24 

Some aspects of the histology and physiology of luminescence in "railroad worms." 
JOHN B. BUCK. 

In the Uruguayan "railroad worm," Phrixothrix, the lateral photogenic organs are small 
compact ovoid masses of small dense cells near the posterior edges of the segments somewhat 
above the spiracular level. The organ is apparently supplied by one trachea ramifying profusely 
between the cells. There are no end-cells. Large oenocyte-like cells are present near some of 
the lateral photogenic organs and elsewhere. 

In Phengodcs, a close American relative of Phrixothrix, the lateral organs are in the pos- 
terior ends of horizontal rolls of tissue which extend along the segments ventral to the spiracles. 
Light is also emitted along the dorsal posterior edges of most of the segments. Both lateral 
and dorsal organs apparently consist of loose aggregations of very large oenocyte-like cells with- 
out end-cells or special tracheal supply. Similar cells are present in small numbers in parts of 
the body not regarded as luminous but not in the lateral tissue roll except in the region which 
emits light. Further evidence is furnished by the observation that the light of Phcnyodcs can be 
seen microscopically to come from clusters of round or oval spots corresponding in shape, posi- 
tion, number, and size to the oencyte-like cells. 

The photogenic organ of Phyri.vothrix is very similar to that in the larval firefly and agrees 
with the generalization that luminous beetles which produce a lingering glow rather than a short 
flash, have organs of relatively simple structure without end-cells. 

The photogenic organs of Phcngodes are the simplest yet known in insects and represent 
the first time, that bioluminescence has been ascribed to eonocytes. A corresponding physiological 
simplicity may be the fact that the light is continuous. 

Phengodcs dims in the vapor of KT 2 and 1CT 3 M. HCN at about the same rate as luminous 
bacteria, and faster than fireflies. 

Effect of caffeine concentration upon retardation of Arbacia development. RALPH 
HOLT CHENEY. 

Sea urchin eggs and sperm were subjected to eight different concentrations of caffeine-in- 
sea-water for 15 minutes, then mixed for fertilization and the developmental rates in S.W. and 
S.W.C. were compared with the normal rate of untreated ova and sperm. Observations were 
made at intervals during a three-day 'period. Normal time rates were accepted as stated by E.B. 
Harvey, 1940 (Biol. Bull, 79, (1) "Plate II, photographs 16-32 inclusive). 

All eggs utilized in a single experiment were obtained from the same female and all sperm 
from one male. Eggs and sperm were shed directly into S.W. or S.W.C. prior to mixing. The 
series of six combinations presented in 1942 (Biol. Bull., 83) were repeated and extended to a 
full three day period as follows : N? X Nrf,' N? X Crf, C? X Nrf, C? X Cd, all developed in 
normal sea water after the original immersion of fifteen minutes after shedding as indicated 
into S.W. or S.W.C. In the cases of C? X NJ 1 and C$ X Cd 1 , each was developed also in S.W.C. 

Results indicated that the period of immersion (15 min.) in the caffeine concentrations em- 
ployed prior to mixing the gametes did not render the ova non-fertilizable subsequently nor 
destroy the ability of the sperm to fertilize. Eggs and/or sperm, however, were not unaffected 
at least by higher concentrations, since C? X Cd cultures, although they did form the fertilization 
membrane when mixed and developed in uncaffeinized S.W., the fertilized ova never survived 
longer than the early cleavage stages. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 227 

Plutei developed in normal time and form in all of the six combinations of 0.002 per cent and 
0.004 per cent S.W.C. Gametes shed into two per centum S.W.C., in each of the four combina- 
tions developed in S.W. formed the P.M. but showed retarded development and in no case 
reached the pluteus stage before death. In the two combinations developed in S.W.C., the P.M. 
was not formed. Intermediate percentages used between these extremes of concentration gave 
intermediate effects indicating that in general, the effects were directly proportional to the con- 
centration of the caffeine. 

Other experimental series subjected normally fertilized ova (N? X N^ 1 in S.W.) which had 
developed to a desired stage of development, to the different concentrations of caffeine-in-S.W. 
Results here indicated similarly that the retardation effect upon the development time was pro- 
portional to the concentration. 

Shape changes in flic denuded Nereis egg preceding first cleavage. ALBERTA T. 
JONES. 

It is known from Hoadley (1934) that Nereis eggs undergo a series of amoeboid changes 
prior to first cleavage. Neither the reason for this phenomenon nor the exact pattern followed 
has been completely described. It is the purpose of this paper to report the principal findings on 
the pattern of shape changes up to first cleavage in the egg of Nereis limbata. The denuded egg 
was used to eliminate the complications of membrane and external jelly. 

Gametes were taken from animals caught the previous night in Eel Pond (Woods Hole) and 
artificial insemination was carried out. The fertilized eggs were denuded by treatment with 
alkaline 0.53 molar NaCl solution brought to pH 10.5 by addition of Na L ,CO 3 . This is the method 
used by Costello (1939 and 1945). Observations began when the denuded eggs were rinsed free 
of alkali and the first polar body had formed. Outline drawings of the eggs were made with a 
camera lucida at three minute intervals. To serve as reference points, the position of polar 
bodies and oil droplets was indicated. 

Hoadley states: "pulsations of the (Nereis) egg are of two sorts, one of which is quite 
extensive and results in general distortion of the sphere, and the other of which results in surface 
irregularities which appear more or less localized." The shape changes discernible in denuded 
eggs seem to correspond to Hoadley's first category. A consistent pattern of sequences, dif- 
ferent from those described by Hoadley for the intact egg, has been found. The sequences in- 
clude such general distortions as : ( 1 ) polar flattening followed by rerounding ; (2 ) elongation 
in the polar axis followed by rerounding; and, (3) elongation in the equatorial axis followed by 
formation of the first cleavage plane. The magnitude of these changes, in comparison with the 
pulsations observed by Hoadley, may be attributed to the absence of jelly mass and membrane. 

It may be concluded that shape changes in the denuded Nereis egg prior to first cleavage 
proceed (1) according to a definite pattern; and (2) always with a particular relation to the 
polar axis of the egg. 

'Hormone control of dchydrogenasc activity of Crustacean tissues. ELOISE KUNTZ. 

Sea water extracts of the sinus glands of Libinia cmarginata, Honiants americanus, Uca 
pugilator and U. pugnax and similarly prepared extracts of the central nervous system of Libinia, 
Homarus and the arachnoid, Limulus polyphcmus, were made. These were boiled and centri- 
fuged and the supernatant fluid was used. The extracts were tested for their effect upon de- 
hydrogenase activity of gastric gland and muscle of Libinia, Honiants and Limulus, which were 
measured in Thunberg tubes with methylene blue as the hydrogen acceptor. 

The effect of sinus gland extract was dependent upon the concentration. Half of a 
Libinia sinus gland doubled the rate of methylene blue reduction, but the reduction rate rapidly 
fell to slightly above that of the controls with increasing concentrations. Half of a Uca pugilator 
sinus gland also doubled the reduction rate, but activity remained high for concentrations of I 
sinus glands, falling to the level of the controls at 6 sinus glands. Here it remained. Uca 
pugnax showed strong inhibitory action in concentrations of 6 to 12 sinus glands. The character 
of the curves suggests the possibility of two active substances which vary in relative proportions 
in different species. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 

Central nervous system extracts of Homarus, Libinia and Limuhts in concentrations of 0.7 
mg. tissue per cc. strongly stimulated dehydrogenase activity. Extracts of other tissues were 
ineffective at several times this concentration. 

Localization of hormone production within the nervous system was demonstrated. In 
Homarus the circumoesophageal ganglia and second ventral ganglion were most effective. The 
brain and suboesophageal ganglion were ineffective. The remainder of the ventral cord had a 
relatively weak action. All parts of the circumoesophageal ring of Limulus were effective. 

An antagonistic action of sinus gland and nervous system extract was demonstrated. The ad- 
dition of sinus gland hormone to a system stimulated by central nervous system extracts de- 
pressed dehydrogenase activity to that of the controls. 

A comparative study of cholinest erase activity in normal and gcneticallv deficient 
strains of Drosop/iila melanogaster. DR. F. POULSON AND E. J. ROELL. 

Cholinesterase activity has been determined in late embryos of several strains of Drosophila 
melanogaster by means of the cartesian diver technique which measures the evolution of CO^ 
from Ringer-bicarbonate solution following hydrolysis of acetylcholine in the presence of an 
atmosphere of 95 per cent N 2 and 5 per cent CO 2 . Timed eggs from a stock of the deficiency 
known as Notch 8 crossed to Canton-S wild strain (N s /+) were dechorionated by Slifer's hypo- 
chlorite method and classified as normal or Notch-deficient. At 24 hours normals are larvae 
ready to hatch, while the deficient male embryos are strikingly abnormal and possess a nervous 
system about three times normal size. The ratio of types is 3 normal : 1 abnormal. Embryos 
were cut up and placed in divers containing 1 mm. :f Ringer-bicarbonate and 0.5 mm. 3 of 1.5 per 
cent acetylcholine. Although it is possible to carry out measurements on single embryos, two 
to five embryos per diver were used in most experiments. Readings were made at ten minute 
intervals for one hour after the divers had reached thermal equilibrium. 

A series of four determinations on 24-hour normals gave an average of 12.8 m ju.l. CO 2 /em- 
bryo/hour. A series of five determinations on Notch 8 deficient male embryos gave an average of 
34.0 m./z.l. COo/embryo/hour. The cholinesterase activity of Notch embryos is 2.7 times that of 
normal, which is nearly the same as the volume ratio of Notch/normal nervous systems, 3.3 as 
determined by planimeter from camera lucida outlines of sections. Thus cholinesterase activity 
is proportional to volume of nervous tissue. Notch-deficient embryos of other strains have given 
similar results. Thus the Notch male nervous system while abnormal in size and morphology 
is biochemically normal with respect to cholinesterase. A first step has been made in studying 
the rate of increase with development of cholinesterase activity in both normal and deficient em- 
bryos. At 18.5 hours the activity of the Notch embryo is 8.3 m.ju.l. CO 2 /hour, that of normal 3.8 
m.ju.l. COo/hour. In the unhatched Notch embryo at 48 hours the activity increases to 51.0 
m.fji.l. COn/hour. 

As checks, cholinesterase activity of unfertilized eggs and methyl butyrase activity of normal 
and Notch embryos were measured and found to be negligible. 

To determine the location of cholinesterase in normal embryos, central nervous systems were 
dissected out and their cholinesterase activity measured separately from the remaining portion of 
the embryos. One determination has given a value of 17.0 m. .l./N.S./hour. The value for the 
remnant is 2.6 m./i.l./hour. Since the central nervous system makes up not more than one- 
sixth the embryonic volume the cholinesterase activity there is roughly forty times that in the 
remnant. 

Possible metabolic and pliysical chemical factors in the production of the injury 
potential in spider crab nerve* A. M. SHANES. 

In contrast to frog sciatic nerve, spider crab nerve is permeable to both potassium and 
chloride ions and to a lesser extent to sodium. The relationship between metabolism and the 
potentials therefore cannot be the same as in frog nerve which is highly impermeable to chlo- 
ride and other small anions as well as to sodium. This is confirmed by the following observa- 
tions : (1) Although 0.002 to 0.0006 M iodoacetate (IAA) produces a continuous slow fall in 

* Aided by grants from the Penrose Fund of the American Philosophical Society and from 
the American Academy of Arts and Sciences. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 229 

potential in oxygen, 0.02 M pyruvate is unable to counteract this inhibition; (2) pyruvate 
accentuates IAA inhibition of the post-anoxic recovery of potential ; (3) the decline of potential 
in nitrogen is more rapid than in frog nerve and is not hastened by IAA ; (4) glucose does not 
retard the fall in potential during anoxia and inhibits a recovery in oxygen. The inhibitory 
effects of pyruvate and glucose may be the result of acid production, for 5 per cent CO 2 lowers 
the potential in this system in contrast to its effect in frog nerve. 

These results provide a basis for understanding some peculiarities of carbohydrate metabo- 
lism in spider crabs. For example, blood sugar levels average only 1 mg. per cent, nerve gly- 
cogen ranges from 500 to 2,000 mg. per cent wet weight (Kleinholtz, unpublished), and the 
breakdown of glycogen is reported to be largely to simple sugars as well as to lactic acid. 

The potassium content of these fibers is known to be high and the potential is inversely 
related to the extracellular potassium concentration. Consequently the injury potential is 
probably a potassium concentration potential as in frog nerve. In yeast (Rothstein and Haege, 
1943) potassium retention is stoichiometrically related to hydrogen ions lost to the medium when 
glucose is assimilated to form reserve carbohydrate. This mechanism may explain the high 
levels of both glycogen and potassium, and hence the metabolism-potential relationship, in spider 
crab nerve. 

Some effects of tannic acid on osmotic hemolysis. T. H. WILSON AND M. H. 
JACOBS. 

Human erythrocytes are less easily hemolyzed in hypotonic solutions of NaCl in the pres- 
ence of tannic acid than in its absence. The salt solution employed in the present experiments 
ranged from 0.091 to 0.069 M and those of tannic acid from 1/800 to 1/51,200 per cent. Even 
at the lowest of these concentrations of tannic acid there was a marked protective effect. In 
hypotonic solutions of Na 2 SO 4 ranging from 0.045 to 0.031 M and with the same concentrations 
of tannic acid as before, the effect was the exact opposite, hemolysis invariably being increased 
except at the lowest concentrations of tannic acid. Very similar effects, somewhat complicated 
by the permeability of the erythrocyte to ammonium salts, were obtained with NH 4 C1 and 
(N,H 4 ) 2 SO 4 , respectively. 

In the light of results obtained with molecular films of proteins by Schulman and others, 
the action of the tannic acid in the chloride solutions might be explained either by a strengthen- 
ing of the cell surface or by a decrease in its permeability to hemoglobin. Such an action in the 
case of the sulfate solutions is not necessarily excluded, but it seems to be overshadowed by an- 
other effect of a different nature, namely, the decreased permeability to anions produced by 
tannic acid, described elsewhere by Jacobs, Stewart and Butler. Since swelling of the erythro- 
cyte is known to be opposed by the exchange of bivalent sulfate ions from the outside for uni- 
valent anions from the inside, tannic acid, by hindering this exchange, might in this particular 
case indirectly favor hemolysis, despite its more direct protective effect on the cell surface. 

The effect of roentgen radiation on protoplasmic viscosity changes during mitosis. 
WALTER L. WILSON. 

Roentgen radiation has a marked effect on the protoplasmic viscosity of the dividing sea- 
urchin egg. If the eggs, or sperm, or both the eggs and sperm of Arbacia punctulata are ir- 
radiated before fertilization, then the normal pattern of viscosity change is altered. In the 
control the viscosity was low shortly after fertilization, then increased to a peak at 15 minutes 
(23 C.). It remained high for 6-10 minutes and then decreased. This decrease was markedly 
retarded by irradiation of the sperm or eggs (11,300 r at 6400 r/m), or both the sperm and 
eggs (5,000 r at 6400 r/m) before fertilization. In these experiments the viscosity remained 
high two or three times longer than in the controls. In three experiments out of ten in which 
irradiated eggs were fertilized with normal sperm, the viscosity increased to a value almost 
twice that of the controls. 

Biological specificity and the synthesis, oj native proteins. D. WRINCH. No ab- 
stract submitted. 



230 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

PAPERS READ BY TITLE 

The effects of massive doses of ergonovine male ate * upon the smaller blood 
vessels as seen directly with the microscope in the living mammal. 1 RICHARD 
G. ABELL. 

In these experiments, as in those described above, the blood vessels were studied in trans- 
parent 'moat' chambers in rabbits' ears. All injections were of 3.0 mgm., and all were made 
intravenously. Three mgm. in a rabbit corresponds to 60 mgm. in a man, which is 600 times 
the clinical intravenous dose. In these experiments the injection of 3.0 mgm. caused complete 
arteriolar constriction for 3 to 4 minutes. The larger arterioles (80 to 90 microns in diameter) 
remained narrowed to approximately one-half of their control diameters for 3 to 4 hours. A 
temporary constriction of the venules to from 0.5 to 0.8 of their control diameters occurred. 
In addition, this amount of ergonovine caused thicking of leukocytes to the walls of the arte- 
rioles, capillaries and venules. The degree of sticking varied widely in different rabbits ; in 
some cases it was slight ; in others large numbers of leukocytes stuck to the walls of the capil- 
laries and venules, and emigrated into the surrounding tissue. In one rabbit injections of 3.0 
mgm. were followed by the formation of leukocytic emboli, which blocked many of the capil- 
laries and venules and formed thrombi. The reaction was reversible and the thrombi usually 
disappeared within approximately 4 hours following the injections. This is in accord with the 
flow toxicity of ergonovine, and its failure to produce gangrene on repeated injections. 

As shown by the work of numerous investigators, two other ergot alkaloids, ergotoxine and 
ergotamine, do produce gangrene on .repeated injection. Such gangrene also occurs in ergot 
poisoning and is due to obliterative endarteritis and thrombosis. The formation of these 
thrombi is usually attributed to prolonged constriction of the small arteries, and interruption of 
the blood flow, but this is entirely hypothetical. 

In the present experiments thrombi were formed due to the increase in stickiness of the 
endothelium toward leukocytes and of the leukocytes toward each other. 

Perhaps gangrene produced by ergot and its more toxic alkaloids may be caused by a more 
severe and prolonged reaction of the type described above. 

Secretory cells in the branchial epithelium of fislics. GERRIT BEVELANDER. 

It was shown by Smith (1930, 1931, 1932) that the osmotic regulation of the body fluids in 
fresh and salt water teleosts and in elasmobranchs is effected considerably by the extrarenal 
excretion of salt (NaCl and KC1) under conditions that probably involve considerable osmotic 
work. It was further inferred that this exchange occurred in the gills. A previous study of 
the branchial epithelium in an extensive and widely divergent group of fishes (Bevelander 
(1935), led this writer to conclude that the only specialization occurring in the branchial epi- 
thelium of fishes consists in a thicker epithelium in the elasmobranchs than in teleosts and the 
presence of numerous mucous cells in all species examined. The cells which we described as 
mucous, were alleged to be "chloride secreting" cells in Anguilla and in some fresh water tele- 
osts, but not in elasmobranchs by Keyes and Willmer (1932). 

A re-examination of this problem included the experimental stimulation of secretion of the 
cells in dispute in representative teleosts and elasmobranchs. These cells were then subjected 
to a number of histochemical tests and were shown to be positive for mucin. Further, the oral 
and opercular membranes were also examined and it appears unlikely on the basis of structure 
that they are concerned with osmotic regulation. 

In order to comply with the observed physiological data, the cells which are responsible 
for extrarenal excretion must be in intimate relation with the blood supply and the external 
milieu, they must be very extensive to account for the considerable work performed, and finally 
they must be present in teleosts and elasmobranchs. Our observations reaffirm the absence of 
any specialized structures ; the only cells which comply with the three criteria required are the 

* 'Ergotrate' (Ergonovine Maleate, U.S. P., Lilly). 

1 This work was aided by a grant made by Eli Lilly and Company to the Department of 
Anatomy of the University of Pennsylvania Medical School. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 231 

respiratory epithelial themselves. It is further suggested that the observed conservation of 
urea in the elasmobranch gills may be affected by the relatively thick respiratory epithelium 
which covers the gill filaments. 

A modified Crompton formula for the latent heat of vaporisation. ALBERT P. 
MATHEWS. 

The general formula for the latent heat, L, which I have found is 

(1) L = CR'Thi e (d/D) 

R' is the actual value of the gas constant in the liquid phase, constantly falling as molecular co- 
aggregation increases with falling temperature, d is the liquid density and D that of the vapor, 
C is a constant peculiar to the substance, often not far from 2. 

The values of C and R' are obtained from the following general formulas which apply to all 
non-associating substances with the possible exceptions of hydrogen and helium. 

(2) C =C(R/R' e )(L/(L-E)-) a 

) 

(3) C - 1.1292- (3/8)5-+ (9/64 ) S 2 = 0.8792 + ( (3/8)5 - 0.5) 2 

5 in (3) is the critical coefficient: RT c /p c Vc in which R has its ideal value. 5 may be com- 
puted by (4) : 



(4) S = [(d m RT c /Mp c ) + 16((7 c -D/r o ) -12((T C -T)/T C Y] 

/[I + 5.158((7\, - T)/T e ) - 3.158 ((r. - T)/T c y] 



d m is the mean density of saturated vapor and liquid at temperature, T ; pc and T c the critical 
pressure and temperature. 



(5) (L/(L-))= 

L-E is the internal latent heat of vaporization. (5) is obtained from (6). 

(6) ((T/p)(dp/dT)) e = (L/) c =l + (27S 2 tf' c )/64 R") 

(7) R'c/R = (512 - 64S + 216S 2 - 27S 3 )/512.S 
R' T in (1) is obtained from (8). 

(8) (L/(L-E) ) r = R' T /R'. = 1 + ( (L/(L-E) ) - 1) ( (9/16) (T/T C ) + 7/16) (7YT C ) 2 

(9) R', = R 



At absolute zero L/ (L-E) is 1 and it advances with temperature as co-aggregation diminishes. 
In the ideal state C, 5 and (L/) c are 1. When 5 has its highest value of 4 in a normal sub- 
stance (L/E) C will be 7.3346 and, when 5" has its lowest value of 3/8, (L/E) C will be 4. 5 may 
be calculated also from the latent heat of vaporization at any temperature, C' being equal to 
(L-E)/RTln.(d/D) by (10): 

(10) 5= (8/3) (0.5 + V (C- 0.8792)) 

Obtained from (3) above; E being taken, with small error usually, as equal to p(V-v). 

Formula (1) above is an easier and more accurate way of computing the latent heat of 
vaporization than by the thermodynamic equation: L (Tdp/dT) (V-v). The results by (1) 
agree usually within 1 per cent with the experimental determinations at the normal boiling point 
as made by J. H. Mathews and others. The derivation of all the foregoing formulas will be 
given in the full papers together with examples of application to specific cases and also the general 
formula for the Cailletet and Mathias law (11) : 

(11) rf m = rf.[l + (5.158- 16/5) ((T,-T)/Tc) + (12/5 - 3.158) ((7. - 



232 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

Heat Death. PAUL R. ORR. 

I. Time-temperature relationships in marine animals. 

Temperature as an intrinsic ecological factor which determines, to a great extent, the abun- 
dance, life cycle, and distribution of marine organisms, is a well-established fact. However, the 
duration of exposure required to produce death at each temperature in the effective series has 
not been taken into consideration. Thus, in order to state accurately the conditions of heat 
death, it is necessary to plot a curve in which both variables, temperature and time, are 
represented. 

Heat death curves have been plotted for Uca pugilator, Asterias forbesi, Ophiodenna 
brevispuium, Arbacia punctulata, Nassa obsolcta, Fiindulus hctcrnclitus. 

All of the curves have approximately the same shape. For a relatively slight rise in tem- 
perature there is a marked drop in the length of exposure necessary to cause death. This rela- 
tionship is not one of direct proportionality. 

II. Differential response of the entire animal (Rana pificns) and several of its organ systems. 

Whether we are dealing with cells or multicellular organs and tissues, or the organism as 
a whole, we are confronted with the fact that not all of the cells, organs, etc., have the same 
sensitivity to heat. An animal exposed to excessive heat for a length of time to cause complete 
loss of excitability might well be pronounced dead, for it never again will show any signs of 
life as a complete organism. Yet there are parts of the complex animal that are "alive." 

The animal as a whole, the tadpole, sciatic nerve, sartorius and gastrocnemius muscles, and 
heart were separately studied, and curves were plotted for the heat death points of each. The 
data show that in the adult animal the order of death is: (1) the organism as a whole; (2) the 
muscular system; (3) heart, and (4) nervous tissue. 

All heat death curves plotted are of the same shape, showing a sudden drop followed by a 
gradual approach to a constant level. 

III. The effect of high temperatures on heart rate in Venus mercenaria. 

In the clam heart (Venus mercenaria} we have an automatic mechanism by which the effect 
of heat can be studied. By subjecting excised hearts to a series of high temperatures and noting 
the heart rate it was possible to determine the lethal point for each temperature and thus plot 
a curv.e showing time/temperature relationship. 

For the clam heart the same general type of curve was found as shown in previous studies 
on marine animals and frogs. That is, there is a point at which the hearts will beat for a rela- 
tively long period of time; then as they are subjected to higher temperatures there is a rapid 
decrease in heart rate, followed by a leveling off to a constant rate. 

Penetration glands in tapeworm Onchosphcres. \Y. MALCOLM REID. 

Although various types of cystogenous and penetration glands have long been figured and 
studied as a part of the internal structure of trematode cercariae and miracidia, they have not 
been recognized in the onchosphere stage of cestodes. A pair of such glands has been found in 
the fowl cestodes Raillietina cesticillus (Molin), and Choanotaenia infundibulum (Bloch) and 
in a herring gull cestode Hymcnolepis sp. Although these glands may be seen under favorable 
conditions without special stains, they respond in the same manner to vital stains as do trema- 
tode glands, showing up best with Nile blue sulfate and neutral red. The gland stretches to the 
posterior end of the larva, where it appears to be anchored. The secretion pores are located 
near the anterior and slightly to the side and above the middle pair of hooks when these hooks 
are oriented with the points directed anteriorly and downward. The granular contents may be 
seen to move about as the general contour of the glands is changed by the violent contractions 
associated with hook movements and at times some of the secretion may be seen exuding from 
the pores. A single nucleus is located near the middle of each gland, and the two glands are 
connected by a narrow isthmus near the posterior end. 

The nature of the secretion has not been determined but it is possible that it assists the 
larva in penetration since this granular substance is given off at a time in the life cycle when 
the six-hooked embryo must break out of the covering membranes of the egg and penetrate the 
gut of an arthropod intermediate host. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 233 

Intensity-duration relation in stimulation by light. F. J. M. SICHEL AND P. B. 
ARMSTRONG. 

The excised sphincter pupillae of many vertebrates will respond by constriction to stimula- 
tion by visible light of suitable wave-lengths. In these experiments the sphincter pupilla of the 
eel, Anguilla rostrata, was used. The sphincter was excised from small adults, 15 to 18 inches 
in length. 

The sphincter was pinned out, anterior surface uppermost, on white beeswax. It was 
illuminated for observation by transmitted red light, to which the preparation is insensitive. 
The source of light for stimulation was a tungsten filament lamp maintained at constant voltage. 
This was focussed on the preparation obliquely from above. The intensity of the stimulating 
light was varied by Wratten neutral niters and a neutral wedge. The duration of the stimu- 
lating flash was controlled by a shutter manually operated and timed by a stop watch. The 
criterion of threshold was the smallest contraction visible through a low-power microscope. 
An eyepiece filar micrometer was used to advantage in determining the threshold stimulus. 
The preparation was bathed in a Ringert's fluid and permitted to become dark-adapted before 
each experiment. 

The threshold was found to be a function of the duration and of the intensity of the stimu- 
lating flash. The intensity-duration relation conforms with Hill's theory of excitation for rec- 
tangular stimuli. The chronaxies averaged about 12 seconds, the range being from about 6 
seconds to 20 seconds. In terms of the reciprocity law this would mean that the law holds 
reasonably well for flashes shorter than, say, 10 seconds. At longer durations the deviation is, 
in direction and amount, what would be expected on the basis of Hill's equation for excitation. 
There is a definite rheobase, or minimal intensity of the stimulating flash below which excitation 
is never produced, even for very long exposure times. 

The pattern of flic intrinsic palmar musculature. WILLIAM L. STRAUS, JR. 

The intrinsic palmar musculature of tetrapod vertebrates comprises two fundamental series : 
(1) a superficial, arising from fascia or tendon, and showing variable tendency toward strati- 
fication, and (2) a deep, arising from bone and always arranged in two layers separated by the 
deep palmar nerves and vessels. Between the two series lies the mid-palmar space. 

In urodeles (Nectunts inaculosus, Cryptobranchus alleghaniensis), the superficial series is 
a single layer (flexores breves superficiales) arising from the dor sum of the long flexor tendon. 
The deep series is composed of a superficial (contrahentes or adductores) and a deep (flexores 
breves profundi, intermetacarpales, interphalangeus III?; in Cryptobranchus also flexores breves 
minimi) layer. 

In lizards (Sceloporus spinosus, Ctenosaura siinilis}, the superficial series tends to form 
two layers a superficial (flexores breves superficiales, marginal abductors), arising from the 
transverse carpal ligament, and a deep (lumbricales), arising from the long flexor tendon; in 
Sccloporus, however, such lamination is incomplete, for fibers of the superficial layer also arise 
from the long flexor tendon. The deep series again exhibits superficial (contrahentes) and 
deep (flexores breves profundi) layers. 

In mammals (Didclphis virginiana, Macaco mulatto, Homo}, the superficial series forms 
two distinct layers a superficial (abductor pollicis brevis, flexor pollicis brevis, opponens pol- 
licis?, palmaris brevis, flexor V brevis, abductor V; in Didclphis also a flexor brevis manus), 
largely from palmar aponeurosis and transverse carpal ligament, and a deep (lumbricales), from 
the deep long flexor tendon separated by the superficial palmar vessels and nerves. The deep 
series again has superficial (contrahentes ; only adductor pollicis in man) and deep (interossei, 
opponens V) layers. 

Muscular homologies, at least between vertebrate classes, cannot be reasonably extended 
beyond comparison of entire palmar layers. Direct homology of individual muscle units is 
profitless and probably invalid. 



234 PRESENTED AT MARINE BIOLOGICAL LABORATORY 

The toxicity of a mixture of high molecular alkyl-dimethyl-benzyl ammonium 
chlorides to Fundulus. CHARLES H. TAFT. 

The mixtures of high molecular alkyl-dimethyl-benzyl ammonium chlorides used is sold by 
the Winthrop Chemical Company under the trade name Zephiran Chloride * for use as an anti- 
septic or disinfectant. 

Taft and Strandtmann (1945. Fed. Proc., 4: 136) showed that under laboratory conditions 
this material is an efficient larvicide for the mosquito Culex quinquefasciatus and Aedes aegypti 
in dilutions up to 1 : 250,000. It seems desirable to determine its toxicity to some of the animals 
it might be brought in contact with if used for this purpose. Taft (1946. Texas Rpts. on Biol. 
and Mcd., 4: 25) has reported its toxicity for various invertebrates. 

To determine the toxicity by injection fundulus were injected intraperitoneally with different 
doses of one per cent solution; 0.25 cc. killed 17 out of 22, 0.05 cc. killed 15 out of 17 while 0.1 
cc. killed 24 out of 24 fundulus. When these fish died they were darker than the controls and 
in many of them the abdomen was red about the site of injection. When the abdomen was 
opened there was frequently a greenish fluid present and the viscera had the appearance of 
having been cooked. The liver, gall bladder, heart, kidneys, and gills appeared normal. 

Other fundulus were placed in finger bowls containing 225 cc. aerated sea water with dif- 
ferent concentrations of the drug. When the fish were placed in dilutions of from approxi- 
mately 1 : 2,500 to 1 : 100,000 all the fish died in from 35 to 105 minutes. On autopsy there 
were no significant gross changes. A dilution of 1 : 225,000 killed 25 per cent of the fish while 
1 : 500,000 did not kill any of the fish exposed to it. 

To determine the effects of longer exposure to the drug several fundulus were placed in 
battery jars in aerated sea water solution of from 1 : 100,000 to 1 : 400,000 and observed at the 
end of 24 hours. All the fish exposed to 1 : 100,000 and 1 : 200,000 were found dead. Twenty- 
five per cent of those exposed to 1 : 300,000 died while the 1 : 400,000 solution failed to kill any 
fish. It is evident that the effective range of this drug when employed as mosquito larvicide 
might be deleterious to fundulus. 

Further evidence of polypoidy in the conjugation of green and colorless Paramecium 
bnrsaria. RALPH WICHTERMAN. 

In a study of the time-relations of the nuclear events in living and Feulgen-stained prepara- 
tions through conjugation, instances of polyploidy were encountered. Polyploidy was first re- 
corded in Paramecium by Chen (1940, Proc. Nat. Acad. Sci. V: 26) for P. bursaria and this 
represents the second report of the phenomenon. Pure-line races of the colorless (255) and 
green (B9) paramecia were mated. The individuals of each race have well-defined micronuclei 
of approximately equal size. 

The three pregamic divisions were found to be remarkably constant in respect to time and 
micronuclear behavior at a given temperature. However, in the cytological examination of 
many hundreds of joined pairs, approximately 2 per cent were observed in which the micro- 
nuclear behavior resulted in the polyploid conditions only after the pregamic divisions. The 
crucial stage where polyploidy occurs is found during the period of pronuclear transfer, ap- 
proximately 16-18 hours after the animals have been mated. It follows the third suggestion 
made by Chen in accounting for polyploidy ; namely, the failure of a migratory pronucleus in 
one of the conjugants to migrate to the other conjugant. The result is an individual with one 
small pronucleus (the "stationary") which is haploid, and the conjugant with three pronuclei 
(two "migratory" and one "stationary") which fuse and form a larger triploid synkaryon. 

What is the fate of each nuclear body that is now comparable to the normal synkaryon ? The 
subsequent micronuclear stages show a conspicuous and persisting size difference in all later 
stages and hence are recognized easily. In the haploid conjugant, late anaphase stages (com- 
parable to postgamic ones) measure 10.8 /z in length and are very narrow; similar stages in 
the triploid co-conjugant measure 27 /JL in length and are proportionately wider. Their division 
products measure 8 (j. in the haploid and 15.5 fj. in the triploid individuals respectively. 

While polyploidy occurs in only 2 per cent of the cases in this material, it nevertheless cre- 
ates variation in micronuclear composition and is therefore of evolutionary significance. 

* Kindly furnished by the Winthrop Chemical Company. 



PRESENTED AT MARINE BIOLOGICAL LABORATORY 235 

The Lipids in Pelomyxa carolinensis. CHARLES G. WILBER. 

In 1942 the author demonstrated that the cytoplasm of Pelomyxa carolinensis contains lipid 
material, that this lipid comes from digested food, and that it is composed of a high proportion 
of fatty acid. In the latter respect the stored fat differs from that in Amoeba proteus in which 
fat is stored in the neutral form. In the previous work, Nile blue sulfate was used to distinguish 
neutral fat from fatty acid. This dye has been criticized as a reagent for fat tests. Conse- 
quently it seemed desirable to use specific chemical procedures to ascertain the nature of the 
lipid material in Pelomyxa. 

Ninety mg. (wet weight) of pelomyxae were thoroughly washed in boiled culture fluid. 
By repeated centrifugation the cells were broken up and then the lipids were extracted in hot 
alcohol. The quantities of phospholipid, cholesterol, and fatty acid were ascertained by the 
Bloor method. It was found that in the amount of cellular material used there was no meas- 
urable phospholipid or cholesterol. The total weight of fatty acid in 54 mg. of cells was 2.05 mg. 
or 3.8 per cent fatty acid. 

These results are in agreement with the results previously obtained using Nile blue sulfate. 
It seems that in Pelomy.ra carolinensis the lipid material occurs chiefly as fatty acid and that 
the amount of other lipids is very small. 

The presence of lip as c in Pdomyxa carolinensis. CHARLES G. WILBER. 

The digestion of fat in rhizopods has been demonstrated by several investigators. More- 
over, it has been shown that the fats digested are incorporated into the cytoplasm. However, 
none of the investigations so far has given direct evidence for the presence of lipase in the cyto- 
plasm of rhizopods. 

Pelomyxae were starved and then ground up in a drop of water. A drop of this solution 
was added to a drop of 0.2 per cent emulsion of castor or olive oil and a drop of pure water 
was added to another drop of the emulsion as a control. After 30 minutes both drops were 
treated with hydroxylamine hydrochloricle and potassium hydroxide. Then after acidification 
each drop was treated with 1 per cent ferric chloride solution. In each case a violent brown 
color was produced in the control, whereas no color was produced in the drop containing the 
ground up pelomyxae. 

The above reaction is a test for esters. Lipases are known to be ester ferments. Since the 
oil emulsions mixed with ground pelomyxae did not give the characteristic ester reaction, it can 
be concluded that the esters were broken down by something in the cytoplasm. We therefore 
have direct evidence for the presence of lipase in Pelomyxae carolinensis. 



PAPERS PRESENTED AT THE MEETING OF THE SOCIETY 
OF GENERAL PHYSIOLOGISTS 

SEPTEMBER 5 AND SEPTEMBER 6 
FIRST SESSION S. C. BROOKS, CHAIRMAN 

The effect of cold on capillary permeability and fluid movement in the frog. 
ELLEN BROWN, M.D.* AND EUGENE M. LANDIS, M.D. 

Micro-manipulative methods were used to study the relationship between capillary blood 
pressure and the rate of fluid movement through the walls of single capillaries in the frog's 
mesentery (a) at ordinary room temperatures of 22.5 to 25.5 C. and (b) when the mesentery 
was cooled to between 2 and + 2 C. Cooling the mesentery decreased capillary perme- 
ability, reduced the observed rates of nitration and increased the observed rates of absorption. 
The nitration constant of the capillary wall was reduced from the control value of 0.0070 
i"- r '/i". 2 /sec./cm. water pressure to 0.0019, a decrease of 73 per cent. The effective osmotic pres- 
sure of the blood within the capillaries was elevated from 10.5 to 13.8 cm. water, an increase 
of 31 per cent. 

Four possible causes for the increase in apparent or effective osmotic pressure were con- 
sidered. (1) An increase of absolute colloid osmotic pressure was excluded because plasma 
protein concentrations, calculated from specific gravities of plasma samples, were the same in 
the two series of frogs. (2) An increase in effective colloid osmotic pressure due to greater 
retention of plasma protein during cooling could also be excluded because the control experi- 
ments showed that plasma proteins were already retained completely, or almost completely, even 
at room temperature. (3) It is possible, however, that the effective non-protein osmotic pres- 
sure might rise if the passage of smaller molecules, e.g., glucose, amino acids, urea or certain 
electrolytes, was impeded more than that of water as the permeability of the capillary wall 
decreased during chilling. (4) Tliermosmosis might also be responsible because relatively warm 
blood was circulating through capillaries surrounded by cooler tissues. Studies are in progress 
to determine whether or not this factor modifies the movement of fluid through membranes in 
vitro using a schema which simulates the conditions existing in vivo. 

The effects of cold on the capillaries of the frog differ from those observed in mammalian 
capillaries because the former become less permeable at 2 C., whereas the latter become more 
permeable as temperature falls below 10 C. However, actual freezing of the frog's capillaries 
at temperatures of 5 to 10 C. increased capillary permeability conspicuously, as shown 
by the appearance of stasis during thawing. If the duration of actual freezing was brief this 
stasis usually disappeared within a few minutes as the capillary wall regained its normal relative 
impermeability to protein. 

Bubble formation within single cells. ~f E. NEWTON HARVEY, K. W. COOPER, A. 
H. WHITELEY, D. C. PEASE, AND W. D. MCELROY. 

Normal living isolated cells (Amoeba sp., Chaos chaos, Paramoecium, Arbacia and Aste- 
rias eggs and Nitella) do not form internal gas bubbles if saturated with nitrogen gas at 80 to 
120 atmospheres pressure and then suddenly decompressed. Bubbles may form on the outside 
of the cells due to contamination with gas nuclei (minute gas phases sticking to hydrophobic 
spots). Cells which have been killed by chloroform or formalin likewise form no bubbles within 
but sometimes spontaneously dead cells or those previously injured by twisting before subjec- 

* Research Fellow in Physiology, Commonwealth Fund. 

t Part of the work described in this Abstract was done under a contract recommended by 
the Committee on Medical Research between the Office of Research and Development and 
Princeton University. 

236 



PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 237 

tion to the high gas pressures do form bubbles within after decompression. A living Nitella 
cell just after decompression from high gas pressures and still free of bubbles, will immediately 
form a bubble inside if the cell wall is gently pinched (not enough to penetrate the wall) or 
twisted. Such bubbles are believed to result from local decreased tensions that tear the liquid, 
forming a space or cavity (a vapor phase) into which gas diffuses, forming a gas nucleus 
that immediately grows to a bubble. Such cavities can form inside or outside of cells even at 
atmospheric pressures. They are believed to be formed during muscular exercise in man, 
when the incidence of aviator's bends is greatly increased. 

The action of various cations of muscle protoplasm. L. V. HEILBRUNN AND F. J. 
WIERCINSKI. 

There are two ways to study the colloidal behavior of muscle protoplasm. One way is to 
isolate pure proteins and follow their reactions in test tubes; the other way is to subject the 
protoplasm itself to reagents and observe the results. In our studies, we injected solutions of 
various salts into the interior of isolated muscle fibers of the frog. We then noted the degree 
of shortening of the constituents of the muscle. With the aid of a micrometer eyepiece, we were 
able to determine the effect of the injections on the length of the fiber. In numerous experiments 
we found that rather dilute calcium chloride solutions invariably caused an immediate and pro- 
nounced shortening of the protoplasmic constituents of the muscle. On the other hand, potassium 
and sodium chloride had very little effect. Even when injected in concentrations isotonic with 
the muscle, they ordinarily caused no shortening whatsoever. Rarely, a shortening did follow 
injection of isotonic sodium or potassium chloride. This we believe was due to the release of 
calcium ion. Magnesium ion likewise causes no shortening of the protoplasmic constituents. 
Barium acts like calcium. The results support the calcium ion theory of stimulation and they are 
opposed to Szent-Gyorgyi's opinion that potassium is the ion primarily responsible for the con- 
traction of muscle. 

Further observations on an oligodynamic action of copper and mercury on eryth- 
rocytes. M. H. JACOBS AND DOROTHY R. STEWART. 

The specific effect of copper in decreasing the permeability of erythrocytes to glycerol seems 
to be absent in all species whose erythrocytes show a low degree of permeability to this solute. 
It is also lacking in a number which show a very high permeability both to glycerol and to other 
hydrophilic solutes of comparable molecular volume. It has so far been found only in those 
species whose erythrocytes show a disproportionately great permeability to glycerol, thus sug- 
gesting that some special mechanism of penetration may be involved, which is reversibly in- 
activated by copper. This generalization is supported by the behavior of the erythrocytes of a 
number of birds in which the specific permeability to glycerol is particularly great. 

The effects of HgCL in some ways resemble and in others differ from those of CuCL. One 
of the most important differences is that HgCU forms a double salt with NaCl, and its activity 
is therefore greatly reduced by the presence of any considerable quantities of the latter salt. A 
second difference is that HgCL readily enters the erythrocyte, while CuCU does not. These two 
fundamental differences are responsible for a number of secondary ones. 

That copper may hinder the escape of glycerol from human erythrocytes, as well as its en- 
trance into them, is suggested by the following experiment. To a suspension of erythrocytes in 
an isotonic salt solution, small amounts of copper and of concentrated glycerol are added, and 
the resulting mixture is then diluted with a properly chosen hypotonic salt solution. If the copper 
is added before the glycerol, it decreases hemolysis by preventing the entrance of glycerol into the 
cells. If the copper is added 30 seconds or more after the glycerol, it increases hemolysis by 
preventing the escape of the glycerol that has entered the cells. 

The prolytic loss of K from red cells. ERIC PONDER. 

The prolytic loss of K, i.e., the loss of K which takes place from red cells exposed to hypolytic 
concentrations of lysin, has been measured by means of the flame photometer in systems containing 
distearyl lecithin, sodium taurocholate, sodium tetradecyl sulfate, saponin, and digitonin. The 



PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 

lysins are added in various concentrations to washed red cells from heparinised human blood, 
and the K in the supernatant fluids is determined after various intervals of time at various tem- 
peratures. This prolytic loss of K, K P , is compared in every experiment with the loss K s into 
standard systems containing one per cent NaCl alone, without lysin. 

The losses K,, and K s increase with time, so that new steady states are approached logarith- 
mically. The values of K p which correspond to the new steady state depends on the lysin used, 
being greatest with taurocholate and smallest with powerful lysins such as digitonin (confirming 
an observation of Davson and Danielli). The extent and course of the K losses seem to have no 
simple relation to the prolytic phenomenon of the disk-sphere transformation. 

Just as the prolytic loss of K occurs without the loss of any Hb, so in concentrations of lysin 
sufficient to produce hemolysis the loss of K, expressed as a percentage of the total red cell K, 
increases much more rapidly with lysin concentration than does the loss of Hb, expressed as a 
percentage of the total Hb. The explanation of these relations depends on whether the loss of K 
is treated as being all-or-none in the case of the individual cell, or as being the result of the loss 
of part of the K from all the cells. This point has yet to be decided. 

SECOND SESSION L. MICHAELIS, CHAIRMAN 

Effect of fluoroacetate on the metabolism of baker's yeast. E. S. GUZMAN BARRON 
AND GEORGE KALNITSKY. 



Among the organic halogen compounds, those containing fluorine occupy a special position 
regarding their chemical and physiological properties. Because of the high value of the energy 
of the C-F bond and of the electro-negativity of F, the introduction of F into the C atom pro- 
duces a greater stability, specially in alyphatic compounds. This is shown on measuring the rate 
of combination of cysteine with halogen acetates. At 23, half-reaction with iodoacetate took 
place in 4.4 minutes ; with bromoacetate in 6.2 minutes ; with chloroacetate, in 125 minutes. 
With fluoroacetate it did not react at all. There is a certain relationship between the rates of 
reaction and the bond-energy values of the C-halogen bonds as well as the electronegativity 
values of the halogens. On studying the effect of these halogen acids on the rate of oxidation 
of acetate by baker's yeast it was found that 0.001 M of fluoroacetate inhibited it 90 per cent; 
bromoacetate, 17 per cent; iodoacetate, chloroacetate, and trifluoroacetate, none at all. On com- 
paring the interatomic distances between C and the halogen it can be seen that the C-F bond 
with a distance of 1.41 A approaches most closely the distance of the C-H bond, 1.09 A. By in- 
creasing the size of the fluoroacetate molecule through the replacement of the other two hydrogens 
with fluorine (trifluoroacetate) the inhibiting effect was destroyed. This inhibition is a sub- 
strate competitive inhibition, the fluoroacetate occupyng the place of acetate in the protein moiety 
of the acetate metabolism enzyme. Increase of the length of the molecule as in fluoropropionate, 
fluorobutyrate, and fluorocrotonate destroyed the inhibition. Inhibition was partially reversed 
on addition of large amounts of acetate (0.08 M). Inhibition occurs in the first step of acetate 
metabolism, namely, condensation with oxaloacetate to give citrate. The formation of citrate 
from acetate was completely inhibited with 0.005 M fluoroacetate. In the presence of ethanol, 
the rate of O 2 uptake was not affected by fluoroacetate up to 42 per cent of the total O? uptake, 
when the inhibitory effect appeared. This is indication that ethanol oxidation occurs in three suc- 
cessive steps : -oxidation of ethanol to aldehyde ; and of aldehyde to acetate, both unaffected by 
fluoroacetate ; and oxidation of acetate, inhibited by it. At the end of the experiment there were 
in the control 1572 cmm. O 2 used, and 150 cmm. of acetate formed from 940 cmm. of ethanol; in 
the presence of fluoroacetate there were 975 cmm. O... used and 890 cmm. of acetate formed. 

The effect of sodium aside on Parameciuwi calkinski. E. J. BOELL. 

Sodium azide is generally regarded as an inhibitor of respiration by virtue of its inactivation 
of cytochrome oxidase. In Parameciiim calkinsi, experiments have shown that this compound, in 
a concentration of 0.001 to 0.01 molar, reversibly depresses respiratory activity by 50 to 60 per 
cent. Under certain circumstances, however, azide instead of inhibiting respiration serves as a 
powerful respiratory stimulant. The stimulating effect of azide seems to depend primarily upon 
the pH of the medium. For example, a 0.01 molar solution of NaN 3 at pH 6.02 will depress 
respiration to a value about 30 per cent of normal ; at pH 6.24 respiration is 70 per cent of nor- 



PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 239 

mal, while at pH 6.59 the same concentration of azide stimulates respiration of 238 per cent of 
normal. Calculation of the hydrazoic acid concentration at these pH values shows that the ef- 
fect produced depends, within certain limits, upon the concentration of undissociated HN 3 . 

A study has been made of the mechanism of azide stimulation. It has been found that the 
respiratory quotient of normal animals averages 0.99 ; that of animals in the presence of a 
stimulating dose of azide averages 1.05. The increased oxidation thus involves the metabolism 
of organic substrate. It is also apparently mediated by the normal enzymic mechanisms for it 
is sensitive to cyanide. Carbon monoxide, however, exerts only a slightly depressing effect. 

The metabolism of Paramecia under normal circumstances is accompanied by the production 
of large quantities of ammonia nitrogen. On the assumption that such ammonia production 
represents protein breakdown, approximately 75 per cent of the total oxygen consumption of 
control animals can be accounted for in this way. Although Paramecia treated with azide show 
increased ammonia production, only 22 per cent of the extra oxygen uptake induced by azide can 
be accounted for as protein breakdown with ammonia as the end product. 

In addition to the effects already noted, azide interferes with the ability of Paramecium 
calkinsi to maintain normal water balance. The activity of the contractile vacuoles is greatly 
reduced and supernumerary vacuoles are frequently formed. 

The oxygen consumption concerned with growth in bacterium coli. KENNETH 
FISHER. No abstract submitted. 

Enzymatic acetylation and the co enzyme of acetylation. FRITZ LIPMANN. 

The mechanism of enzymatic acetylation of aromatic amines has been studied in pigeon liver 
homogenates and extracts (Lipmann, F., 1945. /. Biol. Chcm., 160: 173). In this enzymatic 
system the condensation of an aromatic amine, like sulfanilamide, with acetate, is effected 
through a transfer of phosphate bond energy from adenylprophosphate. (Cf., Nachmansohn, 
D. and Machado, A. L., 1943. J. Neurophysiology, 6: 397 for a similar system of choline acety- 
lation in brain). 

A heat stable and dialysable coenzyme was recently found necessary in this reaction, besides 
the energy donor adenylpyrophosphate. The characterization of this new coenzyme is now in 
progress in this laboratory in collaboration with Dr. Nathan O. Kaplan. We find the same 
coenzyme necessary to complement dialyzed brain extracts for acetylation of choline, although 
the brain enzyme is specific for choline and the liver enzyme specific for amines. The coenzyme 
is present in largest amounts in brain, liver, and kidney. Appreciable amounts are present in 
all tissues tested, including carcinoma. Therefore its action must be a very general one and 
probably not merely restricted to acetylation. 

The coenzyme is destroyed by intestinal phosphatase with liberation of phosphate. It is 
inactivated by a rather general tissue enzyme without liberation of phosphate. The link at- 
tacked by the latter enzyme is unknown. The compound follows the general pattern of nucleo- 
tide precipitation. Our most active preparations showed sporadic crystals on microscopic ex- 
amination. This quite uniform fraction contained adenine, ribose, and phosphate in the propor- 
tion 1 to 1 to 2. Acid hydrolysis showed the second phosphate not to be in pyrophosphate 
linkage. If we assume the presence of some crystals to indicate near purity, which it not neces- 
sarily does, then the content of approximately 50 per cent of adenylic acid in our best prepara- 
tions should mean the coenzyme to be of dinucleotide structure. Therein the adenylic acid 
should be linked through the second phosphate to an as yet unidentified part. Electrotitration 
and cleavage experiments seem to support the outlined constitution. 

Penetration and action of cholincsterasc inhibitors. DAVID NACHANSOHN. No 
abstract submitted. 

The metamorphosis of visual systems in amphibia. GEORGE WALD. 

In the rods of the vertebrate retina two visual systems are found. One is based upon the 
red photosensitive pigment rhodopsin, engaged in a cycle with vitamin A, ; the other involves 
the purple photopigment, porphyropsin, bound in a similar cycle with vitamin A 2 . 



240 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 

The porphyropsin system appears to be the more primitive in vertebrate evolution. The 
cyclostome, Petromyzon marinus, possesses only this system. The same is true of all types of 
freshwater fish so far examined. 

Vertebrates have followed two pathways out of fresh water, one into the sea, the other 
to land. Both have led them to the use of vitamin A, in vision. Thus all marine fishes which 
have been examined, with the single exception of certain Labridae, have the rhodopsin system 
alone ; so also do all the birds and mammals investigated. 

Interpolated between freshwater and marine fishes are euryhaline forms, which can exist 
as adults in either environment. Among them, the salmons and the "freshwater" eel have mix- 
tures o'f the rhodopsin and porphyropsin systems ; while the alewife and white perch have only 
the latter. In all these forms the visual system is predominantly or exclusively that normally 
associated with the environment in which the fish develops embryonically, and is relatively inde- 
pendent of the environment in which it is found as an adult. 

Interpolated between freshwater fishes and true land vertebrates are the amphibia. Their 
life histories for the most part are closely analogous with those of euryhaline fishes, amphibian 
migrations to land replacing fish migrations into the sea. 

Adult frogs possess the rhodopsin system and vitamin A, alone. The tadpole of the common 
bullfrog, Rana catesbiana, however, has exclusively the porphyropsin-vitamin An system just. 
prior to metamorphosis. During metamorphosis it transfers completely to the rhodopsin sys- 
tem, which is found alone in the new emerged frog. Partly metamorphosed animals have mix- 
tures of both systems, such as have been found otherwise only in euryhaline fishes. 

The common newt, Triturus viridescens, begins its life as a gilled larva in fresh water. 
After several months it metamorphoses to the land-living red eft; then after 1-2 years of growth 
it undergoes a second metamorphosis to the sexually mature newt, returning to the water for 
the remainder of its life. The eye of the red eft contains a mixture of vitamins Aj and A 2 , 
predominantly the former; while that of the water-phase adult presents just the reverse propor- 
tions of both vitamins. This is a change opposite in direction to that in the frog, but associ- 
ated in the same way with the chemical metamorphosis of visual systems. 

Amphibia, therefore, like euryhaline fishes possess as a group both the rhodopsin and 
porphyropsin systems ; but in amphibia these systems succeed one another as the animal goes 
through its basic metamorphoses. 

THIRD SESSION J. H. BODINE, CHAIRMAN 

X-ray effects in mixtures of compounds. RUBERT S. ANDERSON. 

It has been reported previously that ascorbic acid, as shown by experiments in plasma, has 
a preferential ability to react with the materials produced in water by x-rays. Much of the 
ascorbic acid reaction is not observed in irradiated muscle. Non-uniform distribution of the 
ascorbic acid in muscle would tend to make the observed result too low. Another possibility 
is that other compounds are present in muscle which take some of the reactive material away 
from the ascorbic acid. 

Evidence has been obtained that the ascorbic acid reaction consists in part of a reversible 
oxidation, presumably to dehydroascorbic acid. When present during the irradiation, glutathi- 
one and cysteine gave substantial, although variable, protection of ascorbic acid against x-rays. 
Alanine was much less effective, suggesting that the sulfhydryl grouping is largely responsible, 
whether it is a true competitive protection or a reversal of oxidation. Glutathione and cysteine 
and possibly protein sulfhydryl groups could thus account for a part of the protective effect of 
muscle on ascorbic acid. 

There is no evidence that the destruction of a small amount of these compounds woulc 
damage a cell. However, the work shows that, in principle, a compound through which the 
water reaction might damage the cell could exist. 

Ascorbic acid, glutathione and cysteine partially protect pepsin from the inactivating effect 
of x-rays. Alanine is much less active. 

If at least a part of the reaction in water is distributed randomly throughout the proteins 
of the cell and nucleus, the occasional loss of a molecule or two from compounds represented 
by hundreds of molecules need have little effect on the cell although the products formed, such 
as denatured proteins, might secondarily be harmful to the cell. However, if there are in the 



PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 241 

cell or nucleus some thousands of different protein compounds or structures each form of which 
is essential to the cell and each one of which is represented by but one or two molecules or 
particles, then randomly distributed products of irradiated water might destroy one of these 
entities and so damage or kill the cell. This is essentially the argument used by Lea in reaching 
the conclusion that his theory led to the expectation of gene and chromosomal effects from 
irradiation. 

Electrical studies of acetylcholine and choline estcrase. T. C. BARNES. 

Acetylcholine passes through the thin oil layer of a bubble of guaiacol-resin-cholesterol so 
rapidly that the spike potential must be recorded by an oscillograph. First the acetylcholine 
produces a negative phase boundary potential on one side of the oil layer but on reaching the 
opposite side of the oil a new potential is established which produces the descending part of the 
spike (no esterase required). At the suggestion of Osterhout, solutions were shaken 5 hrs. 
with guaiacol with these results : oil with saline 5 X 1(T 7 mhos ; same with 0.002 M acetylcholine 
35 X 10" 7 mhos (conductivity was determined of oil separated from aqueous solution). Apply- 
ing the Nernst equation, 0.058 times log conductivity difference (6) gives 40 mv. (observed 
phase boundary potential). At the suggestion of Loewi, tetramethylammonium iodide was 
found to give no potential on nitrobenzene but 0.05 per cent gave 25 mv. negative on guaiacol. 
The type of oil and not the tertiary or quarternary nature of the compound determines electro- 
genie effects. Thus prostigmine produces 85 mv. negative on guaiacol compared with 35 mv. 
generated by acetylcholine (both 0.002 M). Prostigmine inhibits the cord in spasticity by 
flooding with high negative potential which may also act as a stimulus on muscle in myasthenia. 
Dialantin and phenobarbital produce positive potential (20 mv. at concentration of 0.05 per cent) 
which probably neutralizes the excess negativity of acetylcholine in the brain in epilepsy. 
Lyovac plasma reduces the phase boundary potential of 0.05 per cent acetylcholine from 35 to 
15 mv. (residual potential is produced by choline). Potential of benzyol choline is destroyed 
by serum and part of the mecholyl potential by one per cent ground cat brain. Eserine and 
DFP preserve the potential of acetylcholine in the oil-cell in the same manner as in the nerve. 

One per cent DFP increases the specific conductance of guaiacol 100 per cent which explains 
part of its blocking action on nerve and muscle. 

The action-current in cholinergic nerve is probably a phase-boundary potential of acetyl- 
choline (sympathin is the electrogenic amine in adrenergic nerve). 

Two schools of thought in electrophysiological theory. R. BEUTNER AND T. C. 
BARNES. 

The older school, entrenched as the hypothesis of sieve membranes retaining negative but 
not positive ions, explains everything but solves no problems. The newer school omits hypotheses 
and proposes searches for electrogenic materials in tissues by setting up artificial battery systems 
composed of lipoid layers (oils) inserted between aqueous salt solutions. Some of these resemble 
analogous battery systems containing a tissue in place of the lipoid. One type of system studied 
i s : concentrated saline/tissue or lipoid (oil) /diluted saline +. Tissue, in such a set-up, may pro- 
duce the maximum e.m.f. of 58 millivolts if the concentrated solution is 1/10 mol. ; the diluted one, 
1/100 mol. Only few oils show such an effect, as e.g., fatty acid dissolved in a phenol-derivative, 
but not neutral fats, gelatin, etc. 

The production of bio-electricity does not depend on such aqueous salt concentrations but on 
metabolic processes in tissues, chiefly oxidation. A search for suitable electrogenic systems has 
led to the following one (Beutner, Loznerea, 1930) : saline/reduced substance e.g., a higher 
alcohol or lower fatty acid as in dying tissue/oxidized substance e.g., corresponding acid or cor- 
responding higher fatty acid as in respiring tissue saline +. For the action current one possible 
electrogenic substance is acetylcholine since even dilute solutions produce an e.m.f. in contact 
with oils in a system such as : + saline without addition/oil/saline with acetylcholine added 
1 : 100,000 to 1 : several million (Beutner and Barnes, 1941). 

'Tissue extract can be used in the place of the oil, also frog's nerve by the Netter technique. 
Adrenergic amines produce similar negative potentials but on different oils which are inactive in 
contact with choline esters. A difference in chemical composition may therefore be responsible 
for the specific function of cholinergic and adrenergic fibers. The rapid disappearance of the 



242 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 

negative potential, which occurs even in the absence of choline esterase, may be explained by a 
penetration of acetylcholine through a thin lipoid layer (membrane) creating a potential dif- 
ference in the opposite direction on the other side. Physico-chemical studies are not needed for 
the search for electrogenic substances, but when performed on oil cells, they show the existence 
of phase boundary potentials depending on electrolyte distribution ; the charged pore theory fails 
to explain the phenomena and is contradictory. 

The frequency of x-ray-induced chromated breaks in Tradescantia as modified by 
near infrared radiation. C. P. SWANSON AND ALEXANDER HOLLAENDER. 

The frequency of x-ray-induced chromatid breaks in Tradescantia can be significantly in- 
creased by treatment of the inflorescences with near infrared radiation. Pretreatment with near 
infrared radiation for seven hours prior to x-radiation inceased the frequency of single deletions, 
double (isochromatid) deletions, and translocations between and within chromosomes; post-treat- 
ment increased only single deletions and translocations. A delay of 21 hours between treatment 
with infrared and x-rays did not appreciably decrease the effectiveness of the infrared, suggesting 
that the changes within the cell induced by the infrared were of a relatively permanent nature. 
At the present time, the nature of the effect of infrared is not clearly understood. 



Vol. 91, No. 3 December, 1946 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 




A STRONGLY INTERSEXUAL FEMALE IX HABROBRACON 



P. W. WHITING 

University of Pcmisyk-ania, Philadelphia, and the Marine Biological Laboratory, Woods Hole 

In the parasitic wasp Habrobracon fuglandis (Ashmead), diploid males have 
never shown any tendency toward intersexuality ; they are as definitely male as 
their normal haploid brothers. When a "diploid male with female genitalia" was- 
found, it was therefore regarded with especial interest. The specimen, designated 
freak 994, developing from a heavily x-rayed ( 29,300 r) egg, occurred among the 
offspring of a treated wild type (stock 33) female crossed with an untreated lemon 
honey male (Experiment by A. R. Whiting. 1945). 

Freak 994 shows the heterozygous condition of the semidominant body color 
gene lemon inherited from its father. (Note light base of antennae in Figure 1.) 
The number of its antennal segments and its large ocelli are male characteristics. 
It was to be expected, therefore, that male reproductive reactions would occur. 
Several tests at different times failed to evince any response toward females although 
the specimen appeared healthy, drank honey water and lived for several days until 
fixed in Carnoy fluid. Since- it likewise failed to give any response (female) to 
caterpillars, its indifference was probably not due to its sex type but to some un- 
known factor. 

Because of the small "feminized" genitalia on the "male" body, freak 994 was 
at first recorded as a "diploid gynandroid male." ( 1\ nandroids, however, have 
always been haploids. They are mosaic males in which the two sexually different 
types of male tissue react in a complementary way to feminize the external genitalia 
(Whiting, Greb, and Speicher, 1934). Their mosaicism is shown by their asym- 
metry, not only in body color, in number of antennal segments, in mutant traits, 
and often in wing length, but especially in the external genitalia which are a mixture 
of normal male and feminized male structures with much reduplication and irregu- 
larity. In freak 994 there are no male genital structures and the female genitalia, 
consisting of a pair of sensory gonapophyses with no visible sting, are symmetrical 
and larger than in gynandroids. They are much smaller, however, than the female 
genitalia found in gynanders which are male-female mosaics with clearly separated 
male and female regions. That freak 994 is not a sex mosaic is shown by its 
symmetry in body coloration, in antennal flagella with nineteen segments in each 
and in length of wings and legs. 

Two types of intersexes have hitherto been reported in Habrobracon. (1) 
Gynoid, dependent upon a single mutant gene, is a weakly intersexual male, func- 
tioning normally as a male, but having certain external traits, including antennae, 
feminized. (2) Nine intersexual females were reported (Whiting. 1943) occurring 
in a single fraternity. "Superficially, these appear to be the reverse of the gynoid 

243 



244 



P. W. WHITING 



males, being more masculine anteriorly, feminine posteriorly." They resemble 
freak 994 in head and thorax and in the anterior part of the abdomen which are 
altogether like those of the male. In the posterior region, however, the sclerites are 
thickened, there is a normal sting and the sensory reproductive appendages are of 
full leneth characteristic of the female. "The nine intersexual females must be 




FIGURE 1 



regarded as UK ire strongly intersexual than gynoid males since antennae, ocelli and 
instincts are completely sex reversed." Freak 994 is an intersexual female, com- 
parable to these nine but still more strongly intersexual because of greater restric- 
tion of the "female" region and reduction of the genitalia. 

In Habrobracon, normal haploid males have cells almost as large as the corre- 
sponding cells of diploid females and in some stocks they are actually larger (Grosch, 



INTERSEX IN HABROBRAO )\ 245 

1945). Cells of diploicl males are much larger than are those of females or of 
haploicl males. These relationships have heen determined by counts of micro- 
chaetae within a given area on the upper surface of the wings, each microchaeta 
corresponding to a single cell. Study of the dispersion of microchaetae in freak 
994 showed its cell size to be within the range for the female or haploid male and 
therefore much smaller than that characteristic of the diploid male. The marked 
shift of the intersex in the male direction does not then affect the size of its cells. 
It may be fundamentally female, heterozygous for the sex factor. This condition 
perhaps prevents the abnormal expansion of cell size while permitting development 
of antennae and ocelli of normal male type. 

The nine intersexual females previously reported had internal abdominal struc- 
tures as in the female with normal poison sac and glands and seminal receptacle. 
Each ovary, however, appeared to be a pair of sacs of oogonia showing no differen- 
tiation of nurse cells and ova. Serial sections were made of the abdomen of freak 
994 and the internal structures were studied. The digestive tract is entirely normal 
with the crop greatly distended from honey water feeding. A poison apparatus is 
present but imperfectly developed and situated near the median plane, directly 
dorsal to the compound posterior nerve ganglion instead of being shifted laterad to 
the digestive tract. The poison glands are normal although of somewhat small size. 
Their ducts converge to a common duct connecting distally with an imperfect poison 
"sac" and proxiinally traversing the very short distance to the region where nor- 
mally lies the root of the sting. The poison ''sac," of approximately normal length, 
is reduced in diameter to an irregularly scleroti/.ed strand. It is surrounded In- 
longitudinal muscles as in a normal female. Nothing corresponding to a seminal 
receptacle could be located, nor were any gonads to br found. The tat body appears 
normal, surrounding the digestive tract and the poison apparatus dorsally and 
laterally. 

DISCUSSION 

In the report on the nine intersexual females, it was suggested that they might 
be accounted for by a dominant mutation in a sex allele changing .vb to xb m . The 
intersexes would then be modified females, .ni .vb" 1 . A similar hypothesis would 
cover freak 994. but here the mutation may have been x-ray induced and more 
potent than in the previous case so that the intersexuality would be more extreme 
with turning-point earlier in development. 

Failure to find gonads in freak 994 does not necessarily mean that they were 
lacking from the beginning for they may have begun development and then disin- 
tegrated. 

Comparison may be made between freak 994 and certain types of "deficient" 
individuals previously reported in Habrobracon (Whiting, 1926). Some of the 
"deficient" had external genitalia lacking but gonads present. Others had testes 
of reduced size, or present on one side, lacking on the other. Some of the "de- 
ficient" females with no trace of poison apparatus had well differentiated ovaries 
with eggs and nurse cells. This is just opposite to the condition found in the 
intersexual female, freak 994. There was no intersexuality among the "deficient." 



24f) P. W. WHITING 

SUMMARY 

An inter sexual female developed from a heavily x-rayed egg fertilized by an 
untreated sperm. The specimen is more strongly intersexual than a group of nine 
previously reported, for its external female genitalia are much reduced, its poison 
apparatus defective and its ovaries altogether lacking. Externally, it appears like 
a diploid male with small female genitalia. 

It is suggested that the x-radiation may have caused a change within a sex- 
differentiating allele, so that the heterozygote would develop into an intersex rather 
than a normal female. 

LITERATURE CITED 

GROSCH, D. S., 1945. The relation of cell size and organ size to mortality in Habrobracon. 

Growth, 9: 1-17. 
WHITING, P. W., 1926. Influence of age of mother on appearance of an hereditary variation 

in Habrobracon. Biol. Bull.. 51 : 371-385. 
WHITING, P. W., 1943. Tntersexual females and inUTst-xuality in Habrobracon. Biol. Bull,. 

85: 238-243. 
VYnnrxr,, P. W.. RAYMOND J. GREB AND B. I\. Sri K HKR, 1934. A nr\v type of sex-intergrade. 

Biol Bull.. 66: 152-165. 



LOCI OF ACTION OF DDT IN THE COCKROACH 
(PERIPLANETA AMERICANA) 

J. M. TOBIAS AND J. J. KOLLROS * 
University of CJiicago To.ricity Laboratory^ and the Department of Physiology 

In the cockroach. DDT produces symptoms which clearly reflect involvement 
of the neuromuscular apparatus. These are qualitatively much the same in all 
arthropods which have been studied, though there are important quantitative dif- 
ferences. Thus, in any given animal the time course of the poisoning is a function 
of dose, and for a dose of comparable toxicity in terms of final mortality, the symp- 
toms unfold and death occurs much more rapidly in some insects (the fly) than in 
others (the roach) (Tobias, Kollros, and Savit, 1946a). In the roach, the sequence 
of symptoms is initiated by hyperextension of the legs, elevation of the center of 
gravity and development of postural instability. The hyperextension then decreases 
and is superseded by increasing and generalized tremulousness, involving the head, 
body, and all appendages ; the gait becomes ataxic, and minor stimuli of sound or 
touch result in great hyperactivity, exhibited mainly in running and climbing. The 
animal falls on its back time after time until finally it can no longer right itself. Leg 
movements continue in the supine insect with two components, a high frequency 
intermittent tremulousness and a slower incoordinated flexion and extension. These 
two types of activity possibly reflect the double innervation which has been described 
for cockroach muscle (Pringle, 1939), one fiber type producing relatively slow tonic 
contractions, the other producing relatively fast twitches. It will be seen later that 
after poisoning these two types of movement can be independently altered. Activity 
finally diminishes progressively. The fast tremors disappear first and finally there 
remain only occasional isolated movements of body wall, tarsi, palpi, cerci, or an- 
tennae. When no further somatic movement can be detected, the heart usually 
continues to beat for some time, and electrical stimulation of the nerve cord may still 
evoke muscle responses. The animal may live in this condition for a day or so and 
finally die. 

Mammals exhibit a similar symptomatology up to a point. In the rat and dog, 
given DDT intravenously or orally, muscular fibrillations and excessive blinking are 
followed by tremulousness, ataxia, falling and gross convulsive seizures. The ani- 
mal may have a number of convulsions and die in the tonic phase of one or recover 
after gradual subsidence of symptoms. There is no period of prostration and nearly 
complete immobility as in the insect, because death occurs when systematic respira- 
tory movements cease. In the insect, the small amount of body movement and 
twitching sufficiently augment diffusion for respiratory exchange. Then too, the 
insect is far more resistant to anoxia than is the mammal (Wigglesworth. 1939). 

* Department of Zoology and The College. Present address. Zoology Department, Uni- 
versity of Iowa. 

t This work was carried out under contract with the Medical Division of the Chemical 
Warfare Service. 

247 



248 J. M. TOBIAS AND J. J. KOLLROS 

The frog, as might be expected, responds more like the insect than the mammal 
(Tobias, Kollros, and Savit, 1946b). Respiratory exchange through the skin can 
sustain life, and, after a period of hyperirritability, the animal lies prostrate and more 
or less immobile. Such symptomatology has prompted a number of investigations 
designed to discover a locus of action of DDT. As will be seen, there probably are 
a number of sites of action depending largely on dosage, but this point of view was 
only gradually attained. 

In mammals (Crescitelli and Oilman, 1946), DDT apparently does not act di- 
rectly on either muscle, myoneural junction or spinal cord. Since tremors persist 
after decerebration and mesencephalic transection, and since abnormal cerebral and 
cerebellar electrical activity persists after atlanto-occipital transection, neither cere- 
bral cortex nor basal ganglia can be a critical site of action, and intact spinal afferents 
are obviously not necessary lor the central effect. The cerebellum is considered, by 
these authors to be the most likely critical site of action in the mammal. Locus of 
action has also been investigated in insects. In Drosophila (Bodenstein, 1946). 
DDT seems not to act on muscle or myoneural junction, but does act on peripheral 
nerve and may act on the central nervous system. In the cockroach (Periplaneta 
americana}, DDT has been found to act on nerve in high concentrations (Yeager 
and Munson, 1945), and, in low concentrations, on peripheral receptors (probably 
proprioceptors) (Roeder and Weiant, 1946). The latter workers also have evi- 
dence which they interpret to mean that high concentrations may act directly on 
either the myoneural junction or muscle itself. In the crab (Cancer irroratus) there 
is evidence for action on motor nerves (Welsh, 1946). 

It was the purpose of this study to further investigate loci of action of DDT in 
an insect. Because of its large size and ready availability, the cockroach (Peri- 
planeta americana} was used throughout. 

METHODS 

Cockroaches were immobilized by exposure to 100 per cent CO 2 for 20-60 sec- 
onds or by etherization. After CO 2 , anesthesia seldom lasted over a minute. Once 
anesthetized, the roach was fastened to a bit of cardboard by pins passed through 
either side of the pronotum. Appendages were held in any desired position by 
pins crossed over the body. 

Decapitation was easily achieved by simply cutting the neck with a small scissors. 
The exposed stump was sealed with low melting-point paraffin. Ligation of the 
neck prior to decapitation to prevent loss of hemolymph did not prolong survival 
time. Such animals live about 60 hours (Table I). 

To expose a thoracic ganglion, the spinasternum just caudal to the ganglion 
was cut through, and the incision extended along the sides of the sternal plate. After 
the plate was reflected forward, removal of superficial fatty tissue and tracheal tubes 
fully exposed the ganglion. The connectives anterior to the ganglion were held in 
a jeweler's forceps and cut with iridectomy scissors. Traction on the connectives 
exposed the lateral nerves, which were sectioned. Finally, the posterior connectives 
were cut and the ganglion removed. Simple isolation of a ganglion from the rest 
of the nerve cord can be achieved without excising it by cutting the connectives 
through slits in the cuticle. Complete transection of the entire roach between sets 



LOCI OF ACTION OF DDT 



249 



TABLE J 

Effect of lesions of the central nervous system on symptoms of DDT poisoning in the cockroach 



Operation 


No. roaches 


Aver- 
age 
sur- 
vival, 

hours 


General results 


Legs which showed 
DDT 
effects 


Con- 
trols, 
no 
DDT 


Operated 


Nature 


Site 
(See Fig. 1 ) 


Before 
DDT 


After 
DDT 


Hyperactivity, tremors 
and convulsions 


Decapitation 


At A 


14 


14 


14 


61 

56 
56 


Rare tremors in 3 ani- 
mals no convulsive 
activity 
Typical DDT effects 
in all animals 
Typical DDT effects 
in all animals 


None 

All 
All 


Transaction of 
ventral nerve 
cord 


At C and D (both 
anterior and pos- 
terior to thoracic 
ganglion no. 2) 


16 


15 





104 
60 


None in any animals 

Typical DDT effects 
in all animals 


None 

All in 13 animals; 2nd 
and 3rd pairs in two 
animals 


Destruction of 
ganglion 


Th. 2 (thoracic 
ganglion no. 2) 


15 


IS 





77 
71 


None in any animals 

Typical DDT effects 
in all animals 


Leg 2 paralyzed in all 

1 and 3 in 13 animals. 
1, 2 and 3 in two ani- 
mals 



of legs results in an isolated segment containing a ganglion, nerves and the attached 
legs. Such a preparation, if kept moist, is viable for at least 6 to 8 hours. 

Excision of the heart largely prevents circulatory removal of substances applied 
to structures to elicit a local effect. Longitudinal incisions through the cuticle, on 
either side of the heart tube along its entire length, isolate a strip whose removal 
carries the heart with it. The heart may be cauterized with equal ease (Yeager and 
Munson, 1945). 

Methods for the administration of measured doses of DDT to insects have been 
described elsewhere (Tobias, Kollros, and Savit, 1946a). 

RESULTS 
Localisation experiments with uncontrolled DDT doses 

Except where otherwise specified, contact poisoning was carried out by confining 
the roach for 5-15 minutes within a glass cylinder coated with DDT previously pre- 
cipitated from acetone solution. 

Roaches decapitated before or after such contact with DDT behaved like intact 
poisoned animals (Table I). Therefore, neither the supra- nor the sub-oesophageal 
ganglia are essential for the development or maintenance of DDT-induced motor 
activity in the legs or body. Ventral nerve cord connectives were transected both 
anterior and posterior to the mesothoracic ganglion (Fig. 1, levels C and D). Ani- 
mals so prepared but given no DDT showed incoordination of the mesothoracic legs 
when walking, but there were no symptoms which could be confused with those 
of DDT poisoning. When such animals were subsequently poisoned, however, the 
mesothoracic as well as the other legs exhibited typical abnormal activity (Table I). 
After complete transection of the whole body of the poisoned roach, at both these 
levels (excised segment Fig. 1), leg tremulousness and hyperactivity continued un- 



250. 



J. M. TOBIAS AND J. J. KOLLROS 



abated in the isolated segment. The application of DDT emulsion or DDT in ace- 
tone to the cut surface of such segments obtained from normal roaches evoked typical 
DDT effects in the attached legs within a few minutes. The same was true of DDT 
applied directly to the exposed ganglion in the otherwise intact animal. Emulsion 
or acetone without DDT had no such effect. 

The cells of origin of the leg nerves lie within the lateral halves of the thoracic 
ganglia, each ganglion in the adult being formed by the midline fusion of two em- 
bryonic ganglion masses. Median sagittal section of the ganglion in a poisoned 
roach (Fig. 1. level F) did not stop hyperactivity in either of the legs innervated 




FIGURE 1. Levels of section in cockroach nervous system. 

from the resulting ganglion halves. Therefore, even half a segment contains all the 
structures necessary for the maintenance of DDT symptoms in a leg. 

If, however, the entire ganglion was removed the results were generally quite 
different. Mesothoracic ganglia were removed from thirty normal roaches. The 
corresponding legs of all were paralyzed and failed to respond to touch or pressure. 
Shortly after the operation, fifteen of the animals were contact poisoned. All 
showed typical DDT effects in the pro- and metathoracic legs, but the ganglionecto- 
mized mesothoracic legs remained entirely quiet in thirteen and showed only occa- 
sional tarsal twitching and some slight movement of the other joints in two. Simi- 
larly, ganglionectomy after the development of hyperactivity, rather than before 



LOCI OF ACTION OF DDT 



251 



TABLE II 

Experiments on isolated roach segments containing local ganglion, nerves, and legs 



No. of 
segments 



6 
3 

7 



4 



Material applied 



Nothing 

Emulsion* without DDT 

Acetone without DDT 



1 Per cent DDT emulsion* 
10 Per cent DDT in acetone 

DDT powder 



Route 



On cut surface 
Injected 



On ganglion 
Injected into vicinity 

of ganglion 
On ganglion 



Number of segments in which 

there was persistent DDT 

leg activity 



None 
None 
None 



Occurred in all 
Occurred in all 

Occurred questionably in 
one 



* Emulsion 1 per cent DDT, 10 per cent peanut oil, 1 per cent lecithin and 88 per cent 
0.90 percent NaCI (5). 

poisoning, either stopped or markedly reduced symptoms in the corresponding legs 
(Table IV). As was to be expected from these experiments, section of leg nerves 
lateral to the ganglion stopped or markedly reduced activity in many (65 per cent) 
of the legs (Table III). 

These experiments tentatively suggested that the ventral cord ganglion was 
critically involved in the motor action of DDT and might itself be a site of action. 
Conflicting data, however, were also obtained. It was possible, as also reported by 
others (Yeager and Munson, 1945; Roeder and Weiant. 1946). to produce motor 



TABLE III 

Visible effect of DDT on amputated legs 
(Dose not controlled) 



No. of 
legs 



30 



58 



12 
22 
35 



Source of legs 



Normal roaches 



DDT poisoned 
roaches tremulous 
and hyperactive 



Normal roaches 
Normal roaches 
Normal roaches 



Treatment after amputation 



Normal controls 



Emulsion without DDT in- 
jected into cut end 

Emulsion with 1 per cent 
DDT applied to cut end 

Emulsion with 1 per cent 
DDT injected into cut end 



Results after amputation 



No spontaneous movement 



Continued activity in 20. No 
movement in 38 



No movement in any 
Movement in 1, others all quiet 
Movement in 25, other 10 quiet 



9 
13 



Normal roaches 
Normal roaches 



Acetone without DDT in- 
jected into cut end 

Acetone with DDT injected 
into cut end 



Movement in 1, other 8 quiet 
Movement in 3, other 10 quiet 



252 



J. M. TOBIAS AND J. J. KOLLROS 



activity in a large percentage of amputated legs by the injection of DDT emulsion 
(1 per cent DDT, 1 per cent lecithin, 10 per cent peanut oil, and 88 per cent 0.9 
per cent NaC! solution). It will also he recalled that ganglionectomy failed to en- 
tirely quiet the legs in two of fifteen experiments (Table T). 

Such conflicting data were difficult to interpret. Ganglionectomy or denerva- 
tion usually stopped leg activity, but this was not invariably the case, and it was 
possible to produce activity in the amputated legs by injection of DDT. It was 
suspected that such results might be resolved in terms of DDT dose. Further 
experiments were then done with measured doses of DDT. 

Localization experiments with controlled doses of DDT 

It was immediately found that the effectiveness of ganglionectomy in abolishing 
motor effects was inversely related to dose (Table IV). That is, as the dose of 
DDT was increased ganglionectomy stopped movement in progressively fewer cases. 

TABLI-: IV 

Effect of ganglionectomy on symptoms after various doses of DDT 







Results of ganglionectomy 


No. 






experi- 


DDT* 






ments 




Number resulting in complete 


Number resulting in a 






cessation of activity 


reduction of activity 


10 


Usual moderate contact dose (5-10 mins. 


70% 


30% 




in DDT coated tube) 






5 


Excessive contact dose (approximately 


20% 


80% 




2 hours in DDT coated tube) 






25 


5-30 mg. DDT per kg. injected intra- 


68% 


32% 




abdominally in emulsion*" 






12 


60-70 mg. DDT per kg. injected intra- 


50% 


50% 




abdominally in emulsion** 






15 


130 mg. DDT per kg. injected intra- 


7% 


93% 




abdominally in emulsion** 







* LD-50 for DDT injected intra-abdominally in emulsion is 20 mg. per kg. (Tobias, Kollros, 
and Savit, 1946a). 

** Emulsion 1 per cent DDT, 1 per cent lecithin, 10 per cent peanut oil, 88 per cent of 
0.9 per cent NaCl. 

In all cases, however, even when movements were not entirely stopped they were 
both qualitatively and quantitatively changed. The high frequency tremulousness 
was always markedly reduced or entirely abolished and the slower movements 
were much diminished. 

Nicotine, in low concentrations, is known to block synaptic transmission cen- 
trally as well as peripherally (Libet and Gerard, 1938; Pringle, 1939) but not 
axonal transmission. When applied to the cockroach ganglion there is an initial 
burst of electrical hyperactivity (100-800 impulses per sec.) followed by electrical 
silence (Pringle, 1939). As would be expected, such application of nicotine to a 
ganglion also produces great motor hyperactivity in the attached leg which can be 
abolished by amputating the leg (Yeager and Munson, 1945). 

Now then, if nicotine applied to a ganglion in a concentration which did not 



LOCI OF ACTION OF DDT 



253 



affect peripheral nerve were to stop DDT symptoms, this would be added evidence 
for the importance of the ganglionic cell bodies or synapses in the development and 
maintenance of such symptoms. After poisoned roaches became hyperactive the 
heart was excised. This did not decrease activity, but served to greatly diminish 
circulatory transport of solutions applied for local effects. Solutions were then 
applied as small droplets to the ganglion or a region of leg nerve exposed by cuticle 
excision. 

Dilute nicotine solutions (0.01 per cent in insect Ringer) applied to the leg 
nerves of the normal or poisoned roach did not paralyze the leg. Typical DDT 
induced activity could not be stopped in this fashion. This was almost surely not 
due to failure of nicotine to reach the nerve since spontaneous movement as well 
as that following electrical stimulation of the ganglion could be stopped by a similar 

TABLE V 

Effect of locally* applied nicotine and novocaine on motor symptoms of 
DDT poisoning after various doses of DDT 



No. 
experi- 
ments 


DDT** 


Number of experiments in which activity was modified 


1.0% novocaine 


0.01% nicotine 


Injected into tibia 


Injected into tibia 


Applied to ganglion 


Complete inactivity 


Complete inactivity 
or reduced activity 


Activity 
stopped 


Activity 
reduced 


4 


10-30 mg. per kg. applied 
to body surface 18 hours 
before 


100% 


o% 


100% 


o% 


9 


100 mg. per kg. applied to 
body surface 18 hours be- 
fore 


100% 


0% 


77% 


22% 


13 

4 


500 mg. per kg. applied to 
body surface 
1000 mg. per kg. applied to 
body surface 






23% 
0% 


77% 
100% 



* All experiments on cardiectomized roaches to prevent circulatory removal of substances 
applied for local effect. 

** DDT applied to surface in acetone. LD-50 for DDT so applied is 10 mg. per kg. (Tobias, 
Kollros, and Savit, 1946a). 

administration of 1 per cent novocaine. When, however, this nicotine solution was 
applied to a ganglion (in the same normal or poisoned animal in which it was in- 
effective on peripheral nerve) there was a short-lived burst of great activity in the 
legs of the segment, followed by complete immobility or markedly decreased activity. 
Since the nicotine was effective in concentrations which did not block peripheral 
nerve, it was concluded that it was acting by blocking ganglionic synapses and not 
by spill-over to the emerging nerve roots. It is clear (Table V) that, as in the 
case of ganglionectomy, the immobilizing effect of nicotine decreased as the dose 
of DDT increased, and, as was also true after ganglionectomy, if nicotine did not 
stop activity it considerably decreased and modified it. 



254 J. M. TOBIAS AND J. J. KOLLROS 

DISCUSSION 

It is clear that DDT can produce motor symptoms l>y effects peripheral to the 
ganglion. It is equally clear, however, that the ganglion plays a role in the 
initiation and maintenance of symptoms and that this role is to some extent 
dependent upon DDT dose. 

Roecler and Weiant (1946) found that, in the cockroach, very low concentra- 
tions of DDT can initiate centripetally directed, high frequency (300-400 per sec.), 
temporally irregular bursts of nerve impulses, presumably excited by action of 
DDT on the campaniform sensilla (presumptive proprioceptors). There was no 
evidence of any muscle movement which might have initiated such centrally directed 
impulses. Welsh (1946) has demonstrated that DDT in very low concentrations 
can also favor repetitive response of motor fibers (Cancer irroratus] to a stimulus 
normally evoking single responses, and Yeager and M tin son (1945) have concluded 
that high concentrations can produce similar changes in the cockroach. 

The results of ganglionectomy, in the cockroach (surgical or nicotine inacti- 
vated), after various doses of DDT, support the view that the initiation and con- 
tinuation of the hypermotor symptoms of DDT poisoning after low doses of DDT 
require an intact sensori-motor reflex arc, and that random afferent impulses in 
sensory nerves may indeed, as suggested by Roeder and Weiant, excite motor 
neurones in the ganglion to initiate incoordinated muscular activity. From the 
experiments here reported, this would appear to be a part of the common sequence 
of changes in the roach poisoned by uncontrolled contact doses. The fact that 
ganglionectomy becomes less and less effective as the dose of DDT is progressively 
increased would suggest that larger amounts of DDT may act directly on motor 
nerves. Obviously, these data do not rule out a possible direct action on muscle. 
Within the dose ranges which have been used there is, however, no conclusive evi- 
dence for a direct action on muscle. This is not to say that such action could not 
occur at some sufficient dosage level. In addition, ganglionectomy is seen to have 
stopped the rapid tremulousness after any dose which was tried, suggesting that 
the high frequency movements may be initiated reflexly rather than by direct action 
on motor fibers at large as well as at low doses of DDT. This general picture is 
compatible with the subsidence of high frequency tremulousness before subsidence 
of slower muscular activity. If the former is reflex and the latter due to direct 
nerve action one might expect this order of dropping out on the basis of much 
greater fatigability for the reflex arc than for the nerve trunk. 

Pattern development of symptoms 

It has been claimed (Laiiger, Martin, and Miiller, 1944) that if DDT be put 
on one leg of a fly the development and progression of symptoms follow a definite, 
orderly and reproducible path from leg to leg. Such a phenomenon might be very 
important indeed for an understanding of the mechanism of DDT action. The 
authors have not been able to confirm this finding. 

CONCLUSIONS 

1. Neither decapitation, section of one or several nerve cord connectives nor 
complete transection of the entire insect body at one or several levels between nerve 
cord ganglia prevents the development of the typical motor effects of DDT in any 



LOCI OF ACTION OF DDT 255 

of the legs of the cockroach. After combined antero-posterior isolation of a nerve 
cord ganglion, even median sagittal section of the ganglion does not prevent motor 
symptoms in the legs still attached to the lateral ganglionic cell masses. There- 
fore, the anatomical elements necessary for development of the motor symptoms of 
DDT are contained within the lateral half of a body segment which contains the 
lateral half of a ganglion, leg nerves and peripheral structures. 

2. Since the motor symptoms of DDT poisoning can occur in amputated legs, 
in legs whose nerves have been cut, and in legs whose segmental ganglia have been 
destroyed, it is possible for DDT to produce its motor effects by action on some 
structure or structures peripheral to the segmental ganglion. 

3. The motor symptoms of DDT poisoning can be stopped or diminished in a 
leg by ganglionectomy, leg nerve section, or ganglion synaptic block with nicotine. 
The effectiveness of these procedures is in inverse relation to the dose of DDT 
administered. These findings suggest that, in the cockroach, low doses of DDT 
may excite motor fibers reflexly by impulses fired into the ganglion over afferent 
nerve fibers, whereas high doses may act on elements on the motor side of the 
ganglion and thus not require an intact reflex arc. Since ganglionectomy stops the 
fast component of the hypermotor activity, however, equally well after large or 
small doses of DDT, this component may be reflexly initiated and maintained 
after all doses of DDT. 

LITERATURE CITED 

BODENSTEIN, D., 1946. Investigation on the locus of action of DDT in flies (Drosophila). 

Biol. Bull, 90: 148. 
CRESCITELLI, F. AND A. OILMAN, 1946. Electrical manifestations of the cerebellum and cerebral 

cortex following DDT administration in cats and monkeys. Amer. Jour. Physiol., 

147: 127. 
LAUGER, P., H. MARTIN AND P. MULLER, 1944. The constitution and toxic effect of botanicals 

and new synthetic insecticides. Hclv. Chim. Acta, 27. 
LIBET, B. AND R. W. GERARD, 1938. Automaticity of central neurones after nicotine block of 

synapses. Proc. Soc. Exp. Biol. and Mcd., 38 : 886. 

PRINGLE, J. W. A., 1939. The motor mechanism of the insect leg. Jour. Exp. Biol., 16 : 220. 
ROEDER, K. D. AND E. A. WEIANT, 1946. The site of action of DDT in the cockroach. Sci- 
ence, 103 : 304. 
SAVIT, J., J. J. KOLLROS AND J. M. TOBIAS, 1946. The measured dose of gamma hexachloro- 

cyclohexane (y 666) required to kill flies and cockroaches and a comparison with DDT. 

Proc. Soc. Exp. Biol. and Mcd., 62 : 44. 
TOBIAS, J. M., J. J. KOLLROS AND J. SAVIT, 1946a. Relation of absorbability to the comparative 

toxicity of DDT for insects and mammals. Jour. Pharm. and Exp. Ther., 86 : 287. 
TOBIAS, J. M., J. J. KOLLROS AND J. SAVIT, 1946b. Acetylcholine and related substances in 

the cockroach, fly and crayfish and the effect of DDT. Jour. Cell. Comp. Physiol. 

(In press.) 

WELSH, J. H., 1946. Personal communication. 
WIGGLESWORTH, V. B., 1939. The principles of insect physiology. Dutton and Co., Inc.. New 

York. 
YEAGER, J. F. AND S. C. MUNSON, 1945. Physiological evidence of a site of action of DDT 

in an insect. Science, 102 : 305. 



TILLINA MAGNA: MICRONUCLEAR NUMBER, ENCYSTMENT 

AND VITALITY IN DIVERSE CLONES; CAPABILITIES 

OF AMICRONUCLEATE RACES 

C. D. BEERS 

Department of Zoology, University oj North Carolina, Chapel Hill 

It is well established that the number of micronuclei in Tillina magna is highly 
variable. For example, Gregory (1909) found 6-10, and Ilowaisky (1921), in a 
ciliate which he called Pseudocolpoda cochlearis cicnkoivskii, reported 2-6. An ex- 
amination of Ilowaisky's text and figures shows conclusively that his ciliate was T. 
magna Gruber (1879) as Kahl (1931, p. 282) pointed out. Kahl apparently re- 
garded six as the typical number. Bresslau (1922) observed the nuclei in sufficient 
detail to note the extrusion of macronuclear material at division and the presence of 
several micronuclei, though he reported no counts of their actual number. The 
writer (1946), in a study dealing chiefly with the history of the nuclei during divi- 
sion and encystment, counted the number of micronuclei in 100 individuals (50 active 
and 50 encysted) of each of three clones, and found that it varied from 6 to 11 in one 
clone and from 4 to 6 in the other two. Thus the number was found to vary in dif- 
ferent individuals of the same clone, and the mean number was found to vary in dif- 
ferent clones. Active specimens and resting cysts of any particular clone had on the 
average like numbers of micronuclei. Contrary to statements in the literature, it 
was shown that the micronuclei divide at the time of cell division, and not indiscrimi- 
nately or without regard to cell division. The mechanism by which two daughter 
cells may receive unlike numbers of micronuclei at division, thus accounting for vari- 
ations in number within a clone, was described. 

The significance of the wide variation in micronuclear number is unexplained. 
Structurally and physiologically an individual having only 4 micronuclei does not 
appear to be fundamentally unlike one having 11 micronuclei. The same condition 
prevails in the closely related species, T. canalijcra, which I was formerly disposed 
to regard (1945) as identical with T. magna. However, on the basis of informa- 
tion furnished me by Dr. George W. Kidder, of Amherst College, it appears that T. 
canalifera merits recognition as a valid species, chiefly because of the very conspicu- 
ous nature of its canal system. In T. canalifera, Turner (1937) reported 414 
micronuclei, though Burt, Kidder, and Gaff (1941), in specimens obtained from the 
late Dr. Turner, found only one. Hence, it is clear that the micronuclear number 
may vary from 1 to 14, yet the evidence indicates that the uninucleate and multi- 
nucleate races were equally cultivable, vigorous, and capable of producing normal 
resting cysts. 

The present study of T. magna was undertaken in order to obtain additional in- 
formation concerning two points : ( 1 ) the normal variation in micronuclear number 
in various natural races and (2) the significance of such variation. The investiga- 
tion of the first point is readily feasible, in that the micronuclei may be counted with 
absolute certainty in Feulgen preparations of favorably oriented resting cysts or 

256 



MICRONUCLEI OF TILL1NA MAGNA 257 

medium-sized trophic specimens. The investigation of the second point, though less 
suited to direct approach, is not impracticable. A number of questions arise, some 
of which submit to experimental analysis. For example, is the number of micro- 
nuclei related in any way to size, whether of trophic specimens, division cysts or 
resting cysts ; to division rate ; to vitality, meaning capacity to endure with undimin- 
ished vigor as generations pass ; to ability to produce resting cysts ; to the viability 
of such cysts ; or to ability to excyst ? Of the foregoing measurable characters, the 
following were selected as being most readily amenable to experimental investiga- 
tion : ability to produce resting cysts, size and viability of such cysts, capacity to 
excyst, division rate and vitality. These then, will be considered in relation to 
micronuclear number, though not all of them will receive equal consideration. The 
study assumed unlooked-for interest when it became evident that three of the races 
were amicronucleate. Thus a comparison of the potentialities of micronucleate and 
amicronucleate clones became possibile. 

MATERIALS AND METHODS 

Twenty clones of T. inagna, to be designated numerically, were used in the study. 
The progenitors of these respective clones were collected in a meadow, known locally 
as Sparrow's Pasture, in the vicinity of Chapel Hill, North Carolina. Comparisons 
with clones from other sources were desirable, but unfortunately attempts to collect 
Tillina elsewhere in the Chapel Hill region, and in the vicinity of Stanford Univer- 
sity, California, and Woods Hole, Massachusetts, were unavailing. In this study 
a clone refers to all the progeny which were derived asexually from a single resting 
cyst or trophic specimen. The intervention of encystment and subsequent excyst- 
ment is not considered to be a valid reason for changing the clonal designation, since 
there is no evidence that encystment in Tillina involves a sexual process which 
might change the genetic constitution of the clone. (It should perhaps be recalled 
that Tillina, like its near relative Colpoda, reproduces within a thin-walled tempo- 
rary cyst, from which usually four progeny emerge shortly as a result of two succes- 
sive divisions. The term encystment, as used in this study, does not refer to these 
temporary division cysts, but to the protective or resting cysts.) It is not defi- 
nitely established that all of the twenty clones were genetically different, since their 
histories prior to their period of laboratory life were unknown. 

The progenitors of clones 1, 2, 6, 8, 9, 11, 12, 13, 15, 17, 18, and 19 were taken 
as active specimens on Sept. 10, 1945, and these clones were therefore cultured 
simultaneously in the early part of the study. Eight of the foregoing clones, namely, 
6, 11, 13, 18, 15, 19, 1, and 17, have already been reported on briefly under the 
numerical designations 1 to 8, respectively (Beers, 1946a). In the present paper 
my original numerical designations of all clones have been changed for the con- 
venience of the reader in using the accompanying tables. The progenitors of clones 
3, 14, 16, and 20 were isolated on Feb. 4, 1946, when dried leaves and debris, after 
8 months of storage at 19 C., were immersed in weak hay infusion; these clones 
were therefore cultured simultaneously. It is evident that they were derived from 
dried cysts. The progenitors of clones 4, 5, 7, and 10 were isolated on April 8, 
1946, when moist leaves and debris, which had recently washed against the bases of 
willow saplings in the meadow, were immersed in hay infusion; these clones were 



258 C. D. BEERS 

maintained in culture simultaneously toward the end of the study. They were un- 
doubtedly derived from wet cysts. 

An attempt was made to maintain each of the clones in pure-line culture for a 
period of 60 days. Sixteen of the clones were readily cultivable and continued with 
undiminished vigor throughout the period ; four were intractable in that their divi- 
sion rates declined and the lines encysted well before the end of the period. Thus 
the laboratory histories of the clones varied, although the conditions of culture -were 
uniform. The details, in relation to micronuclear number, will follow. 

Each clone was cultured in depression slides in the form of four sub-lines. 
These were maintained at 23 C. in 0.05 per cent lettuce infusion to which suitable 
quantities of Psciidomonas fluorescent, grown on nutrient agar, were added as food. 
Previous experience has shown that this general procedure, combined with daily 
isolations and transfers to fresh environments, meets adequately the cultural needs 
of Tillina (Beers, 1944, 1945). Records were made daily of fission rates and other 
points of interest. 

Surplus animals from the lines were stained on cover glasses by the Feulgen 
method in order to make micronuclear counts of active specimens. Small stock 
cultures of each clone furnished precystic specimens when the food supply neared 
depletion. These specimens were removed and allowed to encyst on cover glasses 
in the manner described by Beers (1946). Thus convenient preparations were 
available, first for making measurements of living cysts, and then for Feulgen stain- 
ing. All measurements and micronuclear counts of cysts were made on single 
resting cysts. These are the common type. They are practically spherical and 
therefore well suited for making accurate measurements. 

NORMAL VARIATION IN NUMBER OF MICRONUCLEI 

The data bearing on diversity in micronuclear number in the twenty clones are 
summarized in Table I, in which the clones are arranged and numbered in the order 
of decreasing mean numbers of micronuclei. The data, ignoring for the present 
the mean diameters of resting cysts, are largely self-explanatory. A few points 
deserve special mention. 

In any particular clone both active specimens and resting cysts showed practi- 
cally the same extremes of variation (range) in micronuclear number and had essen- 
tially the same mean number of micronuclei. 

In different clones the mean numbers of micronuclei were extremely variable. 
Some clones (e.g., 1 and 2) had consistently high mean numbers; others (e.g., 16 
and 17), consistently low numbers, with many intergrades between these extremes. 

Clones 18, 19, and 20 were amicronucleate. This statement is not based on 
casual observation, but on an intensive study of these clones. In trophic specimens 
and resting cysts the micronuclei of Tillina are not disposed toward secretiveness. 
They are never imbedded in the macronucleus. Each has an endosome which stains 
intensely and conspicuously by the Feulgen method. In mature resting cysts only 
the rod-shaped or ellipsoid macronucleus and the micronuclei stain to any appre- 
ciable extent; there is nothing in the cytoplasm to conceal the micronuclei. In 
trophic specimens it is true that the food vacuoles also stain, but the micronuclei 
always lie in the clear peri-macronuclear space and are not in a position to be con- 
cealed by the vacuoles. Moreover, considerable numbers of individuals of clones 



MICRONUCLEI OF TILLINA MAGNA 



259 



18, 19, and 20 were stained. These included not only the usual resting cysts and 
medium-sized trophic specimens, but also young cysts, cysts in the process of excyst- 
ment, and individuals just excysted. None showed a micronucleus, whereas indi- 
viduals of the remaining seventeen clones, stained at the same time by the same 
method, invariably showed micronuclei. 

The individuals of some clones (e.g., 1, 3, 5, 12) showed great diversity in 
micronuclear number within the clone. This fact is brought out clearly by the 
range which is cited for these clones, and it is further emphasized by the high 
standard deviations in the clones. Clone 12 showed the greatest degree of hetero- 
geneity in that the range in micronuclear number extended from 2 to 11, with all 
intervening numbers being represented. On the other hand, some clones (e.g., 
2, 4, 8; 13, 15, 16, 17) were relatively homogeneous, with narrow ranges and low 
standard deviations. Other clones lay between these extremes. Only the amicro- 
nucleate clones showed complete homogeneity. 

Thus, it is seen that individuals of a clone exhibit varying numbers of micro- 
nuclei, that clones differ with respect to their mean number, and that amicronncleate 
clones exist in nature. 

The mean number of micronuclei in the 850 micronucleate active specimens of 
Table I (representing 17 clones) was 7.06; the mean number in the 850 micro- 
nucleate cysts was 7.08. Unfortunately, the number in the progenitor of each clone 

TABLE 1 

Tillina magna. Variation in number of micronuclei in twenty clones; relation of micronuclear 
number to size of cysts. The clones are numbered and arranged as the mean number of micronuclei 
(average of means for fifty active specimens and fifty resting cysts} decreases. 



Numerical 
designation 
of clone 


Range in number of micronuclei 


Mean number of micronuclei 
standard deviation 


Mean diameter of 
50 resting cysts 
in microns 
standard 
deviation 


50 active 
specimens 


50 resting 
cysts 


50 active 
specimens 


50 resting 
cysts 


1 


10-16 


9-16 


12.901.87 


12.321.93 


85.76 9.65 


2 


8-12 


9-12 


10.480.94 


10.960.87 


82.94 7.65 


3 


7-14 


6-13 


9.622.30 


9.242.08 


79.68 8.72 


4 


7-10 


7-10 


8.52 0.82 


8.600.95 


88.62 7.25 


5 


6-14 


6-12 


8.171.95 


8.281.66 


88.36 9.61 


6 


6-10 


6-10 


7.561.28 


7.781.24 


93.60 6.21 


7 


5-10 


5-10 


7.251.28 


7.381.36 


92.44 7.08 


8 


6- 9 


6- 9 


7.160.85 


7.060.92 


78.50 7.82 


9 


5- 9 


4- 9 


6.521.15 


6.741.21 


84.16 8.62 


10 


4- 8 


4- 8 


5.981.52 


5.941.46 


85.42 5.28 


11 


5- 8 


5- 9 


5.901.03 


5.760.96 


86.14 5.34 


12 


2-10 


2-11 


5.742.41 


5.901.97 


85.64 5.32 


13 


4- 6 


4- 6 


5.120.42 


5.100.45 


80.64 8.92 


14 


4- 8 


4- 8 


5.121.51 


4.981.42 


84.18 4.76 


15 


4- 6 


4- 6 


4.860.53 


5.200.57 


81.60 8.41 


16 


4- 6 


4- 6 


4.92 0.63 


4.980.75 


80.92 7.37 


17 


3- 5 


3- 5 


4.280.75 


4.140.74 


91.42 6.87 ' 


18 














86.28 5.63 


19 














84.32 10.36 


20 














81.52 5.34 



260 C. D. BEERS 

is unknown, since the micronuclei cannot be identified in living specimens. It 
seems reasonable to assume that the progenitor of each had a number approximately 
equivalent to the mean which was determined for the clone, and that it produced 
some offspring having fewer, and some having more, than its own number. 

It is well known that the number of micronuclei is variable in many species of 
ciliates. Thus, Paramecium multimicronucleatwn has 2 to 7 (Powers and Mitchell, 
1910), though usually 4 (Wenrich, 1928) ; Spathidium spathula, 6 to 9 (Maupas, 
1888, p. 247) ; Urostyla grandis, 10 to more than 40 (Tittler, 1935) ; and S tent or 
coeruleus, 10 to 42 within a single clone (Schwartz, 1935). On the whole, such 
variations within a species appear to have little effect on the structure or behavior 
of the individuals and to be without functional significance. This general conclu- 
sion is supported by the observations on T. umgiia which follow immediately. 

NUMBER OF MICRONUCLEI IN RELATION TO VARIOUS ASPECTS OF CYSTMENT 

All the clones produced normal resting cysts upon the depletion of the food 
supply in small stock cultures prepared with surplus animals from the lines. Fur- 
thermore, all the specimens in such cultures encysted ; none persisted in prolonged 
swimming, thereby to perish of starvation. Hence, it is clear that the ability to 
encyst is not dependent on the presence of the micronucleus, since amicronucieate 
as well as micronucleate clones were able to encyst. Moreover, the cysts of all 
the clones remained viable for many months. They could be activated at any time 
after the fourth day by immersion in distilled water or 0.05 per cent lettuce infusion. 
From 2 to 2.5 hours were required for emergence at 23 C., and practically 100 per 
cent of the specimens excysted. No precise figures are given here, since the per- 
centage of excystment under various conditions has been dealt with in a previous 
paper (Beers, 1945) and the present study contributes nothing new on this point. 
'However, the present results show clearly that the viability of resting cysts and 
their capacity to excyst are in no wise related to the presence of a micronucleus, or 
to the number of micronuclei. Well over 90 per cent of the cysts produced in the 
various clones were single ones ; double cysts appeared sporadically, some in ami- 
cronucieate clones, some in micronucleate. Amicronucieate cysts undergo the usual 
colpodid type of macronuclear reorganization, involving the extrusion of a portion 
of the macronuclear substance into the cytoplasm (Taylor and Garnjobst, 1941 ; 
Burt, Kidder and Claff, 1941; Beers, 1946). 

The size of the cysts in different clones was made the subject of special study, 
for it was thought that the number of micronuclei might affect the size of the cysts. 
The diameters of fifty living single cysts of each clone were measured, each measure- 
ment extending from the outer surface of the ectocyst of one side to the correspond- 
ing surface of the other. The results of these measurements are included in Table 
I. An inspection of the table shows at once that the calculation of coefficients of 
correlation between micronuclear number and cyst size would be of little value, 
since cyst size is independent of micronuclear number. For example, if we con- 
sider certain extremes in micronuclear number, it is seen that the cysts of clone 1 
had a mean diameter of about 85 /A, and those of clone 19 a diameter of 84 p, with 
approximately equivalent standard deviations. Clones 2 and 20 and clones 4 and 
18 constitute other examples of extreme disparity in micronuclear number with 
close agreement in cyst size. Among the micronucleate clones, other examples of 



MICRONUCLEI OF TILLINA MAGNA 261 

wide divergence in micronuclear number, yet with general uniformity in cyst size, 
are furnished by clones 3 and 16 and by clones 6 and 17. On the other hand, some 
clones having widely divergent micronuclear numbers produced cysts of dissimilar 
sizes, e.g., clones 3 and 17 and clones 6 and 13. Thus it is seen that clones having 
widely different micronuclear numbers may produce cysts of equivalent mean sizes 
or of dissimilar mean sizes. It must be concluded that there is no relation between 
the number of micronuclei and the size of the cysts. 

The same conclusion is reached if we adopt another approach and consider cyst 
size in clones which had similar, or only slightly different, micronuclear numbers. 
For example, clones 6 and 7 had similar mean numbers of micronuclei and they 
produced cysts of equivalent mean sizes; clones 15 and 16 constitute a second ex- 
ample. On the other hand, clones 7 and 8 had similar micronuclear numbers, but 
they produced cysts which differed significantly in mean size; clones 16 and 17 
furnish another example. Hence, clones having similar mean micronuclear num- 
bers may produce cysts of equivalent mean sizes or of different mean sizes. Again, 
it is evident that there is no relation between number of micronuclei and size of 
cysts. The mean diameter of the 850 micronucleate cysts of Table I was 85.23 p.; 
that of the 150 amicronucleate cysts was 84.04 p.. 

A word concerning the size of individual cysts may be of interest. In any 
particular clone of T. mayna, whether micronucleate or amicronucleate, there is 
usually wide variation in cyst size, even though the cysts under immediate consid- 
eration are all produced in the same small stock culture meaning a Columbia 
culture dish containing 1 cc. of fluid. Cysts in such a culture often vary in size 
from 75 ^ to 95 p. ; extremes of 64 /*, and 104 /A have been noted. The factors which 
affect cyst size appear to be of a complex physiological nature and are therefore not 
readily identifiable. 

The point of greatest interest in this consideration of various aspects of cystment 
is the fact that amicronucleate and micronucleate clones behaved alike ; clearly the 
micronucleus of T. magna plays a negligible role, if any, with reference to encyst- 
ment, viability of cysts, excystment, macronuclear reorganization within the cysts, 
and cyst size. 

NUMBER OF MICRONUCLEI IN RELATION TO DIVISION RATE AND VITALITY 

The cultural histories of the twenty clones are presented in Table II. Clones 
1, 2, 3, and 5 could not be maintained in culture for the arbitrary period of 60 days. 
The remaining clones were maintained with undiminished vigor and were discon- 
tinued at the end of the period. 

First, the division rates of the sixteen vigorous clones will be considered in 
relation to micronuclear number. Clones 4, 6, 7, and 8, as has been seen, had rela- 
tively high micronuclear numbers (means, about 7 to 8.5). The total average 
number of generations produced by the four sub-lines of these respective clones 
varied from 149 to 174. Thus the clones themselves varied with respect to mean 
division rate. Clones 9-12 had intermediate micronuclear numbers (means, 6 to 
6.5) ; these clones produced from 149 to 167 generations. Clones 13-17 had low 
micronuclear numbers (means, 4 to 5) ; they produced from 160 to 176 generations. 
Thus clones having intermediate or low micronuclear numbers produced in general 
as many generations, and had therefore the same division rates, as clones having 
relatively high micronuclear numbers. 



262 C. D. BEERS 

Amicronucleate clones 18-20 produced from 154 to 172 generations. Thus the 
amicronucleate clones had approximately the same division rates as the micronu- 
cleate clones; e.g., clone 19 produced almost as many generations as clone 4; clone 
18 produced more than clone 10 ; clone 20 produced about as many as clone 9 or 
13. Many kinds of comparisons are possible, and the reader may choose to make 
other comparisons between amicronucleate and micronucleate clones. For example, 
clones 18-20 had higher division rates (i.e., produced more generations) than clones 
8, 10, and 14; but clones 1820 had lower division rates than clones 4, 7, and 16. 
Thus the general conclusion that amicronucleate clones have the same division rates 
as micronucleate clones is not invalidated. Sections of amicronucleate division cysts 
showed that the macronucleus undergoes the usual reorganization after each of its 
divisions, as in normal micronucleate cysts (Burt, Kidder and Gaff, 1941; Beers,, 
1946). 

Next, the vitality of the sixteen vigorous clones must be considered. An ex- 
amination of the number of divisions produced in the successive 5-day periods in 
any particular clone shows that the clone was dividing as rapidly at the end of the 
experiment as at the beginning. Within the time limits of the experiment, the 
clones showed no decrease in vitality as measured by the division rate. How long 
the sixteen clones would have continued without diminution in vitality is a question 
that cannot be answered on the basis of the available data. The important findings 
are these : Some clones which have relatively high micronuclear numbers are as 
vigorous as those which have low numbers ; amicronucleate clones are fully as 
vigorous as many micronucleate clones. 

Clones 1, 2, 3, and 5 must receive special consideration. As has been said, these 
clones could not be maintained in culture for the duration of the 60-day experi- 
mental period. Clone 1 showed a rapid decrease in fission rate and encysted on 
the fourth clay. Some of the cysts were activated and new lines were established. 
These in turn declined shortly and encysted. Three additional attempts were made 
to culture clone 1 ; each time the lines encysted after 3-5 days. Indeed, clone 1 
was so refractory that without these repetitions it would have been impossible to 
obtain sufficient specimens for the usual number of micronuclear counts. Clones 
2, 3, and 5 likewise could not be maintained in culture for 60 days. Their histories 
are presented in sufficient detail in Table II. Following the encystment of the 
original lines of these clones, new lines were established with excysted specimens, 
but they also declined and encysted after 3-4 weeks of culture. It may be main- 
tained that the decline and encystment of these four clones resulted from a failure 
to meet their cultural needs. However, the conditions of culture were adequate for 
a total of sixteen clones, and it seems not unreasonable to assume that they were 
likewise adequate for clones 1, 2, 3, and 5 and to conclude that these clones declined 
as a result of intrinsic factors. 

It has been shown that clones 1, 2, and 3 had higher micronuclear numbers than 
any of the other clones. Clone 5 likewise had a high number, though slightly lower 
than clone 4, which was cultivable. The evidence indicates that a large number of 
micronuclei may be detrimental to the welfare of the organism and incompatible 
with high vitality, but the number of such clones studied was too small to justify 
a general conclusion. On the whole, the results show that in T. inagna the rate 
of division and the vitalitv of the race are in no wise related to the number of micro- 



MICRONUCLEI OF TILLINA MAGNA 



263 



V. 1 

<. 8 




o 

(N 


IO to to to IO to to IO IO IO to to 







^> s 






<nfN"-H^tCN't'lO-3<r'5<-siroio 






^^ 












c -^ 
a g 




2 


toioiootoioiooioiooo 


o 




3 * 






<Nt5ttloOvO-t | f*5'*' v 5>0->* 


r-i 




<u 












a 

S '.S 




00 


totoioioootoioiooioo 


O 




$1 






.i^l\O01--irO'^0--t | 0)-HfNPO 






VJ V) 












50 8 

a <u 




r- 


lOlOtOlOlOlOOOtOlOOlO 


to 




<^ ^> 
~-^ *< 




- 


cs ^H o) ro rf i^; -f to rt< rf. r^ r^ 


o 




i .* 

v- 




U 




-H 


t/3 (/) 


^ S 3 







ooo-oiotoioot^toioto 


O 


-S -5 


8 
8 'S 







T f<TfiOlO't\O-t~OTfvO'*" P 5 




1^ vO 


-S a 
* 




U 






ro CN 


e 
g 




vr> 


to to otoototoiotoo to to 


LO 


flj <D 

-M -4-* 


a 
** *- 






^CNiDvOTtlO^tN-^vO^-f 


O 




a 


tn 


U 






o -a 


S^ 

Q 




re 


toototoioiotototooioo 


IO 


U iD 


c* * 

<Q 


G 





^fir^<-HTfCNro-rt<^H-trslfO 




>> x 


- 




U 






c c 


8-8 


"55 


n 


IO <O to to O 5 tO to to to to to ^> 


10 


W U 


a - 


<u 
u 




*~p to f^ 'rt' to fN ^ | *^ T i r*^ ^f to 


vO 




^ H 

fs 2? 


u 


U 






PC to 


i? ' 

S'o' 




ft 


O to to to to to to O to to to O 


to 


U U 


o 






c<; -^ 10 f"O CN] (V} *fi LO <"*"; CN Tf LO 




* * 


** 5 

O \o 


c 


U 








8 -2 to 
g ^ 


z 


- 


ootoiooioioioooioio 


10 




*'.&. a 


G. 




ro-^r^^^rotvirfrOiOC-JCN 


OO 




^ ^< t 
"a 1? ^ 




U 








~ <ii 
; 'S < ~> 







lOlOLOlOtolOlOlOtOlOOlO 







> i ~i "<3 

^3 ** .^ 






^O^itO^fr^cN^CSOfOO 






W a ^> v 




U 








a ? 

W ^> V> 


u 





Loioooiootoootoioo 


o 




< a -o >-< 




: 


^^-.ror^^ftoiO^t^Oi^OCNtN 


fM 




^ "S-2 




U 








s 4s *> 

2 Z.r 


V ' 


00 


IO to IO O LO to LO IO to to O to 







Z ^^ 


M 

rt 


_ 


-H^lCNrO i ^* <N c*5 -H <N <T> PO 






K> 8 ^> 

^J s 


V 


U 








^ ^ -^ 

^ 


a 


^ 


o o to o to to o to to o to to 


10 




S *> o 

" fe 


a 





O'Or^^Csl'tLOvOrO^LO'* 


f*j 


t/5 


g s^ 


H 


U 






tn >> 


~ 2 s 

s K a 







looioiootootoootoo 


c 


03 -O 


a ^ a 




_: 


CN^O-rp^HiOrN^frorO'OrNTt 




** CN 


>^ <a v. 

Vi "-s ej 




U 






U. U 


S <o ^3 




U) 


* 
IO IO to O O to 


o 


M -4-> 

' ' 


*- o S 
^.fe 1 




U 


IO CM -> O t O 


LO 


rt c3 

"^ 'O 
<1> 1* 


^ s-g 




-* 


lOLOLOOOtOOtOtOOtOO 


to 


en tn 


s^ s 






-fvOrOTj'POOtOOOTf'^lO-rf 


-f 


U 


a -^ s 








-H 


W UJ 


. < 

^ ^ 5 




W) 


# 


LO 




C> g K 

'SJ V. 
^- t^ *^i 
O M J 




c 


,-j OS cs 00 Ox t- CM 





> t^i 


I-8JJ 

* SS-f 

rv jg CvO 

^ s 




U 


* 
to o to LO 


LO 


UU 

* * 


^'S 
* b 

& S k 




U 


* 
LO 

to 


LO 
LO 




Tillina ma, 

sixty days. F 
diminished mg( 


Successive 


>. VI 

ST3 
3.0 


- ?r ^.o. Ssa 


Total nuniber 
of generations 





264 C. D. BEERS 

nuclei, or even to the presence of a micronucleus, since amicronucleate clones proved 
to be as vigorous as micronucleate clones. 

The origin of the amicronucleate races cannot be accounted for with certainty. 
It is agreed that such races may arise at any of four periods in the life of ciliates 
generally : at endomixis, by the transformation of all the reconstruction micronuclei 
into macronuclei ; at autogamy or conjugation, by an analogous transformation of 
all the derivatives of the synkaryon or amphinucleus into macronuclei ; or at division, 
by an unequal distribution of the micronuclei to the daughter cells. By studying 
dividing individuals of an unusual race of Paramecium caudatum, Wichterman 
(1946) observed the simultaneous production of bimicronucleate and amicronu- 
cleate daughters. In T. inagna conjugation is of rare occurrence, ahd endomixis 
and autogamy are unknown. Therefore, it is likely that amicronucleate races usu- 
ally take their origin in an unequal distribution of the products of division. How- 
ever, it should not be concluded from the present results that 15 per cent of all 
Tillina clones are amicronucleate ; actually such clones appear to be very exceptional. 
I have stained many specimens during a period of 8 years; all these specimens had 
micronuclei, except for the members of clones 18-20. 

DISCUSSION 

The functional significance of the nuclear dimorphism of the Euciliata has long 
held the attention of protozoologists. In general, the dimorphic condition has been 
viewed as representing a segregation of idiochromatin and trophochromatin, the 
former in the micronucleus. the latter in the macronucleus. It was originally as- 
sumed, since ciliates normally possess both types of nuclei, that both are necessary 
for the continued survival of the individual. Although the precise functions of the 
respective nuclei are difficult to determine, two lines of investigation have supplied 
pertinent findings, namely, a study of the capabilities of amicronucleate ciliates of 
natural occurrence and a study of regenerative capacity, survival, and reproduction 
in ciliates which have been deprived experimentally of either nucleus, or of both, 
whether by merotomy or other operative procedures. 

The existence of naturally occurring amicronucleate races has long been conceded 
by such authorities as Woodruff (1921), Calkins (1930), and Reichenow (1929, 
p. 29) and is now accepted as a fact. The potentialities of these races, as revealed 
by intensive laboratory study, have demonstrated that the micronucleus is not at all 
necessary for the maintenance of the essential vital processes of the individual, 
whereas the macronucleus is indispensable. Aside from the absence of a micro- 
nucleus and the manifest inability to carry to completion such processes as endo- 
mixis, autogamy and conjugation, amicronucleate races of many ciliates do not differ 
structurally or physiologically from micronucleate ones. 

Thus, Dawson (1919; 1920) maintained an amicronucleate race of Oxytricha 
liyinenostonia in pure-line culture for 289 generations (4 months) and in small mass 
cultures for 5 months longer. The absence of micronuclei did not prevent the ani- 
mals from attempting to conjugate, but these attempts were abortive. Woodruff 
(1921) cultured amicronucleate races of Oxytricha jallax and Urostyla grandis for 
246 and 128 generations, respectively, and maintained a race of Paramecium can- 
datum in pure-line culture long enough to determine that it was definitely amicro- 



MICRONUCLEI OF TILLINA MAGNA 265 

nucleate. A few pairs of conjugants occurred in mass cultures of O. falla.v, but 
they failed to live when isolated. 

Amicronucleate races of other ciliates have likewise been isolated a,nd cultured 
long enough to demonstrate not only their viability but their sustained vigor and 
good health. Among these are the following: (1) Spathidmm spathula. Moody 
(1912) was unable to find micronuclei in her specimens, though she was able to 
culture them for 218 generations. It is evident that they were amicronucleate, since 
the micronuclei of Spathidium were observed and counted by Maupas and were 
found regularly by Woodruff and Spencer (1922). (2) Didimuin nasutum. Pat- 
ten (1921) cultured an amicronucleate race, which was derived from an exconjugant 
of a normal micronucleate race, for 652 generations. Conjugation occurred in the 
amicronucleate race, but the exconjugants invariably died. The resting cysts were 
likewise inviable. It is evident that the race arose through the faulty reorganiza- 
tion of the exconjugant. (3) Paramecium bursaria. Woodruff (1931) cultured 
a race characterized by micronuclear instability for 7 years. Neither endomixis nor 
conjugation was observed. The race was originally bimicronucleate ; later it was 
variable, exhibiting from 1 to 4 micronuclei ; then for about 4 years it was unimicro- 
nucleate ; finally, a derived race showed no micronucleus, although this race was 
apparently as healthy and vigorous as its bimicronucleate ancestors which were iso- 
lated 7 years earlier. Woodruff (p. 543) aptly points out that "whatever function 
the micronuclear apparatus plays, the somatic life of the animals is not obviously 
influenced by profound variations in volume or in distribution of micronuclear 
material." (4) Urostyla grandis. Tittler (1935) found amicronucleate individuals 
in stock cultures which previously contained only micronucleate specimens. They 
were indistinguishable externally from their micronucleate progenitors, and they 
flourished in mass cultures for 2 years. Their macronuclear divisions followed the 
usual complex pattern characteristic of the species. The race produced resting 
cysts, some of which could be excysted, although endomixis, which usually occurs 
in the precystic forms, was absent. Evidently some of the cysts \vere not entirely 
normal, since they showed a tendency to disintegrate, perhaps because of the omis- 
sion of endomixis. (5) Colpoda steini. Piekarski (1939) studied comparatively 
the structure and reproduction of a micronucleate and an amicronucleate race and 
was able to culture the latter for approximately 6 years. Both races were equally 
cultivable and vigorous. They reproduced within division cysts from which four 
progeny regularly emerged and they produced normal resting cysts. They showed 
the same Sequence of events in the division of the macronucleus. These events are 
of special interest, in that eight chromatic (Feulgen-positive) bodies appear in the 
macronucleus of the young division cyst. Ultimately each of the daughters receives 
two of them, and thus the behavior of these bodies suggests an equational distribu- 
tion of chromosomes. Piekarski concludes that the absence of a micronucleus had 
no recognizable effect on the activities of the animals. 

The present study of amicronucleate races of Tilliim inagna likewise demon- 
strates the adequacy of the macronucleus, not only for long-continued reproduction 
accompanied by sustained vigor, but also for encystment and excystment. Thus 
endowed with the ability to produce viable resting cysts, these races would seem to 
be capable of indefinite survival, even under the changeable conditions of a natural 
environment. 



266 C. D. BEERS 

Still more remarkable, in connection with the capabilities of amicronucleate races, 
are the observations of Sonneborn (1940) and Kimball (1941) on mating types. 
In certain races of Paramecmm aurelia Sonneborn found a small percentage of ani- 
mals which, upon undergoing autogamy or conjugation, developed a new macro- 
nucleus from a fragment of the old macronucleus. Since the micronuclei commonly 
disappeared in clones produced by these animals and since the mating type never 
changed at macronuclear reorganization, Sonneborn concludes that "hereditary 
characters (including mating type) of clones from macronuclear regenerates cannot 
be directly determined by micronuclei, for they persist in the absence of micronuclei. 
Mating type must be determined by the macronucleus. . . ." Kimball was able to 
assign amicronucleate specimens of Euplotcs patella to definite mating types, since 
they paired readily with individuals of known mating type. He concludes that "the 
micronucleus is thus unnecessary for an animal to be of a definite mating type." 

With reference to the role of the nuclei in the regeneration of ciliates following 
merotomy, the results obtained with different species are not always in complete 
accord. The physical properties of the cytoplasm constitute an experimental vari- 
able ; a fluid cytoplasm or a rigid pellicle may interfere with the closure of an injury 
and thus affect regeneration adversely. Balamuth (1940) has presented an ex- 
cellent review of the extensive literature on this subject. For the present only a 
few representative examples of regeneration in ciliates will be considered, with spe- 
cial reference to the nuclear components of the merozoa (cell fragments). 

It is well known that enucleate fragments of ciliates can neither regenerate nor 
continue to live, whereas nucleate fragments regenerate successfully and pursue 
normal lives. These conclusions are particularly evident in Balamuth's six-page 
tabular summary of the findings on regeneration in the ciliates. As a rule the 
macronucleus and micronucleus cannot be separated, and a nucleate fragment, as in 
Dembowska's work (1925) on Stylonychia mytilus, usually means one having both 
macronuclear and micronuclear material. 

However, some investigators have succeeded in obtaining nucleate merozoa of 
the two types desirable for an experimental analysis of the role of the individual 
nuclei in regeneration; namely, macronucleate (without micronuclei) and micro- 
nucleate (without any part of the macronucleus). Reynolds (1932), for example, 
in work on an amicronucleate Oxytricha jallax, found that various types of macro- 
nucleate merozoa could regenerate their missing parts and resume their normal 
physiological activities. Schwartz, using microdissection techniques, was able to 
remove the entire macronucleus from Stcntor and yet leave a number of micro- 
nuclei in the specimens. These micronucleate individuals never survived. By 
means of successive operations, he was also able to remove all the micronuclei from 
a few specimens, leaving a portion of the beaded macronucleus in place. These 
macronucleate individuals regenerated and could be cultured as pure lines. Thus 
he produced experimentally an amicronucleate race, which as regards size and divi- 
sion rate was not different from the normal controls. Bishop (1943), employing 
the ultra-centrifuge as a means of obtaining merozoa of Oxytricha jallax, obtained 
sixty-seven macronucleate fragments, all of which regenerated, and seven micro- 
nucleate fragments, none of which regenerated. Twelve of the regenerated macro- 
nucleate individuals were cultured as amicronucleate pure lines. Bishop concluded 
(p. 451) that "the lack of micronuclear material makes no difference in the regen- 
erative capacity, division rate, motility or morphology of Oxytricha jallax" 



MICRONUCLEI OF TILLINA MAGNA 267 

On the other hand, there is evidence that in some forms the micronucleus, as 
well as the macronucleus, is necesesary for regeneration and survival. Thus Rey- 
nolds found that both nuclei are necessary for the regeneration of merozoa of Eu- 
plotcs patella. This observation is in accord with the results of Taylor and Farber 
(1924), who, by means of a micro-pipette, removed the micronucleus from fifty 
specimens of E. patella, all of which died within a few days. None produced more 
than four progeny. Hence, these authors conclude that "the micronucleus plays 
more than a purely germinal role in the life history of Eiiplotes patella." However, 
the situation in E. patella is confused, for some of Kimball's unimicronucleate double 
animals produced viable amicronucleate individuals at division. Some of them sur- 
vived as clones, though with a low division rate ; one such amicronucleate clone sur- 
vived for 341 days. The results show, according to Kimball (p. 30), "that the 
micronucleus is not essential for continued life in at least some clones of Euplotes 
patella, though its absence results in a marked decrease in vigor." In various spe- 
cies of Uronycliia (Young, 1922), and in Uroleplus mobilis (Tittler, 1938), Spa- 
thidium spathula and Blepharisma undulans (Moore, 1924) both types of nuclei ap- 
pear to be necessary for the complete regeneration, growth and division of merozoa. 

Thus the evidence afforded by the long-continued culture of a number of natu- 
rally occurring amicronucleate races demonstrates conclusively that the macro- 
nucleus alone suffices for the maintenance of the vegetative life of the organism- 
meaning by vegetative life such diverse activities as locomotion, food capture, diges- 
tion, assimilation, growth, excretion, respiration, reproduction, and maintenance of 
cell proportions and form. On the basis of this evidence the normal role of the 
micronucleus in vegetative life appears to be one of relative passivity. The evidence 
from operative procedures shows that in many ciliates the macronucleus alone is 
adequate for complete regeneration, as well as for subsequent growth and division, 
whereas in other ciliates the micronucleus also is necessary. There is no authenti- 
cated case on record in which the micronucleus alone is adequate for the maintenance 
of vegetative functions or for regeneration. Balamuth's thorough survey of the 
literature leads him to make the following comment in his summary : "Of the dual 
nuclear apparatus, only the macronucleus can be shown to function in the actual 
regenerative process. The role of the micronucleus in this connection is as yet 
unclear ; apparently it is more important in the viability of some forms than in oth- 
ers." On the whole, the evidence tends only to emphasize the importance of the 
macronucleus and to attest to the validity of Calkins' comment (1930, p. 161) on 
this organelle : "Far from being negligible it is on the contrary probably the most 
important element of the cell in matters of metabolism, reorganization, and continued 
cell life." 

The tendency to underestimate the importance of the macronucleus in the life 
of the organism results probably from its apparent monotony of structure. Lacking 
chromosomes, its division is usually unspectacular. Nevertheless, its mode of origin 
is not an incidental phenomenon in the life of the ciliate, for both macro- and micro- 
nucleus almost invariably have a common and simultaneous origin. Usually they 
develop from the synkaryon of the conjugant, by divisions which appear to be equa- 
tional. Again, they develop from the synkaryon of autogamy or from the endo- 
mictic micronucleus. Thus they inherit equally from a common nucleus of origin, 
and each receives equivalent chromatin elements, chief among which are presumably 
the genes. In the definitive micronucleus these elements retain the ability to or- 



268 C. D. BEERS 

ganize periodically as chromosomes, and thereby to arrest the attention of the ob- 
server. Once in the definitive macronucleus, on the contrary, they never again 
assemble in the form of chromosomes. However, it is possible that they are dis- 
tributed at macronuclear division by a mechanism which is fully as effective as the 
mitotic distribution of chromosomes, though less conspicuous. For example, it is 
not impossible that they are represented in multiplicate in the macronucleus and 
distributed at random throughout its substance. Thus each daughter at division 
would be reasonably assured of receiving representatives of every type of chromatin 
element. The behavior and the potencies of the macronuclear fragments of Sonne- 
born's unusual specimens of Paranieciuin aurelia indicate a multiplicate representa- 
tion of the chromatin elements. In these specimens the forty or more macronuclear 
fragments grew and segregated during subsequent cell divisions, until there was only 
one in each cell. Thus each fragment was adequate for the regeneration of a com- 
plete macronucleus and for the continued life of the organism, even in the absence of 
micronuclei. Hence, Sonneborn concludes that "the normal macronucleus must con- 
tain at least forty complete and discrete genomes." A more precise mechanism for 
the distribution of the chromatin elements, involving, for example, a differential 
streaming of genetically equivalent elements toward opposite ends of a polarized 
macronucleus, may be postulated. Regardless of the type of mechanism involved 
in the distribution of the chromatin elements of the macronucleus at division, the 
fact remains that inheritance in an amicronucleate ciliate is no less precise, to judge 
by the structure and physiological activities of the offspring, than in a micronucleate 
ciliate. The fact that the behavior of the macronucleus does not conform to the 
chromosome theory of heredity in sensu strict o, in that chromosomes are absent, 
may mean simply that a different mechanism for the distribution of the genes is 
involved. 

Whether the macronucleus of amicronucleate ciliates may justifiably be regarded 
as an amphinucleus one containing idiochromatin as well as trophochromatin, as 
Woodruff (1921), Moore and others have suggested seems doubtful in the light 
of recent investigations. Thus it has been shown by Schwartz and by Bishop that 
viable amicronucleate races of S tent or and Oxytricha may be derived by experi- 
mental means from normal individuals in which the idiochromatin and trophochro- 
matin were presumably segregated in the two types of nuclei. The macronucleus 
of these individuals, following removal of the micronuclei, was adequate to maintain 
all the usual vegetative activities in the derived amicronucleate races. 

In conclusion, the evidence shows that the macronucleus is the essential nuclear 
element in the vegetative life of ciliates. The micronucleus functions largely, if not 
solely, in the periodic replacement of the macronucleus and in the production of 
new genetic combinations, some of which undoubtedly render the species better 
adapted to survival. The nature of the physiological conditions which call for a 
renewal of the macronucleus is not clear; that such renewal meets an imperative 
physiological need is shown by the widespread occurrence of the phenomenon in 
the Euciliata. 

SUMMARY 

The number of micronuclei was examined in 50 trophic specimens and 50 resting 
cysts of each of 20 clones of Tillina magna, three of which were amicronucleate. 



MICRONUCLEI OF TILLINA MAGNA 269 

In any particular clone trophic specimens and resting cysts contained approxi- 
mately equivalent mean numbers of micronuclei. In different micronucleate clones 
the mean number varied from 4.21 to 12.61. The mean for 1,700 specimens of 17 
clones was 7.07. 

The number in the individuals of any particular micronucleate clone was vari- 
able ; some clones showed relatively little variation, e.g., 3 to 5 micronuclei ; others, 
considerable variation, e.g., 2 to 11 micronuclei. The smallest number observed in 
any micronucleate individual was 2; the largest, 16. 

All the clones produced normal resting cysts upon depletion of the food supply 
(Pseudomonas fluorescent} . The cysts of different clones were equally viable and 
capable of excystment. Their size was unaffected by the number of micronuclei. 
Amicronucleate cysts showed the usual macronuclear reorganization. Hence, 
neither the number of micronuclei nor the absence of micronuclei affected encyst- 
ment, viability and size of cysts, excystment or macronuclear reorganization. 

An attempt was made to maintain each clone in pure-line culture for 60 days 
and thereby to examine the division rate and vitality. Four clones were refractory 
and encysted before 60 days expired. The remaining 16 clones, including the three 
amicronucleate ones, survived with undiminished vigor and were discontinued. The 
13 micronucleate clones produced from 149 to 176 generations during the 60-day 
period; the three amicronucleate clones produced 154, 164, and 172 generations, 
respectively. Hence, the 16 surviving clones showed slight differences in their 
average daily division rates, but neither the divison rate nor the vitality of these 
clones was correlated with variations in micronuclear number or with the absence 
of micronuclei. Division cysts of amicronucleate clones showed the usual macro- 
nuclear reorganization after each division of the macronucleus. The four refractory 
clones had high micronuclear numbers. 

Since conjugation is rare and endomixis and autogamy are unknown in Tillina, 
it is probable that amicronucleate races arise at division by an unequal distribution 
of the daughter micronuclei. 

Some of the literature on amicronucleate ciliates and on the regeneration of vari- 
ous types of nucleate merozoa is reviewed. The evidence shows that the macro- 
nucleus is the indispensable nuclear element in the so-called vegetative life of the 
organism, whereas the micronucleus during this period appears to be a relatively 
passive organelle. Its chief function concerns the periodic replacement of the macro- 
nucleus and the production of new hereditary combinations. Special attention is 
directed to the fact that inheritance in an amicronucleate race is no less precise than 
in a typical micronucleate race, although the division of the macronucleus is amitotic 
and usually reveals no suggestion of true chromosomes. It is evident that the 
hereditary mechanism of amicronucleate races, and perhaps of ciliates generally, 
differs radically from the conventional chromosomal mechanism of metazoa. 

LITERATURE CITED 

BALAMUTH, W., 1940. Regeneration in protozoa: a problem of morphogenesis. Quart. Rev. 

Biol, 15: 290-337. 
BEERS, C. D., 1944. The maintenance of vitality in pure lines of the ciliate Tillina magna. 

Amer. Nat., 78: 68-76. 
BEERS, C. D., 1945. Some factors affecting excystment in the ciliate Tillina magna. Physiol. 

ZooL, 18 : 80-99. 



270 C. D. BEERS 

BEERS, C. D., 1946. History of the nuclei of Tillina magna during division and encystment. 

Jour. Morph., 78: 181-200. 
BEERS, C. D., 1946a. Micronucleate and amicronucleate races of Tillina magna. Anat. Rec., 

94 (3) : 92-93. 

BISHOP, E. L., JR., 1943. Studies on the cytology of the hypotrichous infusoria. I. The rela- 
tion of structure to regeneration. Jour. Morph., 72 : 441-475. 
BRESSLAU, E., 1922. tiber Protozoen aus Rasenaufgiissen. Verh. Deutschen Zool. Gesell, 

27 : 88-90. 
BURT, R. L., G. W. KIDDER, AND C. L. CLAFF, 1941. Nuclear reorganization in the family 

Colpodidae. Jour. Morph., 69: 537-561. 
CALKINS, G. N., 1930. Uroleptus halseyi Calkins. II. The origin and fate of the macronuclear 

chromatin. Arch. f. Protistenk., 69: 151-174. 
DAWSON, J. A., 1919. An experimental study of an amicronucleate Oxytricha. I. Study of 

the normal animal, with an account of cannibalism. Jour. Exp. Zool., 29 : 473-513. 
DAWSON, J. A., 1920. II. The formation of double animals or "twins." Ibid., 30: 129-157. 
DEMBOWSKA, W. S., 1925. Studien iiber die Regeneration von Stylonychia mytilus. Arch. f. 

Mikr. Anat. u. Entwmech., 104: 185-209. 
GREGORY, L. H., 1909. Observations on the life history of Tillina magna. Jour. Exp. Zool., 

6: 383-431. 

GRUBER, A., 1879. Neue Infusorien. Zeit. f. Wiss. Zool, 33 : 439-466. 
ILOWAISKY, S. A., 1921. Zwei neue Arten und Gattungen von Infusorien aus dem Wolgabassin. 

Arb. d. Biol. Wolga-Station, 6: 95-106. 
KAHL, A., 1931. Die Tierwelt Deutschlands, Part 21, 181-398. Jena: Verlag von Gustav 

Fischer. 
KIMBALL, R. F., 1941. Double animals and amicronucleate animals in Euplotes patella with 

particular reference to their conjugation. Jour. Exp. Zool., 86: 1-33. 
MAUPAS, E., 1888. Recherches experimentales sur la multiplication des infusoires cilies. 

Arch, de Zool. Exp. et Gen., Ser. 2, 6 : 165-277. 
MOODY, J. E., 1912. Observations on the life-history of two rare ciliates, Spathidium spathula 

and Actinobolus radians. Jour. Morph., 23 : 349-407. 
MOORE, E. L., 1924. Regeneration at various phases in the life-history of the infusorians 

Spathidium spathula and Blepharisma undulans. Jour. Exp. Zool., 39: 249-316. 
PATTEN, M. W., 1921. The life history of an amicronucleate race of Didinium nasutum. Proc. 

Soc. Exp. Biol. and Med., 18 : 188-189. 
PIEKARSKI, G., 1939. Cytologische Untersuchungen an einem normalen und einem Micro- 

nucleus-losen Stamm von Colpoda steini Maupas. Arch. f. Protistenk., 92: 117-130. 
POWERS, J. H., AND C. MITCHELL, 1910. A new species of Paramecium (P. multimicro- 

nucleata) experimentally determined. Biol. Bull., 19: 324-332. 
REICHENOW, E., 1929. Lehrbuch der Protozoenkunde, Part 1. Jena: Verlag von Gustav 

Fischer. 
REYNOLDS, M. E. C., 1932. Regeneration in an amicronucleate infusorian. Jour. Exp. Zool., 

62: 327-361. 
SCHWARTZ, V., 1935. Versuche iiber Regeneration und Kerndimorphismus bei Stentor coeruleus 

Ehrbg. Arch. f. Protistenk., 85 : 100-139. 
SONNEBORN, T. M., 1940. The relation of macronuclear regeneration in Paramecium aurelia 

to macronuclear structure, amitosis and genetic determination. Anat. Rcc., 78 (Suppl.) : 

53-54. 
TAYLOR, C. V., AND W. P. FARBER, 1924. Fatal effects of the removal of the micronucleus in 

Euplotes. Univ. Calif. Publ. Zool, 26: 131-144. 
TAYLOR, C. V., AND L. GARNJOBST, 1941. Nuclear reorganization in resting cysts of Colpoda 

duodenaria. Jour. Morph., 68: 197-213. 
TITTLER, I. A., 1935. Division, encystment and endomixis in Urostyla grandis with an account 

of an amicronucleate race. La Cellule, 44: 187-218. 
TITTLER, I. A., 1938. Regeneration and reorganization in Uroleptus mobilis following injury 

by induced electric currents. Biol Bull, 75: 533-541. 
TURNER, J. P., 1937. Studies on the ciliate Tillina canalifera n. sp. Trans. Amer. Micr. Soc., 

56: 447^56. 
WENRICH, D. H, 1928. Eight well-defined species of Paramecium. Trans. Amer. Micr. Soc., 

47: 275-282. 



MICRONUCLEI OF TILLINA MAGNA 271 

WICHTERMAN, R., 1946. Unstable micronuclear behavior in an unusual race of Paramecium. 

Anal. Rcc., 94 (3) : 94. 
WOODRUFF, L. L., 1921. Micronucleate and amicronucleate races of infusoria. Jour. Exp. 

Zool., 34 : 329-337. 
WOODRUFF, L. L., 1931. Micronuclear variation in Paramecium bursaria. Quart. Jour. Micr. 

Sci., 74 : 537-545. 
WOODRUFF, L. L., AND H. SPENCER, 1922. Studies on Spathidium spathula. I. The structure 

and behavior of Spathidium, with special reference to the capture and ingestion of its 

prey. Jour. Exp. Zool., 35: 189-205. 
YOUNG, D. B., 1922. A contribution to the morphology and physiology of the genus Uronychia. 

Jour. Exp. Zool., 36 : 353-395. 



THE EFFECT OF LOW TEMPERATURE AND OF HYPOTONICITY 
ON THE MORPHOLOGY OF THE CLEAVAGE FURROW 

IN ARBACIA EGGS * 

ALLAN SCOTT 

Department of Biology, Union College, Schenectady, N. Y. and The Marine Biological 

Laboratory, Woods Hole, Massachusetts 

When Arbacia punctulata eggs are exposed to low temperature during the first 
cleavage, a pronounced stalk develops between the daughter blastomeres. A stalk- 
also develops at room temperature if the eggs are made to divide in hypotonic sea 
water or in sea water lacking calcium ion. The development of a conspicuous 
cleavage stalk is not a normal feature of the first cleavage in Arbacia, although it 
does occur regularly in some cells ; for example, when fibroblasts divide. The 
object of the work reported here was to examine the conditions under which the 
stalk is formed in Arbacia and to relate these facts to current theories of the mech- 
anism of cleavage. These particular experimental treatments were used because 
they were found to affect the appearance of the cleavage stalk. 

METHODS 

Eggs of Arbacia punctulata in the first cleavage served as experimental material. 
Ovulation was induced by the removal of the oral half of the test; eggs emerging 
from the genital pores were collected in a dish of sea water. The eggs were al- 
lowed to settle and the sea water was decanted after which fresh sea water was 
added. Two such washings were carried out to minimize contamination by 
coelomic fluid. Fertilization was effected by the use of diluted "dry" sperm, and 
the sperm were never more than one hour old. 

The fertilization membranes were removed by shaking. A heavy suspension 
of eggs was placed in a five-inch test tube, one-half full of the suspension, and 
shaken rapidly thirty times. Eggs so treated cleave in time with the controls. 
The best time for treatment is at 2% minutes after fertilization, for if shaken earlier, 
many exovates are formed, and if shaken later, many eggs retain the fertilization 
membrane. The alternative method of removing the fertilization membrane by 
treatment with the hatching enzyme (Ishida, 1936) was not attempted. 

The hyaline layer was removed in a few experiments by washing the eggs in 
calcium-free artificial sea water. This was accomplished by several decantations 
and additions of the calcium-free mixture. It was found that the hyaline layer 
regenerates somewhat if the eggs are returned to a solution possessing calcium 
ions ; hence if eggs are to lack the hyaline layer, they must be allowed to cleave in 
the calcium-free mixture. 

This study was largely accomplished by photographic means. Photomicro- 

1 This investigation was aided by a Grant-in-Aid from the Sigma Xi Alumni Research 
Fund. 

272 



CLEAVAGE FURROW IN ARBACIA EGGS 273 

graphs taken at intervals with Leica-Ibso apparatus, were projected as negatives 
(1,000 X) and measurements made with dividers. 

Temperature was maintained by means of a thermostatically controlled, water 
jacketed, glass well, mounted on the microscope stage and connected through a 
centrifugal pump to a water bath. By this means temperature could be maintained 
within 0.2 C. at or about 20 and within 0.4 C. at or about 10 C. 

Artificial sea water lacking calcium ions was compounded according to the 
method of Shapiro (1941). This solution has an osmotic pressure and pH closely 
similar to that of normal sea water. 

The hypotonic solutions were prepared either by the dilution of normal sea 
water or of the calcium-free mixture. 

A few observations are presented on polyspermic eggs cleaving to three or to 
four cells in one division. Polyspermic development was induced by the method 
of Smith and Clowes (1924) which involves fertilization in pH 7.2 sea water and 
the return of the eggs to the normal pH of 8.4 within two or three minutes. 

RESULTS 
Morphology of the cleavage furrow 

The shape of the deepening furrow is markedly different under different con- 
ditions; it is influenced by temperature, concentration of calcium ion, tonicity and 
by presence or absence of the fertilization membrane. 

Temperature 

At temperatures between 20 C. and 30 C. there is normally no stalk in 
cleaving eggs whose fertilization membrane has been removed (free cleavage). 
The furrow is peaked at the apex (Figs. 1 and 2). At low temperatures, 6 to 
12 C., a real stalk is formed during the latter part of the furrowing. This occurs 
whether the egg is enclosed in the fertilization membrane or not. At these low 
temperatures eggs undergoing membrane-free cleavage, come to resemble a dumb- 
bell with a handle (Figs. 3 and 4). 

Calcium ion or urea 

Chambers (1938) described the short cleavage stalk which develops when Ar- 
bacia punctulata eggs divide in calcium-free solutions at room temperature. 
He used isotonic mixtures of sodium chloride and potassium chloride. In the pres- 
ent study also a short stalk occurred when the eggs were exposed to calcium-free 
sea water. Similarly a short stalk was figured by Moore (1930a and b) and by 
Motomura (1934), after treatment with urea solutions. 

Fertilization membrane 

It is a common practice to remove the fertilization membrane either by dis- 
solving it in urea solutions or by shaking an egg suspension rather violently. These 
techniques allow the mitotic axis to become much longer and the furrowing is thus 
more readily followed. If the eggs are confined in the fertilization membrane at 
10 C., the blastomeres tend to stay apart and the walls of the furrow are almost 
vertical (Figs. 11 and 12). At the end of the cleavage a stalk connects the two 
blastomeres. If this same experiment is varied so that the eggs divide within their 



274 ALLAN SCOTT 

fertilization membranes at 10 C. and in calcium-free sea water, a cleavage stalk like- 
wise develops. In this case, however, the stalk moves eccentrically until it is close 
to the fertilization membrane (Figs. 30 through 34). The difference is presumably 
due to the fact that the hyaline layer is dissolved in solutions lacking calcium ion 
and when the hyaline layer is missing the egg is able to slide around inside the 
fertilization membrane. 

Membrane-free cleavage in polyspermic eggs 

Polyspermic eggs may undergo free cleavage to form four cells in the first divi- 
sion. In such cases they frequently divide so that a symmetrical figure is seen from 
above. In this circumstance the four blastomeres each rest upon the bottom of the 
glass container (Figs. 15, 16, 17, and 18). Frequently one blastomere rests upon 
the other three at the end of the cleavage (Fig. 21). The former, more symmetrical 
type of cleavage is more readily followed. When such an egg begins to cleave it 
first flattens like a biscuit; at this stage it resembles a balloon around which two 
rubber bands have been placed at right angles. Such a balloon is flattened on the 
two surfaces where the rubber bands cross. Perhaps the egg, like the balloon, is 
subject to greater elastic tension in the region where the incipient furrows cross, 
and therefore flattens on these surfaces. 

As seen from above, the egg periphery is roughly square, with corners rounded 
(Fig. 15) ; the wide furrows (at 10 C.) gradually sink towards the center with the 
apices of the furrows approaching one another. The upper and lower surfaces 
meanwhile remain relatively flat although the two flat surfaces slowly come together. 
The whole figure at the stage illustrated in Figure 16 resembles a balloon stretched 
closely over four tennis balls with two rubber bands placed at right angles. Finally 
a definitive stalk is formed (Fig. 18). 

When the furrows first appear, the egg is to be considered as having two equa- 
torial furrows; that is, two constricting rings (Fig. 29a), which cross each other. 
The quasi-independence of the furrows is demonstrated by some eggs which cleave 
in a similar way but in zvhich the furrows incise at different rates (Figs. 19 and 20). 
In Figure 29b the furrow separating ab from cd is well in advance of the furrow 
separating ad from be. This type of cleavage leads to a figure like Figure 20. 

It appears that this curious type of cleavage is brought about by the develop- 
ment of two new ring-like tensions which develop around the necks of the indi- 
vidual blastomeres after the deeper furrow is well established. As a result of the 
deep primary furrow, four new isthmuses are established about the necks of the 
four incipient blastomeres (cf., Fig. 29b). Perhaps the most significant feature of 

PLATE I 

FIGURES 1 AND 2. Egg cleaving at 20 C. in sea water, fertilization membrane removed. 

FIGURES 3 AND 4. Egg cleaving at 10 C. in sea water, fertilization membrane removed. 

FIGURES 5 AND 6. Egg cleaving at 20 C. in 65 per cent sea water, fertilization membrane 
removed. 

FIGURE 7. Egg cleaving within the fertilization membrane at 20 C. in sea water. 

FIGURE 8. Late cleavage at 20 C. in 65 per cent sea water, fertilization membrane removed. 

FIGURES 9 AND 10. Polyspermic egg fertilized in sea water at pH 7.2, transferred to nor- 
mal sea water at room temperature until cleavage began. Cleaving at 10 C. in sea water. 

FIGURES 11 AND 12. Eggs cleaving within fertilization membrane at 10 C. 



CLEAVAGE FURROW IN ARBACIA EGGS 



275 




I 



276 



ALLAN SCOT I 



this type of cleavage is the bridge-like stalk which results (Figs. 23 and 24). In 
these latter figures note that one circumferential furrow deepened symmetrically 
and more rapidly than the other. The furrow which started later is very asym- 
metrical, being much deeper on one side (cf., at the arrow) than the other. Egg^ 
cleaving to three cells show a similar behavior (Fig. 22) and when cleavage is com- 




FIGURE 13. 
in sea water. 



Series showing late cleavage and development of the cleavage stalk at 10 C. 



plete they may have a Y-shaped stalk, or if one furrow deepens more rapidly than 
the others, two stalks may connect to one blastomere (Figs. 9 and 10). 

The speed of furrowing in polyspermic eggs cleaving to four cells may be as rapid 
as when two cells are being formed, yet it should be remembered that the amount 
of new surface formed is much greater when a sphere divides into four equal smaller 
spheres. The surface of a sphere divided into two spheres increases 26 per cent, 



CLEAVAGE FURROW IN ARBACIA EGGS 



277 







f 





l/V 




FIGURE 14. Series showing late cleavage and continued activity of cleavage stalk, during 
fourteen minutes in calcium-free sea water at 11 C. 



ALLAN SCOTT 

if divided to four spheres the surface increases 58 per cent. A polyspermic egg 
cleaving to four cells forms about 26 per cent more surface than the normal first 
cleavage but it may do so in the same amount of time. 

fifiembrane-jree cleavage in hyputonic sea water and in hypotonic calcium-free sea 
water 

Dilution of the sea water causes a swelling of the egg ; it also causes an unusually 
wide furrow to develop during the cleavage and leads to the formation of a stalk 
at the end (Figs. 5. 6, and 8). This effect occurs at room temperature (20 C.). 
The stalk may become very long if the sea water has been diluted sufficiently. In 
mixtures of 65 parts sea water and 35 parts distilled water, for example, the stalk 
may finally be 30 micra long. This effect occurs either in the presence or absence 
of calcium ion. The stalk region is certainly a relatively rigid gel, for it has suffi- 
cient rigidity to push the daughter blastomeres far apart. Figure 8 and Figures 40 
through 42 show the process of elongation in these extreme cases. Enlarged photo- 
graphs of the stalk at these stages are shown in Figures 43, 44, and 45 with dimen- 
sions noted. In Figure 43 the stalk is only 4.4 micra in diameter at one point. In 
Figure 44 its minimum width is about 2 micra and it is over 22 micra long. In 
Figure 45 the constriction is completed. The stalk is still 5 micra wide at some 
points, but it is less than 3 micra in diameter for a third of its length. Chambers 
(ibid.) relates that a spherical oil drop lying within the egg in the furrow region is 
not deformed until the "external surface of the advancing furrow is 4 to 5 \n from 
the surface of the oil." If the egg pictured in Figure 43 has a cortex comparable 
in thickness, then the stalk must certainly be all gel by the time its diameter is re- 
duced to 7 or 8 fj.. One blastomere sometimes ruptures when eggs cleave in 65 
per cent sea water. Xo endoplasm escapes if the stalk has closed. One such closed 
stalk is shown in Figure 28; it is 5 micra in diameter. The conclusion that the stalk 
is all gel (and yet it continues to constrict) is a most important conclusion for it 
strongly supports the contraction theory of cleavage of W. H. Lewis. Close in- 
spection at high magnification fails to show any movement of granules located in the 
stalk. The active constriction of a 5 /A stalk is recorded in Figures 26 and 27. 

PLATE II 

FIGURES 15, 16, 17, AXI> 18. Cleavage of a dispermic egg, cleaving in calcium-free sea 
water at 10 C. Egg fertilized in pH 7.2 sea water, transferred to sea water at room tem- 
perature until beginning of cleavage. Time after fertilization: Figure 15, 72 min. ; Figure 16, 
74 min.; Figure 17, 88 min.; Figure 18, 190 min. 

FIGURES 19 AND 20. Egg snowing dispermic cleavage. Treatment as in Figures 15-18. 
Time after fertilization: Figure 19, 86 min.; Figure 20, 88 min. 

FIGURE 21. Dispermic egg. Treatment as in Figures 15-18. One blastomere out of the 
horizontal plane. 

FIGURE 22. Diagram illustrating two types of cleavage to three cells. 

FIGURES 23 AND 24. Egg in 70 per cent sea water at 25 C., after accidental polyspermy. 
Time after fertilization: Figure 23, 50 min.; Figure 24, 52 min. 

FIGURE 25. Dispermic egg cleaving in sea water at 11 C., following fertilization in pH 
7.2 sea water. 

FIGURES 26 AND 27. Late cleavage of egg in 65 per cent sea water. Room temperature. 
Time after fertilization : Figure 26, 83 min. ; Figure 27, 85 min. 

FIGURE 28. Closed stalk following rupture of one blastomere ; 65 per cent calcium-free 
sea water. 

FIGURE 29. See text. 



CLEAVAGE FURROW IN ARBACIA EGGS 



279 












DIAM. if 







b 

29 



PLATE II 



280 ALLAN SCOTT 

The stalk 

The mitotic axis (greatest length) of eggs undergoing free cleavage becomes 
progressively longer at 10 than at 20 C. (compare Figs. 1 and 2 with 3 and 4) ; 
moreover the early furrow at 10 C. is much more blunt in contour than is the 
furrow of eggs at higher temperatures. A study of the final phase of cleavage under 
high power (Fig. 13) shows how the wide furrow is transformed into a stalk. 

In Figure 13a the deepening furrow is still blunt with a diameter of about 14 
micra. In Figure 13/>, however, the stalk is beginning to square off. The arrows 
(Figs. 13d and r) indicate the region where the constriction is most active. The 
details are similar and are very clear in eggs cleaving in calcium-free sea water at 
10 C. The series of diagrams shown in Figure 14, a to /. again show that the 
broad furrow first deepens until the diameter of the waist is about 7 or 8 micra (a 
and b), then the stalk is elongated by the constriction of the subequatorial cortex 
(c and d, see arrows) ; meanwhile the entire stalk is diminishing in diameter. The 
minimum diameter of the stalk is about 4 micra at 10 C. and in calcium-free sea 
water ; in hypotonic solutions the diameter is often less. When the diameter of the 
stalk diminishes below 4 micra, it does so in local areas only (cf., Fig. 14<y, h, /). 

Amoeboid activity and cleavage activity 

Many workers have noted that the polar surface of the cell bubbles actively dur- 
ing cytokinesis (Bowen, 1920 in Eucliistits spcnnatocytcs; and Lewis 1942 in 
tissue culture fibroblasts). This is not the case with the egg of the sea urchin dur- 
ing the first cleavage, instead the polar surface remains smooth and inactive. How- 
ever, a variety of agents, will cause the formation of blebs in the sub-furrow region. 
One such agent is hypotonic calcium-free sea water. The blebs usually begin to 
form after the completion of the major furrowing and they give rise to sizable 
spherules which are cut off by a process very much like cleavage (Figs. 46a, b, c}. 
The inactivity of the polar surface may indicate that the cortex there is different 
from the equatorial cortex in Arbacia." 

Eggs that have been in the hypotonic medium for some time may show a sud- 
den rush of endoplasm from one blastomere to the other, often causing the blasto- 
meres to become very unequal in size (Fig. 47 and Figs. 35 to 39). 3 This endo- 
plasmic flow is a very rapid one, usually lasting only two or three seconds. It is 
remarkable, however, that the flow is accompanied toy a rapid deepening of the fur- 
row, appearing as though a tension has been suddenly overcome, allowing the fur- 
row to constrict much more rapidly than usual. The sub-cortical flow in such eggs 
may be down one side of the furrow, through the constricted stalk and up the other 
side of the furrow, yet the furrowing continues normally to completion (Fig. 47). 

- The view that there is a special substance (a special type of plasmagel) located around 
the equator has been espoused by a number of workers. Marsland (1942) and Lewis (1942) 
among others. Beams and King (1937) are of the opinion that they have removed the "surface 
active material" of Ascaris eggs by centrifugation at 150,000 X gravity. 

3 The rush of endoplasm described is in this case related to cleavage. It resembles the 
amoeboid activity described by Moser (1940) after urea treatment. Moser, p. 77, cites other 
cases from the literature. 



CLEAVAGE FURROW IN ARBACIA EGGS 



281 




lit 



HO 





PLATE III 

FIGURES 30, 31. 32, 33, AND 34. Eccentrically placed cleavage stalk. Eggs in fertilization 
membrane at 10 C, in calcium-free sea water. 

FIGURES 35, 36, 37, 38, AND 39. Volume changes of individual blastomeres. Calcium-free 
sea water. 

FIGURES 40, 41, AND 42. Elongation of the cleavage stalk; in 65 per cent sea water at 
room temperature. Time intervals: Figures 40-41, 1 min. and 40 sec.; Figures 41-42, 1 min. 
and 35 sec. 

FIGURES 43, 44, AND 45. Enlargements of Figures 40, 41, and 42. The edges of the nearly 
transparent stalk have been, retouched in Figures 4, 8, 26, 27, 28, 43, 44, and 45. 



282 



ALLAN SCOTT 



DISCUSSION 



The stalk 



The occurrence of a stalk during the cleavage of the Arhacia egg is correlated 
.with the degree of gelation of the furrow cortex. Both the observations made in 
this paper and those of other workers who have concerned themselves with the de- 
gree of gelation of the egg cortex confirm this. The results of several workers are 
summarized below : 

Brown (1934) : Cortical pigment granules are especially resistant to displace- 
ment by centrifugation during the division phase. 

Chambers (1938) : The furrow cortex resists disintegration after the two in- 
cipient blastomeres have been punctured at the poles. 






FIGURE 46 

Brown and Marsland (1936) : There is a quantitative decrease in the gel value 
of dividing eggs as the hydrostatic pressure is increased. Under high pressures the 
furrow regresses. 

No one has yet recorded the effect of temperature, hypotonicity and lack of 
calcium ion upon the cortex of the dividing Arbacia egg, although these observations 
have been made upon the unfertilized egg. A brief summary of this work follows: 

Costello (1938) : It takes progressively longer to fragment the eggs as the tem- 
perature is lowered. 

Cole (1932) and Harvey (1943) : It takes longer to fragment eggs in hypotonic 
than in isotonic solutions. 

Harvey (1945) : Arbacia eggs break less readily in solutions possessing calcium 
ions than in solutions lacking calcium ions. 

These treatments (low temperature, hypotonicity and calcium ion) are pre- 
cisely the ones which favor the development of a cleavage stalk. It is possible that 



CLEAVAGE FURROW IN ARBACIA EGGS 



283 



these treatments may increase the elastic strength of the egg surface by toughening 
the extra cortical structures, but it is probable that they favor cortical gelation as 
well. 

Hypotheses concerning the mechanism of cleavage; surface tension 

Chambers and Kopac (1937) found that clean oil drops of the proper inter- 
facial tension with sea water, will coalesce spontaneously with a naked egg (Arbacia, 
Lytechinus, and Echinometra). They state: "The tendency to coalescence in the 
furrow and polar zones of cleaving eggs (late amphiaster and later) was investi- 
gated and no difference was found." They used oils whose approximate tensions 
in contact with sea water were 30, 10, and 3 dynes per cm. The fact that coalescence 
occurs at all indicates a fluid layer around the egg periphery. Spontaneous coales- 




FlGURE 47 



cence does not occur in Amoeba protcus (Kopac and Chambers, 1937), which in- 
dicates a non-fluid surface. In view of these observations any surface tension 
hypothesis is untenable. 

Subcortical currents 

Chambers (1938) has hypothesized that cleavage results from the activity of 
"the sub-cortical currents (which) sweep around the two asters and add gelating 
material to the inwardly growing cortex." In this hypothesis he combines his own 
observations with those of Schechtman (1937) on localized cortical growth during 
the cleavage of the amphibian egg. It was shown in the present paper (page 280) 
that normal furrowing may be associated with abnormal currents, which argues 
against the importance of such currents for division; moreover Lewis (1942) found 
no currents in the dividing fibroblast. 



284 ALLAN SCOTT 

Astral cleavage 

An astral theory of cleavage, much modified from Gray (1931), has been elabor- 
ated by Katsuma Dan (1943). He believes that the asters are composed of radiate 
fibers with intrinsic rigidity ; he considers them to be anchored to the cortex ; he 
believes that the rays cross at the equator ; and he believes that the spindle elongates 
autonomously. The following quotation (Dan 1943) summarizes his theory of 
cytokinesis : ". . . it was also shown that this concept of the mechanism of cell 
division is adequate to explain the stretching phase of the furrow surface. That is, 
when two such radiate asters are pushed apart, they can in turn, push the cell mem- 
brane of the polar region somewhat as a paint brush would push some object. As 
they travel away, how r ever, since they enclose the fluid endoplasm within the inter- 
spaces of their rays, the fluid endoplasm is carried away from the equatorial 
region and the cortex there is sucked in, giving rise to a furrow. The cortex is 
stretched as it is pulled in by the suction." 

The strength of Dan's hypothesis lies in its ability to explain the differential 
stretching and shrinkage of the surface which he and his coworkers observed (Dan, 
Yanagita, and Sugiyama, 1937; Dan and Yanagita, 1938; Dan, 1943) and for which 
no other explanation has been forthcoming. It appears that Dan's hypothesis will 
explain such unusual cleavages as are pictured in Figures 9 and 10 of the present 
paper. It could be assumed that one element of the tripolar spindle elongated be- 
fore the others causing the asters to move apart, and by the suction mechanism, caus- 
ing the development of the initial furrow (Fig. 9 at a). On this assumption the de- 
velopment of the other furrows (Figs. 9b and c ) begins later, presumably because 
the other two spindles begin their elongation later. The secondary furrow (Fig. 10 
at a') is presumably caused by the suction resulting from the separation of the lower 
two asters. Similar explanations would doubtless serve for the tetra-astral cleav- 
ages shown in Figures 23 and 24 of the present study. One can imagine also that 
the crossing rays from all four asters, if they became attached to the cortex, would 
explain the flattening of the upper and lower surfaces of the egg observed in Figure 
15. 

Dan's hypothesis is not in accord with the observations presented here concerning 
the continued elongation of the cleavage stalk in hypotonic sea water for it is impos- 
sible to see how the astral suction mechanism could explain the further constriction 
of a long, completely gelated stalk. 

The main weakness of the astral suction hypothesis lies in its limited scope. It 
fails to explain undoubted cases of anastral cleavage (tissue culture, for example) 
frequently noted in the literature. Dan's easy conclusion that all of these anastral 
cases are explainable by his astral suction hypothesis (". . . it is possible to imagine 
that in cells of the anastral type, similar gelation systems may be existing although 
they cannot be discerned morphologically") is unconvincing. 

One of Wilson's observations is discordant with Dan's hypothesis. Wilson ob- 
served, in a form which normally has asters, that a spindle need not be present for 
complete cleavage to occur. He found that a cleavage furrow may cut in around 
the base of an isolated aster and result in a complete cleavage. Compare Wilson 
(1901), page 376 and Figure 11. 

In one of Chamber's microdissection experiments he bisected the partially cleaved 
egg in a plane at 45 to the plane of the furrow (1924, Fig. 36). The cut resulted 



CLEAVAGE FURROW IN ARBACIA EGGS 

immediately in two cells. However, the original furrow remained on each artificially 
produced blastomere and, on each, the furrow gradually cut through forming two 
small "cells" as well as two large ones. This continued cleavage seems to be quite 
unexplainable by Dan's hypothesis which requires crossed astral rays, an elongat- 
ing spindle and a suction produced by the separation of the asters. 

Cortical grozt'th or cortical contraction? 

Schechtman has proposed another theory of the mechanism of cytokinesis. He 
suggested (1937) that the furrow cortex grows by the "intussusception of clear 
cytoplasm," but simple growth of the equatorial cortex would not be expected to cut 
the egg in half. Other factors must account for the inwardly directed furrow and 
its narrowing. It seems clear that there is a stretching of the egg cortex at the time 
of furrowing as concluded by Dan et al. (1937, 1938), by Schechtman (1937) and 
by Motomura (1940), but whether the stretching is active (the result of growth) 
or whether it is passive and due rather to a contracting ring at the head of the fur- 
row (Lewis, 1942), is not easy to decide. Schechtman is of the opinion that 
"Cleavage is initiated by a contraction of the egg cortex at the site of the future 
furrow." And he notes that the "'Cortex becomes thicker and bulges toward the 
egg interior." He therefore uses both contraction and cortical growth in his com- 
plete hypothesis. The observations made in this paper on the continued constric- 
tion of small stalks after they consist entirely of gelated material are taken as strongly 
favoring the constricting ring theory of Lewis. For if the gelated stalk is able to 
contract at that late stage of cleavage, it seems reasonable to suppose that it possesses 
contractile power earlier. The direction of contraction is ringwise about the equa- 
tor (Fig. 29c) and it is to be expected that such contraction would draw stained 
areas out into fine lines as Schechtman observed, if such areas are located in the fur- 
row or subfurrow region. 

It would be illuminating to know whether or not kaolin particles placed around 
the equator would be brought closer together during the furrowing but no one has 
made these observations. 

Amoeboid activity and bleb formation 

One can scarcely observe the amoeboid behavior of eggs in hypotonic media and 
particularly the "normal" false cleavages which occur during the amoeboid phase 
preceding pronuclear fusion in the nematode egg (Spek, 1918), without being con- 
vinced that a fundamental similarity exists between amoeboid motion and cleavage. 
Moreover the abscission of blebs is strikingly similar to cleavage. 4 It is suggested 
that any deforming force which establishes an isthmus about the cell or a part of 
the cell will result in the development of a contracting ring disposed around the 
isthmus, provided that the egg is in the cleavage phase. This view would explain 
why the normal egg, deformed by the elongating spindle, cleaves at the equator. It 
would explain why cleavage planes cut in around the base of cytasters which are 
unconnected to a spindle (Wilson, 1901) and it would explain why blebs formed in 

4 Very recently Holtfreuter (1946) has suggested "that in normal cytoplasmic division the 
activity of the nucleus and of the endoplasm are of a mere secondary importance." He observed 
that isolated, embryonic amphibian cells may develop annular constrictions which lead to the 
fragmentation of the cell. He considers, however, that the contraction occurs in the membrane 
rather than in the plasmagel layer. 



286 ALLAN SCOTT 

the sub-furrow region may cut off from the remainder of the egg as reported above. 
This hypothesis also agrees with the idea that the enlarging gelated asters play a 
mechanical role in localizing the furrow. 

SUMMARY 

1. Under certain conditions the eggs of Arbacia punctulata develop a cleavage 
stalk between the first two hlastomeres. No stalk forms in sea water if the tempera- 
ture is in the 20 C. to 30 C. range; low temperature (10 C.) causes the develop- 
ment of a stalk in sea w r ater; a short stalk develops in isotonic calcium-free sea 
water at 20 C. ; a very long stalk develops if eggs are cleaving in hypotonic sea 
water (65 per cent). 

2. The effect of the above treatments on the appearance of cleaving dispermic 
eggs is described. 

3. Evidence indicates that stalks of 8 micra diameter are all gel, yet in hypotonic 
sea water they continue to constrict and elongate. This is good evidence that the 
cortical gel has inherent contractile properties. 

4. It is hypothesized that any event which deforms the Arbacia egg (if it is in the 
"cleavage phase") leads in some way to an orientation of contraction around the 
isthmus. The deforming force may be an enlarging aster, an elongating spindle, or 
an endoplasmic flow. 

LITERATURE CITED 

BEAMS, H. W. AND R. L. KING, 1937. The suppression of cleavage in Ascaris eggs by ultra- 

centrifuging. Biol. Bull, 73: 99-111. 

BOWEN, R. H., 1920. Studies on insect spermatogenesis. I. Biol. Bull., 39: 316-362. 
BROWN, D. S., 1934. The pressure coefficient of "viscosity" in eggs of Arbacia punctiilata. 

Jour. Cell. Comp. Physiol., 5 : 335-346. 
BROWN, D. S. AND D. A. MARSLAND, 1936. The viscosity of Amoeba at high hydrostatic 

pressure. Jour. Cell. Comp. Physiol., 8: 159-165. 
CHAMBERS, R., 1924. The physical structure of protoplasm as determined by micro-dissection 

and injection. General cytology, pp. 237-309. The University of Chicago Press. 
CHAMBERS, R., 1938. Structural and kinetic aspects of cell division. Jour. Cell Comp. Physiol., 

12: 149-165. 
CHAMBERS, R. AND M. S. KOPAC, 1937. The coalescence of sea urchin eggs with oil drops. 

Ann. Report Tortugas Laboratory, Carnegie Inst. of Washington. No. 36: 88. 
COLE, K. S., 1932. Surface forces on the Arbacia egg. Jour. Cell. Comp. Physiol., 1 : 1-9. 
COSTELLO, D., 1938. The effect of temperature on the rate of fragmentation of Arbacia eggs 

subjected to centrifugal force. Jour. Cell. Comp. Physiol., 11 : 301-307. 
DAN, K., 1943. Behavior of the cell surface during cleavage. VI. On the mechanism of cell 

division. /. Fac. Sci., Tokyo Imp. Univ., Sec. IV, 6 : 323-368. 
DAN, K., J. C. DAN AND T. YANAGITA, 1938. Behavior of the cell surface during cleavage. 

II. Cytologia, 8: 521-531. 
DAN, K., T. YANAGITA AND M. SUGIYAMA, 1937. Behavior of the cell surface during cleavage. 

I. Protopl., 28 : 66-81. 

GRAY, J., 1931. A text book of experimental cytology. Cambridge University Press. 
HARVEY, E. B., 1943. Rate of breaking and size of "halves" of the Arbacia punctulata egg 

when centrifuged in hypo- and hyper-tonic sea water. Biol. Bull., 85: 141-150. 
HARVEY, E. B., 1945. Stratification and breaking of the Arbacia punctulata egg when centri- 
fuged in single salt solutions. Biol. Bull., 89 : 72-75. 

HOLTFREUTER, JOHANNES, 1946. Structure motility and locomotion in isolated embryonic am- 
phibian cells. Jour. Morph., 79: 27-62. 
ISHIDA, JURO, 1936. An enzyme dissolving the fertilization membrane in sea urchin eggs. 

Annotationes Zoologicae Japoncnses, 15 : 449-459. 



CLEAVAGE FURROW IN ARBACIA EGGS 287 

KOPAC, M. S. AND R. CHAMBERS, 1937. The coalescence of living cells with oil drops. Jour. 

Cell. Comp. Physiol, 9: 345-361. 
LEWIS, W. H., 1942. The relation of the viscosity changes of protoplasm to amoeboid motion 

and cell division. The structure of protoplasm, pp. 163-197. Iowa State College Press. 
MARSLAND, D., 1942. Protoplasmic streaming in relation to gel structure in the cytoplasm. 

The structure of protoplasm, pp. 127-161. Iowa State College Press. 
MOORE, A. R., 1930a. Fertilization and development without membrane formation in the egg of 

the sea urchin Strongylocentrotus purpuratus. Protopl., 9: 9-17. 
MOORE, A. R., 1930b. Fertilization and development without membrane formation in the egg of 

Dendraster eccentricut. Protopl., 9 : 18-24. 
MOSER, F., 1940. Studies on a cortical layer response to stimulating agents in the Arbacia egg. 

III. Biol. Bull., 78: 68-91. 
MOTOMURA, I., 1934. On the mechanism of fertilization and development without membrane 

formation in the sea urchin egg, with notes on a new method of artificial partheno- 
genesis. Science Reports of the Tohoku Imp. Univ., 4th scries, Biol., 9: 33-45. 
MOTOMURA, I., 1940. Studies of cleavage. I. Science Reports of the Tohoku Imperial Unir., 

4th series, Biol., 15: 123-130. 
SCHECHTMAN, A., 1937. Localized cortical growth as the immediate cause of cell division. 

Science, 85: 222-223. 
SHAPIRO, H. H., 1941. Centrifugal elongation of the cell and some conditions governing return 

to sphericity, and cleavage time. Jour. Cell. Comp. Physiol., 18 : 61-78. 
SMITH, H. W., AND G. H. A. CLOWES, 1924. The influence of hydrogen ion concentration on 

the fertilization process in Arbacia, Asterias and Chaetopterus eggs. Biol. Bull., 47: 

333-344. 
SPEK, J., 1918. Die amoboiden Bewegung und Stromungen in die Eizellen einiger Nematoden 

wahrend der Vereinigung der Vorkerne. Arch. f. Entw. Mcch., 44: 217-254. 
WILSON, E. B., 1901. Experimental studies in cytology II and III. Arch. /. /'. Mech., 

13 : 353-395. 



DEVELOPMENTAL RELATIONS BETWEEN GENITAL DUCTS 
AND GONADS IN DROSOPHILA 

DIETRICH BODENSTEIN 

Medical Division, Edge-wood Arsenal, Maryland 

While studying the reproductive system of Drosophila sinmlans gynandromorphs 
Dobzhansky (1931) made the following interesting observation: If female genital 
ducts and testes were present in the same individual and if the female ducts were 
attached to the testes, the latter underwent extreme degeneration. Yet, the attach- 
ment of male genital ducts to ovaries did not affect the development of these organs. 

Now we know that in normal development the attachment of the ducts to the 
gonads takes place during the early period of pupal life. We know further that by 
transplantation of gonads from one individual to another it is possible to obtain at- 
tachment of the transplanted organ to the host ducts. This knowledge makes it 
possible to attack experimentally the question whether the degeneration of testes 
when attached to the female ducts, as observed in Drosophila sinmlans gynandro- 
morphs, is a peculiarity of this special case, or whether the phenomenon is a general 
one and always occurs when female ducts and testes are brought into contact with 
each other. 

The problem can be approached experimentally in two ways : the larval testes 
can be transplanted into female host larvae or the female genital disc from which 
the ducts originate can be transplanted into male larvae. By transplanting two or 
three testes into one host, the chance that one transplantal will attach itself to the 
host duct is quite good. The chances for attachment of the testes to the female duct 
are even better when the six oviducts which arise by outgrowths from the three 
transplanted imaginal discs compete with the two host ducts for attachment. In 
the following studies both these methods were used. 

EXPERIMENTAL 

Transplantation of testes into female ducts 

Two or three testes of mature virilis larvae were transplanted together into the 
abdominal cavity of female host larvae of the same age. After the hosts had 
emerged, the condition of the transplants and their relationship to the female genital 
system was studied in careful dissections. This series consisted of ten cases. The 
following was found. All transplants had failed to assume their characteristic spiral 
shape. This was to be expected, since the work of Dobzhansky (1931) and Stern 
(1941a and b) had shown that the testes have to be attached to the vas in order to 
accomplish their spiral growth. In six out of ten cases one of the transplanted 
testes had attached itself to one of the oviducts of the hosts. The attached testis was 
always greatly reduced in size and appeared degenerate. Figure 1 shows camera 
lucida drawings of five representative cases of this series. It will be noted that the 
degenerative reduction occurs only when the testis is attached to the oviduct of 

288 



DEVELOPMENT IN DROSOPHILA 



289 



the host (Fig. I A, B, 1) and E). Testes that lie free in the body cavity (Fig. IA 
and B) or testes that have been attached to the ovary (Fig. 1C and B) are un- 
affected. Thus the principle which produces degeneration is apparently given off 
only by the oviducts and depends for its action on a close cellular contact with the 
testes. This principle, moreover, seems unable to penetrate larger cell barriers, for 
testes which were connected to ovaries which in turn had their normal oviduct con- 
nection remained unaffected (Fig. 1C). Yet two testes which had established close 




FIGURE 1. The spatial and developmental relations of transplanted testes to the reproductive 
system of their female hosts. O, ovary ; OD, oviduct : 7\ to T x , transplanted testes. 

contact with each other had both suffered degenerative reduction, although only one 
of these fused organs has actually established contact with the oviduct (Fig. IA 
and E). 

Transplantation of female genital discs into male hosts 

In a second series of experiments, two or three female genital discs from mature 
virilis larvae were transplanted together into the body cavity of hosts of the same 
age. The condition of the host testes and their relationship to the transplanted 
female structures was again studied by dissection. Several of the affected testes 



290 



DIETRICH BODENSTFIN 



were also sectioned and studied histologically. Thirty successful cases were avail- 
able for investigation. In seven of these cases, the host testes were not connected 
to the transplanted ducts, although the latter had developed well and were found 
in the immediate neighborhood of the male gonads. The testes of these seven hosts 
were normal in size, shape, and histology. One testis in each additional animal was 
not connected with the vas of the host, nor to any of the transplanted ducts. These 
testes were not coiled but were otherwise normal. The other testis in two of these 
individuals was connected to the vas of the host and was normal, while the other 
testis of the third individual, although connected to the vas, was not coiled. The 
non-coiling of an attached testis is rare, but has been observed at times in otherwise 
normal animals. Whether the inabilitv of attached testes to coil is a result of faultv 

* * 

connections with the vas or whether the vas in these cases has lost its growth in- 
ducing capacity is not known. 

TABI.K I 

Transplantation of female genital ducts into mule hosts 



Number of 
discs trans- 
planted 


Testis free (round) 


Testis (spiral). 
Normal attached 


Testis attached 
to 9 duct 


State of degeneration of testis 
when attached to 9 duct 


One side 


Both sides 


One side 


Both sides 


One side 


Both sides 


One side 


Other side 


3 






Yes* 




Yes 




+ 




3 






Yes 




Yes 




+ + + + 




3 






Yes 




Yes 




+ + + + 




3 






Yes 




Yes 




+ + + + 




3 








Yes 










3 








Yes 










3 






Yes 




Yes 




+ + + + 




2 






Yes 




Yes 




+ + + 




2 






Yes 




Yes 




+ + + + 




2 












Yc-s 


+ + + + + 


+ + + + + 


2 


Yes 




Yes 








+ 




3 






Yes 




Yes 




+ + 




3 




. 




Yes 










3 






Yes 




Yes 




+ + + + 




3 






Yes 




Yes 




+ + + + +.+ 




3 






Yes 




Yes 




+ + + + + 




2 








Yes 










2 






Yes 




Yes 




+ + + 




2 




Yes 














2 




Yes 














2 




Yes 














2 








Yes 










2 








Yes 










2 












Yes 


+ + + + + 


+ + + + + 


2 






Yes 




Yes 




+ + + + 




2 












Yes 


+ + + 


+ + + + + 


2 h. 








Yes 










2 h. 






Yes 




Yes 




+ + + + 




2 h. 






Yes 




Yes 




+ + + + 




2 hv. 






Yes 




Yes 




+ + + + 





h. = hydei discs into hydei hosts, 
vas, but not spiral. 



hv. = hydei discs into virilis host. * = testis attached to 



DEVELOPMENT IN DROSOPHILA 



291 



IT- 







u 13 

II 

2 
o 5 



t.S 

3 u 



1- C. 

u * 



u 3 

t * 



rt 



rt 

5 

(L) 



CD 



I'i 



CL- 






i -*- 1 . 

Cr^ 03 

H -t- 

rt rt a. 



_ 
t -*- 1 rt 



OJ L- (/) 

p o c 



O rt 



- P 
cr ^ nf 

>rt Nv 
"T^ E 



rt M-, 
rt t : 



H t<-c 

o ;/. 

rt 

^ > 



<n c 

-t- 1 ^ 

O J? 



DIETRICH BODENSTEIN 

In twenty individuals one or both totes were connected to the oviduct of the 
transplant. In some cases one testis was even found to have connection with the 
oviducts of two discs. All testes that were attached to oviducts showed a more or 
less pronounced degree of reduction and appeared degenerate. Table I summarizes 
'the results of this experimental series. The state of degenerative reduction of the 
testes in this table is indicated by crosses. One cross signifies slight ; six crosses, 
extreme size reduction. Figure 2 shows camera lucida drawings of four representa- 
tive cases of this series. Figure 2A is a case listed in Table I having one cross. 
Figure 2C is listed by having six crosses and Figure 2D and B are listed by having 
five crosses each. Figure 3 illustrates by microphotography an extremely reduced 
testis. 




> 
t 





B 



FIGURE 3. A, normal adult testes. B, (arrow) testis of the same individual degenerated under 

the influence of a transplanted and attached oviduct. 

It will be noted from Table I that the reduction of the testes is in most cases 
very pronounced. Only six of the testes attached to oviducts were reduced to state 
"3" or less, while sixteen testes were degenerated to state "4" or more. 

This experimental series thus confirms strikingly the original observation that 
testes attached to female oviducts suffer degenerative changes and that it is the 
oviduct that elicits the principle causing degeneration. The observation from the 
previous experiments that the presence of oviducts f>cr sc has no effect on testis 
development is also confirmed, for testes in the presence of as many as three pairs 
of oviducts in the immediate organic environment remained normal if they were not 
attached to the oviduct. In comparing the attached testes of the two series with 
each other, a difference in their general shape was noted. While the transplanted 



DEVELOPMENT IN DROSOPHILA 



293 



testes in the first experimental group were small, roundish bodies, the testes in the 
second experimental group were in most cases thin and elongated in shape (com- 
pare Fig. 1 with Fig. 2). Now it was found in the second group that in all cases 
when the attached testis was thin and elongated it was attached not only to the trans- 
planted oviduct but also to the vas efferens of its host (Fig. 2). In those cases, 
however, where the testis attached only to the transplanted oviduct, had not estab- 
lished connection with the vas, it was roundish. This situation is well illustrated 
in Figure 2C. The left testis in this case, a small roundish degenerated organ, is 
attached only to the oviduct while the right testis. which is attached to the vas ef- 
ferens of the host and to one transplanted oviduct, has become an irregularly elon- 
gated structure. The elongated shape of such a degenerate testis is thus due to the 
stimulating influence of the vas on the growth of the testis, which in normal develop- 
ment leads to the coiling of this organ, while the observed degeneration is caused 
by the influence of the oviduct. 





FH.IUE 4. Sections through two extremely reduced testes. </, degenerating cells. 



Not only virilis but also hydei oviducts cause degeneration of hydei testes at- 
tached to them. This was shown by three cases in which larval hydei female discs 
were transplanted into hydei male larvae (see Table I). 

The factor in the oviduct causing degeneration of the testis by contact is not 
species specific, for hydei oviducts will cause virilis testes to degenerate (see Ta- 
ble I). 

Sections of reduced testes were made and their histology studied. It was found 
that, depending upon the degree of reduction of size, the testes contained various 
amounts of spermatogonia and spermatocytes in all stages of degeneration. The 
remnants of disintegrated cells in the form of granular picnotic masses together with 
quite normal appearing cells were observed. Figure 4 shows the condition found 
in an extremely reduced testis. 

CONCLUSION AND SUMMARY 

By transplanting female genital discs into male hosts, attachment to the host 
testes of oviducts developed from transplanted genital discs is obtained. In these 
cases the attached testes suffer extensive degeneration. Only cellular contact of 



294 DIETRICH BODENSTEIN 

the oviducts to the testes brings about this phenomenon. Unattached female ducts 
do not affect the development of the testis. The principle causing degeneration is 
not species specific. The findings indicate that the phenomenon encountered is no 
unique instance, but representative when oviduct and testis establish cellular contact 
during pupal development. 

LITERATURE CITED 

BODENSTEIN, D., 1946. The post-embryonic development of Drosophila. In Biology of Dro- 
sophila. Pub!. Carnegie lust. IT ash. (in press). 

DOBZHANSKY, T., 1931. Interaction between female and male parts in gynandromorphs of 
Drosophila simulans. Roux' Arch. Entzv. Mech., 123: 719-746. 

STERN, C, 1941a. The growth of testes in Drosophila. I. The relation between vas deferens 
and testis within various species. Jour. E.\-p. ZooL, 87: 113-158. 

STERN, C., 1941b. The growth of testes in Drosophila. II. The nature of interspecific- 
differences. Jour. Exp. ZooL, 87: 159-180. 



cV 1 

r^Xo* s *; 

( L I B ? ( A R V 



J*4S' 



A HISTOLOGICAL STUDY OF SYNDISYRINX FRANCISCANUS. 

GEN. ET SP. NOV., AN ENDOPARASITIC RHABDOCOEL 

OF THE SEA URCHIN, STRONGYLOCENTROTUS 

FRANCISCANUS 1 

H. E. LEHMAN 

Department of Zoology of the I'nhrrsity of Xorth Carolina - 

I NTRODUCTION 

Up to the present time eight genera of worms endoparasitic in echinoderms and 
sipunculids have been described that belong to the rhabdocoel family Umagillidae 
Wahl, 19101). Schneider described the first species, Anoplodinm parasita, in 1858. 
Since then six questionable and three valid species of this genus have been re- 
ported from widely separated localities as parasites of holothurians. Their distribu- 
tion extends from the Mediterranean, Ionian, and North Seas to Japan and the 
Philippines (Bock, 1926). Syndesinis ccliinoniin Francois, 1886, the only spe- 
cies of the genus, is found in echinoids. It has been collected in the Mediterranean 
(Russo, 1895), Norway (Westblad. 1926), and the English Channel (Braun. 1889). 
Three species of the genus C'olUishuna are found in sipunculids at Roscoff (Dorler, 
1900), the Gulf of Kola (Beklemischev, 1916), and the Bay of Naples (Wahl, 
1910a). The genus Dcsiuotc is represented by one species, D. vora.v, discovered in 
a crinoid collected in the Gulf of Kola (Beklemischev, 1916). A single species 
parasitic in holothurians has been described in each of four genera, i.e.. a Japanese 
form, Xcnoiuctra arbor a Ozaki, 1932, and three reported from the'coa.st of Nor- 
way, Wahlia tiiacrostylijera Westblad. 1930, Anoplodlcra valuta \Yesthlad. 1930. 
and type genus Uiiiagilla forskalensis Wahl, 1909. 

The only reference to a member of the Umagillidae from the Western Hemis- 
phere was made by Powers in 1936. He reported the presence of a Syndesmis- 
like worm in the coelomic cavity of the echinoid, Ccntrccliinns antillantin. at Tortu- 
gas. A complete description was not given; however, as compared with Syndesinis. 
noticeable differences were observed in details of the copulatory apparatus and the 
arrangement of the shell glands. While the endoparasitic rhabdocoel of Sirongylo- 
centrotiis jranciscanns. the large common sea urchin of the California coast, is well 
known to some investigators who have worked at Pacific Grove, a description of 
tli is worm has not been recorded in the literature prior to the present account. 

1 This work was done at the Wilson Zoological Laboratory of the University of North 
Carolina in partial fulfillment of the requirements for the degree of Master of Arts. The 
author is indebted to Professor D. P. Costello for suggesting the problem, for the slide prep- 
arations upon which this study was essentially based, and for the invaluable suggestions and 
criticisms rendered during the preparation of this paper. The author wishes to acknowledge his 
appreciation to Dr. L. H. Hyman for many valuable recommendations and for permission to 
introduce her revised and hitherto unpublished terminology relating to this group. To Miss 
Catherine Henley the .author expresses his gratitude' for the translation of a number of the 
references cited herein. 

- Now in the School of Biological Sciences of Stanford University. 

295 



296 H. E. LEHMAN 

Systematic position :; 

Order Rhalxlocoela 

Suborder Lecithophora 

Section Dalyellioida 

Family Umagillidae Wahl, 1910 

Subfamily Umagillinae Wahl. 1910 

Genus Syndisyrinx. gen. nov. 
Genotype Syndisyrinx franciscanus, sp. nov. 

Holotypc. A whole mount in the United States National Museum, Washington 
D. C. 

Repositories oj type material. In each of the following repositories a whole 
mount, a transversely sectioned, and a sagittally sectioned preparation selected from 
the type material have been deposited : U. S. National Museum, Washington. D. C. ; 
American Museum of Natural History, New York City ; British Museum. London ; 
California Academy of Science, San Francisco ; Wilson Zoological Laboratory of 
the University of North Carolina ; and Museum of Natural History, Stanford Uni- 
versity. Additional preserved material may be obtained from the author or from any 
of these institutions. 

Type locality. Mussel Point. Monterey Peninsula. California, Lat. 36. 37', 
20" N., Long. 121, 54', 15" W. 

Collectors. D. P. Costello, 1937 and H. E. Lehman, 1945. 

Distinguishing characteristics. Umagillinae with a single intestine, paired and 
branched ovaries, cuticular penis, and a bursa seminalis connected by cuticular ducts 
to the seminal receptacle and bursal canal. 

MATERIALS AND METHODS 

Fifty-four rhabdocoel parasites were obtained from two specimens of the sea 
urchin Strongylocentrotus franciscanus (A. Agassiz) by Dr. D. P. Costello in Au- 
gust 1937 at Pacific Grove, California. These specimens were fixed in Heath's, 
Boveri's, Lillie's and Worcester's solutions. Five of the individuals were sectioned 
serially at 10/i and stained with Heidenhain's iron hematoxylin and orange G. 
One of these preparations was exceptionally fine and the majority of the accompany- 
ing figures \vere made from it. Unfortunately this preparation, which the author 
intended to designate as the holotype, was lost when a microscope was stolen. 
This material, including the slide preparations, was turned over to me by Dr. Cos- 
tello. The morphological study was based on this material. 

In the summer of 1945 during June, July, and August, the author collected sev- 
eral hundred additional specimens from the same locality. Over sixty urchins were 
examined and all were found to be infested ; frequently three dozen or more parasites 
were obtained from the intestine of a single host. These worms were fixed in 
Heath's and Beauchamp's solutions. Seventy were sectioned serially at 10 ,u and 
stained with Mayer's acid hemalum and triosin. Thirty whole mounts stained 
with paracarmine were also made. The type material was selected from these prep- 

3 Classification according to Bresslau (1933), with the exception of "Family Anoplodiidae 
Graff, 1913," which has been rejected in favor of "Family Umagillidae Wahl, 1910b," inasmuch 
as no reason is given by Graff for discarding the older name or for selecting Anoplodium as 
type genus. The subfamily Umagillinae has been retained as designated by Wahl, 1910b. 



A PARASITIC RHABDOCOEL 297 

parations. At this time another parasite of Str. jranciscanus was discovered which 
differed from Syndlsyrin.v in shape, manner of locomotion, and color. A description 
of this worm is being prepared and preliminary examination of sectioned material 
indicates a close relationship to Syndesmis cchinorum. Upon the suggestion of 
Prof. A. R. Moore, who had occasionally observed parasitic worms in Str. purpur- 
atus (Stimpson), forty-seven of these urchins were examined. In twenty-nine of 
them, worms that are very similar to, and may be identical with Syndisyrin.r fran- 
ciscanits were present in small numbers. 

GENERAL MORPHOLOGY 

The living animals are bright red with a dark brown or yellow median longi- 
tudinal line which marks the extent of the intestine. The worms are flattened 
dorsoventrally and have a leaf-like appearance, being rounded at the anterior end 
and slightly pointed posterad. Individuals vary in size from 2 to 3 mm. long and 
1.6 to 2.5 mm. wide. The body is thickest at approximately one-fourth of the dis- 
tance from the anterior end and at this level measures about 0.5 mm. in the dorso- 
ventral axis. Laterally and posteriorly the thickness of the body diminishes gradu- 
ally to about 0.2 mm. at the periphery. A ciliated epithelium covers the entire 
surface ; rhabdites and cuticle are lacking. 

The mouth is situated on the ventral surface about one-fourth of the distance 
from the anterior end and a common genital pore opens ventrally at the posterior 
extremity of the body. The musculature and parenchyma are typical of other 
Umagillidae. No excretory system was observed. The strongly muscular pharynx 
is typically doliiform and possesses pharyngeal glands ; it communicates by a short 
oesophagus with the gut. The intestine, possessing a number of small lateral diver- 
ticula, extends posterad under the dorsal epidermis along the mid-line and termi- 
nates one-quarter of the distance from the posterior end of the body. The gut con- 
tains no permanent lumen and food masses lie in temporary cavities surrounded by 
large digestive cells. The brain, composed of two cerebral ganglia connected by a 
wide commissure, lies anterior to the pharynx and gives off paired anterior, lateral, 
and posterior nerves. 

Lobed testes lie lateral to the mid-line in the anterior half of the body. Acces- 
sory glands empty into the sperm duct that arises from each testis and passes 
anteracl. These paired tubes unite mesially and enter a small spermiducal vesicle 
that is continued posterad as a muscular common sperm duct which lies dorsal to 
the uterus along the mid-line. This tube terminates in an elongated cuticular stylet, 
the penis, which is enlarged and funnel-like at the base. The penis stylet enclosed 
in the male antrum extends through the posterior third of the body to the common 
genital antrum and over most of its length does not exceed 3 p. in diameter. 

Paired vitellaria are found immediately posterior to the testes ; they are greatly 
ramified and fill most of the ventrolateral spaces in the middle third of the body. 
Posterior to the vitellaria a pair of ovaries is located, one on each side of the 
mid-line. Laterally each branches into five or more finger-like lobes. Three or 
four collecting ducts from the vitellaria empty with the ovaries and seminal recep- 
tacle into the anterior end of the ovovitelline duct. The seminal receptacle is oval 
and filled with sperm. Located posterodorsad to this organ is a vesicular, sperm- 
filled bursa seminalis connected to the seminal receptacle by a fine cuticular insemi- 



298 



H. E. LEHMAN 



ph.g. 




.5 



bur. c. 



FIGURE 1. Semidiagrammatic median sagittal section. 



A PARASITIC RHABDOCOEL 299 

nation canal. Arising in close association with this tubule is a similar duct, the 
cuticular proximal part of the bursal canal that passes posterad from the bursa 
seminalis approximately 60 p, before widening into the posterior muscular portion 
of the bursal canal (vagina). A cuticular sheath surrounds the openings of these 
two ducts into the bursa seminalis. The composite structure, consisting of this 
sheath and the canals passing through it, makes up the bursal valve. 

An ovovitelline duct, into which accessory glands empty, arises ventrally at the 
anterior end of the seminal receptacle. It passes posterad and unites with the female 
antrum. The uterus, lying close to the ventral epidermis, extends anteriorly from 
the female antrum almost to the pharynx. At the anterior end of the uterus an egg 
capsule containing from one to five ova and numerous yolk cells is generally found. 
The capsule is continued posterad as a long coiled whip similar to those found in 
related forms. Most of the ventrolateral spaces of the posterior third of the body 
are filled by cement glands ; they communicate by many small ducts with the female 
antrum. The common genital antrum is an elongated cavity at the posterior end 
of the body into which the female antrum enters ventrally, the male antrum and penis 
open mesially and the bursal canal is given off dorsally. At its posterior end is the 
common genital pore which opens ventrally to the exterior. 

HlSTOLOGICAL STRUCTURE 

Epidermis 

A ciliated epithelium covers both dorsal and ventral surfaces of the body. No 
pigment or special gland cells were observed in this layer and a cuticle and rhabdites 
are lacking. The cytoplasm of the cells in the epidermal layer is granular and cell 
boundaries, though faintly stained, are distinct. The cells covering the dorsal sur- 
face are cuboidal and measure 10 /A from basement membrane to external surface. 
The cytoplasm of these cells stains moderately with hematoxylin. On the ventral 
surface the cells are flattened and are about 7 /A thick and from 12 to 35 ^ wide; 
they have little affinity for hematoxylin. Cilia of the ventral epidermis are about 
6.5 p. long and are almost twice the length of those found on the dorsal surface. 
Cells possessing the staining properties and short cilia characteristic of the dorsal 
layer extend for a short distance ventrally around the lateral edges. A zone 4 to 6 
cells wide of intermediate nature accomplishes the transition between typical dorsal 
and ventral epithelium. 

Musculature and parenchyma 

The arrangement of the musculature is essentially the same as that described for 
other Umagillidae. Under the basement membrane of the surface epithelium is 

Abbreviations for Figures 1 and 2. 

a.o.d. accessory glands of ovovitelline duct, a.s.d. accessory glands of sperm duct, br. 
brain, b.c. buccal cavity, bur. c. bursal canal, bur. c'. cuticular end of bursal canal, b.s. 
bursa seminalis, b.v. bursal valve, e.g. cement glands, c.s.d. common sperm duct, e.c. egg 
capsule, f.a. female antrum, g.a. common genital antrum, g.p. genital pore, int. intestine, 
i.e. insemination canal, 1. int. lumen of intestine, m.a. male antrum, oe. oesophagus, ov. 
ovary, ov'. ovum, o.d. ovovitelline duct, p. penis, p'. base of penis, ph. pharynx, ph. g. 
pharyngeal glands, s.d. sperm duct, s.r. seminal receptacle, s.v. spermiducal vesicle, te. 
testis, u. uterus, vit. vitellaria, vit. d. vitelline ducts, w. whip of egg capsule, y. yolk cells. 



300 



H. E. LEHMAN 



c.s.d. 



a.s.d. 



vit. d. 




(7. O. d. 



g.a 



m 



-9-P- 2 

FIGURE 2. Semidiagrammatic median frontal section, intestine omitted. 



A PARASITIC RHABDOCOEL 301 

found a thin layer of suhepidermal muscles (Figs. 3-5, 7, 8). The superficial mus- 
cles are circular ; these overlie a longitudinal sheet, and interposed at intervals be- 
tween these layers are well-developed oblique fibers. In addition to these, bundles 
of fibers attached to the internal organs or the basement membrane of the epidermis 
pass dorsoventrally through the parenchyma (Figs. 1-3). The special muscles of 
the reproductive and digestive systems will be described in connection with the 
organs with which they are associated. 

A parenchyma, composed of large, irregularly shaped cells with coarsely granular 
or vacuolated cytoplasm, fills most of the spaces between the internal organs and 
epidermis. A histologically distinct parenchymatous mass of cells enclosed in a 
fibrous capsule extends posterad along the mid-ventral line from the posterior level 
of the pharynx to the region in which the female antrum enters the common genital 
antrum. The flattened, nonvacuolated cells of this tissue possess finely granular 
cytoplasm and are arranged in concentric layers around the reproductive ducts, most 
of which pass through the mid-ventral parenchyma (Figs. 3-5, 7). Nowhere within 
the parenchyma were flame cells or collecting ducts of an excretory system observed. 

Nervous system 

The brain is similar in all respects to those described in other members of the 
family. It is located just anterior to the pharynx and consists of two ganglia con- 
nected bv a wide commissure. Around the central fibrous mass of the brain are 

j 

numerous ganglionic cells that stain quite evenly with hematoxylin. Poorly devel- 
oped anterior, lateral and posterior pairs of nerves leave the brain and can be traced 
for short distances into the parenchyma. No theca separates the brain or nerves 
from the parenchyma and no special sensory organs were found. 

Digestive system 

The mouth lies on the ventral surface about one-fourth of the distance from the 
anterior end of the body. It opens into a very small buccal cavity lined by flattened 
ciliated cells that are continuous externally with the ventral epithelium (Figs. 1, 8). 
A sphincter underlying the epithelium regulates the size of the oral opening. Lying 
immediately dorsal to the mouth and opening into the buccal cavity is the doliiform 
pharynx which has the appearance of a dorsally compressed sphere. Its dorso- 
ventral axis is about 0.1 mm. long and its greatest diameter is about 0.17 mm. 
Passing dorsoventrally through the pharynx is a funnel-shaped lumen that is nar- 
rowest at the oral or ventral end. The musculature of the pharynx is similar in 
most details of its organization to that found in Syndcsuiis as described by Russo 
(1895). A thin superficial layer of vertical fibers overlies the well-defined muscles 
encircling the lumen of the pharynx. In addition to the circular and vertical mus- 
cles, radial fibers pass from the lumen to the peripheral surface of the pharynx. 
Nonmuscular cells with heavily staining reticular cytoplasm fill the spaces between 
the radial fibers (Fig. 8). Surrounding the pharynx is a sharply defined basement 
membrane to which are attached numerous short, radially arranged, protractor mus- 
cles that extend to the basement membrane of the ventral epidermis. The more 
oblique of these fibers serve also as dilators of the pharynx. Poorly developed re- 
tractors are attached to the equator of the pharynx and pass to the dorsal surface. 
Pharyngeal glands are present encircling the dorsal end of the pharynx. The 



302 H. E. LEHMAN 

peripheral contours of these glands are lobular and a thin basement membrane sepa- 
rates them from the parenchyma. The cells which make up these glands have in- 
distinct cell boundaries and dense cytoplasm containing numerous granules that 
stain darkly with hematoxylin. Cytoplasmic continuations of the cells extend ven- 
trally and line the lumen of the pharynx (Fig. 8). Leading dorsad from the 
pharynx is a short oesophagus which passes through the pharyngeal glands and 
opens into the anterior end of the intestine. 

The intestine lies along the mid-line under the dorsal epidermis and extends pos- 
terad from the level of the brain to about one-fourth of the distance from the pos- 
terior end of the body (Fig. 1). The width of the gut varies from 0.1 to 0.2 mm. 
at the anterior end and diminishes gradually posteriorly. Short diverticula ex- 
tend laterally on each side. The epithelium of the intestine is made up of large ir- 
regularly shaped cells containing moderately granular cytoplasm. The basal end 
of most cells reaches the fibromuscular investing sheath of the intestine that sepa- 
rates it from the parenchyma. The lumen of the intestine can only be observed when 
ingested material is present ; this condition is similar to that found in some alloeo- 
coels. In an animal that has been feeding, food masses often lie in cavities that have 
lost all direct communication with the oesophagus (Fig. 1). Food vacuoles of 
varying sizes are generally present in the cells surrounding the ingested material 
and digestive cells were occasionally observed that had apparently migrated into 
the food masses by amoeboid movement. 

Male reproductive system 

The paired testes lie lateral to the mid-line in the anterior half of the body. 
They are approximately 0.5 mm. long and from 0.3 to 0.5 mm. wide.. Each is made 
up of four to six vesicular lobes, the lumina of which are in direct communication 
with one another (Fig. 2). Separating the testes from the parenchyma is a fibrous 
sheath that pentrates and partially subdivides the lobes. The chambers so formed 
are filled with developing germ cells and tangled masses of mature spermatozoa 
(Fig. 3). Mature sperm are present in all lobes but are more numerous midway 
between the anterior and posterior ends of the testes near the wide openings of the 
sperm ducts. These ducts run mesially from the testes and enter the mid-ventral 
parenchyma, whereupon they diminish to about 10 //, in diameter and generally con- 
tinue their course anterad, dorsolateral to the uterus (Figs. 1-3). A thin epithelium 
surrounded by loose fibromuscular elements makes up the walls of the sperm ducts. 
Near the origin of these ducts from the testes, glandular cells that probably possess 
some accessory function are found in the mid-ventral parenchyma adjacent to the 
ventral walls of the tubes (Fig. 2). 

At varying distances posterior to the pharynx the sperm ducts unite mesially 
and enter the anterior end of a common sperm duct which at this point is somewhat 
enlarged to form a small spermiducal vesicle (Figs. 1-3). The slightly coiled com- 
mon sperm duct continues posterad from the vesicle through the mid-ventral paren- 
chyma. It gradually diminishes in diameter from 45 ^ to 12 (j.. Its walls are com- 
posed of connective tissue cells surrounded by a sheath of circular, oblique, and 
longitudinal muscle fibers. The lumen of the tube is lined by a thin squamous 
epithelium that is separated from the theca by a thick basement membrane. Pos- 
teriorly, the common sperm duct unites with the enlarged base of the penis at 



A PARASITIC RHABDOCOEL 303 

about one-third of the distance from the posterior end of the body (Figs. 1, 2, 4). 
The penis lies in a muscular sheath, the male antrum, which is a diverticulum of the 
genital antrum. Histologically this sheath is similar in most details of its structure 
to the common sperm duct ; however, the lining epithelium of the male antrum is 
thicker, in some regions almost occluding the lumen, and a thick basement mem- 
brane is lacking (Fig. 5). The copulatory organ is a cuticular tubule that extends 
through the posterior third of the body and is about 3 ^ in thickness over most of 
its length. The lumen of the stylet does not exceed 2 /* in diameter except at the 
anterior end of the penis which is enlarged to 12 p, at its union with the posterior end 
of the common sperm duct (Figs. 13). The rim of the funnel-like base of the 
penis is thickened to form a collar ; longitudinal muscles in the walls of the male 
antrum and common sperm duct attach to this collar and function as protractors 
and retractors of the penis. 

Female reproductive system 

The paired ovaries lie in the posterior third of the body. Each is made up of 
from five to ten lobes that branch dichotomously from common trunks arising 
near the anterior end of the seminal receptacle. The lobes of the ovaries are di- 
rected posterolaterad and are separated from the parenchyma by a very poorly de- 
veloped theca. The branches are made up of dovetailed chains or rouleaux of com- 
pressed ova that are proliferated from primordial cells at the distal ends of the lobes 
(Fig. 2). Mature ova are approximately 75 /A in diameter and vary in thickness 
from 20 to 60 \n. The cytoplasm of immature eggs is at first homogeneous, 'but as 
development continues many small peripherally distributed granules appear that 
are probably stored nutrient materials. During the period of growth the nuclei of 
the ova increase from 7 to 25 p. in diameter and the chromatin granules gradually 
lose their affinity for basic dyes. In mature ova only the spherical or oval nu- 
cleolus stains deeply with hematoxylin (Fig. 4). 

A pair of greatly branched vitellaria lie anterior to the ovaries and fill most of 
the ventrolateral spaces in the middle third of the body (Figs. 1, 2). Many of the 
dorsoventral muscles of the parenchyma contribute fibers to the diffuse sheath that 
encloses these ducts. Primordial cells at the distal ends of the branches give rise to 
yolk cells. As the cells increase in size, the cytoplasm which at first is homogeneous, 
becomes filled with refractile granules that coalesce to form amber-colored droplets 
(Figs. 3, 4). From each side three or four collecting ducts packed with mature 
yolk cells pass posterad from the vitellaria and unite near the mid-line shortly be- 
fore emptying into the anterior end of the ovovitelline duct (Fig. 2). 

The seminal receptacle is somewhat oval and lies ventral to the intestine within 
the sheath that surrounds the gut. Its anterior extremity is about one-third of the 
distance from the posterior end of the body. The posterior part of this organ is 
thin walled and masses of mature spermatozoa are observable in its extensive lumen. 
Anteriorly the seminal receptacle opens with the paired ducts of the vitellaria and 
ovaries into the ovovitelline duct which arises ventrally in this region (Figs. 1, 2). 
The wall of the anterior third of the seminal receptacle is lined by large gland-like 
cells that restrict the lumen to a narrow channel 6 to 10 /A wide which connects the 
posterior vesicular portion to the ovovitelline duct (Fig. 4). 

The bursa seminalis lies dorsal to the vesicular portion of the seminal receptacle. 
It is enclosed in the same sheath that surrounds the seminal receptacle and the pos- 



304 H. E. LEHMAN 

terior end of the intestine (Figs. 1, 2). The large lumen of the bursa seminalis is 
lined by an epithelial layer very similar to that lining the posterior part of the seminal 
receptacle. In every specimen examined spermatozoa were found in the bursa ; 
frequently they were aggregated into roughly spindle-shaped masses in which degener- 
ating sperm were observable (Figs. 5, 6). Arising ventrally, or in some cases later- 
ally, from the wall of the posterior half of the bursa seminalis is the insemination 
canal, a fine cuticular tubule about 4 ^ in diameter connecting the lumina of the 
bursa seminalis and seminal receptacle. In close association with the insemination 
canal, a second cuticular tube of the same dimensions arises from the wall of the 
bursa seminalis and connects the bursa posteriorly to the bursal canal (Figs. 1, 2, 
5, 6). Surrounding the ends of the ducts as they penetrate the lining epithelium of 
the bursa is a cuticular sheath, 7 /JL in diameter and 10 /x long. The inner end of 
this sheath is involuted and fused to the ends of the two ducts (Fig. 6). To desig- 
nate this composite cuticular structure made up of the insemination canal, the proxi- 
mal end of the bursal canal and the sheath surrounding the ends of these ducts, the 
term, "bursal valve," is suggested. 

The bursal canal (vagina) is a tubular structure about 0.1 mm. long and 20 ^ 
in diameter that arises as an anterodorsal continuation of the common genital an- 
trum. Its wall is composed of an inner epithelial layer surrounded by a strong 
fibromuscular sheath. At the posterior end of the canal the epithelium possesses 
cilia-like projections characteristic of the lining of the common genital antrum. 
Anteriorly the lumen of the canal is reduced and the thin basement membrane un- 
derlying the epithelium becomes continuous with the cuticular wall of the tubule 
leading into the bursa seminalis. 

A flattened muscular ovovitelline duct (ductus communis) arises ventrally near 
the anterior end of the seminal receptacle and receives the ducts of the ovaries and 
vitellaria. It passes posterad through the mid-ventral parenchyma to about the level 
of the posterior end of the bursa seminalis and here enters the anterior end of the 
female antrum (Figs. 1, 2, 5). The ovovitelline duct is approximately 35 ^ wide 
but is capable of considerable expansion to allow ova and yolk cells to pass into the 
uterus. Circular, oblique and longitudinal muscles are observable in contact with 
the thin basement membrane that underlies the lining epithelium ; no fibrous sheath 
separates this duct from the cells of the mid-ventral parenchyma. Running parallel 
to the ovovitelline duct in the lateral parenchyma are paired accessory glands which 
enter the posterior part of the duct prior to its union with the female antrum (Figs. 
1, 2, 5). Generally the cytoplasm of these gland cells stains evenly; however, in 
some cases the cells were observed to be filled with eosinophil granules. 

The uterus arises ventrally from the anterior end of the female antrum. It ex- 
tends anterad almost to the pharynx through the mid-ventral parenchyma and 

FIGURE 3. Transverse section through egg capsule and spermiducal vesicle (X350). 
FIGURE 4. Transverse section through entrance of ovary into seminal receptacle (X500). 

Abbreviations for Figures 3 and 4. 

a.o.d. accessory glands of ovovitelline duct, e.g. cement glands, e.c. egg capsule, int. 
intestine, mu. muscle sheath, mu'. subepidermal muscles, mu". dorsoventral muscles of 
parenchyma, ov. ovary, ov'. ovum, p'. base of penis, pa. mid-ventral parenchyma, s.d. 
sperm duct, s.r. seminal receptacle, s.v. spermiducal vesicle, te. testis, u. uterus, \v. whip 
of egg capsule, y. yolk cells. 



A PARASITIC RHABDOCOEL 



305 




.?s -ii/**' 4 -'.. . - 




FIGURES 3-4. 



306 H. E. LEHMAN 

through its entire course lies very close to the ventral surface of the body (Figs. 1, 
2, 4). The anterior end of the uterus is enlarged and encloses an amber-colored, 
oval egg capsule containing numerous yolk cells and from one to five spherical eggs 
.(Figs. 1, 2, 3). The egg capsule is cuticular and possesses a whip-like prolongation 
that extends posterad through the entire length of the uterus and female antrum. 
Over most of its length the whip is about 10 /A thick. In the middle portion of the 
uterus the whip is often coiled back upon itself a number of times so that its total 
length may greatly exceed that of the uterus (Figs. 1, 2). The uterine wall is very 
similar in structure to the ovovitelline duct and is able to enlarge greatly to accom- 
modate the egg capsule and the folded part of the egg whip (Figs. 3, 4). 

The female antrum extends from the posterior ends of the uterus and ovovitelline 
duct to the common genital antrum (Figs. 1, 2). The walls are lined by columnar 
epithelial cells surrounded by a thin basement membrane and a muscular layer that 
is continuous with the fibers enclosing the uterus and ovovitelline duct. The lu- 
men is about 12 /A in diameter and the posterior end of the egg whip, when present, 
almost completely fills this space (Figs. 5, 7). The ventrolateral spaces of the pos- 
terior third of the body contain numerous unicellular cement glands. The cyto- 
plasm of these cells is generally uniformly filled with small granules that have a strong 
affinity for hematoxylin. Throughout the entire length of the female antrum many 
ducts from these glands enter the lateral walls (Figs. 1, 2, 5, 7). The secretions of 
the cement glands are believed to be associated with the attachment of the egg cap- 
sules to the substrate when expelled. Living animals compressed under a cover 
glass were occasionally observed at low magnification to undergo a series of rapid 
contractions which resulted in the extrusion of the egg capsule and whip. How- 
ever, nothing is known about the normal deposition and attachment of the capsules, 
nor are other details of the life cycle understood. 

The common genital antrum lies at the posterior end of the body. It is an 
elongated tube lined by flattened cells that appear to have cilia about 20 // long which 
extend into the lumen (Figs. 1, 2, 7). A diffuse fibrous sheath separates this or- 
gan from the parenchyma. The common genital antrum receives the terminal 
ducts of both male and female reproductive systems : the bursal canal arises from it 
as a dorsal diverticulum ; the male antrum enclosing the penis stylet is given off as 
a mesial evagination ; and the female antrum enters it ventrally. The common geni- 
tal pore opens on the ventral surface at the posterior end of the body. At this point 

FIGURE 5. Transverse section through bursa seminalis and bursal valve (X350). 
FIGURE 6. Bursal valve (X 1,050). 

FIGURE 7. Transverse section through the entrance of female antrum and male antrum 
into the common genital antrum (X 350). 

FIGURE 8. Transverse section through pharynx (X 200). 

Abbreviations for Figures 5 through 8. 

a.o.d. accessory glands of ovovitelline duct, a.o.d'. ducts of accessory glands of ovovitel- 
line duct, b.c. buccal cavity, bur. c. bursal canal, bur. c'. cuticular end of bursal canal, b.s. 
bursa seminalis, b.v. bursal valve, cil. cilia, e.g. cement glands, d.c.g. ducts of cement 
glands, f.a. female antrum, g.a. common genital antrum, int. intestine, i.e. insemination 
canal, 1. int. lumen of intestine, m.a. male antrum, mu. muscle sheath, mil'. subepidermal 
muscles, mu". pharyngeal protractor muscles, n. nerves, oe. oesophagus, o.d. ovovitelline 
duct, pa. mid-ventral parenchyma, p. penis, ph. pharynx, ph. g. pharyngeal glands, s. sper- 
matozoa, s'. degenerating spermatozoa, u. uterus, w. whip of egg capsule. 



A PARASITIC RHABDOCOEL 



307 



CIS&-V 

r 1 ^ ' 7 '- A v ', 

J. . .. > : .-'-,! ' : ,', : 

6 












" 




7 



FIGURES 5-8. 



308 H. E. LEHMAN 

the ciliated ventral epithelium is invaginated and forms a short bulb-like canal which 
meets an outpocketing of the common genital antrum. Sphincters encircle both ends 
of this canal and regulate the size of the pore. 

DISCUSSION 
Comparison of genera 

Although the parasite described here is similar in many respects to all genera in 
the family Umagillidae, there are certain structural characteristics that do not cor- 
respond to those of any previously reported genus of this family. Therefore, it is 
considered necessary to establish a new genus to be designated by the name Syndi- 
syrinx. This name is intended to describe the complex bursal valve which is not 
present in any other genus of the family. The specific name, Syn. franciscanus, is 
given to designate the host, Strongylocentrotus franciscanus, in which it was first 
found. 

For the sake of uniformity in the following comparison of genera of the family 
Umagillidae, the morphological nomenclature used by the various authors in their 
original descriptions of genera and species has been altered to conform with the 
terminology employed in the preceding analysis of Syndisyrinx. 

In addition to the fact that both Syndisyrinx and Syndcsmis are found in the in- 
testine of echinoids, the morphological characteristics of Syndisyrinx indicate a 
closer relationship to Syndcsmis than to the other genera of the family. The loca- 
tion and appearance of important organs, viz., muscular pharynx, lobed testes, small 
spermiducal vesicle, muscular common sperm duct, ramified vitellaria, dichotomously 
branched ovaries, elongated uterus and egg capsule with whip, are very similar in 
Syndcsmis and Syndisyrinx and strongly suggest a close relationship between these 
two genera. Syndisyrinx differs from Syndcsmis chiefly in the structure and re- 
lationships of the bursa seminalis and seminal receptacle. In Syndcsmis a single 
vesicle is present for the reception of sperm and cuticular structures such as the 
parts which make up the bursal valve of Syndisyrin.\- are lacking. In addition to 
these differences, the stucture of -the penis is markedly dissimilar in these tw r o forms. 
The penis of Syndisyrinx is a cuticular hollow stylet attached only at the base, 
whereas the copulatory organ of Syndcsmis is a muscular eversible tube with a cu- 
ticular lining (Russo, 1895; Fig. 16). 

Structures corresponding to the cuticular canals in the bursal valve of Syndi- 
syrinx are found in Anoplodicra valuta, Wahlia macrostylifcra, and Dcsmote vorax. 
In A. valuta the relationships of the two cuticular canals to the bursa seminalis, as 
described by Westblad (1930), are very similar to the arrangement of these struc- 
tures in Syndisyrinx. However, the cuticular sheath that surrounds the entrance 
of these ducts into the bursa is lacking in A. valuta. There do not appear to be 
grounds for concluding that Syndisyrinx and Anoplodiera are closely related since 
the appearance and location of the testes and vitellaria, the presence of a single ovary, 
and the absence of a female antrum connecting the ovovitelline duct and uterus to 
the common genital antrum in A. valuta differ strikingly from the arrangement 
found in Syndisyrinx, 

In W. macrostylifcra, described by Westblad (1930), and D. vorax, according 
to Beklemischev (1916), the proximal end of the bursal canal is cuticular but an in- 
semination canal is lacking. In other respects W. macrostylifcra differs from 



A PARASITIC RHABDOCOEL 309 

Syndisyrinx chiefly in regard to the morphology of the male reproductive system. 
The penis stylet is greatly elongated, and paired sperm ducts arising from compact 
testes unite and communicate by means of a single duct with the large spermiducal 
vesicle situated anterior to the pharynx. Many points of difference are likewise 
found by comparing the morphology of Syndisyrinx and Dcsmotc. The most 
evident of these are the bipartite gut and the presence of two genital pores, the an- 
terior pore by which the uterus opens to the exterior and the posterior pore which 
serves for copulation in D. vorax. 

The other genera of the family lack cuticular parts in the copulatory complex 
comparable to those in the bursal valve of Syndisyrinx and to a greater or less de- 
gree exhibit dissimilarities in the location, distribution, number, arrangement and 
relationships of organs in the body. In these genera the most conspicuous differ- 
ences with respect to Syndisyrinx are : the single ovary and absence of a cuticular 
copulatory stylet in the genus Anoplodiuin ; the unbranched ovaries and double- 
walled cuticular penis stylet in the genus Umagilla; the absence of a cuticular penis 
and the general arrangement of testes and vitellaria in the genus Xcnoinctra; and 
the single testis in the genus Collastoma. A manuscript is in preparation which will 
deal at greater length with the structural relationships of these forms. 

Bursal valve 

There is a superficial similarity between the bursal valve of Syndisyrinx and the 
cuticular nozzle-like mouthpieces of acoels. In the acoel, Amphichoerus, described 
by Graff (1891), and many allied forms, one end of the mouthpiece is generally con- 
nected to a vesicular sac or bursa filled with sperm ; the other end is directed toward 
the ovary. L. H. Hyman (1937) points out that the function of these mouthpieces 
is apparently to direct sperm toward the ova to help insure fertilization. This func- 
tion can hardly be ascribed to the insemination canals of Anoplodiera and Syndi- 
syrinx which conduct sperm from the bursa seminalis to the seminal receptacle and 
not directly to the ova; nor does it seem probable that the insemination canals of 
Umagillidae are homologous to these mouthpieces. Noncuticular ducts connect the 
bursa seminalis to the seminal receptacle and bursal canal in most genera of Umagil- 
lidae, which suggests that cuticular structures are probably of relatively recent 
rather than primitive origin. In an analysis of the existing genera, Wahl (1910b) 
presents evidence which leads him to conclude that Umagilla is the most primitive 
and least modified genus of the family. If one accepts this view, it lends support 
to the opinion expressed above, inasmuch as Umagilla lacks any cuticular structures 
that might be considered homologous to the bursal valve. It is possible that the 
absence of cuticular parts in some of the species is due to a greater degree of simpli- 
fication associated with a parasitic existence. However, there is no direct evidence 
for this supposition, since in the most closely related free-living families, Grarfillidae 
and Dalyelliidae, cuticular structures such as these are not found. This suggests 
that these tubules have arisen independently, and until additional information is 
available, the insemination canals and bursal canals of Umagillidae should not be 
considered as mouthpieces in a true sense. 

Although copulation has not been observed in Syndisyrinx, it is believed that the 
sperm of one animal are injected by means of the protrusible penis into the bursal 
canal of another. Before fertilization can take place, sperm must migrate from the 



310 H. E. LEHMAN 

bursal canal through its narrow proximal end into the bursa seminalis, there re- 
maining until able to find their way through the insemination canal into the seminal 
receptacle. Evidently many sperm are unable to accomplish this migration and de- 
generate in the lumen of the bursa seminalis. Sperm that do reach the seminal re- 
ceptacle must then pass through the constricted anterior part of this organ to fer- 
tilize the mature ova that enter the ovovitelline duct at the anterior end of the 
seminal receptacle. 

It is difficult to explain any selective advantage for the presence of the fine canals 
that make up the bursal valve of Syndisyrinx. It was thought at first to be a mech- 
anism for the prevention of polyspermy. However, this explanation is negated by 
the presence of large masses of spermatozoa in the seminal receptacle. The simplest 
explanation for the presence of these ducts is that they act as valves which regulate 
the number of spermatozoa entering the bursa seminalis and seminal receptacle. If 
this interpretation is correct, it is probable that the function of the bursal valve is 
to insure a necessary aging of the sperm in the bursa before fertilization. The cu- 
ticular walls are necessary to prevent the collapse of these narrow tubes. It is 
evident that the bursal valve restricts the free passage of sperm from the bursa sem- 
inalis and therefore as the result of a single copulation, a continuous supply of 
sperm may be maintained over a long period of time. 

SUMMARY 

After completing a histological study of an endoparasitic rhabdocoel from the 
Pacific Coast sea urchin, Strongylocentrotus jranciscanus, the following conclusions 
have been reached : 

1. This parasite belongs to the rhabdocoel family Umagillidae but differs in cer- 
tain characteristics from the eight known genera of the family. 

2. The distinguishing characteristics are a single intestine, paired and lobed 
ovaries and testes, a tubular single-walled ctiticular penis stylet, and cuticular ducts 
connecting the bursa seminalis to the bursal canal and seminal receptacle. 

3. A characteristic structure typical of this parasite and not present in other 
genera of the family is the bursal valve composed of two cuticular tubes, the in- 
semination canal and proximal end of the bursal canal, which enter the bursa sem- 
inalis through a cuticular cup-like sheath. 

4. The parasite here described is given the name Syndisyrinx frandscanus, gen. 
et sp. nov. 

LITERATURE CITED 

BEKLEMISCHEV, W., 1916. Sur les Turbellaries parasites de la cote Mourmanne, II. Rhabdo- 

coela. Trav. Soc. Imp. Nat. Petrograd, Zool. et Physiol., Sect. 4, 45: 1-59 (Resume, 

60-79). 
BOCK, S., 1926. Anoplodium stichopi, ein neuer Parasit von der Westkiiste Skandinaviens. 

Zool. Bidrag, Uppsala, 10: 1-30. 
BRAUN, M., 1889. Uber parasitische Strudehvurmer in Rostok. Ccntralbl. Bakt. Parasit., 

Abt. 1,5: 41-44. 
BRESSLAU, E., 1933. Turbellaria. Kiikenthal und Kntmbach, Handbnch der Zoologic, 2: 

264-269. 
DORLER, A., 1900. Neue und wenig bekannte rhabdocole Turbellarien. Zcitschr. zviss. Zool., 

68 : 1-42. 



A PARASITIC RHABDOCOEL 311 

FRANCOIS, P. H., 1886. Stir le Syndesmis, nouveau type de Turbellaries decrit par, W. A. 

Silliman. C. R. Acad. Sci. Paris, 103 : 752-754. 

GRAFF, L. v., 1891. Die Organisation der Turbellaria acocla, Leipzig (Engelmann), p. 73. 
GRAFF, L. v., 1913. Turbellaria, II. Rhabdocoelida. Das Tierreich, Berlin (F. E. Schulze), 

35: 152-163. 

HYMAN, L. H., 1937. Reproductive system and copulation in Amphiscolops langerhansi (Tur- 
bellaria acoela). Biol. Bull., 72: 319-326. 
OZAKI, Y., 1932. On a new genus of parasitic Turbellaria, Xenometra, and a new species of 

Anoplodium. Jour. Sci. Hiroshima Univ. (Series B, Zoo/.), 1: 81-89. 
POWERS, P. B. A., 1936. Studies on the ciliates of sea urchins. A general survey of the 

infestations occurring in Tortugas echinoids. Pap. Tortugas Lab. Carnegie hist. 

Washington, 29: 319-320. 
Russo, A., 1895. Sulla morfologia del Syndesmis echinorum Francois. Ricerche Lab. Anat., 

Roma, Fasc. 1,5: 43-68. 
SCHNEIDER, A., 1858. Uber einige Parasiten der Holothuria tubulosa. I. Anoplodium parasita. 

Miillcr's Arch. f. Anat. Phys. und u-iss. Mcd., Berlin: 324-329. 
WAUL, B., 1909. Untersuchungen liber den Bau der parasitischen Turbellarien aus der Familie 

der Dalyelliiden (Vorticiden). II. Teil, Die Genera Umagilla und Syndesmis. Wien, 

Sitz.-Bcr. kais. Akad. u'iss. Math.-nat., Abt. 1, 118: 943-965. 
WAHL, B., 1910a. Untersuchungen iiber den Bau der parasitischen Turbellarien aus der 

Familie der Dalyelliiden (Vorticiden). III. Teil, Das Genus Collastoma. Wien, 

Sitz.-Bcr. kais. Akad. iviss. Math.-nat., Abt. 1, 119: 363-391. 
WAHL, B., 1910b. Beitrage zur Kenntnis der Dalyelliiden und Umagilliden. Fcstschr. f. R. 

Hertivig, Jena (G. Fischer), 2: 41-60. 
WESTBLAD, E., 1926. Das Protonephridium der parasitischen Turbellarien. Zoo!. Anz., 67 : 

323-333. 
WESTBLAD, E., 1930. Anoplodiera voluta und Wahlia macrostylifera, zwei neue parasitische 

Turbellarien aus Stichopus tremulus. Zcitschr. f. Morph. u. Okol. Tierc, 19 : 397-426. 



A QUANTITATIVE STUDY OF THE RELATIONSHIP BETWEEN 
THE ACTIVITY AND OXYGEN CONSUMPTION OF THE 
GOLDFISH, AND ITS APPLICATION TO THE MEAS- 
UREMENT OF RESPIRATORY METABOLISM 

IN FISHES 

W. A. SPOOR 

Department of Zoology, University of Cincinnati 

INTRODUCTION 

The fact that fish consume more oxygen when active than when quiescent has 
been observed by many investigators (Krogh, 1916; Bowen, 1932; Clausen, 1933, 
1936; Wells, 1935; Schlaifer, 1938; Smith and Matthews, 1942), but apparently 
no attempt has been made to determine the exact relationship between oxygen con- 
sumption and activity in fishes. It is the purpose of this paper to present data 
which are believed to provide an objective and quantitative basis for the relationship 
between activity and oxygen consumption in the goldfish, and to describe a method 
for making the necessary measurements. The method is based on the use of a re- 
cording activity detector (Spoor, 1941) combined with a continuous flow system for 
measuring oxygen consumption. 

The lack of definite information on the activity of fish under experimental condi- 
tions has been one of the chief sources of difficulty in work on the respiratory metab- 
olism of fishes, and attention has been called to the need for an experimental method 
which would make it possible to distinguish between "standard metabolism" and 
the increased metabolism due to muscular movements (Wells, 1935). In view of 
the fact that the oxygen consumption is affected by changes in the basal metabolic 
rate as well as by changes in activity, the importance of such a method is apparent. 
The method employed in the present work seems to meet this need, inasmuch as 
the state of activity is recorded continuously and periods of inactivity can be selected 
for measuring basal oxygen consumption. 

Szymanski (1914) and Spencer (1939), using other types of activity detectors, 
have reported that goldfish show considerable individual variation in activity and 
that the activity pattern is affected by light. Spencer (1939) also found- activity 
to be influenced by food. Knowledge of the behavior of the fish under the experi- 
mental conditions is of importance in the collection of data on oxygen consumption 
in the method to be described, as well as in the interpretation of these data. For 
this reason further observations on the patterns and rates of activity and on the ef- 
fects of food, light and disturbances are included in the present paper. 

THE ACTIVITY OF THE GOLDFISH UNDER EXPERIMENTAL CONDITIONS 
Method 

Several dozen goldfish (Carassius auratus) ranging between 24 and 96 grams 
in weight were selected at random from a stock obtained from a local goldfish farm 

312 



ACTIVITY AND OXYGEN CONSUMPTION 



313 



and studied individually in experimental chambers, each of which was equipped with 
a recording activity detector. The experimental chambers were set up in a ground 
floor aquarium room which was seldom entered except for the purposes of this 
study, so that the fish could be left for long periods with relatively little disturbance. 
The recording apparatus was kept in another room. Records of the activity of each 
fish were started shortly after its introduction into a chamber and continued for pe- 
riods ranging from a few days to many months in length, during which the patterns 
and rates of activity and the effects of food, light and disturbances upon them were 
studied. With a few exceptions, oxygen consumption was not measured in this 
series of observations. 

The experimental chamber (Fig. 1) consisted of a one-gallon brown glazed 




FIGURE 1. Diagram of apparatus for measuring oxygen consumption and activity. (1) 

paraffin oil (this was omitted when activity alone was being measured), (2) glass plates, (3) 

No. 44 copper wire, (4) to sensitive relay, (5) resistor, (6) wire screen, (7) glass tube, (8) 
wire frame protecting paddle. Explanation in text. 

crock fitted with a galvanized iron wire screen of % mcn mesh to prevent the fish 
from reaching the surface of the water. A glass tube about 3 cm. in diameter was 
fitted into an opening in the center of the screen so that it extended 3.5 cm. above 
and 3 cm. below the screen ; its purpose will be considered in a later section. The 
surface of the water stood about 3 mm. above the screen, the total volume to this 
level being 2,600 cc. Water entered the chamber from a constant level reservoir 
through 8 mm. glass tubing and left by way of a siphon of 8 mm. glass tubing which 
dipped into a constant level drain, the rate of flow (between 70 and 100 cc. a minute) 
being regulated by means of a glass stopcock in the inlet. The intake of the siphon 
was placed about 5 cm. above the bottom of the chamber, so that feces and other 
debris that fell to the bottom did not enter the siphon until they had been broken 
into small pieces in the course of their passage upward to the intake. The chamber 
was practically self cleaning under these conditions, the flow of the water and the 
movements of the fish being sufficient to move debris into the siphon. The fish 



314 W. A. SPOOR 

could therefore be maintained in the chamber for months without cleaning. A 
thistle tube entering the inlet provided for the introduction of food, being closed off 
at all other times. The water supply consisted of tap water passed through an ac- 
tivated charcoal filter, brought to the desired temperature and aerated until it ap- 
proached equilibrium with the atmosphere. Most of the observations were made at 
temperatures between 20 and 24 C. The fish seldom extracted more than one- 
third of the oxygen from the w^ater at the rates of flow employed, and they usually 
took less than this. In view of the findings of Crozier and Stier (1925), Toryu 
(1927), and Schlaifer (1938), it seems unlikely that behavior was influenced by 
the oxygen tension of the water. 

The chamber was enclosed in a wooden case to minimize disturbances and to 
make it possible to control the light. The top of the case was fitted with a pane of 
glass for natural illumination, and with a wooden cover when either complete dark- 
ness or constant light was desired. A ventilator in the side of the case, with baffles 
to prevent light from entering, permitted some circulation of the air. The water 
inlet, outlet siphon, and a tube leading to a U tube indicating the water level in the 
chamber passed through the wall ; a coat of black paint over each tube prevented 
light from entering the chamber through these openings. 

The detector consisted of a light-weight aluminum paddle suspended in the water 
in the experimental chamber by a fine copper wire in such a way that a silver rod 
at the top of the paddle shaft passed through a small hole in a fixed silver plate. 
Water currents set up by the movements of the fish moved the paddle, causing the 
rod to make and break contact with the sides of the hole and thus to activate a sen- 
sitive relay. This relay operated the recording apparatus. The blade of the paddle 
consisted of aluminum foil (5 cm. long and 2.5 cm. wide) with the corners bent in 
at right angles so that the water currents struck a flat surface regardless of their 
points of origin. The shaft (10 cm. long) consisted of no. 22 aluminum wire ce- 
mented to the blade and imbedded at its upper end (7 cm. above the blade) in a 
bakelite insulating rod (2 cm. long and 0.2 cm. in diameter) in the upper end of 
which the silver rod (1 cm. long and about 0.04 cm. in diameter) was imbedded. 
This silver rod was soldered to a 14.5 cm. length of no. 44 enameled copper wire 
held in an insulated binding post attached to a wooden supporting shaft. A 
wooden bracket rising from the case supported this shaft in a vertical position 
so that the paddle hung in the water through the glass tube in the center of the 
screen. A cylindrical frame of galvanized iron wire protected the paddle from 
contact with the fish. A small lead weight (about 0.1 gm.) clamped to the 
paddle shaft below the bakelite helped to bring the paddle back to the resting 
position after displacement by the water currents. The silver plate (about 0.5 
cm. square) was attached to the supporting shaft and held in a horizontal posi- 
tion about 6 cm. above the screen. The hole in the plate was between 0.08 and 0.1 
cm. in diameter. The current to operate the sensitive relay was supplied by a 6 volt 
storage battery; the coil of the relay had a resistance of 1,000 ohms. A 5,000 ohm 
resistor across the detector contacts prevented sparking and welding without caus- 
ing an observable reduction in the sensitivity of the detector. The plate was kept 
warm by means of a small insulated heating coil in order to prevent water from 
condensing upon it from the humid atmosphere above the chamber. 

The sensitivity of the detector could be controlled somewhat by adjusting the 
position of the silver rod with respect to the sides of the hole in the plate. The nor- 



ACTIVITY AND OXYGEN CONSUMPTION 315 

mal movements of the operculum and the position-maintaining fin movements of a 
quiescent 25- to 30-gram goldfish were usually sufficient to move the paddle slightly, 
and when the rod was close to the plate these movements were recorded. For the 
observations to be described, however, the rod was centered in the hole so that the 
ordinary respiratory movements did not move the paddle enough to make contact, 
these movements being considered as among the basal functions of the fish. Vigor- 
ous respiratory movements and any movement that resulted in a change in the posi- 
tion of the fish moved the paddle enough to make and break contact, slow swimming 
movements causing few, and vigorous activity causing many impulses to be recorded. 
At the flow rates used in these experiments the flow of water through the chamber 
did not move the paddle. 

The sensitive relay activated a counter which in turn caused signal magnets to 
record every tenth and hundredth impulse on a long paper kymograph moving about 
30 mm. an hour. The frequency of the impulses was such (ranging up to 6,000 an 
hour) that they usually could not be counted when recorded individually. The 
counter was capable of following and recording at least 10 impulses a second. Time 
was recorded in hours beneath the activity record. 

Patterns and rates of activity 

In agreement with the results of Szymanski (1914) and Spencer (1939), the 
goldfish used in this study proved to be quite variable in their patterns and rates of 
activity, even when they were maintained under almost identical conditions of light, 
feeding, temperature, water supply and disturbance. Three general types of be- 
havior appeared when the fish were kept under natural conditions of light: (1) 
arhythmic activity, in which no relation to clay or night could be detected; (2) 
rhythmic activity, in which the fish were active by day and quiescent at night; (3) 
rhythmic activity, in which they were quiescent by day and active at night. 

Fish showing the first, arhythmic, type of behavior were extremely variable. 
A few were vigorously active da)' and night for periods as long as ten days, others 
were moderately active throughout the 24-hour period for weeks at a time, and still 
others remained practically inert for similar periods. Some of these arhythmic fish, 
particularly those in the last group, showed irregular bursts of activity now and then, 
with no apparent relation to the time of day, feeding, or disturbance. 

An example of the second type of behavior, diurnal activity and nocturnal quies- 
cence, is shown in Figure 2, which is based on the number of impulses recorded by 
a 35-gram male goldfish during each hour between 1 P.M. January 11 and 1 P.M. 
January 13, 1946. The fish was fed at 11:05 A.M. on January 12, otherwise the 
room was not entered between 2:45 P.M. January 11 and 7:15 P.M. January 13. 
Aside from the feeding, the effects of which are discussed below, light was the only 
known variable, the temperature, rate of flow and aeration of the water being the 
same at the end as at the beginning of the period. Most of the fish showing rhythmic 
changes in activity 'followed patterns of this type, although the active phase varied 
considerably, sometimes being interrupted by several hours of quiescence during 
the day, sometimes beginning later in the day, and occasionally continuing well into 
the night. The most constant period of quiescence occurred between midnight and 
4 A.M., which is in agreement with Spencer's (1939) observations. 

The third type of behavior, diurnal quiescence and nocturnal activity, was found 
less frequently than the second, although it was not uncommon. An example is 



316 



W. A. SPOOR 



shown in Figure 3, which is based on records of the activity of a 32-gram male gold- 
fish between 1 P.M. July 3 and 1 P.M. July 5, 1945. The room was not entered be- 
'tween 4 P.M. July 3 and 8 A.M. July 5, and aside from the daily changes in light the 
environmental conditions apparently remained constant throughout the period. 

The patterns of rhythmic fish did not seem to be fixed, however, even when the 
environmental conditions remained unchanged. After several weeks of rhythmic 
behavior the fish frequently became arhythmic for several weeks or months, occa- 
sionally becoming rhythmic again in the course of extended periods of observation. 
This suggests that those fish which did not show daily activity rhythms under the 



3000- 

2500 

2000- 

P 1500- 
o 

1000- 
500- 





6RM. 
I/ /46 



2M. 



6A.M. I2N. 
I/I2/46 



6P.M. I2M. 



6 AW. 
I/I3/46 



!2N. 



FIGURE 2. Activity pattern of 35-gram male goldfish between 1 P.M. January 11 and 1 
P.M. January 13, 1946. Activity is expressed as number of impulses recorded each hour. Tem- 
perature 21.5 C. Fed at 11 :05 A.M. January 12. 

experimental conditions may have done so eventually had they been studied for lon- 
ger periods, and that by chance the observations were made during arhythmic 
periods. 

Activity and food 

The fish were fed rolled oats, commercial fish foods, shredded shrimp, ground 
liver or chopped earthworms about three times a week, usually 0.5 to 1 gram at each 
feeding. The effects of daily feeding, larger amounts of food and starvation were 
also studied. Under the conditions of the experiments the type of food given had 
no consistent effects upon activity, but the quantity of food had pronounced effects, 
particularly on the total amount of activity. A well fed fish was usually sufficiently 
active that the number of impulses recorded in the course of a 24-hour period aver- 
aged between 500 and 1,500 an hour, and averages in excess of 2,500 impulses an 



ACTIVITY AND OXYGEN CONSUMPTION 



317 



hour were not uncommon. Starvation caused this rate to decrease markedly, some- 
times to fewer than 100 impulses an hour, although as a rule the lowest rates did not 
appear until the fish had been starved for a week or so. No fish was observed to 
become completely inactive for periods of more than an hour or two, however, even 
when starved for two weeks. The effects of feeding after a period of starvation 
were striking, activity increasing to normal "well fed" rates within a few minutes. 
Doubtless the swimming movements associated with feeding accounted for some of 
the activity recorded following the introduction of food, but it seems that the nu- 
tritional state also affected the amount of activity. Food given in amounts of one 



3000- 



2500- 



^2000- 

H 

p I 500- 
o 

1000- 



500- 



TJ 






J" 



6RM. I2M. 6A.M. I2N. 
7/3/45 7/4/45 



6 P.M. 



I2M. 
I 



6A.M. 
7/5/45 



2N. 



FIGURE 3. Activity pattern of 32-gram male goldfish between 1 P.M. July 3 and 1 P.M. July 5, 

1945. Units as in Figure 2. Temperature 23.5 C. 

gram or less was usually consumed within three to six hours, but the fish remained 
active (in accordance with their activity patterns) for from several days to a week 
after they had been fed. Similarly, Spencer ( 1939) found the goldfish to maintain 
a'high rate of activity for several hours after feeding, although in his experiments 
the food was usually consumed within 15 minutes or less. 

The effects of feeding upon activity rhythms were not studied in detail, but the 
available data bearing on this question indicate that although the rhythms appearing 
under natural conditions of light were frequently modified by the quantity of food 
and the time of feeding, they were not causally related to food. Feeding modified 
the activity patterns of some fish for part or all of the subsequent 24-hour period, 
usually by prolonging the active phase. A response of this type may be seen in 
Figure 2. The fish was fed 0.5 gm. of rolled oats at 11 :05 A.M. on January 12 (the 
previous feeding being on January 9) ; it will be noted that the activity level re- 
mained relatively high for a much longer period on the night of January 12 than on 



318 W. A. SPOOR 

the preceding night. On the other hand some fish showed no change in activity in 
response to feeding, provided of course that they had not been starved. Variations 
in the quantity of food and in the time and frequency of feeding did not seem to have 
permanent effects on the activity rhythms, and feeding at the same time each day 
did not cause arhythmic fish to become rhythmic. 

Activity and light 

The goldfish did not seem to be much affected by changes in light intensity while 
they were not following daily activity rhythms, but they were usually quite responsive 
to light during their periods of rhythmic behavior. In fact, when the fish were well 
fed and undisturbed the activity rhythms seemed to be closely related to the daily 
changes in natural light, as Szymanski (1914) has reported previously. This view 
is supported by several observations in addition to the fact that the active and quies- 
cent phases of the cycles usually coincided with day and night. Periods of nocturnal 
activity and diurnal quiescence were shown by the 32-gram male goldfish mentioned 
above in July and December of 1945. Although the water temperature and other 
factors except light were the same during both periods, the nocturnal phase of ac- 
tivity usually began earlier in the evening (between 5 and 6 P.M.) and ended later 
in the morning (between 7 and 8 A.M.) in December than in July, when it usually 
began between 7:30 and 8:30 P.M. and ended between 5 and 6 A.M. This suggests 
of course that the nocturnal phase of activity was limited by the setting and rising 
of the sun. This fish also responded readily to experimental changes in light in- 
tensity, particularly during the day, when darkening the chamber caused its ac- 
tivity to increase to levels usually reached only at night. Records were also ob- 
tained in which diurnally active and nocturnally quiescent fish remained active on 
nights when bright moonlight entered the room in which they were kept. Spencer 
(1939) found that the regular diurnal rhythm of the goldfish could be obliterated by 
covering the tank by day and lighting artificially at night. This procedure was ac- 
companied by night feeding, however, so that the change in activity may not be at- 
tributed solely to the reversed lighting. 

On the basis of these observations attempts were made to maintain goldfish at 
definite rates of activity by exposing them to continuous dim light and to continuous 
darkness for periods lasting as long as three weeks, but without success. The fish 
did not maintain constant rates of activity under either condition, but continued to 
alternate periods of increased activity with periods of relative quiescence. In order 
to maintain a low rate of activity it was necessary to starve the fish for about a week, 
the relationship between nutritional state and amount of activity being similar to 
that described in the preceding section. 

Activity and disturbance 

The goldfish proved to be extremely sensitive to disturbances. Noise, slight, 
changes in the water level, sudden lights, the mere presence of the observer in the 
room, or such minor disturbances as the quiet opening and closing of the door to 
the room usually caused a change in the rate of activity. Fish that had been active 
before the disturbance almost invariably became less active, sometimes practically 
motionless, while quiescent fish frequently, although less consistently, became ac- 
tive when disturbed. Whichever the response, the original state of activity was 



ACTIVITY AND OXYGEN CONSUMPTION 319 

usually resumed within a few minutes after the disturbance had ceased. The degree 
of response seemed to be related to the amount of disturbance, for when the observer 
moved slowly and quietly the change in activity was usually less pronounced, and 
recovery more rapid, than after ordinary passage through the room or adjustment 
of the apparatus. The effects of disturbances upon the activity of an otherwise 
quiescent fish may be seen in Figure 3. The room was entered several times in the 
course of the afternoon of July 3 and on July 5, although the experimental chamber 
was not approached and the fish could not see the cause of the disturbance. It is 
obvious that the rates of activity were higher than at corresponding hours on July 4, 
when the room was not entered. Such sensitivity has been observed in other species 
of fish by Clausen (1934), who found that a shadow passing over the aquarium 
caused increases in the body temperatures of perch and members of the sunfish 
group. 

THE RELATIONSHIP BETWEEN ACTIVITY AND OXYGEN CONSUMPTION 
Method 

The activity and corresponding oxygen consumption of individual goldfish were 
measured in observation periods ranging in length from 11 to 210 minutes. Ac- 
tivity was measured in terms of the number of impulses recorded in a given period, 
and the "amount of oxygen consumed by the fish in that period was determined by 
means of a continuous flow system. A control chamber similar to the experimental 
and housed in the same case was supplied with a continuous stream of water from 
the reservoir supplying the fish. The water in each chamber was covered with a 
layer of heavy paraffin oil 2.5 cm. thick to retard the diffusion of oxygen from the 
air, and a sample of the effluent from each chamber was analyzed for oxygen by the 
Winkler method at the beginning and end of each period. The samples were col- 
lected in narrow necked glass stoppered bottles of about 270 cc. capacity arranged 
to serve as constant level drains (Fig. 1). Each line was arranged so that the 
water passed through the outlet siphon to the bottom of the sampling bottle and 
overflowed into a funnel so that it could be collected for flow rate determinations. 
Although the rates of flow ranged from 70 to 100 cc. a minute in the course of the 
study, the rate for any one day's series of samples was held practically constant. 
Due care was taken to prevent the diffusion of oxygen into the samples and to ob- 
tain representative samples from experimental and control lines. Samples that 
were contaminated by participate matter were discarded. The permanganate modi- 
fication was used in most of the analyses, but was omitted during some of the 
shorter periods. The results obtained with and without the modification were 
quite similar, however, which was not unexpected in view of the fact that from four 
to six liters of water passed over the fish each hour. 

The volume of water flowing through the system in the course of an observation 
period being known, together with the oxygen content of the water leaving the 
control and experimental chambers at the beginning and end of that period, the 
oxygen consumed by the fish could be calculated. The calculations took into ac- 
count the change in the amount of oxygen in the constant volume of water in the 
chamber. The volume of water displaced by the fish was too small to affect the 
calculations. 

The samples were collected w T ith the foregoing observations on activity patterns 



320 W. A. SPOOR 

and modifying factors in mind, the periods being timed to yield data at the activity 
rates desired, and the method of sampling being modified as necessary to minimize 
disturbance of the fish. In the latter connection the outlet tubes were lengthened so 
that samples were collected about 10 feet away from the chambers, and the room was 
not entered except for sampling and rate of flow determinations. Precautions were 
taken to prevent changes in the water level in the experimental chamber as there 
were indications that small changes in the level stimulated the fish. These pre- 
cautions were necessary also because the volume of water in the chamber, as well as 
that flowing through it, entered into the calculations of oxygen consumption. 
Samples were discarded if subsequent examinations of the activity records showed 
that they had been collected while the fish was undergoing marked changes in ac- 
tivity as a result of disturbance or in accordance with an activity rhythm. This was 
necessary because although the activity record was instantaneous the change in the 
oxygen concentration of the samples tended to lag somewhat behind that in the 
chamber, the sample drawn at any instant representing the average of the water 
flowing into the bottle in the few minutes preceding its removal. The temperature 
of the water was recorded for each observation period in order to avoid discrepancies 
attributable to the effect of temperature on metabolic rate (Ege and Krogh, 1914). 

Owing to its viscosity and the accumulation of emulsified oil and water at the 
oil-water interface, the layer of oil interfered with the movements of the paddle 
shaft. Its thickness was therefore reduced to 1 cm. within the central glass tube, 
thus permitting the paddle to move about as freely as with a water surface. This 
tube extended below the interface far enough to prevent the emulsion from accumu- 
lating around the paddle shaft. The oil within the tube had to be changed now and 
then, however, to remove the small amount of debris that entered it from beneath. 
The detector contacts and bakelite rod were cleaned every few days as a precaution 
against their becoming coated with oil, which seemed to spread slo\vly up the paddle 
shaft. 

It was established by appropriate tests that the layer of paraffin oil was effective 
in preventing the diffusion of significant amounts of oxygen into the water from the 
atmosphere. In no test did the apparent leakage exceed the limits of error of the 
Winkler method itself (Alice and Oesting, 1934), and it was usually considerably 
less. The average apparent rate of change for the contents of the experimental 
chamber was 0.0015 cc. of oxygen a minute, which was so much smaller than the 
rate at w r hich the fish consumed oxygen that even had the apparent change been 
real it would have had but little effect on the results. It should be mentioned in this 
connection that the oil layer was disturbed relatively little by the movements of the 
fish, inasmuch as the wire screen kept the fish out of the oil and glass plates resting 
on this screen lessened the churning effects of the water beneath it. 

Results 

Three goldfish were studied at several temperatures in a total of 104 observation 
periods. As the results on all three were much alike, data on but one of the fish, 
a 32-gram male on which over two-thirds of the measurements were made, are 
presented here. 

The relationship between activity and oxygen consumption at temperatures be- 
tween 23 and 25 C. is shown in Figure 4, in which oxygen consumption in cubic 



ACTIVITY AND OXYGEN CONSUMPTION 



321 



centimeters per minute is plotted against activity in impulses per minute. Fifty-nine 
observation periods are represented, each point corresponding to one period. The 
line merely indicates the trend, and has not been fitted to the data mathematically. 
As was to be expected, oxygen consumption and activity proved to be closely related, 
the relationship apparently being linear above the basal level of oxygen consumption. 
Although the values for oxygen consumption at any one rate of activity are seen to 
vary somewhat, the trend is clear cut : at high rates of activity the rate of oxygen 



.18 



.16- 



.15- 



.13- 



z 
o 

P .12- 
o. 

01 .1 I ' 



o 





.10- 



.09- 



.08- 



.07- 



06- 



.05- 



.04 



.03 



10 



20 



30 



40 50 60 

ACTIVITY 



70 



80 



90 100 



FIGURE 4. Activity and oxygen consumption of 32-gram male goldfish. Activity in impulses/ 
minute; oxygen consumption in cubic centimeters/minute. Temperature 23 to 25 C. 

consumption is correspondingly high ; at low activity rates less oxygen is consumed. 
The discrepancies that do occur may well have been due to errors in measurement, 
rather than to a lack of correspondence between activity and oxygen consumption. 
In this connection the data on oxygen consumption follow those on activity quite 
closely when the comparison is restricted to one day's series of measurements, thus 
ruling out discrepancies attributable to slight differences in the adjustment of the 
detector contacts. Such a series is shown in Figure 5, which is based on data ob- 
tained with the same fish in a series of thirteen consecutive 15- to 25-minute pe- 
riods at 22 C. 

According to the slope of the data shown in Figure 4, the basal oxygen con- 



322 



W. A. SPOOR 



sumption of this fish was in the vicinity of 0.040 cc. a minute, or 0.075 cc. per gram 
per hour. 

DISCUSSION 

The results of the present study have a bearing on the collection and interpreta- 
tion of data on the respiratory metabolism of fishes, and in the light of these results 
the method described seems to offer a number of advantages not found in previous 
methods which have been employed for this purpose. 

The advantages of the continuous flow method for measuring respiratory metab- 
olism in fishes have been discussed by Keys ( 1930) and need not be reviewed here. 
In view of the relationship between oxygen consumption and activity, however, the 



100 




.03 



150 



215 230 245 300 315 



5OO 



30 



TIME 



FIGURE 5. Activity and oxygen consumption of 32-gram male goldfish in each of thirteen 
consecutive observation periods between 1 :35 P.M. and 5 P.M. November 13, 1945. Units as in 
Figure 4. Each point on the upper line represents the average rate of oxygen consumption for 
the 15- or 25-minute period preceding it. Each point on the lower line represents the average 
rate of activity for the corresponding period. 

observations on the effects of disturbances may be applied to the use of this method, 
inasmuch as the process of sampling may disturb the fish. Should a change in the 
rate of activity (and consequently of oxygen consumption) occur at the time of 
sampling, the sample would not be representative of the volume of water and unit 
of time to which it is related in the calculations. The resulting error could be of 
considerable importance, particularly in investigations in which the samples con- 
sisted of water flowing directly from the experimental chamber and overflowing 
through a sampling bottle. This source of error has been recognized of course, and 
in some investigations the experimental chamber has been covered in attempts to 
minimize stimulation of the fish. It seems very doubtful, however, whether cover- 
ing a goldfish so that it cannot see the investigator is an adequate safeguard against 
disturbance. One advantage of using an activity detector in the continuous flow 
method then lies in the fact that any sudden change in activity occurring at the 
time of sampling can be detected, so that the reliability of the sample may be judged. 
Furthermore, the activity record can be used to test the effectiveness of the steps 
taken to avoid disturbance. 



ACTIVITY AND OXYGEN CONSUMPTION 323 

It is of course well known that fluctuations in activity during the test periods 
constitute a major obstacle to the correct interpretation of measurements of oxygen 
consumption, and numerous attempts to overcome this difficulty have been described 
(Ege and Krogh, 1914; Hall, 1929; Adkins, 1930; Keys, 1930; Wells, 1932, 1935; 
Clausen, 1933; Smith and Matthews, 1942). These measures include the use of 
narcotics, observing that the fish remains quiet, maintaining constant conditions of 
light, sampling at the same time each day, restricting the movements of the fish, and 
maintaining the fish in an experimental chamber until it appears to have come to 
rest or at any rate to have reached a steady state. Although such measures may 
permit the establishment of the reality of a change in oxygen consumption in con- 
nection with an experimental procedure, they do not appear to give a completely 
satisfactory basis for the interpretation of that change. The interpretation must be 
based on knowledge of the activity of the fish, inasmuch as oxygen consumption is 
affected by changes in the basal metabolic rate as well as by activity. The method 
employed must therefore be capable of supplying information on activity and oxygen 
consumption at the same time, so that the fraction of the respiratory exchange as- 
sociated with basal metabolism may be distinguished from that due to muscular ac- 
tivity (Wells, 1935). None of the above methods seems to be adequate for this 
purpose. 

Narcotics are of doubtful value in studies of this type, even for measuring basal 
metabolic rate alone (Adkins, 1930). Among other objections are indications that 
an important fraction of the metabolic functions of the fish may be suppressed to 
such an extent that the oxygen consumption falls below the basal level as it is gen- 
erally understood (Keys and Wells, 1930). In fact, Ege and Krogh (1914) con- 
sidered it necessary to use artificial respiration to insure the survival of their gold- 
fish, the narcotic having interfered with normal respiratory movements. The other 
methods are open to criticism because they are based on the assumption, rather than 
the knowledge, that the fish is quiescent or at a constant level of activity under the 
conditions of the experiment. The results of the present work suggest that for the 
goldfish at any rate this assumption may be unwarranted. The fact that a goldfish 
is quiescent while it can be seen should not be taken as proof that it remains so 
while unobserved, and it does not seem justifiable to assume that constant environ- 
mental conditions mean constant rates of activity. So far as the goldfish is con- 
cerned, the individual variations in activity open to question the reliability of methods 
based on sampling at the same time each day, particularly if several fish are being 
compared. Confining the fish to a small respiration chamber to restrict its move- 
ments gives no assurance that it will remain quiescent or even at a constant rate of 
activity, and the fact that the oxygen consumption varies over a wide range in such 
chambers supports this objection. This method would seem to have a further dis- 
advantage for measuring the basal metabolic rate in that a fish confined to a small 
tube must swim continuously, however slowly, in order to maintain its position in 
the current. The practice of leaving the fish in the respiratory chamber until its 
oxygen consumption has reached a relatively low and constant rate (Keys, 1930; 
Wells, 1935) is far superior to the earlier techniques, but it is limited in its applica- 
tion by the fact that it gives no information as to the amount of activity associated 
with the steady state. 

The requirements of a satisfactory method appear to be met by combining an 
activity detector with the continuous flow system. The rate of activity can then be 



324 W. A. SPOOR 

measured at the same time that oxygen consumption is determined, and the results 
interpreted accordingly. As the activity record is continuous, periods of quiescence 
can be selected for measuring basal oxygen consumption, so that it is not necessary 
to employ special techniques designed to control activity. In this connection, how- 
ever, starvation may be used as a means of prolonging the quiescent state. A fur- 
ther advantage of the present method lies in the fact that the fish can be maintained 
in good health in the experimental chamber for months, so that measurements of 
its respiratory metabolism need not be obscured by the excitement and other effects 
of handling. 

SUMMARY 

1. Apparatus for making continuous records of the activity of isolated and un- 
disturbed goldfish is described, together with a method for measuring oxygen con- 
sumption and activity simultaneously. 

2. The goldfish were quite variable in their patterns and rates of activity under 
the experimental conditions. Some fish were diurnally active and nocturnally 
quiescent, others followed the opposite pattern and still others were arhythmic 
throughout the periods during which they were observed. Moreover, some fish 
showed both rhythmic and arhythmic states of activity when studied for periods 
extending over several weeks or months. 

3. Food, light and minor disturbances had pronounced effects on the activity of 
the goldfish. 

4. Simultaneous measurements of oxygen consumption and activity are pre- 
sented which indicate that the two are closely related above the basal level of oxygen 
consumption. 

5. The bearing of these observations on the collection and interpretation of data 
on the oxygen consumption of the goldfish and on the measurement of its basal 
metabolic rate is discussed, and certain advantages of the method are described. 

LITERATURE CITED 

ADKINS, M., 1930. A method for determining basal metabolism of fishes. Proc. Soc. Exp. 
Biol. Med., 28 : 259-263. 

ALLEE, W. C. AND R. OESTING, 1934. A critical examination of Winkler's method for deter- 
mining dissolved oxygen in respiration studies with aquatic animals. Physiol. Zool., 
7: 509-541. 

BOWEN, E. S., 1932. Further studies of the aggregating behavior of Ameiurus melas. Biol. 
Bull., 63 : 258-270. 

CLAUSEN, R. G., 1933. Fish metabolism under increasing temperature. Trans. Amcr. Fish. 
Soc., 63 : 215-219. 

CLAUSEN, R. G., 1934. Body temperature of fresh water fishes. Ecology, 15: 139-144. 

CLAUSEN, R. G., 1936. Oxygen consumption in fresh water fishes. Ecology, 17 : 216-226. 

CROZIER, W. J. AND T. B. STIER, 1925. Critical increment for opercular breathing rhythm of 
the goldfish. Jour. Gen. Physiol.. 1 : 699-704. 

EGE, R. AND A. KROGH, 1914. On the relation between the temperature and the respiratory 
exchange in fishes. Internal. Revue d. ges. Hydrobiol. u. Hydrog., 7 : 48-55. 

HALL, F. G., 1929. The influence of varying oxygen tensions upon the rate of oxygen con- 
sumption in marine fishes. Amer. Jour. Physiol., 88 : 212-218. 

KEYS, A. B., 1930. The measurement of the respiratory exchange of aquatic animals. Biol. 
Bull, 59 : 187-198. 

KEYS, A. B. AND N. A. WELLS, 1930. Amytal anesthesia in fishes. Jour. Pharm. Exp. Thcrap., 
40: 115-128. 



ACTIVITY AND OXYGEN CONSUMPTION 325 

KROGH, A., 1916. The respiratory exchange of animals and man. Monographs on Biochemistry. 
Longmans, Green and Co., London. 

SCHLAIFER, A., 1938. Studies in mass physiology : effect of numbers upon the oxygen consump- 
tion and locomotor activity of Carassius auratus. Physiol. Zoo/., 11: 408-424. 

SMITH, D. C. AND S. A. MATTHEWS, 1942. The effect of adrenalin on the oxygen consumption 
of the fish, Girella nigricans. Aincr. Jour. Physiol., 137 : 533-538. 

SPENCER, W. P., 1939. Diurnal activity rhythms in fresh-water fishes. Ohio Jour. Sci., 39: 
119-132. 

SPOOR, W. A., 1941. A method for measuring the activity of fishes. Ecology, 22: 329-331. 

SZYMANSKI, J. S., 1914. Eine Methode zur Untersuchung der Ruhe- und Aktivitatsperioden 
bei Tieren. Pfliigcr's Arch. gcs. Physiol., 158: 343-385. 

TORYU, Y., 1927. The respiratory exchange in Carassius auratus and the gaseous exchange of 
the air bladder. Sci. Kept. Tohoku Imp. Univ., 4 Ser. (Biology), 3: 87-96. 

WELLS, N. A., 1932. The importance of the time element in the determination of the respira- 
tory metabolism of fishes. Proc. Nat. Acad. Sci., 18: 580-585. 

WELLS, N. A., 1935. The influence of temperature upon the respiratory metabolism of the 
Pacific killifish, Fundulus parvipinnis. Physiol. Zoo/., 8 : 196-227. 



INDEX 



A BSTRACTS of scientific papers presented 
at the Marine Biological Laboratory, sum- 
mer of 1946, 210. 

Activity and oxygen consumption of the gold- 
fish and its application to the measurement 
of respiratory metabolism in fishes, 312. 

Amaroecium constellatum, II, 66. 

Annual report of the Marine Biological Labora- 
tory, 1. 

Arbacia eggs, the effect of low temperature and 
of hypotonicity on the morphology of the 
cleavage furrow, 272. 

Artemia salina, the space-time pattern of seg- 
ment formation in, 119. 

gEERS, C. D. Tillina magna: Micronuclear 
number, encystment and vitality in di- 
verse clones; capabilities of amicronucleate 
races, 256. 

BODENSTEIN, DIETRICH. Developmental rela- 
tions between genital ducts and gonads in 
Drosophila, 288. 

BROWN, FRANK A., JR., AND LORRAINE M. 
SAIGH. The comparative distribution of 
two chromatophorotropic hormones (CDH 
and CBLH) in Crustacean nervous sys- 
tems, 170. 

("^ARRIKER, MELBOURNE ROMAINE. Ob- 
servations on the functioning of the ali- 
mentary system of the snail Lymnaea 
stagnalis appressa Say, 88. 

CHEN, TZE-TUAN. Temporary pair formation 
in Paramecium bursaria, 112. 

Chromosome movement, hydrostatic pressure 
effects upon the spindle figure and, 145. 

Ciliates of the family Ancistrocomidae Chatton 
and Lwoff, III, 189. 

Ciliates of the family Ancistrocomidae Chatton 
and Lwoff, IV, 200. 

Cleavage furrow in Arbacia eggs, 272. 

T~)DT, loci of action in cockroach (Periplaneta 

americana), 247. 
Drosophila, developmental relations between 

genital ducts and gonads in, 288. 

"pLECTRON microscope observations of the 
trichocysts and cilia of Paramecium, 141. 

("MESE, ARTHUR C. Comparative sensitivity of 
sperm and eggs to ultraviolet radiations, 81. 



JLJJABROBRACON, a strongly intersexual 
female in, 243. 

HALL, C. E. See M. A. JAKUS, 141. 

Histological study of Syndisyrinx franciscanus, 
~-an endoparasitic rhabdocoel of the sea 
urchin, 295 

Hormones (CDH and CBLH) in Crustacean 
nervous systems, the comparative distribu- 
tion of two chromatophorotropic, 170. 

INFLUENCE of texture and composition of 
surface on the attachment of sedentary 
marine organisms, 57. 

JAKUS, M. A., AND C. E. HALL. Electron 
microscope observations of trichocysts and 
cilia of Paramecium, 141. 

L OLLROS, J. J. See]. M. TOBIAS, 247. 
KOZLOFF, EUGENE N. Studies on ciliates 
of the family Ancistrocomidae Chatton and 
Lwoff (order Holotricha, suborder Thig- 
motricha). III. Ancistrocoma pelseneeri 
Chatton and Lwoff, Ancistrocoma dissimi- 
lis sp. nov., and Hypocomagalma phola- 
didis sp. nov., 189. 

KOZLOFF, EUGENE N. Studies on ciliates of 
the family Ancistrocomidae Chatton and 
Lwoff (order Holotricha, suborder Thig- 
motricha). IV. Heterocineta janickii Ja- 
rocki, Heterocineta goniobasidis sp. nov., 
Heterocineta fluminicolae sp. nov., and 
Enerthecoma properans Jarocki, 200. 

[EHMAN, H. E. A histological study of 
Syndisyrinx franciscanus, gen. et sp. nov., 
an endoparasitic rhabdocoel of the sea 
urchin, Strongylocentrotus franciscanus, 
295. 

Lymnaea stagnalis appressa Say, observations 
on the functioning of the alimentary sys- 
tem of the snail, 88. 

IV/f ARINE Biological Laboratory, annual re- 
port, 1. 

Marine Biological Laboratory, program and 
abstracts of scientific papers presented, 
summer of 1946, 210. 

MORRISON, PETER R. Physiological observa- 
tions on water loss and oxygen consump- 
tion in Peripatus, 181. 



327 



328 



INDEX 



/~\XYGEN consumption and water loss in 
Peripatus, 181. 

Oxygen consumption and activity of the gold- 
fish, its application to the measurement of 
respiratory metabolism in fishes, 312. 

DARAMECIUM bursaria, temporary pair 
formation in, 112. 

Paramecium, electron microscope observations 
of the trichocysts and cilia of, 141. 

PEASE, DANIEL C. Hydrostatic pressure ef- 
fects upon the spindle figure and chromo- 
some movement. II. Experiments on the 
meiotic divisions of Tradescantia pollen 
mother cells, 145. 

POMERAT, C. M., AND C. M. WEISS. The in- 
fluence of texture and composition of sur- 
face on the attachment of sedentary 
marine organisms, 57. 

CAIGH, LORRAINE M. .See FRANK A. BROWN, 
JR., 170. 

SCOTT, ALLAN. The effect of low temperature 
and of hypotonicity on the morphology of 
the cleavage furrow in Arbacia eggs, 272. 

SCOTT, SISTER FLORENCE MARIE. The de- 
velopmental history of Amaroecium con- 
stellatum. II. Organogenesis of the larval 
action system, 66. 

Society of General Physiologists, papers pre- 
sented at the meeting of, 236. 

Spindle figure and chromosome movement, 
hydrostatic pressure effects upon, 145. 



SPOOR, W. A. A quantitative study of the 
relationship between the activity and 
oxygen consumption of the goldfish, and 
its application to the measurement of 
respiratory metabolism in fishes, 312. 

Studies on ciliates of the family Ancistro- 
comidae Chatton and Lwoff (order Holo- 
tricha, suborder Thigmotricha). III. An- 
cistrocoma pelseneeri Chatton and Lwoff, 
Ancistrocoma dissimilis sp. nov., and Hy- 
pocomagalma pholadidis sp. nov., 189. 

Studies on ciliates of the family Ancistro- 
comidae Chatton and Lwoff (order Holo- 
tricha, suborder Thigmotricha). IV. 
Heterocineta janickii Jarocki, Heterocineta 
goniobasidis sp. nov., Heterocineta flumini- 
colae sp. nov., and Enerthecoma properans 
Jarocki, 200. 

nPILLINA magna: Micronuclear number, 

encystment and vitality in diverse clones; 

capabilities of amicronucleate races, 256. 
TOBIAS, J. M., AND J. J. KOLLROS. Loci of 

action of DDT in cockroach (Periplaneta 

americana), 247. 



u 



LTRAVIOLET radiations, comparative 
sensitivity of sperm and eggs to, 81. 

EISS, C. M. See C. M. POMERAT, 57. 
WEISZ, PAUL B. The space- time pattern 
of segment formation in Artemiasalina, 119. 
WHITING, P. W. A strongly intersexual female 
in Habrobracon, 243. 



Volume 91 



<LS (J 



Number 1 



THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 



E. G. CONKLIN, Princeton University 
E. N. HARVEY, Princeton University 
SELIG HECHT, Columbia University 
LEIGH HOADLEY, Harvard University 
L. IRVING, Swarthmore College 
M. H. JACOBS, University of Pennsylvania 
H. S. JENNINGS, Johns Hopkins University 



FRANK R. LILLIE, University of Chicago 
CARL R. MOORE, University of Chicago 
GEORGE T. MOORE, Missouri Botanical Garden 
G. H. PARKER, Harvard University 
A. C. REDFIELD, Harvard University 
F. SCHRADER, Columbia University 
DOUGLAS WmTAKER, Stanford University 



H. B. STEINBACH, Washington University 
Managing Editor 



AUGUST, 1946 



Marine Biological lal>\>; 



LIB S* 



SEP 1 

WOODS HOIE, MASS. 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE & LEMON STS. 

LANCASTER, PA. 



BOOKS-WAR VICTIMS 



D 



'URING THE WAR, the libraries of half the world were destroyed in the 
fires of battle and in the fires of hate and fanaticism. Where they were spared 
physical damage, they were impoverished by isolation. There is an urgent need 
now for the printed materials which are basic to the reconstruction of dev- 
astated areas and which can help to remove the intellectual blackout of Europe 
and the Orient. 

There is need for a pooling of resources, for coordinated action in order that 
the devastated libraries of the world may be restocked as far as possible with 
needed American publications. The American Book Center for War Devastated 
Libraries, Inc., has come into being to meet this need. It is a program that is 
born of the combined interests of library and educational organizations, of gov- 
ernment agencies, and of many other official and non-official bodies in the United 
States. 

The American Book Center is collecting and is shipping abroad scholarly 
books and periodicals which will be useful in research and necessary in the 
physical, economic, social and industrial rehabilitation and reconstruction of 
Europe and the Far East. 

The Center cannot purchase books and periodicals ; it must depend upon gifts 
from individuals, institutions, and organizations. Each state will be organized 
to participate in the program through the leadership of a state chairman. Other 
chairmen will organize interest in the principal subject fields. Cooperation with 
these leaders or direct individual contributions are welcomed. 

WHAT IS NEEDED: Shipping facilities are precious and demand that 
all materials be carefully selected. Emphasis is placed upon publications issued 
during the past decade, upon scholarly books which are important contributions 
to their fields, upon periodicals (even incomplete volumes) of significance, upon 
fiction and non-fiction of distinction. All subjects history, the social sciences, 
music, fine arts, literature, and especially the sciences and technologies are 
wanted. 

WHAT IS NOT NEEDED: Textbooks, out-dated monographs, recrea- 
tional reading, books for children and young people, light fiction, materials of 
purely local interest, popular magazines such as Time, Life, National Geographic, 
etc., popular non-fiction of little enduring significance such as Gunther's Inside 
Europe, Haliburton's Royal Road to Romance, etc. Only carefully selected 
federal and local documents are needed, and donors are requested to write 
directly to the Center with regard to specific documents. 

HOW TO SHIP: All shipments should be sent PREPAID via the 
cheapest means of transportation to THE AMERICAN BOOK CENTER, 
C/O THE LIBRARY OF CONGRESS, WASHINGTON 25, D. C. Al- 
though the Center hopes that donors will assume the costs of transportation of 
their materials to Washington, when this is not possible reimbursement will be 
made upon notification by card or letter of the amount due. THE CENTER 
CANNOT ACCEPT MATERIAL WHICH IS SENT COLLECT. Reim- 
bursement cannot be made for packing or other charges beyond actual transpor- 
tation. When possible, periodicals should be tied together by volume. It will 
be helpful if missing issues are noted on incomplete volumes. 



Your Biological News 

You would not go to the library to read the daily newspaper probably 
you have it delivered at your home to be read at your leisure. Why, then, 
depend upon your library for your biological news ? 

Biological Abstracts is news nowadays. Abridgments of all the im- 
portant biological literature are published promptly in many cases before 
the original articles are available in this country. Only by having your 
own copy of Biological Abstracts to read regularly can you be sure that 
you are missing none of the literature of particular interest to you. An 
abstract of one article alone, which otherwise you would not have seen, 
might far more than compensate you for the subscription price. 

Biological Abstracts is now published in seven low priced sections, as 
well as the complete edition, so that the biological literature may be avail- 
able to all individual biologists. Write for full information and ask for a 
copy of the section covering your field. 

BIOLOGICAL ABSTRACTS 

University of Pennsylvania 

Philadelphia, Pa. 



MICROFILM SERVICE 



The Library of The Marine 
Biological Laboratory can 
supply microfilms of ma- 
terial from periodicals in- 
cluded in its list. Requests 
should include the title of 
the paper, the author, peri- 
odical, volume and date of 
publication. 



Rates are as follows: $.30 for 
papers up to 25 pages, and $.10 
for each additional 10 pages or 
fraction thereof. 



LANCASTER PRESS, Inc. 

LANCASTER, PA. 



THE EXPERIENCE we have 
gained from printing some 
sixty educational publica- 
tions has fitted us to meet 
the standards of customers 
who demand the best. 

We shall be happy to have workers at 

the MARINE BIOLOGICAL LABORATORY 
write for estimates on journals or 
monographs. Our prices are moderate. 



INSTRUCTIONS TO AUTHORS 

The Biological Bulletin accepts papers on a variety of subjects of biologi- 
cal interest. In general, a paper will appear within three months of the date of 
its acceptance. The Editorial Board requests that manuscripts conform to the 
requirements set below. 

Manuscripts. Manuscripts should be typed in double or triple spacing on 
one side of paper, 8Vz by 11 inches. 

Tables should be typewritten on separate sheets and placed in correct 
sequence in the text. Explanations of figures should be typed on a separate 
sheet and placed at the end of the text. Footnotes, numbered consecutively, 
may be placed on a separate sheet at the end of the paper. 

A condensed title or running page head of not more than thirty-five letters 
should be included. 

Figures. The dimensions of the printed page, 5 by 7% inches, should be 
kept in mind in preparing figures for publication. Illustrations should be large 
enough so that all details will be clear after appropriate reduction. Explana- 
tory matter should be included in legends as far as possible, not lettered on the 
illustrations. Figures should be prepared for reproduction as line cuts or half- 
tones; other methods will be used only at the author's expense. Figures to be 
reproduced as line cuts should be drawn in black ink on white paper or blue- 
lined co-ordinate paper; those to be reproduced as halftones should be mounted 
on Bristol board and any designating letters or numbers should be made di- 
rectly on the figures. The author's name should appear on the reverse side of 
all figures. The desired reduction should be specified on each figure. 

Literature cited. The list of literature cited should conform to the style set 
in this issue of The Biological Bulletin. Papers referred to in the manuscript 
should be listed on separate pages headed "Literature Cited." 

Mailing. Manuscripts should be packed flat. Large illustrations may be 
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must 
be accompanied by photographic reproductions or tracings that may be folded 
to page size. 

Reprints. Authors will be furnished, free of charge, one hundred reprints 
without covers. Additional copies may be obtained at cost; approximate 
figures will be furnished upon request. 



THE BIOLOGICAL BULLETIN 

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

Subscriptions and similar matter should be addressed to The Biologi- 
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. 
Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, 
$1.75. Subscription per volume (three issues), $4.50. 

Communications relative to manuscripts should be sent to the Manag- 
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, 
between July 1 and September 1, and to the Department of Zoology, 
Washington University, St. Louis, Missouri, during the remainder of 
the vear. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., 

under the Act of August 24, 1912. 



BIOLOGY MATERIALS 

The Supply Department of the Marine Biological Labora- 
tory has a complete stock of excellent plain preserved and 
injected materials, and would be pleased to quote prices on 
school needs. 



PRESERVED SPECIMENS 

for 

Zoology, Botany, Embryology, 
and Comparative Anatomy 

LIVING SPECIMENS 

for 
Zoology and Botany 

including Protozoan and 
Drosophila Cultures, and 
Animals for Experimental and 
Laboratory Use. 

MICROSCOPE SLIDES 

for 

Zoology, Botany, Embryology, 
Histology, Bacteriology, and 
Parasitology. 

CATALOGUES SENT ON REQUEST 



Supply Department 

MARINE 
BIOLOGICAL LABORATORY 

Woods Hole, Massachusetts 



CONTENTS 



Page 
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY. ... i 

POMERAT, C. M. AND C. M. WEISS 

The influence of texture and composition of surface on the 
attachment of sedentary marine organisms 57 

SCOTT, SISTER FLORENCE MARIE 

The developmental history of Amaroecium constellatum. II. 
Organogenesis of the larval action system 66 

GIESE, ARTHUR C. 

Comparative sensitivity of sperm and eggs to ultraviolet 
radiations 81 

CARRIKER, MELBOURNE ROMAINE 

Observations on the functioning of the alimentary system of 
the snail Lymnaea stagnalis appressa Say 88 

CHEN, TZE-TUAN 

Temporary pair formation in Paramecium bursaria 112 



Volume 91 



Number 2 



THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 



Editorial Board 



E. G. CONKLIN, Princeton University 
E. N. HARVEY, Princeton University 
SELIG HECHT, Columbia University 
LEIGH HOADLEY, Harvard University 
L. IRVING, Swarthmore College 
M. H. JACOBS, University of Pennsylvania 
H. S. JENNINGS, Johns Hopkins University 



FRANK R. LILLIE, University of Chicago 
CARL R. MOORE, University of Chicago 
GEORGE T. MOORE, Missouri Botanical Garden 
G. H. PARKER, Harvard University 
A. C. REDFIELD, Harvard University 
F. SCHRADER, Columbia University 
DOUGLAS WHITAKER, Stanford University 



H. B. STEINBACH, Washington University 
Managing Editor 



Marine Biological Labofototy 

L.I Ell A. R Y 

NOV 181946 

WOODS HOLE, MASS. 



OCTOBER, 1946 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE & LEMON STS. 

LANCASTER, PA. 



BOOKS-WAR VICTIMS 



'URING THE WAR, the libraries of half the world were destroyed in the 
fires of battle and in the fires of hate and fanaticism. Where they were spared 
physical damage, they were impoverished by isolation. There is an urgent need 
now for the printed materials which are basic to the reconstruction of dev- 
astated areas and which can help to remove the intellectual blackout of Europe 
and the Orient. 

There is need for a pooling of resources, for coordinated action in order that 
the devastated libraries of the world may be restocked as far as possible with 
needed American publications. The American Book Center for War Devastated 
Libraries, Inc., has come into being to meet this need. It is a program that is 
born of the combined interests of library and educational organizations, of gov- 
ernment agencies, and of many other official and non-official bodies in the United 
States. 

The American Book Center is collecting and is shipping abroad scholarly 
books and periodicals which will be useful in research and necessary in the 
physical, economic, social and industrial rehabilitation and reconstruction of 
Europe and the Far East. 

The Center cannot purchase books and periodicals ; it must depend upon gifts 
from individuals, institutions, and organizations. Each state will be organized 
to participate in the program through the leadership of a state chairman. Other 
chairmen will organize interest in the principal subject fields. Cooperation with 
these leaders or direct individual contributions are welcomed. 

WHAT IS NEEDED: Shipping facilities are precious and demand that 
all materials be carefully selected. Emphasis is placed upon publications issued 
during the past decade, upon scholarly books which are important contributions 
to their fields, upon periodicals (even incomplete volumes) of significance, upon 
fiction and non-fiction of distinction. All subjects history, the social sciences, 
music, fine arts, literature, and especially the sciences and technologies are 
wanted. 

WHAT IS NOT NEEDED: Textbooks, out-dated monographs, recrea- 
tional reading, books for children and young people, light fiction, materials of 
purely local interest, popular magazines such as Time, Life, National Geographic, 
etc., popular non-fiction of little enduring significance such as Gunther's Inside 
Europe, Haliburton's Royal Road to Romance, etc. Only carefully selected 
federal and local documents are needed, and donors are requested to write 
directly to the Center with regard to specific documents. 

HOW TO SHIP: All shipments should be sent PREPAID via the 
cheapest means of transportation to THE AMERICAN BOOK CENTER, 
C/O THE LIBRARY OF CONGRESS, WASHINGTON 25, D. C. Al- 
though the Center hopes that donors will assume the costs of transportation of 
their materials to Washington, when this is not possible reimbursement will be 
made upon notification by card or letter of the amount due. THE CENTER 
CANNOT ACCEPT MATERIAL WHICH IS SENT COLLECT. Reim- 
bursement cannot be made for packing or other charges beyond actual transpor- 
tation. When possible, periodicals should be tied together by volume. It will 
be helpful if missing issues are noted on incomplete volumes. 



Your Biological News 

You would not go to the library to read the daily newspaper probably 
you have it delivered at your home to be read at your leisure. Why, then, 
depend upon your library for your biological news ? 

Biological Abstracts is news nowadays. Abridgments of all the im- 
portant biological literature are published promptly in many cases before 
the original articles are available in this country. Only by having your 
own copy of Biological Abstracts to read regularly can you be sure that 
you are missing none of the literature of particular interest to you. An 
abstract of one article alone, which otherwise you would not have seen, 
might far more than compensate you for the subscription price. 

Biological Abstracts is now published in seven low priced sections, as 
well as the complete edition, so that the biological literature may be avail- 
able to all individual biologists. Write for full information and ask for a 
copy of the section covering your field. 

BIOLOGICAL ABSTRACTS 

University of Pennsylvania 

Philadelphia, Pa. 



MICROFILM SERVICE 

* 

The Library of The Marine 
Biological Laboratory can 
supply microfilms of ma- 
terial from periodicals in- 
cluded in its list. Requests 
should include the title of 
the paper, the author, peri- 
odical, volume and date of 
publication. 



Rates are as follows: $.30 for 
papers up to 25 pages, and $.10 
for each additional 10 pages or 
fraction thereof. 



LANCASTER PRESS, Inc. 

LANCASTER, PA. 



THE EXPERIENCE we have 
gained from printing some 
sixty educational publica- 
tions has fitted us to meet 
the standards of customers 
who demand the best. 

We shall be happy to have workers at 

the MARINE BIOLOGICAL LABORATORY 

write for estimates on journals or 
monographs. Our prices are moderate. 



INSTRUCTIONS TO AUTHORS 

The Biological Bulletin accepts papers on a variety of subjects of biologi- 
cal interest. In general, a paper will appear within three months of the date of 
its acceptance. The Editorial Board requests that manuscripts conform to the 
requirements set Below. 

Manuscripts. Manuscripts should be typed in double or triple spacing on 
one side of paper, SVz by 11 inches. 

Tables should be typewritten on separate sheets and placed in correct 
sequence in the text. Explanations of figures should be typed on a separate 
sheet and placed at the end of the text. Footnotes, numbered consecutively, 
may be placed on a separate sheet at the end of the paper. 

A condensed title or running page head of not more than thirty-five letters 
should be included. 

Figures. The dimensions of the printed page, 5 by 7% inches, should be 
kept in mind in preparing figures for publication. Illustrations should be large 
enough so that all details will be clear after appropriate reduction. Explana- 
tory matter should be included in legends as far as possible, not lettered on the 
illustrations. Figures should be prepared for reproduction as line cuts or half- 
tones; other methods will be used only at the author's expense. Figures to be 
reproduced as line cuts should be drawn in black ink on white paper or blue- 
lined co-ordinate paper; those to be reproduced as halftones should be mounted 
on Bristol board and any designating letters or numbers should be made di- 
rectly on the figures. The author's name should appear on the reverse side of 
all figures. The desired reduction should be specified on each figure. 

Literature cited. The list of literature cited should conform to the style set 
in this issue of The Biological Bulletin. Papers referred to in the manuscript 
should be listed on separate pages headed "Literature Cited." 

Mailing. Manuscripts should be packed flat. Large illustrations may be 
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must 
be accompanied by photographic reproductions or tracings that may be folded 
to page size. 

Reprints. Authors will be furnished, free of charge, one hundred reprints 
without covers. Additional copies may be obtained at cost; approximate 
figures will be furnished upon request. 



THE BIOLOGICAL BULLETIN 

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

Subscriptions and similar matter should be addressed to The Biologi- 
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. 
Agent for Great Britain : Wheldon and Wesley, Limited. 2, 3 and 4 
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, 
$1.75. Subscription per volume (three issues), $4.50. 

Communications relative to manuscripts should be sent to the Manag- 
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, 
between July 1 and September 1, and to the Department of Zoology, 
Washington University, St. Louis, Missouri, during the remainder of 
the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., 

under the Act of August 24, 1912. 



BIOLOGY MATERIALS 

The Supply Department of the Marine Biological Labora- 
tory has a complete stock of excellent plain preserved and 
injected materials, and would be pleased to quote prices on 
school needs. 



PRESERVED SPECIMENS 

for 
Zoology, Botany, Embryology, 

and Comparative Anatomy 

LIVING SPECIMENS 

for 
Zoology and Botany 

including Protozoan and 
Drosophila Cultures, and 
Animals for Experimental and 
Laboratory Use. 

MICROSCOPE SLIDES 

for 

Zoology, Botany, Embryology, 
Histology, Bacteriology, and 
Parasitology. 

CATALOGUES SENT ON REQUEST 



Supply Department 

MARINE 
BIOLOGICAL LABORATORY 

Woods Hole, Massachusetts 



CONTENTS 



Page 
WEISZ, P/vUL B. 

The space-time pattern of segment formation in Artemia 
salina 1 19 

JAKUS, M. A., AND C. E. HALL 

Electron microscope observations of the trichocysts and cilia 
of Paramecium 141 

PEASE, DANIEL C. 

Hydrostatic pressure effects upon the spindle figure and 
chromosome movement. II. Experiments on the meiotic 
divisions of Tradescantia pollen mother cells 145 

BROWN, FRANK A. JR., AND LORRAINE M. SAIGH 

The comparative distribution of two chromatophorotropic 
hormones (CDH and CBLH) in Crustacean nervous systems 170 

MORRISON, PETER R. 

Physiological observations on water loss and oxygen con- 
sumption in Peripatus 181 

KOZLOFF, EUGENE N. 

Studies on ciliates of the family Ancistrocomidae Chatton 
and Lwoff (order Holotricha, suborder Thigmotricha). III. 
Ancistrocoma pelseneeri Chatton and Lwoff, Ancistrocoma 
dissimilis sp. nov., and Hypocomagalma pholadidis sp. nov. . 189 

KOZLOFF, EUGENE N. 

Studies on ciliates of the family Ancistrocomidae Chatton 
and Lwoff (order Holotricha, suborder Thigmotricha). IV. 
Heterocineta janickii Jarocki, Heterocineta goniobasidis sp. 
nov., Heterocineta fluminicolae sp. nov., and Enerthecoma 
properans Jarocki 200 

ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE 

BIOLOGICAL LABORATORY, SUMMER OF 1946 210 

PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GEN- 
ERAL PHYSIOLOGISTS. 236 



Volume 91 







Number 3 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 



Editorial Board 



E. G. CONKLIN, Princeton University 

E. N. HARVEY, Princeton University 

SELIG HECHT, Columbia University 

LEIGH HOADLEY, Harvard University 

L. IRVING, Swarthmore College 

M. H. JACOBS, University of Pennsylvania 

H. S. JENNINGS, Johns Hopkins University 



FRANK R. LILLIE, University of Chicago 
CARL R. MOORE, University of Chicago 
GEORGE T. MOORE, Missouri Botanical Garden 
G. H. PARKER, Harvard University 
A. C. REDFffiLD, Harvard University 
F. SCHRADER, Columbia University 
DOUGLAS WHITAKER, Stanford University 



H. B. STEINBACH, Washington University 
Managing Editor 




DECEMBER, 1946 



JAN -7 -19-47 

WOODS HOLE, 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE & LEMON STS. 

LANCASTER, PA. 



BOOKS-WAR VICTIMS 



D, 



'URING THE WAR, the libraries of half the world were destroyed in the 
fires of battle and in the fires of hate and fanaticism. Where they were spared 
physical damage, they were impoverished by isolation. There is an urgent need 
now for the printed materials which are basic to the reconstruction of dev- 
astated areas and which can help to remove the intellectual blackout of Europe 
and the Orient. 

There is need for a pooling of resources, for coordinated action in order that 
the devastated libraries of the world may be restocked as far as possible with 
needed American publications. The American Book Center for War Devastated 
Libraries, Inc., has come into being to meet this need. It is a program that is 
born of the combined interests of library and educational organizations, of gov- 
ernment agencies, and of many other official and non-official bodies in the United 
States. 

The American Book Center is collecting and is shipping abroad scholarly 
books and periodicals which will be useful in research and necessary in the 
physical, economic, social and industrial rehabilitation and reconstruction of 
Europe and the Far East. 

The Center cannot purchase books and periodicals ; it must depend upon gifts 
from individuals, institutions, and organizations. Each state will be organized 
to participate in the program through the leadership of a state chairman. Other 
chairmen will organize interest in the principal subject fields. Cooperation with 
these leaders or direct individual contributions are welcomed. 

WHAT IS NEEDED: Shipping facilities are precious and demand that 
all materials be carefully selected. Emphasis is placed upon publications issued 
during the past decade, upon scholarly books which are important contributions 
to their fields, upon periodicals (even incomplete volumes) of significance, upon 
fiction and non-fiction of distinction. All subjects history, the social sciences, 
music, fine arts, literature, and especially the sciences and technologies are 
wanted. 

WHAT IS NOT NEEDED: Textbooks, out-dated monographs, recrea- 
tional reading, books for children and young people, light fiction, materials of 
purely local interest, popular magazines such as Time, Life, National Geographic, 
etc., popular non-fiction of little enduring significance such as Gunther's Inside 
Europe, Haliburton's Royal Road to Romance, etc. Only carefully selected 
federal and local documents are needed, and donors are requested to write 
directly to the Center with regard to specific documents. 

HOW TO SHIP: All shipments should be sent PREPAID via the 
cheapest means of transportation to THE AMERICAN BOOK CENTER, 
C/O THE LIBRARY OF CONGRESS, WASHINGTON 25, D. C. Al- 
though the Center hopes that donors will assume the costs of transportation of 
their materials to Washington, when this is not possible reimbursement will be 
made upon notification by card or letter of the amount due. THE CENTER 
CANNOT ACCEPT MATERIAL WHICH IS SENT COLLECT. Reim- 
bursement cannot be made for packing or other charges beyond actual transpor- 
tation. When possible, periodicals should be tied together by volume. It will 
be helpful if missing issues are noted on incomplete volumes. 



Your Biological News 

You would not go to the library to read the daily newspaper probably 
you have it delivered at your home to be read at your leisure. Why, then, 
depend upon your library for your biological news ? 

Biological Abstracts is news nowadays. Abridgments of all the im- 
portant biological literature are published promptly in many cases before 
the original articles are available in this country. Only by having your 
own copy of Biological Abstracts to read regularly can you be sure that 
you are missing none of the literature of particular interest to you. An 
abstract of one article alone, which otherwise you would not have seen, 
might far more than compensate you for the subscription price. 

Biological Abstracts is now published in seven low priced sections, as 
well as the complete edition, so that the biological literature may be avail- 
able to all individual biologists. Write for full information and ask for a 
copy of the section covering your field. 

BIOLOGICAL ABSTRACTS 

University of Pennsylvania 

Philadelphia, Pa. 



MICROFILM SERVICE 



The Library of The Marine 
Biological Laboratory can 
supply microfilms of ma- 
terial from periodicals in- 
cluded in its list. Requests 
should include the title of 
the paper, the author, peri- 
odical, volume and date of 
publication. 



Rates are as follows: $.30 for 
papers up to 25 pages, and $.10 
for each additional 10 pages or 
fraction thereof. 



LANCASTER PRESS, Inc. 

LANCASTER, PA. 



THE EXPERIENCE we have 
gained from printing some 
sixty educational publica- 
tions has fitted us to meet 
the standards of customers 
who demand the best. 

We shall be happy to have workers at 

the MARINE BIOLOGICAL LABORATORY 

write for estimates on journals or 
monographs. Our prices are moderate. 



INSTRUCTIONS TO AUTHORS 

The Biological Bulletin accepts papers on a variety of subjects of biologi- 
cal interest. In general, a paper will appear within three months of the date of 
its acceptance. The Editorial Board requests that manuscripts conform to the 
requirements set below. 

Manuscripts. Manuscripts should be typed in double or triple spacing on 
one side of paper, SVz by 11 inches. 

Tables should be typewritten on separate sheets and placed in correct 
sequence in the text. Explanations of figures should be typed on a separate 
sheet and placed at the end of the text. Footnotes, numbered consecutively, 
may be placed on a separate sheet at the end of the paper. 

A condensed title or running page head of not more than thirty-five letters 
should be included. 

Figures. The dimensions of the printed page, 5 by 7% inches, should be 
kept in mind in preparing figures for publication. Illustrations should be large 
enough so that all details will be clear after appropriate reduction. Explana- 
tory matter should be included in legends as far as possible, not lettered on the 
illustrations. Figures should be prepared for reproduction as line cuts or half- 
tones; other methods will be used only at the author's expense. Figures to be 
reproduced as line cuts should be drawn in black ink on white paper or blue- 
lined co-ordinate paper; those to be reproduced as halftones should be mounted 
on Bristol board and any designating letters or numbers should be made di- 
rectly on the figures. The author's name should appear on the reverse side of 
all figures. The desired reduction should be specified on each figure. 

Literature cited. The list of literature cited should conform to the style set 
in this issue of The Biological Bulletin. Papers referred to in the manuscript 
should be listed on separate pages headed "Literature Cited." 

Mailing. Manuscripts should be packed flat. Large illustrations may be 
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must 
be accompanied by photographic reproductions or tracings that may be folded 
to page size. 

Reprints. Authors will be furnished, free of charge, one hundred reprints 
without covers. Additional copies may be obtained at cost: approximate 
figures will be furnished upon request. 



THE BIOLOGICAL BULLETIN 

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

Subscriptions and similar matter should be addressed to The Biologi- 
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. 
Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, 
$1.75. Subscription per volume (three issues), $4.50. 

Communications relative to manuscripts should be sent to the Manag- 
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, 
between July 1 and September 1, and to the Department of Zoology, 
Washington University. St. Louis. Missouri, during the remainder of 
the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., 

under the Act of August 24, 1912. 



BIOLOGY MATERIALS 

The Supply Department of the Marine Biological Labora- 
tory has a complete stock of excellent plain preserved and 
injected materials, and would be pleased to quote prices on 
school needs. 



PRESERVED SPECIMENS 

for 

Zoology, Botany, Embryology, 
and Comparative Anatomy 

LIVING SPECIMENS 

for 
Zoology and Botany 

including Protozoan and 
Drosophila Cultures, and 
Animals for Experimental and 
Laboratory Use. 

MICROSCOPE SLIDES 

for 

Zoology, Botany, Embryology, 
Histology, Bacteriology, and 
Parasitology. 

CATALOGUES SENT ON REQUEST 



Supply Department 

MARINE 
BIOLOGICAL LABORATORY 

Woods Hole, Massachusetts 







CONTENTS 



' ' 



Page 
WHITING, P. W. 

A strongly intersexual female in Habrobracon ............. 243 

TOBIAS, J. M., AND J. J. KOLLROS 

Loci of action of DDT in the cockroach (Periplaneta ameri- 
cana) ............................................. 247 

BEERS, C. D. 

Tillina magna: Micronuclear number, encystment and vitality 

in diverse clones; capabilities of amicronucleate races ...... 256 

SCOTT, ALLAN 

The effect of low temperature and of hypotonicity on the 
morphology of the cleavage furrow in Arb'acia eggs ........ 272 

BODENSTEIN, DIETRICH 

Developmental relations between genital ducts and gonads 

in Drosophila ........ ^ ................................. 288 

LEHMAN, H. E. 

A histological study of Syndisyrinx franciscanus, gen. et sp. 
nov., an endoparasitic rhabdocoel of the sea urchin, Strongylo- 
centrotus franciscanus ................................. 295 

SPOOR, W. A. 

A quantitative study of the relationship between the activity 
and oxygen consumption of the goldfish, and its application 
to the measurement of respiratory metabolism in fishes. ... 312 



J 

' 






. VVHOI LIBRARY 



UH 17JA D